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Editorial CHICAGO MEDICAL SOCIETY. Jan. 18th. The regular meeting of the Society was almost wholly occupied with the subject of City Health Regulations. Dr. N. S. Davis, from the Special Committee on that subject, made the following report, which, after free discussion, was adopted. The Committee was instructed to present the same to the Common Council, with the draft of an amendment to the present laws, as indicated in the report. REPORT OF THE COMMITTEE ON THE HEALTH DEPARTMENT OF OUR CITY GOVERNMENT. Presented to the Chicago Medical Society, January 18th, 1867. The subject assigned to your Committee for consideration, is certainly one of the most important that can engage the atten- tion of the people of this, or any other large city. The adop- tion and enforcement of proper sanitary regulations is so closely connected with the preservation of health and the prolongation of life and happiness, that no community can ne,gleet them without, sooner or later, suffering the severest penalties. In fulfilling the duties assigned to your Committee, three questions require careful consideration. 1st. What are principles upon which a Municipal Health De- partment should be organized, in order to ensure the highest degree of enlightinent and efficient action? 2d. Is the present organization of the Health Department of our city defective; and if so, in what particulars? 3d. What changes are necessary to remedy its defects, and complete its efficiency? In answer to the first question, we would state, that the basis of a proper Health Organization, should consist of a Board of Commissioners, embodying in its members a high degree of ex- ecutive ability or business capacity, and a thorough knowledge of sanitary science, as applied to the preservation of the health of communities and the prevention of the spread of contagious diseases. No board can embody these qualities, without be- ing composed in part, at least, of thoroughly-educated physi- cians; simply because, In our country, no other class of men are educated to an adequate extent, in reference to those sani- tary subjects on which a Board of Health must continually act. The truth of this proposition is too plain to need either argu- ment or illustration. A Board of Health Commissioners, composed of properly educated and qualified members, should be invested with suffi- cient power to devise and carry into prompt effect, all such measures as the safety and welfare of the community requires. The term of office of its members should be long enough to in- sure a reasonable degree of permanency in whatever sanitary measures or policy may be adopted. In no department of municipal affairs, is a vaccillating, temporizing policy more dis- astrous than in that relating to the preservation of the public health. A show of energy, or a display of zeal, or a prodigal expenditure of money just while some fearful epidemic disease is threatening to commence its work of death, and a negligent indifference at all other times, is but little better than no action at all. Sanitary measures, to be successful in lessening the sickness and mortality of communities, and in protecting them from the ravages of epidemics, must be founded on an accurate knowledge of all those local causes that tend to deteriorate the public health, and then they must be rigidly and perseveringly enforced from year to year. To enable a Board of Health Commissioners to devise such measures, and establish an enlightened and permanent sanitary policy, its members should not only possess the requisite educa- tion and business capacity, with a term of office long enough to add experience to their previous knowledge, but they should be appointed in such a manner as to free them as far as possible from all partisanship, or feelings of dependence on the success or failure of mere political parties. And yet they should be fully accountable to the municipal government for all their official acts. Such are the principles on which a Board of Health Commissioners should be organized. The next question is, how far the present health department of our city government is organized in accordance with these principles, and in what particulars is it defective? Our present Health Department consists of a Board of three Commissioners, who are also at the same time Commissioners of the Police and Fire Departments; a Health-Officer; and a City Physician. The three members of the Board are elected by the people, one from each division of the city, and hold their offices for six years. The Health-Officer is appointed by the Board. The City Physician is elected by the Common Council, and holds office for two years. The present city charter confers on the Common Council power "to do all acts and make all regulations which may be necessary or expedient, for the preservation of health, and the suppression of disease." And, in accordance with this power, we find in the present laws and ordinances of the city full and ample powers conferred and duties enjoined upon the Board of of Health and Health-Officer, for the efficient and thorough regulation of everything relating to the public health and safety. After a full examination of the present laws and ordi- nances, we can see no need of further legislation, so far as relates to the conferring of power upon the Board of Health or its agents. Wherein, then, is the present organization defective? Chiefly in three important particulars. The first and most radical de- fect is, the absence of all provision for securing the selection of members of the Board of Health so thoroughly educated in san- itary matters as to fit them for an enlightened and efficient dis- charge of their duties. To make up a Board of Health, charged with the duty of making and enforcing all such regulations as are necessary to mitigate or prevent the prevalence of diseases in a great city, of men who, neither by education, occupation, nor business habits, have acquired any special knowledge of the nature and causes of disease, or of the laws by which they are developed and diffused, is just as absurd as it would be to elect a practising physician to the office of City Attorney. If there is lack of efficiency in the practical working of our pres- ent health organization, it is not because the members of the present Board of Police, acting as a Board of Health, are defi- cient in integrity or business capacity, or from deficient legal authority to-act, but simply because they have not that special sanitary or medical education which enables them to compre- hend clearly the nature, extent, and importance of the work entrusted to them. The second defect consists in an imperfect and injudicious distribution of duties between the Health-Officer, City Physi- cian, and Clerk of the Board of Health. The Health-Officer shall be simply an intelligent and efficient executive officer, to superintend the prompt enforcement of all the laws and orders of the Board of Health. The City Physician should keep at* all times a free vaccine dispensary for the poor, and have the immediate charge or superintendence of such sick persons as came under the care and authority of the Health and Police Departments of the City Government. And there should be a competent Clerk, who should perform the duties of Secretary to the Board of Health and the Health-Officer, and also the duties of Register of Vital Statistics. The third defect in the present organization is the absence of efficient regulations for the accurate registry of births, mar- riages and deaths, with the causes of the latter. The impor- tance of this needs no comment here. After a somewhat careful and candid examination of the whole subject, we feel certain that whatever inefficiency or defectiveness there is in the practical working of the present health organization of our city, can be traced directly or indirectly to the three sources just enumerated. This leads us to the third and last question' involved in this report, namely: What changes are necessary to remedy the defects to which we have alluded? We answer, so far as State legislation is concerned, we need but a single brief amendment to the laws now in force. If the present law relating to the action of the Board of Police, in the capacity of a Board of Health, was so amended as to provide for the ap- pointment of three thoroughly competent Physicians, (one from each Division of the City,) to act with said Police Commission- ers in all matters pertaining to health or sanitary regulations, to possess coequal powers and duties, and to constitute a part of said Board whenever acting in the capacity of a Board of Health, it would be all the legislation really required. This done, the other defects pointed out could be easily remedied under the authority already possessed by the Common Council and Board of Health. While the proposed amendment should leave the present mode of electing the three Police Commis- sioners undisturbed, it should provide some other and less po- litical method of appointing the three medical members of the Board acting as a Board of Health. Those members of our profession who have acquired that education, experience, and 'reputation, which would enable them to impart that kind of in- telligence and efficiency to a Board of Health, imperatively needed in all large cities, will never be found seeking nomina- tions from political parties, nor lobbying for appointments by a Governor and Senate, or a Mayor and Council. They must be sought ought and invited to accept the position, or their ser- vices will not be obtained. We would suggest whether this would not be done more judiciously and with less reference to anything of a political or partisan character, by the Judges of the Superior Court of this city, than by any other authority. It will be seen that the defects we have pointed out and the remedies proposed are few and simple; yet they are really of vital importance to the practical working and beneficial re- sults of the Health Department of our city. Should the views thus far expressed in this report meet the approbation of this Society, an amendment could be pre- pared in due form, relating to the appointment of three com- petcnt medical men as members of the Board of Health, and an ordinance in relation to the proper registry of vital statistics, and we have no doubt but both would meet the prompt sanction of the Mayor and Council. Before conclud- ing this report, it may be expected that some notice will be taken of the several amendments and health bills which have already been prepared and placed before the Legislature, the Council, and the public. There are three separate projects of this kind. The first consists in amendments to the present laws prepared and recommended by the Common Council. The only item in these, that relates to the important defects we have explained, is the section proposing to give the Common Council power, in cases of imminent danger from the preva- lence of epidemic diseases, to appoint an additional number of members of the Board of Health, to act as such simply while such danger lasts. But it is not provided that even these tem- porary appointees shall be medical men. And hence it does not in any degree remedy the radical defect in the constitution of of the present Board. The second project is in the form of a bill recently introduced into the State Senate by Senator Ward. By the provisions of this bill, everything pertaining to the Health Department, and sanitary regulations, is removed from the control of the people of the city and its municipal govern- ment, and placed in the hands of a Board of Commissioners, consisting of the President of the Board of Police, and two Commissioners appointed by the Governor and Senate, one of whom must be a physician. The two persons thus appointed by the Governor and Senate are to hold office four years, and, with the President of the Board of Police, constitute a Board of Health, with full power to make all needful rules, regula- tions, and appointments of agents, etc., sufficient to devise and execute a complete and independent sanitary system. There are two leading and fatal objections to this bill. The first is, that it deprives the people of this city of all power to regulate and control some of their most important local interests, and assumes that a Governor and Senate, at Springfield, are -better qualified to judge of the capacities and qualifications for office, of citizens of Chicago, than are the local authorities of the city, or the people themselves. The second, and more impor- tant objection is, that it creates a large number of new and ad- ditional officers and employes, requiring a regular annual addi- tional tax upon the city, of from $75,000 to $150,000, without regard to any extraordinary expenditures on account of the prevalence of severe epidemics. And yet it does not provide for the accomplishment of a single valuable object, that would not be as well and certainly accomplished, by simply adding hree thoroughly competent physicians to our present Board of Health. Serious objections could also be made to several items in the details of this bill. For instance, the section relating to the registration of births, marriages, and deaths, is utterly worthless. The third project is that presented by the Citizens' Commit- tee, and entitled the "Metropolitan Health Bill." This is amenable to precisely the same objections as we have made to the bill introduced by Senator Ward; while, practically, it would be far less efficient. Like Ward's bill, it takes from the people and local authorities the selection of a Board of Health, and confers it on the Governor and Senate, at Springfield. Like Ward's bill, it creates a long list of new and additional city officers and employees, requiring a correspondingly large additional expenditure of money annually. But, unlike Ward's bill, it does not fix the salary of the members of the Board of Health, or provide for the appointment of a full corps of San- itary Police. It allows the Common Council to fix their sala- ries, thereby making the officers perform the difficult task of serving two masters; the Governor for their appointment, and the Council for their pay. Again, by depending in part upon the ordinary police for executing the orders of the Health Board, it places the police in the equally embarassing position of obe- dience to the orders of two separate and independent Boards-- a plan that never has and never will work satisfactorily in practice. In conclusion, we cannot refrain from again expressing the opinion, that a single amendment to our present laws, provid- ing for the addition of three competent and thoroughly edu- cated members of the medical profession to the present Board of Health, with the aid of one additional Clerk, as Register of Vital Statistics, would result in rendering our Health Depart- ment as efficient and beneficial to the interests of the city, as it could be by any number of new and expensive schemes. It would require but one police organization, capable of being in- creased or diminished, as public exigencies might require, and practically amenable to but one Board. It would require a larger ratio of medical to the non-medi- cal intelligence in the Board of Health, than is proposed in either Ward's bill, or that of the Citizens' Committee. In- stead of requiring an annual aggregate increased tax upon the city of $75,000 or $100,000 for salaries of new' officers and agents, it would require for the three additional members of the Board of Health and the Register of Vital Statistics an aggre- gate of not over $5,000. It would also,leave our City Govern- ment far less complicated in its details, and, in all its strictly local interests, under the control of its own citizens, where it properly belongs. All of which is respectfully submitted. N. S. DAVIS, ) R. C. HAMILL, V Committee. J. P. ROSS, J Medical Department of University of Michigan.--We have seen in some of our exchanges, a statement of the number of Medical Students in the University of Michigan, accom- panied by the assertion that the large number congregated there could not have been drawn thither by the small pecuniary charges, as the aggregate of attendance is greater, owing to the length of the Lecture-term, than in any other school in the North-west. The entire falsity of such a statement is shown by the following facts:-- The fee for admission to the Medical Department of the Michigan University is, for students in the State $10, from out of the State $20, paid but once. The Lecture-term being six months, allowing $25 per month for board, would make a total necessary expenditure for the full term of $160 for the student of the State, and $170 for students from other States. The Chicago Medical College, in this city, has a Lecture-term of full five months. If we put board here at the same rate as supposed for Ann Arbor, namely, $25 per month, the cost of attending the regular annual course here, would be as follows:-- Five months board, at $25 per month,------$ 125 00 Lecture fees,--------------------------------- 50 00 Matriculation fee,----------------------------- 5 00 Dissecting fee, ------------------------------- 5 00 Hospital fee,---------------------------------- 6 00 Total,_______________________________ $191 00 Clinical Items.--A subscriber wishes to know how to cure "an obstinate, tormenting, intolerable itching, of years stand- ing," either in the genital organs of the female or around the anus of the male. The following cases may answer his purpose:-- Case I. Mrs. B., a married lady, aged about 25 years, had been for several years subject to periodical attacks of puritus pudendi, or intolerable itching of the labia and vulva. She generally suffered most from it after the menstrual periods, and it was generally accompanied by a thin leucorrhoeal discharge. She was placed on the following treatment:-- 1^. Ext. Hyoscyamus,---------------------- SOgrs. Sulph Ferri,------------------------- 30grs. Pulv. Aloes,------------------------- 15grs. Blue Mass, -------------------------- lOgrs. Ext. Nux Vom.,----------------------- lOgrs. Mix and di vide, into thirty pills. One to betaken before breakfast and dinner each day. For local use she was directed a solution of borate of soda (borax), 5iij and sulphate of mor- phia, 20grs., in water, one pint, the vulva to be wet with it of- ten, and a small quantity injected into the vagina each night and morning. Under this treatment the patient recovered, without any return of the disease. Case II. Mrs. W., aged 40 years, had been very severely troubled with the same disease several months, without any re- gard to the menstrual periods. The same solution of borax and morphine, applied locally, and the use of eight drops of Fowlers's arsenical solution, taken before each meal, in a spoonful of sweetened water, and continued for about four weeks, resulted in a cure. Both patients were required to live on plain food and to avoid all stimulating drinks. Case III. Mr. G., aged 30 years, sanguine temperament and full habit, had pruritis of the anus for three years. Some- times the itching would be intolerable for several days and then better for a week or ten days at a time. On examination, the skin, over a circle of an inch, around the opening of the rec- tum, was thickened, slightly fissured, and whiter than usual, lie was directed to take eight drops of Donovan's solution be- fore each meal time, and the affected surface was wet with the liquid persulphate of iron, o.f the strength usually found in the drug stores, every third day. After four local applications he was entirely relieved. He continued to take the drops inter- nally for three weeks, and although more than one year has elapsed the disease has not returned. I recollect treating several cases of old chronic cases of pru- ritis ani sucessfully, by applying locally, each night and morn- ing, the following ointment: ly. Iodide Sulphur,--------------------------- 3ij Oil Tobacco, ----------+----------------2gtts. Simple Crete,--------------------------- SSij Attention should always be paid to the general health, and especially to. the condition of the digestive organs. ^iie Chair of Surgery in Rush Medical College.-- According to statements in the daily papers of this city, the chair of Surgery made vacant by the death of the late Prof. I). Brainard, has been filled by the appointment of Moses Gunn, of Detroit, Professor of Surgery in the University of Michigan. It is also stated that Prof. Gunn has accepted the appointment. City Mortality.--The following is the report of the Health- Officer of the mortality of the City of Chicago for the month of December, 1866: CAUSES OF DEATH. Accidents,---------------------- u Asthma,------------------------- 2 Bronchitis,____________________ 1 Burned,_________________________ 1 Cancer,------------------------- 2 Childbed,----------------------- 2 Cholera Infantum,_______________ 1 Consumption, ------------------ 42 Convulsions,____________________35 Croup,-------------------------- 7 Cold____________________________ 2 Chicken Pox,____________________ 1 Congestion of Brain,____________ 3 Congestion of Lungs,____________ 2 Decline,________________________ 1 Delirium Tremens,______________ 1 Diarrhoea,______________________ 5 Diphtheria,___________________<<. 15 Disease of Heart,-------------- 8 Disease of Liver,______________ 2 Disease of Lungs,______________ 7 Disease of Brain,______________ 6 Dropsy,_________________________ 7 Drowned,________________________ 2 Erysipelas, -------------------- 3 Fever, Childbed,---------------- 2 Fever, Remittent,_______________ 4 rever, scarlet,_______________ 13 Fever, Typhoid,---------------- 4 Fever, Typhus,_________________ 1 Fever, not stated,------------- 1 Hydrocephelus,_________________ 7 Inflammation of Kidneys,_______ 1 Inflammation of Bowels,________ 9 Inflammation of Lungs,_________ 6 Inflammation of Liver,_________ 1 Marasmus,______________________ 1 Old Age,--------k-------------- 15 Palsy,_______________________ 2 Pneumonia,_____________________ 7 Phthisis Pulmonalis,___________ 1 Spasms,________,_______________ 1 Spinal Meningitis,_____________ 1 Suffocation,___________________ 2 Small Pox,_____________________ 2 Suicide, ______________________ 1 Stillborn,____________________ 10 Teething,______*_______________ 3 Whooping-Cough.________________17 Gun shot Wound,________________ 1 #White Swelling,--------------- 1 Unknown,______________________ 33 Total,_________________309 ages of the DECEASED. -- Under o years, loo ; over o and under 1U years, 19; over 10 and under 20, 15; over 20 and under 30, 28; over 30 and under 40, 34; over 4o and under 50, 16; over 50 and under 60, 11; over 60 and under 70, 11; over 70 and under 80, 12; over 80 and under 90, 6; unknown, 4. Total, 309. N \TTVTTTFS Chicago,____________128 Other States,--------63 Belgium,------------- 1 Bohemia,------------- 2 Canada,__________1__ 2 England,____________ 5 Germany,------------47 Ireland,____________38 Norway,------------- 6 On the Sea,_________ 1 Sweden,------------- 5 Scotland,___________ 4 Wales,______________ 1 Unknown,____________ 6 Total,---309 DIVISIONS OF THE CITY. North,..... 75 | South,....105 | West,....129 | Total,... 309 Total number during the month of November,_______________ 382 Decrease from last month,-------------------------------- 73 Total number last year for the month of December,________ 333 Dr. Conneau.--It bus been remarked that nearly every profession but that of medicine was represented in the French Senate. This anomaly has struck the Emperor, it would Appear, as the Evenement announces that his Majesty's physician, Dr. Conneau, is to be promoted forthwith to a seat at the Luxem- bourg.

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CHICAGO MEDICAL EXAMINER. N. S. DAVIS, MD, Editor. VOL. VIII. JANUARY, 1867. NO. 1. wnninat (iHtnmim ARTICLE I. THE MECHANICAL TREATMENT NECESSARY IN INFLAMMATION OF THE KNEE JOINT; WITH A DESCRIPTION OF A NEW APPARATUS FOR MA- KING EXTENSION. By JULIEN S. SHERMAN, M.D., Chicago, Ill. The exposed position of this articulation, the thinness of the tissues surrounding, and the amount of labor performed by it, render it a very frequent seat of those diseases and deformities consequent upon injury. Inflammation of this joint is gener- ally more severe and disastrous than of most others, on account of its large size and the extent of the synovial sack involved in the disease. The well-known pathological fact, that inflamma- tion of joints is always followed by reflex contraction of the muscles in its vicinity, is well exemplified in the knee by the strong contraction of the powerful flexors, bending the leg fre- quently to a right angle with the thigh. The injurious effect of pressure is also well shown, and the necessity for its removal as urgent as in hip-disease. Specific inflammations artf sometimes met with, but are not as frequent as generally supposed, most cases being traumatic in their ori- gin. Yet inflammations occurring in constitutions either scrof- ulous or syphilitic, are more prone to suppuration and caries than when this element is absent. Notwithstanding the advantages of medical treatment, there are indications which must be overcome by mechanical means. They are not only necessary for subduing disease in its early developement, but are indispensable for the correction and prevention of deformities following in severe cases. Inflamma- tion of this joint, even in its first stage, is always accompanied by contraction of the flexors, aggravating the pain and increas- ing the pressure, thereby either wholly preventing, or greatly retarding, spontaneous recovery. This contraction increases with the violence of the inflammation, and should be overcome by tenotomy of all the tendons offering resistance to the exten- sion of the limb. If ether is administered, the particular ten- dons requiring division should be ascertained before anaesthesia is produced, as, when that stage is reached, the muscles become relaxed, and we may be at loss to determine where division is necessary. Special care must be taken to avoid wounding, the peroneal nerve, situated just internal to the biceps tendon. This tendon should be divided from without inwards, at the same time that extension is being made, the sheath and inner fibres of which will then be ruptured before the knife passes completely through, and all danger to the nerve avoided. The limb should then be placed upon an air-cushion, protected by oil-silk, in order that local dressings may be used without soil- ing the bedding. The relief of pressure in inflamed joints is the most important part of the treatment; the diseased surfaces must be separated to allow of recovery or to prevent unfavorable results. The means for accomplishing this desired end are strictly mechani- cal. Adhesive straps may be applied to the leg, below the knee, and the surfaces of the joints separated by making exten- sion with the pulley and weight, as in adhesive strap dressing for fractures of the thigh. The limb may be placed in the hor- izontal position or upon an inclined plane, as circumstances may indicate. This treatment will be found to greatly relieve the pain and entirely remove the pressure. If it be adopted at the outset of the attack, the flexing of the limb will be pre- vented and division of the tendons rendered unnecessary. Should suppuration occur and the joint become greatly dis- tended with pus, it should be carefully evacuated by means of a trochar, avoiding the admission of air into the synovial sack, and the extension persevered in. Moderate pressure upon the femoral artery is advised by good authority, as diminishing the flow of blood to the part, but it is difficult to maintain and fre- quently adds to the discomfort of the patient. The accompanying cuts represent an apparatus for making extension. It consists of a wooden socket, constructed to accu- rately fit the thigh and similar to those used for artificial legs, against this the counter-extension is made, and thus evenly dis- tributed over the thigh and tuberosity of the ischium. A steel rod is attached to each side of the socket, reaching to within a few inches of the ankle, and the two rods are joined behind by a broad band of sheet-iron, which is moulded to fit the posterior part of the leg; on the front, and joining the sheet-iron band, is a strap which, being buckled, holds the leg firmly in the appara- tus. It is applied as follows:--Six adhesive straps are cut, two inches in width at the top and tapering to one at the bot- tom, and should be long enough to reach from about one inch below the knee to the ankle; they are then applied to the.leg, as represented in the cut, and secured by a bandage; the socket is then placed upon the thigh, the strap at the bottom of the instrument buckled, and the lower ends of the adhesive plaster turned over the bottom of the instrument and also attached to buckles upon its sides. The amount of extension is regulated by the degree of tightness to which the straps are drawn. The figure upon the right represents the same apparatus, with the exception that the side rods are extended and fastened to the sole of the shoe, while the adhesive straps are also at- tached to the buckles upon the sides. This will allow the patient to walk about and bear all his weight upon the socket and not any upon the knee-joint. This modification renders it more applicable to the chronic inflammations, when we desire the patient to have out-door exercise, which he can enjoy with as much ease as the wearer of an artificial leg. The advantages gained by this mode of extension are, the large surface to which the counter-extending force is applied, its security, and the ease with which the pa- tient can tolerate it. When the socket is well fitted, there is no tendency for the instrument to rotate or twist upon the leg. This same principle can be used in the construction of numer- ous instruments for the relief of deformities of the knee.

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ARTICLE III. CASE OF OVARIOTOMY. By D. MASON, M.D., Prairie du Chien, Wis. Was called to see Mrs. II., October 21st, 1865, and found her suffering from ovarian dropsy. The history she gave of herself is briefly as follows:--She is now 52 years of age; first noticed a tumor in her right side, 26 years ago, which, at times, seemed to enlarge, and then rap- idly recede, and so continued until October, 1864, about 25 years from its first appearance, when a decided and steady growth commenced. In February, 1865, the growth increased more rapidly, and continued until October, when the distress from distension became almost insupportable. She had been attended during the summer by various eclectics, galvanists, mesmerists, and homoeopaths, but without any benefit. On the 21st of October, 1865, as stated above, I first saw her, inviting my friend, Dr. Conant, to accompany me. The diagnosis was clearly established, and I tapped immediately, drawing off 14 tbs. of gray, turbid fluid, which reduced the tumor to about one-third its former size, very much to the re- lief of the patient. Her general health had been very much reduced by her suffering through the summer, but rapidly im- proved after the tapping. I then explained to her the cer- tainty of its refilling, and proposed its removal; at the same time explaining to her the danger, and probabilities of success of the operation. On the 10th of January, 1866, I was called to see her again, and found her much improved in health, and in good spirits. She expressed herself ready for, and anxious to have the ope- ration done. I proposed the operation for the 18th. On the 17th, gave a full dose of castor oil, which thoroughly emptied the bowels. On the 18th, accompanied by Drs. Andros, Lowe, and IIazeltine, of McGregor, Iowa, and Dr. Conant, of Prairie du Chien, I proceeded to operate. Having the tem- perature of the room at 70deg, gave the patient a mixture of equal parts of chloroform and sulphuric ether to complete anaesthesia; made, the incision on the linea alba, from about an inch above the pubis, upwards about 12 inches in length. The tumor pre- sented itself covered by the omentum and with pretty firm adhesions, the most of which were broken up with the finger, but a few points, more firm, were dissected away. In breaking up these adhesions, a portion of the omentum was slightly torn, and was cut away. Two small arteries were divided in this procedure and threatened to be a little troublesome, but the hemorrhage was arrested by the application of a little persul- phate of iron. Following the tumor down to its pedicle, it was found to be quite small, about four lines in diameter, two inches in length, and round. It was tied by one strong ligature, the pedicle severed and the tumor lifted out, and was found to weigh seven pounds. During this procedure, the exposed bowels were cov- ered by? napkins dipped in warm water; they were now care- fully sponged, all little particles of clots removed, and returned to their natural position. The wound was now closed by eight silver wire sutures, the ligature of the pedicle being brought out of the lower angle of the wound; adhesive straps were placed between the sutures; a lint compress; and a broad ban- dage pinned snugly around the whole. At this time, the effects of the chloroform had pretty wrell passed off; the pulse 98. Sulph. morph., J gr., was given immediately, and repeated in an hour, after which, gr. was given every two hours until the following day. 19th. Has had some vomiting; pulse 100. Sulph. morph. J gr. every four hours. 20th. Pulse 102; respiration 28; vomiting continues at in- tervals; not much pain. Suspend the morph. 21st. Restless; pulse 104; considerable tympanitis. Gave enema of tepid water; bowels moved, which greatly relieved the tympanitis. 22d. Has passed a comfortable night; pulse 104; pain slight. Dressed the wound, which is healing very kindly. 23d. Much tympanitis, though not much pain; pulse 110; tepid water enema relieves the tympanitis. 21flh. Has passed a comfortable night; pulse 108. 25th. Pulse 100; pain slight. R. Sp. vini Gallici SSij. every two hours. 26th. Considerable pain from distension of the bowels; en- ema moves the bowels and relieves all the symptoms. 28th. Has been very comfortable since the last record; remove the sutures; wound pretty firmly united. 30th. Pulse 94; give Rhine wine, SSj. every two hours, in place of brandy, and beef-tea ad libitum. I would here state, that, up to this date, the diet had been toast-water, farina gruel, with small particles of ice to allay thirst. Feb. 7th. Has been very comfortable since last record; pulse 80; ligature gave away upon a little traction to-day. 12th. Wound entirely healed; appetite good. 20th. Patient returned to her home, 10 miles in the country. 26th. Visited patient at her home. I found her moving about the house, and she expressed herself as feeling quite well, though a little weak. I would here tender my great obligations to Dr. Andros, for his assistance in conducting the after treatment.

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ARTICLE VI. CASE OF DEATH FROM CHLOROFORM. By. C. R. PARKE, M.D., Bloomington, Ill. Read to the Illinois State Medical Society, June, 1866. Mr. President:--Permit me to report the following case of leath from the inhalation of chloroform:-- On Saturday, June 2d, I was called to administer chloroform to Miss-------, age about 20 years, apparently in good health. Object, the extraction of teeth. On the previous Wednesday, chloroform had been adminis- tered by a dentist, and six molar teeth extracted, without any deleterious effects. On Saturday, she seated herself in a regular dental chair, was quite cheerful, and anxious to get under the influence of chloroform. I placed about J of a drachm of chloroform on a sponge and applied it to the nose in the usual way, covered by a towel. This quantity not being sufficient to produce the de- sired result, an additional J of a drachm was poured upon the sponge, which, amount, in a few minutes, sufficiently affected her so as to enable the dentist to extract three molar teeth, with but little pain. She sat up and spit out the blood that accu- mulated in the mouth, after which she gave me to understand that I must give her more chloroform before she could submit to the further extraction of any more teeth. The remainder of the drachm of chloroform was then poured upon the sponge and given as before. In a few minutes, she wras considered suffi- ciently under its influence, to have the remaining teeth (three) extracted. Just before the doctor succeeded in extracting the last tooth, I noticed a deathly pallor of the countenance; complete cessa- tion of respiration; pulse scarcely perceptible for a minute, when it ceased entirely. There was also considerable capillary congestion about the skin of the upper portion of the chest, also dilatation of the pupils. Prior to the appearance of the pallor of the face, there was not an unfavorable symptom present. Immediately upon the presentation of the above symptoms, 1 drew the tongue as far out of the mouth as was necessary to allow the air to enter freely, and, with the assistance of Dr. II. Luce, kept up artificial respiration. Applied aqua ammonia to the nostrils and introduced a small quantity of brandy into the mouth. A catheter was also introduced down into the pharynx, through which we blew, thinking the air might the more readily enter the lungs; at the same time we applied the electro-galvanic battery to the spine and muscles of respiration, all of which means were used diligently for three-quarters of an hour without avail, she only making three attempts at inspira- tion during the commencement of our efforts at artificial respi- ration. The pulse acted as it commonly does, during the administration of chloroform, first being excited, then depressed, nothing indicating cardiac trouble of any kind. There was none of that lividity of countenance witnessed in asphyxia. The whole transaction, up to the fatal minute, was not over twenty-five minutes; and the amount of chloroform used, only one drachm. This is the first fatal case I ever witnessed from the inhala- tion of chloroform, and I suppose I have administered it, and seen it administered, in this country and in Europe, nearly a thousand times. Any suggestions thrown out by this Society, as to the partic- ular action of the anaesthetic, and the cause of death in this case, will be thankfully received. My opinion is, it was paral- ysis of the heart, with congestion of lungs.

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ARTICLE II. A PAPER ON EPIDEMICS. By H. NANCE, M.D., Kewanee, Ill. Read to the Military Tract Medical Society. Having been selected by this Association to report, at the. semi-annual meeting, at Galesburgh, on Epidemics and En- demics, I owe an apology for not making this paper more in- teresting. I cannot make it interesting, for the good reason, that we really have not had a severe epidemic, of any disease, for two or three years. In taking a retrospect of the health of our community for the last two years, I would pronounce it un- usually good. Our community, I think, have suffered much less than many others situated quite contiguous to us; this may be owing to the particular topography of our village and its environs. Kewanee is situated on the Chicago,* Burlington, & Quincy Railroad, immediately on the dividing ridge between the Illinois and Mississippi Rivers. The south part of the vil- lage discharges its sewers into the tributaries of the Illinois River, and the north part enter the tributaries of the Missis- sippi River, thus leaving us almost entirely free from any stag- nant pools of water. Physicians advocating a miasmatic ori- gin of the usual autumnal fevers, would promptly come to the conclusion that we would suffer but little from this class of dis- eases, and this conclusion would be nearly correct. Our village is underlaid with an immense field of bituminous coal, being from eight to ten feet below the surface to eighty or ninety feet, according to the grade of the land. How far this mineral or vegetable production may affect the health of our community, I shall not pretend to say; I will only remark that the health of our mining population is equally as good, if not better, than that of the rural population, who are engaged in cultivating the soil. I think they are remarkably exempt from koino-iniasmatic diseases. It is very unusual to see any of them suffering with intermittent diseases; and I can say the same of typhoid fever. I have resided here more than six years, and during that time I do not remember of having treated a case of typhoid fever in the house of any miner. In the fall of 1864, I treated about a dozen cases of genuine ty- phus fever in the mining region, but it was introduced by a patient brought from off a ship sick with the disease, and from this case the others originated. Notwithstanding we are on the dividing ridge between the two great rivers, and that our county is nearly -all underlaid with coal, which may have a modifying influence upon the vari- ous epidemics of our county, yet we occasionally have epidemics. We have had cerebro-spinal meningitis, typhoid fever, measles, pertussis, dysentery, and, probably, some other diseases pre- vailing as epidemics since I have resided in Henry County; and I should not fail to mention the general prevalence of intermit- tents, remittents, infantile diarrhoeas, and general derangements of the alimentary canal of children, usually under two years of age. During the winter, when the weather is mild and the atmosphere is overloaded with a redundancy of moisture, pneu- monia and bronchial affections always prevail. Phthisis pul- monalis is becoming a much more general disease than it was at the first settling of the country, and I am sorry to add, with all our improvements in medicines, including the vaunted rem- edies, oleum jecoris anselli and the phosphites, that tuberculosis advances as rapidly as it did before the introduction of these lauded " specifies." I mention these remedies, not to entirely condemn them, but to put the young and inexperienced prac- titioner on his guard, not to place too much confidence in them when prescribing for his phthisical patient. There is, doubtless, a tendency on the part of some of our leading physicians to eulogize the virtues of some of our medi- cines too much. This excess of praise of these remedies is confined principally to professors in our medical colleges, in order to create a reputation, not only for themselves but for their institutions. I am frequently disgusted in reading in our medical journals of some remedy being recommended so highly for some epidemic which is prevailing at the time. Who of you now believe that the phosphites will eradicate tuberculosis, ren- ovate the general system, and make a healthy, athletic man of a poor anaemic person? Who of you now believe that cod-liver oil is a specific in all pulmonary diseases? I tell you, gentle- men, that we should not be so easily deceived by pretenders in medicine who are trying to build themselves up, nor by profes- sors in our medical colleges who are using every effort, not only to enhance their own reputations, but the reputation of the institution they represent. Look guardedly to the interests of your patient, and let us not vie with those who would rise to fame in their profession, regardless of the good they may do in curing their patients. We would not regard consumption as either an endemic or epidemic disease, but as it frequently becomes a sequel of bron- chitis, pneumonia, pleuritis, etc., especially in persons possess- ing a hereditary taint, I mention it in this connection. Since the introduction of auscultation and percussion by Laennec and Louis, we have had but little difficulty in diagnosing pul- monary diseases. At the early stage of the formation of tuber- cle, some difficulty may be experienced, but when we look to the general symptoms, including quick pulse, general wasting of the fatty tissues, cough, especially in removing the clothing on going to bed, occasional night sweats, and repeated attacks of haemoptysis, we may most certainly come to the conclusion that our diagnosis will be correct if we call the disease con- sumption*. But when we have properly applied the stethoscope, or used immediate auscultation, a doubt no longer remains. I would dismiss the subject of phthisis, by hastily mentioning the treatment in general. Out-door exercise, when the weather is pleasant; good, rich diet, including meat of various kinds, also eggs, butter, and milk; I would give my patient but little medicine if he was comfortable; treat the urgent symptoms as they arise; if the cough troubles him much, give him a mild expectorant combined with an opiate--the latter will do more good than all the expectorants treated of in the U.S. Dispen- satory. My usual form for an expectorant in phthisis is, mor- phia sul. grs.vj., aqua 5'j-, shake and add syrup ipecac, syrup tolu aaSij., tinct. sanguinaria 5j., mix, and take one teaspoon- ful every 20 or 30 minutes, until the cough is relieved. When hectic symptoms make their appearance, I find much advantage from quinine with aromat. sul. acid, given in the stage of apy- rexia. The haemoptysis, I would treat with acetate plumbi et sul. morphia, nit. potash, miqute doses of tart. anti, et pot., or, sometimes, a teaspoonful of common salt, swallowed suddenly, will promptly arrest it. The patient should be kept quiet, with his head and shoulders raised up in bed, jugs of hot water to his feet, and sinapisms to the thoracic region; cold drinks should be used, and the patient cautioned not to exercise much, not to cough, only when involuntarily compelled to, and to avoid the habit which many phthisical patients have of hawking. My general treatment, if any is thought advisable, would, of course, be stimulating and tonic. I would permit the use of wine, good stock ale, and would not discourage the use of good cognac brandy and milk, made in the form of egg-nog or milk- punch. I have sometimes given, with apparent advantage, Nich's prep. cin. and ferri. But I would remark that I have but little confidence in any general treatment. Good warm clothing; flannels next to the skin, and the general hygienic rules which all physicians are supposed to understand, is the safest course, in my opinion, we can pursue in this dreadful disease. My own observation has taught me that patients treated on this plan have lived as long and suffered as little as those who have been the victims of Rushton & Clark's'spurious cod liver oil, or Prof. Churchill's phosphites. In leaving the subject of phthisis, I touch on other pulmo- nary diseases, and the first I mention is hooping-cough. This disease has prevailed somewhat as an epidemic for the last six or eight months; there have been no peculiarities attending it; most of the cases have been light and uncomplicated; but few cases have required the protracted visits of the medical at- tendant. My treatment in uncomplicated cases has been an expectorant composed of com. s. scillae, with syrup tolu, and tinct. opii camph. It has been rarely necessary to administer any laxative or cathartic. I am down on all specifics for this disease. The idea advanced by some of our medical men, that cochineal, belladonna, nitric acid, and several other vaunted remedies, will cut short the disease we are considering, is all bosh, as the politicians say. Treat the disease as a pulmonary, self-limited one, and our rational is correct. Complicated cases of hooping-cough are always serious, es- pecially when connected with diseases of the brain. Arachni- tis, with effusion, is apt to follow; then come spasms, general convulsions, and death. Our treatment should be shaped to meet these symptoms as they arise, and I would refer you all to diseases of the brain, for treatment under their proper heads. During the late epidemic I had the misfortune to have two such cases come under my care, both of which died. All the symptoms of arachnitis presented themselves before death, in- cluding severe convulsions, haemaplegia, blindness, etc., etc. Catarrhal fever, or acute bronchitis, prevails in this section of country every winter and spring. It is confined, principally, to children under eight or ten years of age. The prominent symptoms are dry, hacking cough, quick pulse, flushed face, dry and hot skin, white or yellow fur on the tongue, bowels usually constipated, slight mucus rale. If the disease is ne- glected it results in pneumonia; then we have added to the physical symptoms dullness on percussion, with complete bron- chophony, or in milder cases crepital rale. Occasionally I no- tice rusty colored sputa, though this latter is very rare. My treatment is simple and successful. I very rarely loose a pa- tient with it, and when this does occur it is when complicated with pneumonia, as the bowels are constipated, and the liver in an unhealthy state, which is evinced by the light or green col- ored operations. I give to a child, two or three years old, a laxative composed of grs. iv. hyd. clo. mite., rhei pulv. grs. xv., mag. calc. grs. v., mix and divide in chart No. 5, one every four hours, until the bowels move. After administering one or two of these powders 1 prescribe an expectorant composed of com. syrup scill. oiij., tinct. opi. camph. SSj., mix and give from one-quarter to one-third teaspoonful every hour and a half. If the child has a high grade of fever, which is generally the case, I give one or two drops of the tinct. veratrum in each alternate dose. This treatment, with very little variation, I have found very successful in these catarrhal or pulmonic diseases, usually styled catarrhal fever, pneumonia, or acute bronchitis of chil- dren. Adults are very rarely thus affected, if they have ca- tarrhal fever. It is usually severely complicated with pneumo- nia or acute bronchitis, and sometimes congestion of the lungs, making it a case of serious import, which should be met accord- ing to the type of the disease. The idea of treating all cases of pneumonia with quinine, or all cases with tart. anti, et pot., or with tint. ver. viride and venesection, epispastics, etc., is nothing but empiricism; and he who pursues such a course must expect to hear the church-bell tolling over his misfortunes, and he need not be surprised, in a short time, to find himself pre- paring to emigrate. Congestion of the lungs rarely appears as an epidemic. Let it appear either sporadically or epidemically, it is always a serious disease, and much depends upon the good judgment of the intelligent practitioner to combat it successfully. At one time, it might require the free use of the lancet, and upon this, nearly alone, should we depend; at another time, as we might expect, the free use of quinine, or the more diffusible stimulants, carb, ammo., or wine, or even good brandy; sinapisms, or epi- spastics should be freely applied over the lungs. When the lips become purple, the mind inactive, coma and somnolency making their appearance, the breathing becoming stertorous, let the pulse be either full and slow, or quick and feeble, you may soon expect dissolution. There is one symptom, indicat- ing nearly certain death, not confined entirely to pulmonary diseases, and that is the rising and falling of the pomurn Adami. When you observe this symptom, let other symptoms be as they may, you can quite safely pronounce your patient in, or not far from, artieulo mortis. Twenty-one years ago, when I first entered the profession, pleuritis was quite a frequent disease, but it has grown less and less frequent, until it is almost unknown. The lancet and opium were our sheet-anchors. The disease, in my practice, hardly known any more. Can any of you assign a reason why? In the winter of 1861-2, diphtheria made its appearance, not only in Henry County, but it became a general epidemic throughout a great portion of the United States, in fact, it was not confined alone to our hemisphere, but much of it prevailed in England, France, and many other portions of Europe. It is a disease sui generis, that is, it has many peculiarities belong- ing to itself, unlike any other disease. It has been said by some of our profession, that it is identical with scarlatina. I can see but little resemblance, letting alone the identity. The only thing in the disease that in the least assimilates scarlatina, is the inflammation of the tonsils and fauces, and this, to an acute observer, is easily diagnosed. Diphtheria is a sub-inflam- matory asthenic disease, in which the nervous and circulatory systems are seriously involved. The small and rapid action of the pulse would indicate some serious lesion to the circulation, some general breaking down of the whole system. The general symptoms are indicative of some blood-poison, and such it seems to be. As is the case with other epidemics, so with this! We know not the cause. Why the poison should spend itself locally upon the uvula, tonsils, palate, and, sometimes, the glottis, epi- glottis, and trachea, I leave to wiser men than I am to reason upon. That the nervous system participates largely in this disease, is known by the great debility and irritability, also by the condition the nervous system is left in, in the sequel; for it is not uncommon to find partial paralysis of the organs of de- glutition, and we occasionally find partial or complete amau- rosis. I have also, in a few instances in my practice, observed partial paraplegia. These sequelae, under a continued use of quinine, the ferruginous preparations, or the more powerful tonic and tetanic strychnia, have usually yielded, though I have found it necessary to continue them for weeks, and sometimes several months. As diphtheria is conceded by all to be an asthenic disease, and one of general debility, we would readily come to the con- clusion that a tonic and stimulating course would be the sine qua non in its treatment, and such is my belief. When first visiting patients with diphtheria, I immediately place them upon large doses of quinine every two hours--if the patient is a child six or seven years old, I would give it from 1| to 2 grs. at each dose, and alternate with half a spoonful of the saturated solu- tion of the chlorate of potash, requesting the patient to gargle the latter before swallowing each dose. If there is great debil- ity, as evinced by rapid pulse, general pallor, sordes, etc., I would use brandy in conjunction with the quinine. In 24 hours' time, I have seen very marked effects from this treatment, and I attribute the improvement principally to the quinine. I be- lieve it is worth everything else in the treatment of diphtheria. Of course I would not ignore the use of probangs and gargles, but would use the first very rarely indeed, and the latter pre- pared of some mild astringent or antiseptic. Tannic acid and capsicum, in the proportions of one drachm of each to one tea- cupful of water, makes a very good gargle; and if the patients cannot be taught to gargle, let them swallow it. In this way it will prove equally as effectual as if only used as a gargle, and not prove detrimental to the system. The treatment recommended when diphtheria first made its appearance, of swTabbing out the throat once, twice, or three times a-day with solution of argent, nitratum; muriated tinct. ferri; tinct. iodine; permanganate of potash, etc., etc., was, in my opinion a very bad one, and in many instances, I believe, resulted seriously. For if, by these means, we removed the pseu- do-membranous formation, it only left a raw, granulating sur- face, ready, if the system was in the condition, to very soon pro- duce another membrane probably worse than the one removed. Let me say to you, let the membrane alone until it becomes loose and nearly detached, and then, with a pair of forceps, we can easily remove it from the throat. The main point in the treatment of diphtheria is, to support the general system while the disease is preying upon it, and if we do our duty here, in most cases in a few days, we will have the happiness of visiting our patient in a convalescent state. Local external applica- tions I hold to be of but little value, a flannel cloth moistened with ammonia or camphor liniment, or a piece of fat pork sprinkled with pepper and salt, are probably as good as any- thing. In the winter of 18G3-4, cercbro-spinal meningitis made its appearance in our county. The symptoms are so well known to most, if not all of you, that it is not necessary for me to rehearse them. I had six cases of the disease, well-marked, and out of this number five of them died. Their ages varied from fifteen months old up to ten years. The one that recov- ered was a young man, aged about twenty-one. He had the most aggravated symptoms, including opisthotonos, trismus, coma, entire loss of sensibility, including loss of speech, etc., etc., and these symptoms remained for five or six days, and yet under treatment he recovered. I was called to three other patients during the time the epi- demic was prevailing, with severe pain in the head and cervical i region, face flushed, head drawn back, full and rapid pulse, de- lirium, etc. I immediately bled each one of them, and gave tinct. ver. veridi in full doses; at the same time ordered an active cathartic. Under this treatment they all recovered. The six cases first spoken of were not treated in this active an- tiphlogistic way, for the reason that their symptoms would not admit of such activity. They were treated with laxatives-- quinine, belladonna, mild opiates, carb, amo., etc., etc., with stimulating liniments, epispastics, etc., to the cervico-spinal region. Notwithstanding such authority as Dr. Davis has highly recommended--belladonna in the treatment of cerebro- spinal meningitis--I cannot see the rationality of it, and should feel very much indisposed again to try it. You may urge me for my treatment in this disease. I can frankly reply that I have none. I should treat mv cases according to the symptoms. If active symptoms prevail, indicating congestion of the brain, with full pulse, flushed face, etc., I should cer- tainly use the lancet and ver. viride; but if the type is asthen- ic, a tonic and stimulating treatment would look most plausible. I am firmly of the opinion, knowing the pathology of cerebro- spinal meningitis, that it will never be treated with much suc- cess. How can we successfully combat an inflammatory disease of the brain and spinal marrow, and their membranes, espe- cially when it is an epidemic, and supposed to originate from some unknown aerial cause? Epidemics of all kinds are usually more virulent than when the same disease occurs sporadically, and this remark applies especially to the one under consider- ation. Typhoid fever, which has prevailed in Illinois for a great number of years, and has been so very fatal in many localities, has nearly disappeared from this section of country. How long this immunity may continue none of us can tell. I have seen but five well-marked cases of typhoid fever in the last two years. Two of them were patients of my friend, Dr. Scott. They came under my care for ten or twelve days, during his short absence in Minnesota. They were genuine cases, as marked by petechia, sudamina, tympanites, hebetude, loss of memory, quick pulse, stupor, haemorrhage, etc., etc.; in the sequal, abscess occurred in the perinial region. The other cases I saw in consultation with Dr. Smead, of Lafayette, in Stark County. One of them had haemorrhage from the bow- els, and immediately after convalesced. My treatment in ty- phoid fever is quite simple. If the case is mild, I simply give spts. mindereri and nitre; if the pulse is quick and full I would add two or three drops of ver. viride every two or three hours. When diarrhoea makes its appearance, I would use an emulsion of spts. turpentine, with tinct. opii; would continue this treatment through the disease if the diarrhoea remains. The patient should be well supported on milk-broth, wine, etc.; should the vital forces seem to be giving away, quinine, brandy, etc., are indicated. Typhoid fever is a self-limited disease, and the idea of breaking it up in a few days is untrue. Many empyrics, when this disease is epidemic, call nearly everything typhoid, and hence establish a reputation for treating this dis- ease. I would particularly urge upon the young physician not to give much cathartic medicine. If a laxative is imperatively d 'inanded, (which is rarely the case,) give about half a tea- spoonful of oleun ricini, with from ten to twenty drops of oleum terebinth.; this quantity will rarely fail to operate sufficiently thorough. Typhoid fever being an enteric disease, or dothi- nenteritus, we can easily conceive that a cathartic or laxative would move the bowels very readily, and such is the case. I never gives laxatives so long as the patient feels easy and com- fortable, if the bowels should remain unopened for several days. A species of stomatitis has prevailed as an epidemic since early in the spring, confined to no particular age. It produces ulceration about the roots of the teeth, on the palate, tongue, and, in fact, all over the mucous membrane of the mouth. In some cases the system seems to sympathize, producing a loss of tone, strength, and approaching anaemia. In such cases I have used quinine and the preparations of iron. Locally I have found more good derived from penciling the gums and ulcers on the cheeks with muriated tincture of iron, than from anything else, though I have used, with much benefit, sul. cupri and nitrate of silver. Some cases I treated with chlorate of potash, as a wash, and gave it at the same time as a blood pu- rifier. It has not only acted as an epidemic, but has seemed to me to be contagious, spreading from one member of the family to another, by using the same drinking utensil, or from the parents kissing their children. Many cases have been stub- born, but all have yielded to the above treatment. It remains for me to treat of two more classes of diseases which prevail in our county occasionally as epidemics, which I shall treat of very succinctly (as my essay has already grown too long). I allude to the exanthemata and diseases of the stomach and boivels. In the winter of 1862-3, rubeola prevailed very generally, as an epidemic, not only in Henry County, but, I believe, very generally throughout the State. I think I must have treated more than a hundred patients, and, amongst that number, sev- eral in my own family. The type was remarkably mild, and, consequently, my treatment, to correspond, was equally simple. Where no complications existed, I simply prescribed cool drinks, ad libitum, equal temperature of the room, and the patients to remain indoors for a week or ten days- after the exanthema dis- appeared. I rarely gave even a dose of sul. magnesia. When the catarrhal symptoms were troublesome, I gave a simple ex- pectorant, composed of com. syrup scillee and tinct. opii camp. As a general thing, I discard the idea of prescribing laxatives in rubeola, for the tendency, in the sequel, is diarrhoea in nearly all cases. Under this kind of treatment, I had the satisfaction of consoling myself that all my patients recovered, but one, and he was a boy of 10 or 12 years old, who had been subject to epilepsy from early infancy. Congestion of the brain and, finally, arachnitis made its appearance, and death soon closed the scene. We have occasional cases of variola every year, but our peo- ple are so well protected by vaccination that we need have no fears of its appearing as an epidemic. My treatment is very similar to what it is in rubeola, when uncomplicated, and, con- sequently, need not be rehearsed. 1 would 'especially enjoin upon all the profession the propriety of urging upon the people the benefit of vaccination and re-vaccination, until the system seems to be insusceptible to its effects. This course being rigidly pursued, it would seem that small-pox, in a few years, would be unknown. Would it not be well to urge upon our representatives the propriety of enacting a law compelling the vaccination of all persons? Scarlatina has not prevailed as an epidemic in this section of country since the years 1857--8--9. It then appeared in a ma- lignant form, and many died. I see sporadic cases of it every year, and it is at the present time prevailing to some extent, though in a very mild form. My treatment is remarkably sim- ple, and, since I have been using it, very successful. I would urge upon the members of this Society to give it a fair trial. When called, I order a solution of carb, of ammo, in doses of from 3 grs. to 5 or 7, according to the age of the patient, every hour and a-half or two hours. This treatment I give, regard- less of the rapidity of the pulse, or the redness of the surface, and I must say, that my success has been unequalled since I commenced this mode of treatment. I use gargles of capsicum and chloride of soda, with vinegar and water, or simply a satu- rated solution of chlorate of potash, discarding the use of the swab or probang. Diarrhoeas, with adults and children, make their annual sum- mer and autumnal visits, but their treatment varies so much that time will not permit me to treat of them. Dysentery prevailed as an epidemic in the summer and fall of 1864. In adults, I usually give at the commencement, a good-sized dose of sul. magnesia; if this fails to check the dis- ease, I order a powder composed of sul. morphia, acetate plurabi, and minute doses of calomel given every three or four hours, alternating every day or two with a portion of oleum ricini or sul. magnesia. This treatment will rarely fail to cut the disease short. The treatment in children and infants is not so successful, and I would respectfully refer you to the books. I have seen infantile dysentery as fatal as epidemic cholera, ac- cording to the number of cases attacked. I know of no treat- ment that has been very successful in this fatal form of dysen- tery, and would respectfully urge this Society to give their views on this part of my report. The dreaded epidemic, cholera, has not made its appearance in our village this season, and I have heard of but one case in the county, and that occurred at Galva. The gentleman came through Chicago during the night, arrived in Galva in the morning, and died the same night, or early the next morning. Not having treated any cases of cholera during the present epidemic, I am poorly prepared to give my views; but, notwith- standing this, probably I am as well prepared as many of you, and, possibly, as well as many who have been treating a great number of cases. Who of you would apply ice bags to your patients in collapse with this disease? Who of you w'ould give castor-oil to your patients when the bowels were already pro- fusely running off and the stomach rejecting everything that is swallowed? Who of you would expect to cure your patients when in a state of collapse by the administration of spirits of turpentime and strychnia? And yet such is the treatment recommended by some of the most learned men in our profes- sion. In brief, I have no time to dwell upon this King of Ter- rors. I would treat my patients, if found in the cholerine or diarrhoea stage, with small portions of calomel, acetate of lead, and sul. morphine, given every two or three hours, until the diarrhoea was arrested; would order strict quietude and rest, in the recumbent position; very light diet, and small quantity of drinks of any kind--probably small quantities of iced-water, or even ice itself might be considered preferable. A treatment of this kind will usually succeed, but if we are so unfortunate as to lose this valuable time, or our patient sinks at once into vomiting, purging, cramps, and collapse, what is to be done? "Echo answers, what?" Now the critical time has come, and most of such cases die, regardless of the various treatments which have been proposed and used. Opium, or sul. morphia, so valuable in the first, or diarrhoea, stage, now is of much less value; if it is now given in large doses, or small ones frequently repeated, we produce comatose symptoms, loss of general vital force, a predisposition to arachnitis, and it also enhances the chances for a consecutive fever being set up, in case the patient rallies from collapse. In this stage, I would give tinct. capsicum, tinct. camphor, ess. mentha, ss., and chloroform, combined with a very small quantity of tinct. opii; this preparation I would administer after every spell of vomiting, and if the vomiting ceased, I would give it as often as the cramps, diarrhoea, and pain seemed to indicate. I should be very careful not to produce the nar- cotic effects of this opiate. Stimulating injections might, with propriety, be given. Sinapisms and dry warmth should be used freely. I would give but little, if any water, preferring my patients should use ice in small *pellicles to quench the raging thirst.

PMC10000009

ARTICLE VII. A CASE OF ELEPHANTIASIS--SUCCESSFUL RECOVERY. By R. DEXTER, M.D., Chicago. Carrie G., cet. 20, an inmate of the Erring Women's Refuge, gives the following history of herself and the outgrowth':-- Is a native of Rochester, N.Y.; has resided in the southern part of Illinois several years. A little more than a year ago, she observed that the external labia were somewhat increased in size, and presented an unusually rough appearance. The rapid growth of the parts induced her to seek medical advice. The external application of iodine was prescribed, together with the exhibition of internal remedies, but without any beneficial results. The advice of another physician was sought, but change of treatment brought no relief. Our attention was called to the case about two months since. Found the external labia enormously hypertrophied, presenting every appearance and symptom of elephantiasis. Each lip was about five inches long, and one and a-balf inches through. It seemed that but one alternative remained, viz.:--excision. The counsel of a medical gentleman was obtained, who fully con- curred in the opinions above given. Accordingly, on the 21st of December last, we removed the entire mass. Hemorrhage was controlled during the operation by tying the arteries as they were divided. There were four on each side, but were not enlarged. Recovery will be perfect, with but trifling deformity.

PMC10000010

QUINCY MEDICAL SOCIETY. The semi-annual meeting of the Quincy Medical Society was held-at the office of Dr. Zimmermann, in Quincy, Nov. 18th, 1866. A quorum being present, the President, Dr. C. A. W. Zim- mermann, called the meeting to order. The proceedings of the annual meeting having been read and approved, the President proceeded to deliver his able and elaborate essay upon Bright's Diseasei The manner in which he treated the subject may be inferred from the following extract from the exordium:-- "I discharge this obligation with no inconsiderable degree of pleasure, inasmuch as this subject is classed among the most interesting in pathology; and I intend discussing it by strictly following the medical literature as far as it deals with facts, and particularly as far as it touches the anatomy of this dis- ease, and eschews hypothesis; while I shall sometimes mention my own experience, which is naturally .enough based on obser- vations in view of the sick couch, and the therapeutics of the disease, which are without exception open for discussion." The President entered at length into the consideration of the causes, pathology,- diagnosis, prognosis, and treatment of Bright's disease. On 'motion, resolved, that the thanks of this Society be ten- dered to the President for his able essay, and that a copy is hereby requested for publication. rhe Secretary then read the history and treatment of two cases which had come under his observation since the annual meeting:-- Case I. June 21st, 1866. Was requested to visit Mrs. S., aged 25 years. I learned from her that she had been confined to the bed seven weeks, and had vomited, most of the time, every hour. The matter ejected is described as green and yel- low; urine natural in quantity, specific gravity 1.011, acid, not albuminous. The last menstrual period occurred about four months since, as near as she recollects. She is greatly emaci- ated, very feeble, and takes but little nourishment. The mamma is flaccid and wasted, no change has occurred in the nipple or in the areola about it. On vaginal examination, the uterus was found enlarged, and the os hard and fissured. Prescribed one teaspoonful of Ellis' wine of pepsin in a wineglass of water three times a-day, and one tablespoonful of lime-water and two of milk every two hours, with beef-tea for nourishment. 22d. The symptoms not materially changed. Directed the treatment to be continued. 23d. Patient no better; has vomited every hour, anti has not slept during the night. A consultation was agreed, upon, and Dr. L. II. Baker of this city called. After a careful ex- amination, it was decided that there is no evidence of a living foetus in the uterus, but even if there were, the dangerous con- dition of the mother demands interference. A speculum was introduced and the os brought to view, and a sound passed into the uterus three inches, no fluid followed its withdrawal. A sponge tent was then passed into the os and the patient directed to take the medicine as before. 21f,th. The patient has not vomited as much as formerly, and has taken more nourishment. The tent is not to be found in the vagina, and cannot be seen in the os through the speculum, and the supposition is entertained that it may have passed away unobserved. Introduced a larger tent, and directed treatment to be continued. 25th. The vomiting ceased during the day and returned at night. On examination, the tent cannot be found; introduced another with a small cord attached to it. 26th, 9 o'clock A.M. Has vomited less than formerly. Re- moved the tent and introduced another. 2 o'clock P.M. Quite an offensive discharge has occurred, and the patient complains of pain in the back. Removed the tent and found the os con- siderably dilated; on passing the finger, the two tents first used were found within the os and removed. 27. The patient is more quiet and vomits less. Continue the medicine. 28. The remains of a foetus passed away during the night; it is six inches in length, and weighs one ounce troy; it is much wasted; the skin is wanting; the head collapsed; the walls of the chest and abdomen wanting; no trace is found of the lungs, diaphragm, stomach, or intestines; the heart, liver, and kidneys were observed in their natural, positions. The foetus has, doubt- less, been dead several weeks, and the placenta probably been absorbed, as it has not passed away, and cannot be discovered on examination. No flooding has occurred. Prescribed 5 grs. of pulvis ergot, to be taken every three hours, and a teaspoon- ful of a saturated solution of-sulphite of soda three times a-day. 30th. The vomiting continues frequently, and is supposed to be excited by the medicine, which was discontinued, and the Wine of pepsin and lime-water resumed, and milk freely taken for nourishment. July 1st. The vomiting has now ceased, and the patient's condition satisfactory in every respect. No unfavorable symp- toms afterwards occurred, and the patient made a very rapid recovery. Remarks.--The irritation of a dead foetus in the uterus has been referred to by authors as one of the causes of abortion; but it is evident that- it does not always give rise to this result. Tyler Smith refers to a case, in which the foetus died at the fourth month, but was not expelled until the full term. As the sound of the foetal heart cannot ordinarily be heard until after the fourth month, we have no certain evidence by which we can infer the death of the foetus. In the cases which have come under my observation, I have found the mamma flaccid, the nipple and its areola without any of the ordinary indications of pregnancy, the neck of the uterus harder, and the os more pat- ulous than usual in that condition. It seems desirable that more careful and extended observations be made, as to the symptoms which indicate the presence of a blighted foetus in utero. In this case, if its presence had been known at an ear- lier period, the patient might have been saved several w'eeks of distressing illness. Case II. Was called to visit Mr. B., aged 35, in consulta- tion with Dr. L. H. Baker, of this city, September 3d, 1866, and found the patient suffering from narcotism. The following is the history of the case, as given by the attendants:-- The attention of his wife was called to him by an unusual snoring, and, on going into his room, she found him quite in- sensible, and nearly black in the face and neck. Supposing him to be in a fit, she attempted to give him some wine, spirits of camphor, and other restoratives, of which he swallowed but little, and that at the risk of strangling. Just at that time, she found in his pocket an empty bottle, labelled laudanum. Dr. Baker was called immediately, and gave him, 10 minutes before 4 o'clock, 20 grs. of sulphate of zinc, and repeated the dose in 5 minutes, without effect. I arrived 25 minutes after the emetic had been given. Not having had its effect, it was thought best to use the stomach pump. About one quart of warm water was thrown by it into the stomach and immediately pumped out with the whole of its contents, which gave a strong odor of laudanum. This was repeated three times, and about a gallon of water, in all, was used, the last of which was re- turned nearly clean and devoid of smell of laudanum. During this time, the patient remained entirely insensible, with the eyes closed, the pupils contracted, respiration slow and stertor- ous, pulse slow, with other symptoms of narcotism. After thoroughly emptying the stomach, a galvanic' battery was used for nearly two hours, one pole applied to the upper part of the spine and the other to the front of the chest. About one hour after the stomach was evacuated, and while using the battery, the patient began to sink, the respiration became re- duced to four in a minute. Dr. Baker resorted to artificial JL Uopi 1 cLLlUlL cltlvlltlUll LU L11C UcLLLtH J ) WHICH Wd>>0 KLj/L Lip Uj by Hall's method, about a-quarter of an hour, until an improve- ment occurred in the respiration, which continued until the patient was fully restored. No unpleasant symptoms followed excepting vomiting, which occurred at intervals during the night. It was subsequently ascertained that the patient took two ounces of tincture of opium at 10 o'clock A.M., and also two ounces at 1 o'clock P.M; and that he was not in the habit of taking opium or laudanum; that he took it for a diarrhoea, which he had for three or four days. The apothecary says the tincture of opium sold the patient contained just one-half the officinal quantity of opium. On motion, Dr. J. F. McCormic was appointed essayist for the next meeting. On motion, Dr. A. Niles was appointed delegate to the State Society. On motion, resolved, that the proceedings of this meeting be published in the Chicago Medical Examiner, and an abstract of them in our city papers. ADDISON NILES, Secy.

PMC10000014

Rev Bras Epidemiol Rev Bras Epidemiol rbepid Revista Brasileira de Epidemiologia (Brazilian Journal of Epidemiology) 1415-790X 1980-5497 Associacao Brasileira de Saude Coletiva 10.1590/1980-549720230021 00420 Artigo Original Emergency department use and Artificial Intelligence in Pelotas: design and baseline results Uso servicos de servicos de urgencia e emergencia e Inteligencia Artificial em Pelotas: protocolo e resultados iniciais Delpino Felipe Mendes Conceptualization Formal analysis Methodology Writing - original draft Writing - review & editing I Figueiredo Lilian Munhoz Methodology Writing - review & editing I Costa Andria Krolow Methodology Writing - review & editing I Carreno Iona Methodology Writing - review & editing I da Silva Luan Nascimento Methodology Writing - review & editing I Flores Alana Duarte Methodology Writing - review & editing I Pinheiro Milena Afonso Methodology Writing - review & editing I da Silva Eloisa Porciuncula Methodology Writing - review & editing I Marques Gabriela Avila Methodology Writing - review & editing I Saes Mirelle de Oliveira Methodology Writing - review & editing I Duro Suele Manjourany Silva Methodology Writing - review & editing I Facchini Luiz Augusto Methodology Writing - review & editing I Vissoci Joao Ricardo Nickenig Methodology Writing - review & editing II Flores Thayna Ramos Methodology Writing - review & editing I Demarco Flavio Fernando Methodology Writing - review & editing I Blumenberg Cauane Methodology Writing - review & editing I Chiavegatto Alexandre Dias Porto Filho Methodology Writing - review & editing III da Silva Inacio Crochemore Methodology Writing - review & editing I Batista Sandro Rodrigues Methodology Writing - review & editing IV Arcencio Ricardo Alexandre Methodology Writing - review & editing V Nunes Bruno Pereira Formal analysis Methodology Writing - review & editing I Universidade Federal de Pelotas - Pelotas (RS), Brazil. Duke University School of Medicine - Durham (NC), United States. Universidade de Sao Paulo - Sao Paulo (SP), Brazil. Universidade Federal de Goias - Goiania (GO), Brazil. Universidade de Sao Paulo - Ribeirao Preto (SP), Brazil. CORRESPONDING AUTHOR: Felipe Mendes Delpino. Rua Gomes Carneiro, 1, Centro, CEP: 96010-610, Pelotas (RS), Brazil. E-mail: [email protected] CONFLICT OF INTERESTS: nothing to declare 10 3 2023 2023 26 e23002123 9 2022 05 1 2023 09 1 2023 Este e um artigo publicado em acesso aberto sob uma licenca Creative Commons RESUMO Objetivo: To describe the initial baseline results of a population-based study, as well as a protocol in order to evaluate the performance of different machine learning algorithms with the objective of predicting the demand for urgent and emergency services in a representative sample of adults from the urban area of Pelotas, Southern Brazil. Methods: The study is entitled "Emergency department use and Artificial Intelligence in PELOTAS (RS) (EAI PELOTAS)" ). Between September and December 2021, a baseline was carried out with participants. A follow-up was planned to be conducted after 12 months in order to assess the use of urgent and emergency services in the last year. Afterwards, machine learning algorithms will be tested to predict the use of urgent and emergency services over one year. Results: In total, 5,722 participants answered the survey, mostly females (66.8%), with an average age of 50.3 years. The mean number of household people was 2.6. Most of the sample has white skin color and incomplete elementary school or less. Around 30% of the sample has obesity, 14% diabetes, and 39% hypertension. Conclusion: The present paper presented a protocol describing the steps that were and will be taken to produce a model capable of predicting the demand for urgent and emergency services in one year among residents of Pelotas, in Rio Grande do Sul state. RESUMO Objetivo: Descrever os resultados iniciais da linha de base de um estudo de base populacional, bem como um protocolo para avaliar o desempenho de diferentes algoritmos de aprendizado de maquina, com o objetivo de predizer a demanda de servicos de urgencia e emergencia em uma amostra representativa de adultos da zona urbana de Pelotas, no Sul do Brasil. Metodos: O estudo intitula-se "Emergency department use and Artificial Intelligence in PELOTAS (RS) (EAI PELOTAS)" ). Entre setembro e dezembro de 2021, foi realizada uma linha de base com os participantes. Esta previsto um acompanhamento apos 12 meses para avaliar a utilizacao de servicos de urgencia e emergencia no ultimo ano. Em seguida, serao testados algoritmos de machine learning para predizer a utilizacao de servicos de urgencia e emergencia no periodo de um ano. Resultados: No total, 5.722 participantes responderam a pesquisa, a maioria do sexo feminino (66,8%), com idade media de 50,3 anos. O numero medio de pessoas no domicilio foi de 2,6. A maioria da amostra tem cor da pele branca e ensino fundamental incompleto ou menos. Cerca de 30% da amostra estava com obesidade, 14% com diabetes e 39% eram hipertensos. Conclusao: O presente trabalho apresentou um protocolo descrevendo as etapas que foram e serao tomadas para a producao de um modelo capaz de prever a demanda por servicos de urgencia e emergencia em um ano entre moradores de Pelotas, no estado do Rio Grande do Sul. Keywords: Machine learning Chronic diseases Multimorbidity Urgent and emergency care Palavras-chave: Aprendizado de maquina Doencas cronicas Multimorbidade Urgencia e emergencia pmcINTRODUCTION Chronic diseases affect a large part of the population of adults and older adults, leading these individuals to seek urgent and emergency care. The implementation in 1988 of the Unified Health System (SUS) resulted in a model aimed at prevention and health promotion actions based on collective activities 1 - starting at Basic Health Units (UBS). There is also the National Emergency Care Policy, which advanced in the construction of the SUS, and has as guidelines universality, integrity, decentralization, and social participation, alongside humanization, the right of every citizen 2 . In a study that evaluated the characteristics of users of primary health care services in a Brazilian urban-representative sample, it was found that the vast majority were women and part of poorer individuals, in addition to almost 1/4 of the sample receiving the national income distribution program (family allowance) 3 . Brazil is a country highly unequal in socioeconomic terms; approximately 75% of the Brazilian population uses the SUS and depends exclusively on it, and do not have private health insurance 4,5 . Individuals with multimorbidity are part of the vast majority who seek urgent and emergency services 6 . Multimorbidity is a condition that affects a large part of the population 7 , especially older adults 7 . In addition, the association of multimorbidity with higher demand for emergency services is a challenge to appropriately manage and prevent these problems 8,9 . Innovative approaches may allow health professionals to provide direct care to individuals who are more likely to seek urgent and emergency services. The use of artificial intelligence can make it possible to identify and monitor a group of individuals with a higher probability of developing multimorbidity. In this context, machine learning (ML), an application of artificial intelligence, is a promising and feasible tool to be used on large scale to identify these population subgroups. Some previous studies have demonstrated that ML models can predict the demand for urgent and emergency services 10,11 . Besides, a systematic review showed that ML could accurately predict the triage of patients entering emergency care 12 . However, in a search for studies in Brazil, we found no published article on the subject. In Brazil, urgent and emergency services are a fundamental part of the health care network, ensuring timely care in cases of risk to individuals' lives 9 . Urgent and emergency services are characterized by overcrowding and high demand. In addition, with the current pandemic of COVID-19, updated evidence on the characteristics of the users seeking these services is timely and necessary. The objective of this article was to describe the initial baseline results of a population-based study, as well as a protocol in order to evaluate the performance of different ML algorithms with the objective of predicting the demand for urgent and emergency services in a representative sample of adults from the urban area of Pelotas. METHODS The present cohort study is entitled "Emergency department use and Artificial Intelligence in PELOTAS-RS (EAI PELOTAS)" ). The baseline was conducted between September and December 2021, and a follow-up was planned to be conducted 12 months later. We utilized the cross-sectional study to measure the prevalence of urgent and emergency care and the prevalence of multimorbidity, in addition to other variables and instruments of interest. The prospective cohort design intends to estimate the risk of using and reusing urgent emergency services after 12 months. Contact information, collected to ensure follow-up, included telephone, social networks, and full address. In addition, we also collected the latitude and longitude of households for control of the interviews. Study location and target population The present study was conducted in adult households in the Pelotas, Rio Grande do Sul (RS), Southern Brazil. According to estimates by the Brazilian Institute of Geography and Statistics (IBGE) in 2020, Pelotas had an estimated population of 343,132 individuals ). Figure 1 shows the location of the city of Pelotas in Brazil. Figura 1. Map of Brazil highlighting the city of Pelotas (RS). Source: Pelotas has a human development index (HDI) of 0.739 and a gross domestic product per capita (GDP) of BRL 27,586.96 ). The municipality has a Municipal Emergency Room that operates 24 hours a day, seven days a week, and serves about 300 patients a day, according to data provided by the unit. Criteria for inclusion and exclusion of study participants We included adults aged 18 years or older residing in the urban area of Pelotas. Children and individuals who were mentally unable to answer the questionnaire were not included in the sample. Sample calculation, sampling process, and data collection The sample size was calculated considering three objectives. First, to determine the sample size required to assess the prevalence of urgent and emergency services use, it was considered an estimated prevalence of 9%, with+-two percentage points as a margin of error and a 95% confidence level 13 , concluding that 785 individuals would be necessary. Second, for multimorbidity prevalence, an estimated prevalence of 25%, with +- three percentage points as a margin of error and a confidence level of 95% was used 14,15 ; reaching again, a total of 785 individuals needed. Finally, for the association calculations, similar studies in Brazil were assessed, and the following parameters were considered: significance level of 95%, power of 80%, exposed/unexposed ratio of 0.1, percentage of the outcome in the unexposed 20%, and a minimum prevalence ratio of 1.3. With these parameters, 5,104 individuals would be necessary to study the proposed associations. Adding 10 to 20% for losses and/or refusals, the final sample size would be composed of 5,615-5,890 participants. The process to provide a population-based sample was carried out in multiple stages. The city of Pelotas has approximately 550 census tracts, according to the last update estimates provided by IBGE in 2019. From there, we randomly selected 100 sectors. Since the sectors vary in size, we defined a proportional number of households for each. Thus, it was estimated that, in total, the 100 sectors had approximately 24,345 eligible households. To interview one resident per household, we divided the total number of households by the sample size required, which resulted in 4.3. Based on this information, we divided each of the 100 sectors by 4.3 to reach the necessary number of households for each sector. One resident per household was interviewed, resulting in a total of 5,615 households. If there was more than one eligible resident, the choice was made by a random number generator application. Residents were placed in order, a number was assigned for each one, and one of them was selected according to the result of the draw. The first household interviewed in each sector was selected through a draw, considering the selected jump (4.3 households). Trades and empty squares were considered ineligible, and thus, the next square was chosen. Due to a large number of empty houses, it was necessary to select another 50 sectors to complete the required sample size. The additional households were drawn according to the same methodological criteria as the first draw to ensure equiprobability. Data collection instrument We collected the data with the Research Electronic Data Capture (REDCap), a data collection program using smartphones 16,17 . Experienced and trained research assistants collected the data. The questionnaire from EAI PELOTAS was prepared, when possible, based on standardized instruments, including questions about chronic diseases, physical activity, food security, use of urgent and emergency services, functional disability, frailty syndrome, self-perception of health, COVID-19, in addition to sociodemographic and behavioral questions. Supplementary Table 1 shows the instruments utilized in the present study. Table 1. First descriptive results and comparison with a population-based study. Characteristics EAI PELOTAS* PNS 2019+ Crude % (95%CI) Survey design % (95%CI) % (95%CI) Mean age, years 50.3 (49.9-50.8) 46.2 (45.5-47.0) 46.7 (45.9-47.5) Mean number of household people 2.6 (2.5-2.7) 2.7 (2.6-2.8) 3.0 (2.9-3.1) Female (%) 66.8 (65.6-68.0) 54.2 (52.4-55.6) 54.1 (51.7-56.4) Skin color (%) White 78.2 (77.1-79.2) 77.3 (74.9-79.5) 76.8 (74.6-78.7) Black 15.0 (14.1-16.0) 15.3 (13.5-17.3) 8.3 (7.0-9.8) Brown 6.1 (5.5-6.7) 6.7 (5.7-7.9) 14.5 (12.9-16.3) Other 0.7 (0.5-1.0) 0.7 (0.4-1.1) 0.4 (0.2-0.8) Schooling (%) Incomplete elementary school or less 35.7 (34.5-37.0) 31.3 (28.6-34.2) 30.2 (28.1-32.4) Complete elementary school/incomplete high school 16.2 (15.3-17.2) 16.4 (15.1-17.7) 15.7 (14.0-17.5) Complete high school/incomplete higher education 33.5 (32.3-34.7) 37.6 (35.6-39.6) 36.9 (34.6-39.2) Complete higher education or more 14.6 (13.7-15.5) 14.7 (12.4-17.4) 17.2 (15.7-18.9) *n=5.722; +n=3.002. PNS: Brazilian National Health Survey. PNS 2019 includes residents (selected to interview) from the urban area from the Rio Grande do Sul State; Survey design: weighted for primary unit sampling and post-weight estimates Dependent variables The use of urgent and emergency services was assessed on a baseline using the following question: "In the last 12 months, how many times have you sought urgent and emergency services, such as an emergency room?". This was followed by the characterization of the service used, city of service, frequency of use, and referral after use. One year after the study baseline, we will contact again the respondents to inquire about the use of urgent and emergency care services (number of times and type of service used). Independent variables We assessed multimorbidity as the main exposure using a list of 22 chronic diseases and others (asthma/bronchitis, osteoporosis, arthritis/arthrosis/rheumatism, hypertension, diabetes, cardiac insufficiency, pulmonary emphysema/chronic obstructive pulmonary disease, acute kidney failure, Parkinson's disease, prostate disease, hypo/hyperthyroidism, glaucoma, cataract, Alzheimer's disease, urinary/fecal incontinence, angina, stroke, dyslipidemia, epileptic fit/seizures, depression, gastric ulcer, urinary infection, pneumonia, and the flu). The association with urgent and emergency services will be performed with different cutoff points, including total number, >=2, >=3, and combinations of morbidities. We will also perform network analyzes to assess the pattern of morbidities. Other independent variables were selected from previous studies in the literature 18-21 , including demographic, socioeconomic information, behavioral characteristics, health status, access, use and quality of health services. Data analysis We will test artificial intelligence algorithms, ML, to predict the use of urgent and emergency services after 12 months. The purpose of ML is to predict health outcomes through the basic characteristics of the individuals, such as sex, education, and lifestyle. The algorithms will be trained to predict the occurrence of health outcomes, which will contribute to decision-making. With a good amount of data and the right algorithms, ML may be able to predict health outcomes with satisfactory performance. The area of ML in healthcare has shown rapid growth in recent years, having been used in significant public health problems such as diagnosing diseases and predicting the risk of adverse health events and deaths 22-24 . The use of predictive algorithms aims to improve health care and support decision-making by health professionals and managers. For the present study, individuals' baseline characteristics will be used to train popular ML algorithms such as Support Vector Machine (SVM), Neural Networks (ANNs), Random Forests, Penalized Regressions, Gradient Boosted Trees, and Extreme Gradient Boosting (XGBoost). These models were chosen based on a previous review in which the authors identified the most used models in healthcare studies 25 . We will use the Python programming language to perform the analyzes. To test the predictive performance of the algorithms in new unseen data, individuals will be divided into training (70% of patients, which will be used to define the parameters and hyperparameters of each algorithm) and testing (30%, which will be used to test the predictive ability of models in new data). We will also perform all the preliminary steps to ensure a good performance of the algorithms, especially those related to the pre-processing of predictor variables, such as the standardization of continuous variables, separation of categorical predictors with one-hot encoding, exclusion of strongly correlated variables, dimension reduction using principal component analysis and selection of hyperparameters with 10-fold cross-validation. Different metrics will evaluate the predictive capacity of the models, the main one being the area under the receiver operating characteristic (ROC) curve (AUC). In a simplified way, the AUC is a value that varies from 0 to 1, and the closer to 1 the better the model's predictive capacity 26 . The other metrics will be F1-score, sensitivity, specificity, and accuracy. As measures of model fit, we will perform hyperparameters and balancing fit, as well as K-fold (cross-validation). COVID-19 The current pandemic, caused by the SARS-CoV-2 virus, has brought uncertainty to the world population. Although vaccination coverage is already high in large parts of the population, the arrival of new variants and the lack of other essential measures to face the pandemic still create uncertainty about the effects of the pandemic on people. General questions about symptoms, tests, and possible effects caused by coronavirus contamination were included in our baseline survey. We will also use SARS-CoV-2-related questions to evaluate the performance of ML algorithms. In September 2021, restrictive measures were relaxed due to a decrease in COVID-19 cases in Pelotas, allowing the study to begin. A vaccination passport was required from the interviewers to ensure the safety of both participants and interviewers. In addition, all interviewers received protective equipment against COVID-19, including masks, face shields, and alcohol gel. Finally, the interviewers were instructed to conduct the research in an open and airy area, ensuring the protection of the participants. Quality assurance and control The activities to allow for control and data quality were characterized by a series of measures aimed at ensuring results without the risk of bias. Initially, we developed a research protocol, followed by an instruction manual for each interviewer. Thereafter, interviewers were trained and standardized in all necessary aspects. REDCap was also important to garanteee the control and quality of responses as the questions were designed using validation checks according to what was expected for each answer. Another measure that ensured the control of interviews was the collection of latitude and longitude of households, which was plotted by two members of the study coordination weekly on maps, to ensure that the data collection was performed according to the study sample. With latitude and longitude data, it is also intended to carry out spatial analysis articles with techniques such as sweep statistics and Kernel. The database of the questions was checked daily to find possible inconsistencies. Finally, two members of the study coordination made random phone calls to 10% of the sample, in which a reduced questionnaire was applied, with the objective of comparing the answers with the main questionnaire. Ethical principles We carried out this study using free and informed consent, as determined by the ethical aspects of Resolution No. 466/2012 of the National Council of the Ministry of Health and the Code of Ethics for Nursing Professionals, of the duties in Chapter IV, Article 35, 36 and 37, and the prohibitions in chapter V, article 53 and 54. After identifying and selecting the study participants, they were informed about the research objectives and signed the Informed Consent Form (ICF). The project was referred to the Research Ethics Committee via the Brazilian platform and approved under the CAAE 39096720.0.0000.5317. Schedule Initially, we conducted a stage for the preparation of an electronic questionnaire at the beginning of 2021. In February 2021, we initiated data collection after preparing the online questionnaire. The database verification and cleaning steps occurred simultaneously with the collection, and continued until March 2022. After this step, data analysis and writing of scientific articles began. RESULTS First descriptive results and comparison with a population-based study Of approximately 15,526 households approached, 8,196 were excluded -- 4,761 residents were absent at the visit, 1,735 were ineligible, and 1,700 were empty . We identified 7,330 eligible participants, of which 1,607 refused to participate in the study, totalizing 5,722 residents. Comparing the female gender percentage of the refusals with the completed interviews, we observed a slightly lower prevalence with 63.2% (95%CI 60.7-65.5) among the refusals, and 66.8% (95%CI 65.6-68.0) among the complete interviews. The mean age was similar between participants who agreed to participate (50.3; 95%CI 49.9-50.8) and those who refused (50.4; 95%CI 49.0-51.9). Figura 2. Flowchart describing the sampling process. To evaluate the first descriptive results of our sample, we compared our results with the 2019 Brazilian National Health Survey (PNS) database. The PNS 2019 was collected by the IBGE in partnership with the Ministry of Health. The data are in the public domain and are available in the IBGE website ). To ensure the greatest possible comparability between studies, we used only residents of the urban area of the state of Rio Grande do Sul, aged using the command svy from Stata, resulting in 3,002 individuals (residents selected to interview). We developed two models to compare our data with the PNS 2019 survey: Crude model (crude results from the EAI PELOTAS study, without considering survey design estimates); Model 1 using survey design: primary sampling units (PSUs) using census tracts as variables and post-weight variables based on estimates of Pelotas population projection for 2020 (Table 1). We evaluated another model using individual sampling weight (i.e., the inverse of the probability of being interviewed in each census tract). These models are virtually equal to the above estimates (data not shown). The mean age of our sample was 50.3 years (Table 1), 46.2 for model 1, which was similar to PNS 2019 (46.7 years). Our weighted estimates presented a similar proportion of females compared to the PNS 2019 sample. The proportions of skin colors were similar in all categories and models. Our crude model presented a higher proportion of participants with incomplete elementary school or less compared to model 1 and PNS 2019. Table 2 describes the prevalence of chronic diseases and lifestyle factors in our study and the PNS 2019 sample. Our prevalence of diabetes was higher in the crude model compared to weighted estimates and PNS 2019 sample. In both models, we had a higher proportion of individuals with obesity and hypertension than in PNS 2019. Asthma and/or bronchitis presented similar proportions in our results compared to PNS 2019; the same occurred for cancer. Our study presented a higher proportion of smoking participants in both models than in the PNS 2019 sample. Table 2. First descriptive results and comparison with a population-based study. Chronic diseases and lifestyle factors EAI PELOTAS* PNS 2019+ Crude Survey design 1 % (95%CI) % (95%CI) % (95%CI) Diabetes 14.2 (13.3-15.1) 11.5 (10.6-12.4) 9.0 (8.9-11.1) Obesity 30.4 (29.2-31.7) 29.2 (27.7-30.8) 24.8 (22.6-27.1) Hypertension 39.0 (37.7-40.3) 32.4 (31.0-33.9) 28.1 (25.9-30.5) Asthma or chronic bronchitis 9.3 (8.6-10.1) 9.3 (8.4-10.4) 8.7 (7.3-10.3) Cancer 4.2 (3.7-4.7) 3.4 (2.9-4.0) 3.8 (2.9-4.9) Current smoking 20.6 (19.6-21.7) 20.4 (18.9-22.0) 16.3 (14.6-18.1) *n=5.722; +n=3.002. PNS: Brazilian National Health Survey. PNS 2019 includes residents (selected to interview) from the urban area from the Rio Grande do Sul State; Survey design 1: weighted for primary unit sampling and post-weight estimates. DISCUSSION We described the initial descriptive results, methodology, protocol, and the steps required to perform the ML analysis for predicting the use of urgent and emergency services among the residents of Pelotas, Southern Brazil. We expect to provide subsidies to health professionals and managers for decision-making, helping to identify interventions targeted at patients more likely to use urgent and emergency services, as well as those more likely to develop multimorbidity and mortality. We also expect to help health systems optimize their space and resources by directing human and physical capital to those at greater risk of developing multiple chronic diseases and dying. Recent studies in developed countries have found this a feasible challenge with ML 21,27 . If our study presents satisfactory results, we intend to test its practical applicability and acceptance to assist health professionals and managers in decision-making in emergency services among residents of Pelotas. The baseline and methods used to select households resemble the main population-based studies conducted in Brazil, such as the Brazilian Longitudinal Study of Aging (ELSI-Brazil) 28 , the EPICOVID 29 , and the PNS. The applicability of ML requires suitable predictive variables. Our study included sociodemographic and behavioral variables related to urgent and emergency services, and chronic diseases. EAI PELOTAS study also includes essential topics that deserve particular importance during the COVID-19 pandemic, such as food insecurity, decreased income, physical activity, access to health services, and social support. We also presented one weighting option in order to obtain sample estimates considering the complex study design. All estimates have their strength and limitation. Each research question answered through this study may consider these possibilities and choose the most suitable one. The estimates were similar without weighting and those considering the primary sampling unit (PSU) and sampling weight. Using the census tract in the PSU is fundamental to consider the sampling design in the estimates of variability (standard error, variance, 95%CI, among others). In addition, due to the possible selection bias in the sample, which contains more women and older people than expected, the use of a post-weighting strategy becomes necessary to obtain estimates adjusted for the sex and age distributions of the target population (due to the lack of census data, we used population projections). However, it should be noted that this strategy can produce estimates simulating the expected distribution only by sex and age. Still, we do not know how much this strategy can distort the estimates since the demographic adjustment cannot reproduce adjustment in all sample characteristics, especially for non-measured variables that may have influenced the selection of participants. Thus, we recommend defining the use of each strategy on a case-by-case basis, depending on the objective of the scientific product. Finally, we suggest reporting the different estimates according to the sample design for specific outcomes (e.g., the prevalence of a specific condition) that aim to extrapolate the data to the target population (adults of the city of Pelotas). In conclusion, the present article presented a protocol describing the steps that were and will be taken to produce a model capable of predicting the demand for urgent and emergency services in one year among residents in Pelotas (RS), Southern Brazil. SUPPLEMENTARY DATA: Supplementary data are available at IJE online. DATA AVAILABILITY: All data used in this manuscript are found in the manuscript or in the supplementary material. FUNDING: Research Support Foundation of Rio Grande do Sul, Brazil (FAPERGS) - grant number 21/2551-0000066-0 - Programa Pesquisa para o SUS: gestao compartilhada em saude - PPSUS). Felipe Mendes Delpino received a doctoral fellowship from the National Council for Scientific and Technological Development (CNPq) during the writing of the manuscript. This work was supported by the Research Support Foundation of the State of Rio Grande do Sul (FAPERGS) on the public edict 08/2020 - PPSUS (grant 21/2551-0000066-0). The study was conducted by researchers from the Postgraduate Program of Nursing and the Faculty of Nursing from the Federal University of Pelotas (UFPel). REFERENCES 1. Valentim IVL Kruel AJ The importance of interpersonal trust for the consolidation of Brazil's Family Health Program Cien Saude Colet 2007 12 3 777 88 17680135 2. Brasil. Ministerio da Saude Politica nacional de atencao as urgencias Brasilia Editora do Ministerio da Saude; 2006 3. Guibu IA Moraes JC Guerra AA Junior Costa EA Acurcio FA Costa KS Main characteristics of patients of primary health care services in Brazil Rev Saude Publica 2017 51 suppl 2 17s 29160451 4. Paim J Travassos C Almeida C Bahia L Macinko J The Brazilian health system:history,advances,and challenges Lancet 2011 377 9779 1778 1797 10.1016/S0140-6736(11)60054-8 21561655 5. Castro MC Massuda A Almeida G Menezes-Filho NA Andrade MV Noronha KVMS Brazil's unified health system:the first 30 years and prospects for the future Lancet 2019 394 10195 345 356 10.1016/S0140-6736(19)31243-7 31303318 6. Agborsangaya CB Lau D Lahtinen M Cooke T Johnson JA Health-related quality of life and healthcare utilization in multimorbidity:results of a cross-sectional survey Qual Life Res 2013 22 4 791 799 10.1007/s11136-012-0214-7 22684529 7. Nguyen H Manolova G Daskalopoulou C Vitoratou S Prince M Prina AM Prevalence of multimorbidity in community settings:a systematic review and meta-analysis of observational studies J Comorb 2019 9 2235042X19870934 10.1177/2235042X19870934 8. Nunes BP Thume E Facchini LA Multimorbidity in older adults: magnitude and challenges for the Brazilian health system BMC Public Health 2015 15 1172 10.1186/s12889-015-2505-8 26602756 9. Brasil. Ministerio da Saude Secretaria de Atencao a Saude. Departamento de Atencao Especializada. Manual instrutivo da Rede Atencao as Urgencias e Emergencias no sistema Unico de Saude [Internet] Brasilia Editora do Ministerio da Saude; 2013 [cited on Feb 2, 2023] Available from: 10. King Z Farrington J Utley M Kung E Elkhodair S Harris S Machine learning for real-time aggregated prediction of hospital admission for emergency patients NPJ Digit Med 2022 5 1 104 10.1038/s41746-022-00649-y 35882903 11. Qiao Z Sun N Li X Xia E Zhao S Qin Y Using machine learning approaches for emergency room visit prediction based on electronic health record data Stud Health Technol Inform 2018 247 111 5 29677933 12. Miles J Turner J Jacques R Williams J Mason S Using machine-learning risk prediction models to triage the acuity of undifferentiated patients entering the emergency care system:a systematic review Diagn Progn Res 2020 4 16 10.1186/s41512-020-00084-1 33024830 13. Acosta AM Lima MADS Frequent users of emergency services:associated factors and reasons for seeking care Rev Lat Am Enfermagem 2015 23 2 337 44 10.1590/0104-1169.0072.2560 26039306 14. Rzewuska M Azevedo-Marques JM Coxon D Zanetti ML Zanetti ACG Franco LJ Epidemiology of multimorbidity within the Brazilian adult general population:evidence from the 2013 National Health Survey (PNS 2013) PLoS One 2017 12 2 e0171813 10.1371/journal.pone.0171813 28182778 15. Carvalho JN Roncalli AG Cancela MC Souza DLB Prevalence of multimorbidity in the Brazilian adult population according to socioeconomic and demographic characteristics PLoS One 2017 12 4 e0174322 10.1371/journal.pone.0174322 28384178 16. 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PMC10000015

Cureus Cureus 2168-8184 Cureus 2168-8184 Cureus Palo Alto (CA) 10.7759/cureus.34766 Obstetrics/Gynecology Oncology Clear Cell Ovarian Carcinoma With C1 Lateral Mass Metastasis and Pathologic Fracture: A Case Report Muacevic Alexander Adler John R Sassine Dib 1 Rogerson Daniella 2 Banu Matei 3 Reid Patrick 4 St. Clair Caryn 1 1 Gynecologic Oncology, Columbia University Department of Obstetrics and Gynecology, New York, USA 2 Obstetrics and Gynecology, Columbia University Vagelos College of Physicians and Surgeons, New York, USA 3 Neurosurgery, Columbia University Department of Neurological Surgery, New York, USA 4 Neurosurgery, Columbia University, New York, USA Caryn St. Clair [email protected] 8 2 2023 2 2023 15 2 e347668 2 2023 Copyright (c) 2023, Sassine et al. 2023 Sassine et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This article is available from Osseous metastasis (OM) in ovarian cancer (OC) are rare, with an incidence ranging from 0.8% to 2.6%, and are associated with poor prognosis. The available literature on their management and associated complications is scarce. We report a case of International Federation of Gynecology and Obstetrics (FIGO) stage IVB clear cell epithelial OC (EOC) who presented with neck pain. Imaging revealed multiple cervical spine metastases with left vertebral artery encasement and concurrent C1 lateral mass compression fracture, without neurological deficit, requiring occiput to C2 posterior instrumentation and fusion. Early OM may be associated with shorter overall survival, and survival after OM diagnosis is on the order of months. Management of OM should include a multidisciplinary team and may require surgical stabilization in addition to systemic chemotherapy, local radiotherapy, and osteoclast inhibitors. ovarian clear cell carcinoma vertebral fusion pathological fracture osseous metastasis ovarian carcinoma The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus. pmcIntroduction Ovarian cancer (OC) is the most common cause of gynecological cancer death . Direct spread to adjacent pelvic organs, peritoneal spread, and metastasis via the lymphatic route are common, while hematogenous spread to further sites such as the liver, lung, bone and brain occur less commonly and have a poor prognosis [2-4]. Osseous metastases (OM) in OC are among the rarest, with incidences between 0.82% and 4.0%, and may occur by direct invasion, hematogenous, lymphatic, and transperitoneal spread . The vertebral column is the most common site, though case reports document findings throughout the axial skeleton and pelvis . OMs are associated with shorter overall survival . A recent study found the 1-, 3- and 5-year survival after OM diagnosis were 33%, 15%, and 8%, respectively . Skeletal-related events (SREs) and neurovascular compromise secondary to OM have rarely been described. Here, we report a case of International Federation of Gynecology and Obstetrics (FIGO) stage IVB clear cell epithelial ovarian cancer (EOC) complicated by C1 vertebral metastases with vertebral artery involvement and C1 vertebral fracture, necessitating occiput to C2 posterior instrumentation and fusion and palliative C2 neurectomy. Case presentation A 59-year-old African American P0 (nulliparous) with a history of uterine leiomyoma presented with abdominal pain and bloating. A transabdominal sonogram demonstrated multiple large, complex adnexal masses. Staging computed tomography (CT) chest, abdomen and pelvis was suspicious for metastatic ovarian carcinoma, with bilateral complex adnexal masses, peritoneal carcinomatosis, omental caking, and pericardiac, abdominal, peripancreatic, mesenteric, retroperitoneal, and pelvic lymphadenopathy. No osseous lesions were identified at the time of the initial scan. An initial consult with gynecological oncology revealed an abdominal mass above the umbilicus, pelvic masses, and a palpable left supraclavicular node. Ca-125 was 6,773. Pathological examination of interventional radiology-guided peritoneal mass biopsies had an immunoprofile compatible with a high-grade adenocarcinoma of Mullerian origin, favoring clear cell carcinoma. The tumor showed preserved mismatch repair (MMR) proteins expression (positive for MLH1, PMS2, MSH2, MSH6). Immunostain for human epidermal growth factor receptor 2 (HER-2) was equivocal and difficult to interpret on the scant cell block material with mostly cytoplasmic staining, no convincing membranous staining, score 0-1, and immunostaining for PD-L1 was limited by scant cellularity in the cell block, focal positivity with an estimated combined positive score (CPS) 3-4. Further genetic testing did not show any mutations for possible targeted therapy. The patient was diagnosed with FIGO stage IVB clear cell EOC. She received her first cycle of carboplatin, paclitaxel, and bevacizumab, with a plan for 3-4 cycles of neoadjuvant chemotherapy and interval debulking pending clinical response. After her first cycle of chemotherapy, the patient endorsed persistent neck pain of moderate severity. Magnetic resonance imaging (MRI) cervical spine revealed contrast-enhancing osseous lesions within the anterior arch and left lateral mass of C1, as well as in the C4 and C7 vertebral bodies, left articulating facet of C6 and T4 vertebral body extending into the left pedicle. There was lateral tumor extension with encasement of the vertebral artery at the C1 level. There was no evidence of epidural disease or spinal cord impingement . CT angiogram (CTA) demonstrated multiple osseous lytic lesions and a C1 lateral mass compression fracture extending to the left transverse foramen, with asymmetry of the lateral atlantodental interval measuring 8 mm on the right and 2 mm on the left. There was circumferential tumor encasement of the left vertebral artery in the sulcus arteriosus, with severe narrowing with preserved flow and no evidence of dissection . Metastatic cervical and supraclavicular lymphadenopathy were confirmed. The total body bone scan did not show further OM. Figure 1 Sagittal (left) and axial (right) MRI of the patient A. T2-weighted and B. T1 fat saturation MRI sequences demonstrating C1 lateral mass lesion (red arrow) and displacement of the vertebral artery with no epidural tumor extension or cord compression. C. Parasagittal T1 sequence demonstrating lateral tumor extension with encasement of the vertebral artery (red arrow). D. Coronal (left) and axial (right) CT angiogram demonstrating osteolytic left lateral mass lesion encasing and narrowing the vertebral artery (red arrows). The patient was admitted to neurosurgery with gynecological oncology consultation, where she continued to endorse left-sided cervical pain radiating to the occiput which worsened with movement and improved with opioids, steroids and immobilization with a hard collar. There were no focal neurological deficits, paresthesias, anesthesia, gait difficulties, or bowel or bladder dysfunction. The patient had full strength in the upper and lower extremities and an intact gait. She had no signs of myelopathy and had a negative Hoffman's sign. Given normal cervical alignment, with imaging demonstrating articular facet involvement and lateral mass fracture, this pain was considered to be caused by cervical spine instability . Figure 2 Preoperative (A) and postoperative (B and C) radiographs A. Preoperative anterior-posterior (left) and upright lateral (right) radiographs demonstrating normal cervical spine alignment. B. Postoperative anterior-posterior (left) and lateral (right) cervical spine radiographs demonstrating appropriate instrumentation placement and alignment after posterior hardware fixation of the occiput to C2. C. Postoperative anterior-posterior (left) and lateral (right) standing scoliosis series illustrating normal alignment. Urgent surgery was not indicated without neurological deficits or compressive pathology. A multidisciplinary team discussed the case to determine if surgical decompression was required prior to local radiation, and surgical intervention with occiput to C2 instrumentation and fusion was decided upon. Surgery was performed two days after diagnosis. Intraoperatively, there was extensive tumor involvement of the C1 lateral mass and posterior arch, encasing the vertebral artery and extending towards the occipital condyle. A palliative C2 neurectomy was performed for pain control. A right C1 lateral mass screw, bilateral C2 pedicle screws, and occipital keel plate with three bicortical screws were placed. The fusion bed was prepared by decorticating the occiput and bilateral C1-2 joint spaces with the placement of allograft over the decorticated spaces. Intraoperative monitoring was stable throughout and the patient awoke at neurological baseline. Postoperatively, the patient endorsed significant improvement of her cervical pain. Incisional pain was controlled with methocarbamol, gabapentin and hydromorphone PCA (patient-controlled analgesia). Radiographs demonstrated excellent instrumentation placement and alignment . The patient ambulated without assistance on postoperative day (POD) 1 and was discharged in stable condition on POD3. Radiographs of the spine confirmed stable spinal fusion rods three weeks postoperatively . There was complete resolution of the cervical pain. The patient received her second cycle of carboplatin and paclitaxel four weeks postoperatively; at that time, progression of cervical lymphadenopathy was noted on exam. She since received two additional cycles three and six weeks later, with a downtrending CA-125 of 2,301. Bevacizumab has been held to allow for surgical healing. Denosumab injection of 120 mg every 4 weeks, calcium, and vitamin D were initiated postoperatively, with a plan for palliative radiation therapy (RT) of 20 Gy in 5 fractions to C1-2 and associated hardware to prevent disease progression. Discussion This report illustrates a unique complication of OM in EOC. Given the rare nature of these lesions, the literature is sparse. Four retrospective studies comment on OM in the context of other rare OC metastases . In these, the incidence of OM ranged between 1.2% and 4%. Deng et al. analyzed 1481 patients with stage 4 ovarian carcinoma using the Surveillance, Epidemiology, and End Results (SEER) database. OM was found in 3.74% of the entire cohort with a median survival time of 11 months for the patients with OM. Dauplat et al. examined 255 patients with EOC with stage 4 disease. Only four patients (1.6%) had OM with a median survival of 9 months. Gardner et al. used the SEER database to determine the pattern of the distant metastases at the initial presentation in patients with gynecological cancer and found that OM was present in 4% of the patients with ovarian carcinoma, vs 13% and 23% of patients with uterine and cervical cancer, respectively. Additionally, four retrospective studies looked specifically at OM in OC (Table 1). Table 1 Four retrospective studies examine osseous metastasis (OM) in ovarian carcinoma Publication - Author, Year Cohort - No OM - No (%) Overall Survival - Months Survival After OM Diagnosis - Months Most Commonly Involved Site Ak et al., 2021 (2) 736 19 (2.60) 38.1 13.6 Vertebral Sehouli et al., 2013 (5) 1,717 26 (1.50) 50.5 7.2 Vertebral Zhang, C. et al., 2019 (6) 32,178 352 (1.09) 50.0 5.0 Not Reported Zhang, M. et al., 2013 (7) 2,189 26 (0.82) Not Reported Not Reported Vertebral Ak et al. examined 736 OC patients and found OM in 2.6%; OM was mostly seen with clear cell histology similar to our case report. The vertebrae, as in our patient, were most commonly involved, though patients often had multiple sites of involvement. Pain was the most common presenting symptom, but was absent in over 50%. Unlike our case, out of the two patients presenting with pathologic fractures, one had neurologic deficit. Patients were managed with palliative RT and bisphosphonates. Only advanced, inoperable disease at presentation was associated with a shorter time to development of OM. Median overall survival in OC patients with OM was 38.1 months and median survival after OM diagnosis was 13.6 months. Patients who had OM at the time of diagnosis of their OC had a shorter median OS than those who developed OM later on, 6.1 vs 63 months, respectively. To note, although insignificant, overall survival was shorter in patients with clear cell histology after OM than in patients with a different histologic subtype ( 7 vs 22 months) . Sehouli et al. examined 1,717 patients and found OM in 1.5%. The vertebral column was the most common site, most frequently in the lumbar, followed by the thoracic and cervical regions. Multiple OM were seen in the majority of patients, as in our case. Pain was the presenting symptom in 66%; 9% had impaired mobility unlike our patient, and 4% had neurologic symptoms. Pathologic fractures were reported in 33%. Most patients were treated with bisphosphonates; of those treated, 26% went on to develop pathological fractures and required surgical intervention. RT of OM was performed in a minority of cases for pain control and for SRE prevention. OM was noted to progress in 75% of cases despite systemic and local therapy. The median overall survival of the entire cohort was 50.5 months; patients with early OM had significantly shorter overall survival than those with later OM, 11.2 vs 78.4 months; to note, the overall survival rates were calculated regardless of the histology of the disease. By far, the largest study on this topic, Zhang, C. et al. , examined 32,178 Surveillance, Epidemiology, and End Results (SEER) database OC patients and found the prevalence of OM was 1.09%. OM were more common in women over 65, Black and unmarried women. Similar to our patient, most of those patients had an advanced stage, poorly differentiated grade, non-serous type, elevated CA-125 and had concurrent distant metastasis. The median overall survival for the entire cohort was 50 months, whereas the median overall survival after OM diagnosis was 5 months only. Among examined variables, only surgery at the primary site was associated with significantly longer survival, 18 vs 3 months for primary site surgery versus no surgery. Survival was significantly shorter in patients with a non-serous histology without commenting specifically on the clear cell carcinoma histology . Lastly, Zhang, M. et al. found OM in 0.82% of 2,189 OC cases. The majority of OM were vertebral (12 cervical, 10 lumbar, seven thoracic), eight were pelvic, five were limb, two were sternal, and one was rib. Over half presented with pain, 35% with difficulty walking, and 15% were asymptomatic. Similar to our case report, the majority of cases with OM were in advanced disease and EOC, and most of them were managed with chemotherapy and RT. Cases that were managed with combined chemotherapy and RT had significantly longer survival than those treated with either agent alone, 14.2 versus 11 versus 8.4 months for combined therapy, RT alone, and chemotherapy alone, respectively . There is a paucity of data regarding the management of OM with or without SRE in OC, and the current management of lesions is based on that for other solid tumors. Diagnosis includes history and physical, and imaging options include radiographs, CT and MRI; MRI is the modality of choice for vertebral lesions. A multidisciplinary team approach is often needed for the management of such rare cases, including radiology, orthopedic surgery, neurosurgery, radiation oncology , gynecological oncology, palliative and pain medicine . All of these modalities were utilized as illustrated in this case. Conclusions In conclusion, we present a unique case of clear cell EOC with vertebral OM resulting in pathologic C1 fracture requiring surgical stabilization, further complicated by vertebral artery encasement and narrowing. OM secondary to OC are rare and often present with pain though rarely with neurologic deficit. The risk factors for the development of OM are poorly understood but may include clear cell EOC as in this case, and lesions are more commonly described in those with late-stage disease. Patients who present with OM at the time of diagnosis or early in their disease course may have shorter overall survival than those with later OM. However, survival after OM diagnosis is on the order of months. Surgery at the primary site and combination chemotherapy and RT may prolong survival. These findings are based on limited retrospective studies, and further examination of risk factors and prognostic implications of OM in OC is needed. Human Ethics The authors have declared that no competing interests exist. 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PMC10000017

Rev Bras Epidemiol Rev Bras Epidemiol rbepid Revista Brasileira de Epidemiologia (Brazilian Journal of Epidemiology) 1415-790X 1980-5497 Associacao Brasileira de Saude Coletiva 10.1590/1980-549720230020 00419 Original Article Difficulties in the use of medications by elderly people followed up in a cohort study in Southern Brazil Dificuldades no uso de medicamentos por idosos acompanhados em uma coorte do Sul do Brasil Guttier Marilia Cruz Analise formal Conceituacao Escrita - primeira redacao Escrita - revisao e edicao Metodologia Validacao I Silveira Marysabel Pinto Telis Analise formal Conceituacao Escrita - primeira redacao Escrita - revisao e edicao Metodologia Validacao II Tavares Noemia Urruth Leao Analise formal Conceituacao Escrita - revisao e edicao Metodologia Validacao III Krause Matheus Carrett Analise formal Conceituacao Escrita - revisao e edicao Metodologia Validacao IV Bielemann Renata Moraes Escrita - revisao e edicao Investigacao Metodologia Obtencao de financiamento Supervisao Validacao V Gonzalez Maria Cristina Escrita - revisao e edicao Investigacao Metodologia Obtencao de financiamento Supervisao Validacao VI Tomasi Elaine Escrita - revisao e edicao Investigacao Metodologia Obtencao de financiamento Supervisao Validacao I Demarco Flavio Fernando Escrita - revisao e edicao Investigacao Metodologia Obtencao de financiamento Supervisao Validacao I Bertoldi Andrea Damaso Escrita - primeira redacao Escrita - revisao e edicao Investigacao Metodologia Obtencao de financiamento Supervisao Validacao I Universidade Federal de Pelotas, Programa de Pos-Graduacao em Epidemiologia -Pelotas (RS), Brasil. Universidade Federal de Pelotas, Instituto de Biologia, Departamento de Fisiologia e Farmacologia, Programa de Pos-Graduacao Multicentrico em Ciencias Fisiologicas - Pelotas (RS), Brasil. Universidade de Brasilia, Faculdade de Farmacia - Brasilia (DF), Brasil. Universidade Federal de Pelotas, Faculdade de Medicina - Pelotas (RS), Brasil. Universidade Federal de Pelotas, Programa de Pos-Graduacao em Nutricao e Alimentos - Pelotas (RS), Brasil. Universidade Catolica de Pelotas, Programa de Pos-Graduacao em Saude e Comportamento - Pelotas (RS), Brasil. AUTORA CORRESPONDENTE: Marilia Cruz Guttier. Rua Marechal Deodoro, 1160, 3o Piso, Centro, CEP: 96020-220, Pelotas (RS), Brasil. E-mail: [email protected] CONFLITO DE INTERESSES: nada a declarar 10 3 2023 2023 26 e23002029 9 2022 12 11 2022 18 11 2022 Este e um artigo publicado em acesso aberto sob uma licenca Creative Commons ABSTRACT Objective: This study aimed to assess the need for help by elderly people to take their medications, the difficulties related to this activity, the frequency of forgotten doses, and factors associated. Methods: Cross-sectional study conducted with a cohort of elderly people (60 years and over -- "COMO VAI?" [How do you do?] study), where the need for help to properly take medication and the difficulties faced in using them were evaluated. The Poisson regression model was used to estimate the crude and adjusted prevalence ratios (PR) of the outcomes and respective 95% confidence intervals according to the characteristics of the sample. Results: In total, 1,161 elderly people were followed up. The prevalence of participants who reported requiring help with medication was 15.5% (95%CI 13.5-17.8), and the oldest subjects, with lower educational levels, in worse economic situations, on four or more medications and in bad self-rated health were the ones who needed help the most. Continuous use of medication was reported by 83.0% (95%CI 80.7-85.1) of the sample and most participants (74.9%; 95%CI 72.0-77.5) never forgot to take their medications. Conclusion: The need for help to use medications was shown to be influenced by social and economic determinants. Studies assessing the difficulties in medication use by the elderly are important to support policies and practices to improve adherence to treatment and the rational use of medications. RESUMO Objetivo: Este estudo visou avaliar a necessidade de ajuda dos idosos para tomar seus medicamentos, bem como as dificuldades relacionadas com a sua utilizacao, e a frequencia de esquecimento de doses. Ainda, avaliar fatores associados a necessidade de ajuda dos idosos com os medicamentos. Metodos: Corte transversal em uma coorte de idosos (60 anos ou mais -- estudo "COMO VAI?"), em que foi avaliada a necessidade de ajuda para tomar medicamentos de forma adequada e as dificuldades apresentadas na sua utilizacao. Utilizou-se regressao de Poisson para estimar as razoes de prevalencia (RP) brutas e ajustadas dos desfechos e seus intervalos de confianca de 95% (IC95%) de acordo com as caracteristicas da amostra. Resultados: Participaram 1.161 idosos. A prevalencia de idosos que relataram necessidade de ajuda com os medicamentos foi de 15,5% (IC95% 13,5-17,8), sendo que os mais idosos, com menor escolaridade e em pior situacao economica, em uso de quatro medicamentos ou mais e com pior autoavaliacao de saude foram os que mais necessitaram de ajuda. O uso continuo de medicamentos foi referido por 83,0% (IC95% 80,7-85,1) e a maioria (74,9%; IC95% 72,0-77,5) nunca se esqueceu de tomar seus medicamentos. Conclusao: Observou-se a influencia de determinantes sociais e economicos e de saude sobre a necessidade de ajuda para a utilizacao dos medicamentos. Estudos que estimem as dificuldades no uso de medicamentos por idosos sao importantes para subsidiar politicas e praticas norteadoras de acoes para melhorar a adesao e o uso racional de medicamentos. Keywords: Old age assistance Elderly Cohort studies Drug utilization Palavras-chave: Assistencia a idosos Idoso Estudos de coortes Uso de medicamentos pmcINTRODUCTION Aging with quality of life is an important challenge when it comes to the care for the elderly. Policies have been developed seeking to promote health and aging with autonomy in the elderly population 1 , as well as to help caregivers, as there is an increase in the incidence of chronic diseases and, consequently, the need for medication to treat them 1-3 . In Brazil, the prevalence of use of at least one continuous-use medication among the elderly ranges from 80 to 93% 4-6 . In Italy, this prevalence was similar (88%) 7 . Elderly people make use of multiple medications and are exposed to complex therapeutic regimes 6-11 , which can be unfavorable to treatment effectiveness. Considering that the barriers to accessing health services and medication have been overcome and that the elderly have their drug treatment in hand, there are still other difficulties faced by them. Decline in cognitive status 12 , need for greater attention 13 , loss of visual acuity 14 and loss of ability to handle medication packages 15 , as well as difficulties related to memory and time organization and management, can also be complicating factors for the correct use of medication 16 . In a cross-sectional study carried out in the city of Marilia (SP), 59.8% of the elderly reported difficulties related to the use of medications, with forgetfulness being cited by a quarter of them 16 . A study carried out in Sweden pointed out that the majority (66.3%) of the elderly population had some limitation in the ability to manage their treatments 17 . Another difficulty cited by the elderly was the lack of belief in their efficacy 18 . The main result of these difficulties is the lack of adherence to treatment, but they also contribute to errors in medication administration 16,17 , leading to unsatisfactory clinical results, adverse reactions and drug interactions 9,19 . Bearing in mind that the literature deals with these difficulties within adherence assessment scores 18,20,21 or in studies assessing the instrumental activities of daily living (IADL) of the elderly 13,22-24 , this study aimed to assess the need for help by the elderly to take medications, as well as the difficulties faced when using them, and the frequency doses skipped or forgotten, after having overcome barriers to accessing health services and acquiring medication. Furthermore, the purpose was to evaluate factors associated with the need for help when taking medication at the correct dose and time. METHODS Cross-sectional study with a cohort of elderly people, conducted in the urban area of the city of Pelotas, state of Rio Grande do Sul, Brazil (approximately 340,000 inhabitants in 2016). According to the Brazilian Institute of Geography and Statistics (IBGE) 25 , in 2010, 93% of the population of Pelotas lived in urban areas and approximately 50,000 were aged 60 years or older. The sample recruitment and the first visit of the study called "COMO VAI?" ("How do you do?") took place from January to August 2014. In total, 1,451 non-institutionalized elderly aged 60 years or older were included. The sampling process was carried out in two stages. Initially, clusters were selected using data from the 2010 Census 25 , with census groups being selected by lot. In the second stage, listed and systematically drawn households were selected--31 per sector--to enable the identification of at least 12 elderly people in each of them. The second follow-up took place between November 2016 and April 2017, by telephone interviews; household visits were made in cases where telephone contact was not possible. Calls were made on different days and times, and participants not contacted by telephone had at least four visit attempts at the addresses made available to the study. The understanding of the questions was tested in a pilot study applied in face-to-face and telephone interviews. Demographic, socioeconomic, and behavioral characteristics were the independent variables selected based on studies assessing adherence to treatment 18,20,21 and IADL 13,22-24 . The following characteristics were collected in the first interview, to assist in the description of the sample: biological sex (male, female); age (60-69, 70-79, >=80 years); skin color (self-reported, using the following categories: white, black, brown, yellow and indigenous, with the elderly self-declared as brown, yellow and indigenous grouped under the "mixed" category, due to the low frequency); education, defined as the highest level of education achieved in years of study (later categorized as none, <8 and >=8 years); marital status (married/with a partner, single/divorced/widowed--considered "no partner"); economic situation (A/B -- richer; and C, D/E -- poorer), according to the criteria of the Brazilian Association of Companies and Research 26 . Behavioral and health variables were also considered, given their importance in the evaluation of health care for the elderly. Characteristics such as current smoking (yes, no) were evaluated, considering daily cigarette consumption for more than one month; and alcohol consumption (yes, no), considering consumption of at least one dose of alcoholic beverage in the last 30 days. In addition, the concept of "polypharmacy" was evaluated, that is, simultaneous use of four or more medications 27 . Health perception was measured in 2016 by the question "How do you rate your health?", with the following response options: very good, good, regular, poor and very poor, later recategorized as very good/good, fair, bad/very bad. Outcomes were obtained at the second follow-up with the following filter question: "Do you need help taking your meds at the right dose and time?" (yes/no), which indicated the need for help with medication. Among those who needed help with their treatments, the three outcomes related to difficulties in taking medications were evaluated using the following questions: "Thinking about your medication, could you tell me if the following actions are 'very difficult', 'a little difficult' or if 'not difficult'? removing the medicine from package; reading the medicine package, to assess difficulties with handling, and understanding the package; taking too many medications at the same time, or difficulty with the amount of medications in use. Continuous medication was also evaluated using the question "Do you take any continuous use medicine regularly, with no date to stop?" (Yes/No). For those who were on continuous medication, the following question was asked: a) "Do you sometimes forget to take your medicine?" (Yes/No); b) "How often do you have trouble remembering to take all your medications?", with five response options: never/rarely, from time to time, sometimes, usually, always. Then, the responses were grouped into three categories (never/rarely, occasionally/sometimes, usually/always). These categories have been renamed to never, occasionally, and usually, respectively. Only elderly people who met the outcome and were followed up at both moments were included in the analyses. The analytical sample maintained the characteristics of the original cohort, with the exception of age, since there was a significant decrease in the proportion of elderly aged 80 years or older (p=0.044) (Supplementary Table). Analyses were performed using the Stata statistical package, version 16.0 (Stata Corporation, College Station, USA). First, the sample was described (followed up in 2016 and 2017). Afterwards, the prevalence and 95% confidence intervals (95%CI) of the main outcome were obtained according to the characteristics of the sample. Poisson regression with forward selection was used to estimate the crude and adjusted prevalence ratios (PR), and the adjusted model included the variables that presented p<0.20 in the crude analysis to control for possible confounding factors. The respective 95%CI of each predictor's PR were estimated. Descriptive analyses of the frequencies of outcomes were performed. Proportions were compared using the Pearson's kh2 test. Linear trend was assessed for significant associations between outcomes and exposure to more than two categories. The level of statistical significance was set at p<0.05. The study was approved by the Research Ethics Committee of the Medical School of Universidade Federal de Pelotas--CAAE: 54141716.0.0000.5317. The participants or caregivers signed an informed consent form, guaranteeing data confidentiality. In 2016 and 2017, for the elderly interviewed by telephone, consent was provided verbally with acceptance to answer the questionnaire. RESULTS The initial sample, in 2014, consisted of 1,451 elderly people. Of these, in 2016, 1,306 participants were located (145 obits identified). The follow-up rate was 90%, with the 1,161 elderly people who were alive being followed up. Most interviews (74.4%) took place over the phone. Table 1 shows the analysis of the outcome "Need for help to take medication at the right dose and time", according to demographic and socioeconomic characteristics in 2016. Most participants were females (63.7%) aged between 60 and 69 years (56.0%), white (83.6%), with less than 8 years of schooling (54.2%), married or with a partner (55.9%), and in level C economic status (57.6%). Altogether, 15.5% of the elderly (95%CI 13.5-17.8) reported needing help with medication use. There was no significant difference in the prevalence of help needed according to biological sex and skin color. Age, educational level and economic situation were important predictors for this outcome. The prevalence of elderly aged 80 years or older who reported needing help was 2.3 times higher (95%CI 1.6-3.5) than among subjects aged between 60 and 69 years, and 3.0 times higher (95%CI 1.6-5.4) among participants with no schooling, compared to those with 8 years or more of schooling. The prevalence of elderly people who reports needing help with their medications in economic strata D/E was 70% higher (PR=1.7; 95%CI 1.0-2.8) than among those in economic strata A/B. Marital status, after adjustment, lost statistical significance (Table 1). Table 1 Sample description, with prevalence, crude and adjusted prevalence ratios of help needed to take medication at the right dose and time and respective 95% confidence intervals according to demographic and socioeconomic characteristics. Pelotas (RS), 2016. Sample n Prevalence % (95%CI) Crude PR 95%CI Adjusted PR * 95%CI Sex 0.672 Male 421 (36.3) 14.9 (11.7-18.7) 1.0 Female 740 (63.7) 15.9 (13.4-18.7) 1.1 (0.8-1.5) Age (years) <0.001 <0.001 60-69 648 (56.0) 10.1 (8.0-12.7) 1.0 1.0 70-79 363 (31.3) 17.4 (13.8-21.7) 1.7 (1.2-2.4) 1.3 (0.9-1.9) 80 and older 147 (12.7) 34.5 (27.1-42.7) 3.4 (2.3-4.9) 2.3 (1.6-3.5) Skin color 0.285 White 969 (83.6) 14.7 (12.5-17.1) 1.0 Black 132 (11.4) 18.7 (12.9-26.5) 1.3 (0.8-2.0) Mixed 58 (5.0) 21.8 (12.8-34.6) 1.5 (0.8-2.7) Education (Years of study) <0.001 0.002 None 147 (12.8) 30.7 (26.6-38.8) 5.5 (3.2-9.2) 3.0 (1.6-5.4) Less than 8 625 (54.2) 17.8 (15.0-21.1) 3.2 (2.0-5.1) 2.3 (1.3-3.8) 8 and more 380 (33.0) 5.6 (3.7-8.5) 1.0 1.0 Marital Status 0.021 0.846 Partner 648 (55.9) 13.1 (10.7-15.9) 1.0 1.0 No partner 511 (44.1) 18.5 (15.4-22.2) 1.4 (1.1-1.9) 1.0 (0.7-1.3) Economic status + <0.001 0.029 A/B 311 (28.2) 10.5 (7.5-14.4) 1.0 1.0 C 634 (57.6) 14.0 (11.5-17.0) 1.3 (0.9-2.0) 1.0 (0.7-1.6) D/E 156 (14.2) 30.9 (24.0-38.7) 3.0 (1.9-4.6) 1.7 (1.0-2.8) Total 15.5 (13.5-17.8) * Analysis adjusted for age, education, marital status, economic status, alcohol consumption in the last 30 days, polypharmacy, and self-rated health; + A/B -- richest, C, D/E -- poorest, according to the Brazilian Association of Companies and Research26; PR: prevalence ratio; 95%CI: 95% confidence interval. Table 2 addresses the same outcome according to behavioral and health characteristics of participants. Most did not smoke (88.4%) or drink (76.5%), were under the concept of polypharmacy (53.7%), and perceived their health as very good or good (56.5%). Polypharmacy and self-rated health were important predictors for this outcome. The prevalence of elderly people who needed help was 1.6 times higher (95%CI 1.1-2.3) among those on four or more medications, compared to those on less than four medications. The worse the self-perception of health, the greater the need for help to take the medication, and among those who perceived their health as poor or very poor, the prevalence of help needed was 100% higher (PR=2.0; 95%CI 1.2-3.2) than among those who perceived their health as very good or good. Alcohol consumption in the last 30 days lost statistical significance after adjustment (Table 2). Table 2 Description of the sample, prevalence, crude and adjusted prevalence ratios of help needed to take medication at the right dose and time with respective 95% confidence intervals according to behavioral characteristics. Pelotas (RS), 2016. Sample n Prevalence % (95%CI) Crude PR 95%CI Adjusted PR * 95%CI Smoking currently 0.450 No 1.024 (88.4) 15.8 (13.6-18.2) 1.2 (0.7-2.0) Yes 135 (11.6) 13.1 (8.3-20.0) 1.0 Alcohol intake in the last 30 days 0.002 0.372 No 886 (76.5) 17.3 (14.9-20.0) 1.9 (1.2-2.8) 1.2 (0.8-2.0) Yes 272 (23.5) 9.3 (6.4-13.4) 1.0 1.0 Polypharmacy 0.002 0.006 No 483 (46.3) 11.6 (9.0-14.8) 1.0 1.0 Yes 561 (53.7) 20.9 (17.7-24.5) 1.8 (1.3-2.5) 1.6 (1.1-2.3) Self-rated health status <0.001 0.012 Very good/good 654 (56.5) 9.9 (7.8-12.5) 1.0 1.0 Regular 414 (35.8) 20.4 (16.7-24.6) 2.1 (1.5-2.8) 1.5 (1.1-2.1) Bad/very bad 89 (7.7) 35.4 (25.7-46.5) 3.6 (2.3-5.6) 2.0 (1.2-3.2) Total 15.5 (13.5-17.8) * Analysis adjusted for age, education, marital status, economic situation, alcohol consumption in the last 30 days, polypharmacy, and self-rated health; PR: prevalence ratio; 95%CI: 95% confidence interval. Figure 1 shows the difficulties cited by the 176 elderly people who reported needing help to use medication, stratified by age. No significant difference was observed in the difficulty of removing medications from the package between age groups (p=0.55), to read package instructions (p=0.09) or to take many medications at the same time (p=0.55). For all ages, most participants do not find it difficult to unpack medications and take many at the same time. However, the most prevalent answer for reading the package was "very difficult" at all ages . Figure 1. Level of difficulties faced by the elderly who reported needing help to use medication according to age (n=176). Pelotas (RS), 2016. Pearson's kh2 test to compare the proportions of each outcome with age. In assessing the use of continuous medication, 962 (83.0%; 95%CI 80.7-85.1) participants used them, among which 23.4% (95%CI 20.8-26, 1) reported occasionally forgetting to take doses. Figure 2 shows the proportion of elderly people on continuous medication who reported needing help to take them as forgetting is concerned. Among these users, 17.0% (95%CI 14.7-19.5) reported needing help and 83.0% (95%CI 80.5-85.3) reported not needing help. The proportion of forgetting doses among participants who needed help (35.0%) was significantly higher than among those who did not need help (20.5%; p<0.001) . Figure 2. Proportion of elderly people on continuous medication who reported needing help to take their medications, according to the report of eventually forgetting doses (yes/no) (n=962) Pelotas (RS), 2016 (p<0.001). Pearson's kh2 test. Figure 3 shows the frequency of forgetting doses according to age, for those on continuous medication. Among 956 users, most of them (74.9%; 95%CI 72.0-77.5) never forgot to take any doses. For the 60- 69 age group, 19.3% (95%CI 16.2-23.0) occasionally forget and 2.9% (95%CI 1.7-4.8), usually forget. In the age group 70-79 years old, 26.1% (95%CI 21.4-31.3) occasionally forget and 5.2% (95%CI 1.7-4.7), usually forget. Among the aged 80 years or older, 14.4% (95%CI 9.4-21.5) eventually forget and 8.3% (95%CI 4.7-14.4) usually forget (p=0.002) . Figure 3. Frequency of forgetting medications according to age groups (n=959) Pelotas (RS), 2016 (p=0.002). Pearson's kh2 test to compare outcome proportions with age. DISCUSSION This study shows that 15.5% of the elderly needed help to use their medication in the right dose and at the right time, and the greater the age, the lower the level of education and the worse the economic situation, the greater the proportion of elderly people who reported the need for help. Although there are methodological differences in studies that evaluate outcomes regarding the need for help and difficulty in using medications, in a population-based study carried out with elderly people aged 60 years or older in the city of Sao Paulo (SP), 8.5% of them had difficulty taking their medication and 89.3% received some sort of help in this task 28 . The need for help with medications is a delicate issue, as when misused, they predispose the elderly population to the risks of polypharmacy and the possibility of developing more intense adverse or therapeutic effects, in addition to the likely increase in cost, both individually and for the health system 4 . In addition, the need for help with medication can result in the need to expand the care network for the elderly and, in most cases, this network starts with family members, who leave aside their profession, leisure activities, and self-care to meet the needs of the elderly, often for prolonged periods, often until their deaths, which can lead to damage to the quality of life of the caregiver and the family 29 . Another study, carried out in basic health units in the city of Sao Paulo (SP), used the Lawton Scale to identify the degree of dependence for IADL, and one of the evaluated items was whether the individual was able to take their medication in correct doses and in at correct times. It was observed that 46.8% of the elderly cannot, 28.2% need partial help, and only a quarter can use their medication without any help 24 . Several factors are associated with impairment of functional capacity, such as advanced age, female gender, low income and education 24 . Low educational level was also associated with the inability to take medication in a descriptive study carried out with 95 elderly people treated at a Family Health Strategy (FHS) unit in Goiania (GO) 13 , showing that adverse social and economic conditions negatively influence issues related to health, such as the need for help to use medications at correct dose and time. In that study, 30.0% of the elderly needed reminders to take their medications at the right time and 13.0% were unable to take them by themselves 13 . The need for help from the elderly to deal with their treatments due to difficulty in handling medication packages, reading the packaging or taking too many medications directly interferes with adherence to treatment. Adherence to treatment is a complex, multifactorial matter that is essential for therapeutic results. When the patient does not adhere to treatment, there may be changes of various types, such as reduced benefits, increased risks, or both, which contributes to increased treatment costs for the elderly and for health services 30 . In this sense, understanding the factors that prevent the patient from following the recommendations of health professionals is important. The need for help to take the medication in this study can be explained, in part, by difficulties in activities of daily living identified in the first follow-up, which were also associated with older age, lower education, and presence of multimorbidities 22 ; however, this information was not collected in the follow-up from 2016, not allowing for these analyses. Considering that this is a longitudinal follow-up and aging being a limitation for the use of medication, it is likely that there will be an increase in the difficulties faced while using medication in the upcoming follow-ups. Regarding the difficulties with the therapeutic regimen presented by the elderly who reported needing help, the greater difficulty was related to removing the medication from the package and reading it among elderly people aged 80 years or older. These difficulties may be associated with the loss of fine motor skills and reduced visual acuity in this population, although this study has not found a significant difference. There is evidence that physiological aging can lead to decline in some tasks 31-33 . A systematic review aimed at analyzing factors associated with the autonomy of the elderly showed that the oldest (over 80 years old) are 40% more likely to let other people decide for them, when compared to those aged 60-69 years. That is, with aging, the probability of loss of autonomy increases, as well as the perception of autonomy worsens 34 . Also, visual acuity can decrease with age and this can affect the ability of the elderly to read information on the medicine package, leading to errors or confusions, especially with those whose names are similar. A study carried out with 96 elderly people aged 65 and older from a community in the countryside of Sao Paulo showed a significant increase in the prevalence of low vision, compromising activities of daily living 35 . Other important points refer to continuous medications, polypharmacy, and the self-perception of health. The elderly population lives with chronic health problems, being a great consumer of health services and medicines 36 , especially those for continuous use. This study showed that most elderly people aged 60 and older use this type of medication and that polypharmacy and poorer health perception were also associated with a greater need for help with medication. The high prevalence of polypharmacy among the elderly population points to the importance of identifying the needs of this population in order to make rational use of treatment 37,38 . However, the results of this study showed that, of those on continuous medication, about a quarter eventually forget to take their medication, although most of them reported never forgetting (74.9%; 95%CI 72.0- 77.5). These results were lower than those reported by Bezerra et al. 39 and Rocha et al. 5 , and higher than those reported by Marin et al. 16 Forgetting is a serious problem, as it can directly impact adherence to treatment and, consequently, the effectiveness of medications, leading to unsatisfactory control of multimorbidities 40 . It is estimated that, in high-income countries, adherence to long-term therapies accounts to only 50% on average. In middle-income countries, the rates are even lower, which seriously compromises the efficacy of treatments and has important implications in quality of life, the economy, and public health 1 . One of the limitations are the impossibility of collecting all behavioral and health characteristics in the same follow-up in which the outcome was collected, which may have underestimated or overestimated the relation of these variables with the outcome, even though the interval between follow-ups was of only two years. Not having evaluated the functional limitations of the elderly can also be a limitation, as these characteristics can directly influence the outcomes. However, the study has strengths: a population-based longitudinal study sample was used, with frequent follow-ups; however, hospitalized or institutionalized elderly were not included in the study. Even working with the elderly and the study not being initially planned to have a cohort design, the follow-up rate was high. Social and economic determinants were found to influence on the elderly's need for help to use their medications, and a high prevalence of elderly people on continuous treatment (with a quarter of these forgetting to take doses eventually, significantly higher among those who need help). Studies that estimate the difficulties faced with medications by the elderly are important to support health policies and practices aimed at minimizing issues and guiding actions to improve adherence to treatment and rational use of medication. ACKNOWLEDGMENTS The authors would like to thank all participants of the cohort study "COMO VAI?" ("How do you do?") conducted in 2014 with elderly people living in Pelotas, RS, Brazil, and the entire team, including interviewers, archivists and volunteers FUNDING: the baseline of the study "HOW ARE YOU GOING?" was funded by master's students and the Academic Excellence Program (PROEX) of the Coordination for the Improvement of Higher Education Personnel (CAPES). In addition, the research coordinators (ADB, FFD, ET, MCG, RB) are research productivity fellows from the National Council for Scientific and Technological Development (CNPq). REFERENCIAS 1. World Health Organization Envelhecimento ativo: uma politica de saude Traducao: Suzana Gontijo Brasilia Organizacao Pan-Americana da Saude 2005 2. Shahin W Kennedy GA Stupans I The impact of personal and cultural beliefs on medication adherence of patients with chronic illnesses: a systematic review. Patient Prefer Adher 2019 13 1019 35 10.2147/PPA.S212046 3. Masnoon N Shakib S Kalisch-Ellett L Caughey GE What is polypharmacy? A systematic review of definitions. BMC Geriatr 2017 17 1 230 10.1186/s12877-017-0621-2 29017448 4. Teixeira JJV Lefevre F A prescricao medicamentosa sob a otica do paciente idoso. Rev Saude Publica 2001 35 2 207 13 10.1590/S0034-89102001000200016 11359209 5. Rocha CH Oliveira APS Ferreira C Faggiani FT Schroeter G Souza ACA Medication adherence of elderly in Porto Alegre, RS Cien Saude Colet 2008 13Suppl 703 10 10.1590/s1413-81232008000700020 6. Ramos LR Tavares NUL Bertoldi AD Farias MR Oliveira MA Luiza VL Polifarmacia e polimorbidade em idosos no Brasil: um desafio em saude publica. Rev Saude Publica 2016 50 supl 2 9s 10.1590/S1518-8787.2016050006145 7. Nobili A Franchi C Pasina L Tettamanti M Baviera M Monesi L Drug utilization and polypharmacy in an Italian elderly population: the EPIFARM-elderly project Pharmacoepidemiol Drug Saf 2011 20 5 488 96 10.1002/pds.2108 21264988 8. Kim J Parish AL Polypharmacy and medication management in older adults. Nurs Clin North Am 2017 52 3 457 68 10.1016/j.cnur.2017.04.007 28779826 9. Carvalho MFC Romano-Lieber NS Bergsten-Mendes G Secoli SR Ribeiro E Lebrao ML Polifarmacia entre idosos do municipio de Sao Paulo-Estudo SABE. Rev Bras Epidemiol 2012 15 4 817 27 10.1590/S1415-790X2012000400013 23515777 10. Almeida NA Reiners AAO Azevedo RCS Silva AMC Cardoso JDC Souza LC Prevalencia e fatores associados a polifarmacia entre os idosos residentes na comunidade. Rev Bras Geriatr Gerontol 2017 20 1 143 53 10.1590/1981-22562017020.160086 11. Pereira KG Peres MA Iop D Boing AC Boing AF Aziz M Polifarmacia em idosos: um estudo de base populacional. Rev Bras Epidemiol 2017 20 2 335 44 10.1590/1980-5497201700020013 28832855 12. Gould ON Todd L Irvine-Meek J Adherence devices in a community sample: how are pillboxes used? Canadian Pharmacists Journal/Revue des Pharmaciens du Canada 2009 142 1 28 35 10.3821/1913-701X-142.1.28 13. Costa EC Nakatani AYK Bachion MM Capacidade de idosos da comunidade para desenvolver atividades de vida diaria e atividades instrumentais de vida Diaria. Acta Paul Enferm 2006 19 1 43 8 10.1590/S0103-21002006000100007 14. Daien V Peres K Villain M Colvez A Carriere I Delcourt C Visual acuity thresholds associated with activity limitations in the elderly. The Pathologies Oculaires Liees a l'Age study. Acta Ophthalmol 2014 92 7 e500 6 10.1111/aos.12335 24428942 15. Giordani B Cinelli M Interacao idoso-embalagem: uma reflexao sobre a ergonomia no design de embalagens. In: 13o Congresso Brasileiro de Pesquisa e Desenvolvimento em Design Univille Joinville (SC) 2018 16. Marin MJS Cecilio LCO Perez AEWUF Santella F Silva CBA Goncalves JR Filho Caracterizacao do uso de medicamentos entre idosos de uma unidade do Programa Saude da Familia. Cad Saude Publica 2008 24 7 1545 55 10.1590/S0102-311X2008000700009 18670678 17. Beckman AGK Parker MG Thorslund M Can elderly people take their medicine? Patient Educ Couns 2005 59 2 186 91 10.1016/j.pec.2004.11.005 16257624 18. Borba AKOT Marques APO Ramos VP Leal MCC Arruda IKG Ramos RSPS Fatores associados a adesao terapeutica em idosos diabeticos assistidos na atencao primaria de saude. Cien Saude Colet 2018 23 3 953 61 10.1590/1413-81232018233.03722016 29538575 19. World Health Organization Adherence to long-term therapies: evidence for action: World Health Organization 2003 20. Tavares NUL Bertoldi AD Thume E Facchini LA Franca GVA Mengue SS Fatores associados a baixa adesao ao tratamento medicamentoso em idosos. Rev Saude Publica 2013 47 6 1092 101 10.1590/S0034-8910.2013047004834 24626547 21. Tavares NUL Bertoldi AD Mengue SS Arrais PSD Luiza VL Oliveira MA Fatores associados a baixa adesao ao tratamento farmacologico de doencas cronicas no Brasil. Rev Saude Publica 2016 50 suppl 2 10s 10.1590/S1518-8787.2016050006150 22. Farias-Antunez S Lima NP Bierhals IO Gomes AP Vieira LS Tomasi E Incapacidade funcional para atividades basicas e instrumentais da vida diaria: um estudo de base populacional com idosos de Pelotas, Rio Grande do Sul, 2014 Epidemiol Serv Saude 2018 27 2 e2017290 10.5123/S1679-49742018000200005 29898166 23. Barbosa FDS Melo CL Silva RJS Factors associated with instrumental activities of daily living functional capacity in brazilian older adults. Research, Society and Development 2021 10 4 e39410414144 10.33448/rsd-v10i4.14144 24. Santos GS Cunha ICKO Avaliacao da capacidade funcional de idosos para o desempenho das atividades instrumentais da vida diaria: um estudo na atencao basica em saude. Rev Enferm Cent Oeste Min 2013 3 3 820 8 25. Instituto Brasileiro de Geografia e Estatistica. Censo demografico 2010. Caracteristicas da populacao e dos domicilios: resultados do universo. Rio de Janeiro Instituto Brasileiro de Geografia e Estatistica 2010 26. Associacao Brasileira de Empresas de Pesquisa Criterio de classificacao economica Brasil. Alteracoes na aplicacao do Criterio Brasil, validas a partir de 01/01/2014 [Internet] acessado em 23 nov. 2019 Disponivel em: 27. Patterson SM Hughes C Kerse N Cardwell CR Bradley MC Interventions to improve the appropriate use of polypharmacy for older people Cochrane Database Syst Rev 2012 5 CD008165 10.1002/14651858.CD008165.pub2 22592727 28. Duarte YAO Lebrao ML Lima FD Contribuicao dos arranjos domiciliares para o suprimento de demandas assistenciais dos idosos com comprometimento funcional em Sao Paulo, Brasil. Rev Panam Salud Publica 2005 17 5/6 370 8 16053647 29. Martins OSP A familia do idoso dependente: analise das necessidades/dificuldades no cuidar no domicilio [dissertacao de mestrado] Viana do Castelo Instituto Politecnico de Viana do Castelo 2014 30. Freitas JGA Nielson SEO Porto CC Adesao ao tratamento farmacologico em idosos hipertensos: uma revisao integrativa da literatura. Rev Soc Bras Clin Med 2015 13 1 75 84 31. Summers JJ Lewis J Fujiyama H Aging effects on event and emergent timing in bimanual coordination. Hum Mov Sci 2010 29 5 820 30 10.1016/j.humov.2009.10.003 19931202 32. Jimenez-Jimenez FJ Calleja M Alonso-Navarro H Rubio L Navacerrada F Pilo-de-la-Fuente B Influence of age and gender in motor performance in healthy subjects. J Neurol Sci. 2011 302 1-2 72 80 10.1016/j.jns.2010.11.021 21183189 33. Clares JWB Freitas MC Borges CL Fatores sociais e clinicos que causam limitacao da mobilidade de idosos. Acta Paul Enferm 2014 27 3 237 42 10.1590/1982-0194201400040 34. Gomes GC Moreira RS Maia TO Santos MAB Silva VL Fatores associados a autonomia pessoal em idosos: revisao sistematica da literatura. Cien Saude Coletiva 2021 26 3 1035 46 10.1590/1413-81232021263.08222019 35. Luiz LC Rebelatto JR Coimbra AMV Ricci NA Associacao entre deficit visual e aspectos clinico-funcionais em idosos da comunidade. Braz J Phys Ther 2009 13 5 444 50 10.1590/S1413-35552009005000049 36. Menezes TMO Lopes RLM Azevedo RF A pessoa idosa e o corpo: uma transformacao inevitavel. Rev Eletr Enferm 2009 11 3 598 604 10.5216/ree.v11.47123 37. Santos TRA Lima DM Nakatani AYK Pereira LV Leal GS Amaral RG Consumo de medicamentos por idosos, Goiania, Brasil. Rev Saude Publica 2013 47 1 94 103 10.1590/S0034-89102013000100013 23703135 38. Silva GOB Gondim APS Monteiro MP Frota MA Meneses ALL Uso de medicamentos continuos e fatores associados em idosos de Quixada, Ceara. Rev Bras Epidemiol 2012 15 2 386 95 10.1590/S1415-790X2012000200016 22782104 39. Bezerra TA Brito MAA Costa KNFM Caracterizacao do uso de medicamentos entre idosos atendidos em uma unidade basica de saude da familia. Cogitare Enferm 2016 21 1 1 11 10.5380/ce.v21i1.43011 40. Silva CH Spinillo CG Dificuldades e estrategias no uso de multiplos medicamentos por idosos no contexto do design da informacao. Estudos em Design 2016 24 3 130 44

PMC10000022

The increased attention drawn to the negative environmental impact of the cattle industry has fostered a host of market- and research-driven initiatives among relevant actors. While the identification of some of the most problematic environmental impacts of cattle is seemingly more or less unanimous, solutions are complex and might even point in opposite directions. Whereas one set of solutions seeks to further optimize sustainability pr. unit produced, e.g., by exploring and altering the relations between elements kinetically moving one another inside the cow's rumen, this opinion points to different paths. While acknowledging the importance of possible technological interventions to optimize what occurs inside the rumen, we suggest that broader visions of the potential negative outcomes of further optimization are also needed. Accordingly, we raise two concerns regarding a focus on solving emissions through feedstuff development. First, we are concerned about whether the development of feed additives overshadows discussions about downscaling and, second, whether a narrow focus on reducing enteric gasses brackets other relations between cattle and landscapes. Our hesitations are rooted in a Danish context, where the agricultural sector--mainly a large-scale technologically driven livestock production--contributes significantly to the total emission of CO2 equivalents. critical kinetics planetary boundaries technical solutions feed additives livestock Jevons' paradox Denmark anthropology Independent Research Fund Denmark through the project Cattle Crossroads. Researching Danish Animal Production for the Future0217-00171B This research was funded by the Independent Research Fund Denmark through the project Cattle Crossroads. Researching Danish Animal Production for the Future accessed on 9 February 2023), Grant no. 0217-00171B. pmc1. A Commentary on Feed Additives as Solutions to Cattle's Climate Impact Since 2006, when the much-quoted FAO report Livestock's Long Shadow was published, increasing attention has been paid to the environmental challenges brought about by the cattle industry . Problems pile up. Today, agriculture occupies about 38% of Earth's terrestrial surface . Industrial livestock requires enormous amounts of feed, the production of which takes up more than three quarters of all agricultural land on the planet . This leads to massive deforestation, which proliferates into yet other problems such as soil erosion, nutrient leaching, and loss of biodiversity. Further, the gases emitted from the rumination processes of cattle make up a substantial portion of the greenhouse gases (GHG) that spread through the atmosphere, heating up our globe. In particular, enteric fermentation has been targeted and understood as key to controlling and potentially reducing the climate impact of cattle. In response, a host of market- and research-driven initiatives among relevant actors has emerged. As this special issue suggests, the time has now come to collect knowledge on forage and feedstuff digestion kinetics in ruminants in order to meet the mounting pressures to reduce enteric methane production. The fact that domesticated animals produced around the world (mainly cattle, pigs, and poultry) now outnumber wild mammals and birds by a factor of ten no doubt adds pressure to this predicament . Yet, for all the weight this ratio puts on the world's ecosystems, the scientific knowledge base supporting the industry's transition towards more sustainable futures most often comes from very specific areas of the natural sciences. Nourishing this quantitatively large global production of a few domesticated animal species, then, is the continuous production of scientific knowledge and research related to issues such as feed intensification, genetics, and health. For cattle, the obvious aim is to further optimize and increase dairy and beef production--industries that in Euro-American production systems are premised on principles of high cost efficiency, achieved by keeping large animal herds on small but intensively managed areas, with easy access to feed products that are often both nutritionally optimized and imported from overseas. While the identification of some of the most problematic environmental impacts of cattle is seemingly more or less unanimous, solutions are complex and might even point in opposite directions. As is clear from this special issue, one set of solutions points to interventions into the causal relations of elements kinetically moving one another inside the cow's rumen. More specifically, feed additives of various sorts are developed to decrease the generation of methane, thus targeting climate change. Surely, these interventions are valuable--after all, in these critical, increasingly hot times, why oppose GHG reductions in whatever form and by whatever means? Nonetheless, in this short contribution, we raise two concerns regarding a focus on solving emissions through feedstuff development. Whereas knowledge about the possibilities of technological optimization is important, we suggest that broader visions of the possible negative outcomes of further optimization are also needed. Notably, we are concerned for two reasons: First, the development of feed additives may overshadow discussions about downscaling. Second, a narrow focus on reducing enteric gasses by manipulating a set of kinetic causal processes inside the rumen may bracket other relations between cattle and landscapes. Both of these consequences, we suggest, can potentially limit the scope of climate impact mitigation in relation to cattle. Below, we substantiate these reservations and go on to probe how the development of feed additives sit with ideas about absolute sustainability and safe operating spaces for humanity. Our approach to the issue of feed additives and cattle production is rooted in anthropology and cultural history. We thus work using a method of ethnographic fieldwork among stakeholders engaged in Danish cattle production, and we conduct document analysis of, e.g., policies and public discussions, just as we look to archival and historical literature to trace earlier connections between state making and livestock production in Denmark. 2. Intensified Danish Livestock Production--With Feral Effects Within the agricultural sciences, an answer to the problematic climate impact of cattle has often been to further intensify animal production cf. . This response, however, rests on a modernistic assumption that more-than-human lives are essentially controllable by humans. The ecological crisis that we are presently witnessing does indeed testify to human activities and projects on a massive scale, but not to human control over causal relations in the more-than-human world. This is what we mean by feral kinetics in this opinion's title; we want to indicate that projects in which (causal) relations were once set in motion by humans through intentional projects often spur various unintended effects . Surely, no one set out to change the climate through animals. A brief historical look at the Danish context in which our research is rooted shows how intensification has come about gradually and in response to a host of societal, political, and historical circumstances that jointly make up what livestock came to be. In Denmark, the agricultural sector is responsible for 27.1% of national GHG emissions (excl. land use, land use change, and forestry (LULUCF)), of which intensive livestock production is a prominent contributor . Danish livestock production is often positively framed in public discourse as economically and environmentally cost-efficient when measured at the scale of singular products. Yet, there is reason to question the hidden costs of this mode of calculation, which often fails to include GHG emissions and global environmental impact derived from the livestock industry's dependency on feedstuffs, and thus, on land cultivation and deforestation both in Denmark and overseas, as scientists have also pointed out . In Denmark, as in other European countries, agricultural production has historically informed the organization of government, business, and civil society, and politicians still identify Denmark as "a farming country"--see, for example, . It was through the workings and on-going development of the agricultural sector that Denmark as a nation underwent some of its most significant historical changes, not least that of industrial modernization . More specifically, 19th- and 20th-century industrial modernization in the agricultural sector came about through the emergence of a strong, export-oriented livestock industry--see, for example, . For instance, the number of pigs in Denmark has increased from 301,000 in 1860 to 12.2 million today, which is now more than twice the Danish population . The historical emergence of a strong livestock sector not only changed the welfare and fortuity of rural communities. It also changed the physical appearance of the animal bodies that would increase the reach of Danish industrial interests by yielding more meat and milk per animal than they ever had before, which was achieved, in particular, through the industry's adoption of scientific approaches to rational and optimized feeding . Our point is that during the same historical processes that have made Danish agriculture synonymous with a large livestock industry, the livestock industry has itself become synonymous with high export and feedstuff dependency, as both are integral to how cost efficiency is construed in the industry. However, if the purpose of novel feed additives is to mitigate the climate impact of cattle production, it seems pivotal to ask whether such a high dependency on feeds should itself become a site of scientific intervention in the industry. 3. The Issue of Scale and Re-Bound Effects Indeed, to pinpoint our first reservation, focusing on greening the enteric digestion process by making 'enhanced' rumens less harmful, and politically prioritizing this effort, may engender a so-called rebound effect where decreased methane emission pr. cow is evened out by an increased scale of production--a mechanism sometimes summarized as Jevons' paradox. In other words, addressing the issue of methane emission through conceptualizing the rumen as a discrete and singular site of intervention may occlude the concern both with a potential rise in scale and with other the well-known, environmentally harmful effects of maintaining present-scale livestock production . Inadvertently, we fear, the development of feed additives may contribute to the status quo, thus foreclosing difficult discussions about how we might best use arable land (for feed or food), and about the number of cattle the world's ecologies (including atmospheric greenhouse gasses) can actually support. Aspiring for sustainability--as genuine change--on a planet with limited resources and imminent tipping points requires that we think in absolute terms. Adjustment and improvement miss the mark. 4. The Issue of Other Ecological Relations Our second hesitation is that a narrow focus on kinetics inside the rumen may eclipse wider ecological relations put into motion by the altered processes in the rumen. As the many ecological crises we are witnessing make clear, humans can no longer be seen as being in full control of various processes on earth--what we referred to above as the 'feral' nature of the non-human world in this day and age. Processes once perceived as controllable have proven not to be so, resulting in unintentional global effects that are both distributed and caused by human actions . With regard to feed additives specifically, these may work on other relations than those within the rumen, making it urgent to widen the scope of research. We must be careful not to make yet another potent feral product by limiting our view of what the problems and solutions are. 5. Thinking with Absolute Sustainability To summarize, our hesitations with regard to feed additives concern, first, how additives may maintain status quo in terms of the scale of cattle production, and second, how they may engender unanticipated ecological effects. On both accounts, we suggest, there is a need to consider how technological and natural scientific solutions to the methane issue relate to a planet with boundaries that limit a safe operating space for humanity . In short, we want to question how feed additives as a means of reducing methane emissions sit with ideas about absolute sustainability . If, as we suggest, technological solutions 'work back' on the identification of what the problem even is, the result is too easily a circular argument; we can only see the problems that we think we can solve by way of technology--rather than by a just and green societal transition, sanctioned in progressive politics. In making this argument, and exploring how it works in a Danish context, we draw on the model of planetary boundaries originally suggested by Rockstrom et al. . In 2017, Campbell et al. further worked with the model, arguing that agricultural production is, indeed, a main driver for the eco-system changes occurring, particularly within spheres where the planetary boundaries are transgressed. Our point is simply this: If the impact of agricultural production already exceeds 'permissible' limits, something has to change fundamentally. Making things relatively better is just not enough. This is particularly important in agro-industrially intensified countries such as Denmark where livestock industries are (dis)proportionately large; we must question both issues of scale and of unintended side effects. Choices remain to be made that observe planetary boundaries. Now, the scientists and companies who develop feed additives would probably agree that this is just one tool among many others. We want to stress that it is not feed additive development per se that we take issue with. Rather, looking to the recent political work of pushing for a green transition of the Danish cattle industry, we are concerned with the way feed additives have emerged on the political stage. Here, additives are presented as the obvious solution to a universal problem. However, the problem that additives are key to solving--i.e. a massive cattle industry, nourished by feed that causes deforestation and in a monocultural logic--can remain untouched. Further, the wider effects of the additives have yet to be documented. 6. A Couple of Examples To substantiate our argument, below we will look at a couple of instances where feed additives are discussed in a Danish context. We do so via ethnographic fieldwork, where we engage with stakeholders such as researchers and politicians, as well as with various written sources. Our method is ethnographic in the sense that we trace relations and generate analyses in dialogue with the field; as such, we explore what feed additives may be as they are produced and discussed by people who develop, implement, or entrust them with positive effects for mitigating climate change--see also . In 2021, all political parties but one represented in the Danish parliament signed an agreement on the green transition of agricultural production in Denmark . The agreement, launched as historic and ambitious, commits to reducing GHG emissions from agriculture by 55-65% by 2030 compared to 1990 levels, in addition to reducing nitrogen and phosphorous run-off into waterways in order to comply with EU regulation. To reach this binding target, curiously, the agreement starts with listing a number of caveats, all to the effect that the goal must be achieved without decreasing agricultural productivity, nor compromising public finances and Denmark's competitive edge with regard to agriculture. Instead, the agreement highlights the continued prioritization of developing and implementing new technologies. Indeed, trust in (near) future technological solutions is so great that the historically ambitious deal, as it is now, ensures less than a third of the promised reduction. More precisely, the agreement specifies a reduction of 1.9 tons of CO2e out of the 6.1-8 tons which is the overall target for the agricultural sector, equaling a reduction in GHG emissions of 55-65% from 1990 emission levels. The rest of the required reduction, the agreement states, will be brought about by new technologies that have yet to be developed and implemented. Two overall arenas for technological innovation are singled out in the political agreement: namely, the curbing of emissions from manure from all production animals and enteric fermentation in livestock. The agreement states as follows: "It will be a continued priority that new tools, such as feed additives, are transferred as quickly as possible to the implementation track, and that the demand [for reduced GHG emissions] is adjusted according to what can be realized" (p. 4). What we want to point to here is that the means for a very large proportion of the CO2 reduction that the agreement commits to have yet to be invented, and that reduction targets are adjustable. In other words, the binding and historic agreement on the green transition of Danish agriculture is highly negotiable, and, further, dependent on uncertain technologies. All the while, the agreement lists a number of other priorities that are not up for negotiation--such as productivity, employment, public finances, and rural development. This leaves it up to innovative technologies to find the remaining (majority) of the promised GHG reduction. Accordingly, as we see it, there is a substantial risk that the agreement's limited focus on climate change mitigation will lead to so-called "burden-shifting"--see --as the negative impacts of feed production and consumption are only considered with regard to a single planetary boundary, as opposed to asking how feeds can become sustainable in an absolute sense, heeding all biospheres. This is to say that optimizing feeds as a means to mitigate GHG emissions specifically risks overlooking equally important and environmentally detrimental processes such as the eutrophication or acidification of waterbodies. If global feed production and consumption on the whole are largely left unchanged, the use of feed additives to mitigate GHG emissions risks relocating instead of actually solving the problems caused by livestock production. By leaving it up to hopeful investments in future technologies to reduce GHG, we are not forced to consider the number of cows, nor the other effects of an unchanged scale. Another example from our fieldwork sheds light on the potential unintended effects of implementing feed additive solutions. Below, we provide more detail regarding some of the ways that feed additives work in the practices and discussions of industrial agriculture stakeholders. At the annual Cattle Congress 2022, the head of the Cattle Section in SEGES Innovation--the Danish agricultural interest organization's independent research unit--together with a researcher from the same organization, gave a talk under the headline "Climate Requirements in the Agricultural Agreement"--the same deal mentioned above. From her point of view, climate requirements can be answered by two distinct means of action: the handling of digestion and manure--while not compromising another distinct theme, namely animal welfare. In this way, she framed the problems involved in having an agricultural sector accounting for over one fourth of Danish GHG emissions by setting a very particular triangular frame within which one should think, talk, research, and act in relation to climate requirements: welfare, digestion, and manure. She mentioned that feeding with additives could reduce 20% of the 0.17 million tons CO2 that needed to be reduced by 2025, but also expressed frustration with the limited amount of money reserved to introduce and implement a new product approved by the European Union in the spring of 2022. New routines on farms need to be developed and supported, and potentially skeptical farmers should be convinced that milk yields will not decrease on account of the new additive. The researcher assisted her and elaborated on the tools needed to reach the goals in the climate law and the agreement discussed above. These were tools that altogether confirmed the dictum of 'more for less' (higher yield and more efficiency in feed and in producing bodies) that has made Danish livestock production competitive on a global market despite high production costs. Just as important, the presentation repeated a dictum that has recently become a standard answer to green goals: optimization equals sustainability. The researcher then went on to talk about the possibilities of reducing methane by changing diets--rapeseed and a handful of feed additives were mentioned, along with the possibilities and challenges these feeding options spurred. He singled out one new product and stated that the climate impact of milk will decrease by 17%, adding that if all conventional producers would implement the additive, the reduction targets for 2030 could be met. Thus far, he continued, the additive has only been tested on Holstein cattle. He wrapped up his presentation by saying that if any of the farmers present were interested in testing the product on their animals, they should feel free to get in touch. What interests us here is that the talk can be seen as combining the launch of a solution with calling for further tests, thereby mimicking the agreement above in its expectations for future effects of something yet to be fully developed and tested. Interestingly, a person in the audience raised his hand and questioned the manure from cattle fed with the approved additive, asking whether the emissions from it altered when distributed on the fields. In response, the researcher answered that the amount of product used is so small, and further that the product is processed so quickly in the cow that it was very unlikely that it would have an effect elsewhere, outside the rumen. However, he continued, researchers in Canada have recently conducted a study where they pointed to higher emissions from manure in the fields as an effect of the application of feed additives. Interestingly, this Canadian study--or hesitation, we could call it--did not seem to 'alter' the Danish researcher's hopes for feed additives once they have been further tested. Chatting with the researcher after the talk, it became clear that potentially increased emissions on the fields were understood as a problem for another research field--namely, that which deals with manure handling. For him, it seemed, there were so many kinetic relations to be explored within the rumen, and understanding what happens later, out on the fields, would be a theme to be researched once processes in the rumen are more fully understood. Our point here is to highlight the decoupling of what goes on in the rumen upon applying feed additives from the 'afterlife' of such an intervention. 7. Conclusion: Heeding all Biosphere Domains at Once To conclude, what we argue--and the reason for our reservations towards the prevalent kind of solutionism offered by the development of feed additives--is that it takes a very particular perspective on the cow for its rumen to be the sole target of any intervention. From this perspective, the cow is a singular unit from within which technology can decrease methane emission. To the contrary, as anthropologists, we would see any cow as a set of relations, ranging from the microbial level to global issues of deforestation . While we do not oppose feed additives as such, we do hold that they risk building on and maintaining a tunnel vision, as also described above, that allows for a curious disconnection of cattle's rumen from other cattle-related processes and decisions, including the discussion of scale and other effects than GHG emissions. In Denmark, and elsewhere, other biosphere problems are also apparent as seen from the model of planetary boundaries. Not least, we have huge problems with the leaching of N and P severely affected waterways in Denmark, as a result of the scale of animal production, regardless of its climate efficiency when measured pr. kilogram of, e.g., milk. Put bluntly, as we see it, if we care about the immediate threats to the safe operating space for humanity, it makes little sense to assess the climate impact of cattle pr. singular rumen. One direct insight from the principle of absolute sustainability is that all agricultural resource activities impact many of the nine biosphere domains. Accordingly, solutions need to follow suit. We cannot afford the luxury of solving one problem at a time. Acknowledgments We are very grateful to Hanne Helene Hansen for inviting us to contribute to this special issue, and to the three anonymous reviewers for helpful feedback. We also want to thank Liza Rosenbaum Nielsen, Kari Eriksson, and Camilla Kirketerp Nielsen for sustained and challenging discussions. Author Contributions Conceptualization, N.B., S.B., and F.H.; methodology, N.B., S.B., and F.H.; formal analysis, N.B., S.B., and F.H.; investigation, N.B., S.B., and F.H.; data curation, N.B., S.B., and F.H.; writing--original draft preparation, N.B. and F.H.; writing--review and editing, N.B., S.B., and F.H.; project administration, F.H.; funding acquisition, F.H. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. The article builds on publicly available sources. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000023

Rotational grazing has been mentioned as a potential tool to reduce losses caused by high tick loads. This study aimed: (1) to evaluate the effect of three grazing modalities (rotational grazing with 30- and 45-day pasture rest and continuous grazing) on Rhipicephalus microplus infestation in cattle, (2) to determine population dynamics of R. microplus in cattle under the three grazing modalities mentioned in the humid tropics. The experiment was carried out from April 2021 to March 2022 and consisted of 3 treatments of grazing with pastures of African Stargrass of 2 ha each. T1 was continuous grazing (CG00), and T2 and T3 were rotational grazing with 30 (RG30) and 45 d of recovery (RG45), respectively. Thirty calves of 8-12 months of age were distributed to each treatment (n = 10). Every 14 days, ticks larger than 4.5 mm were counted on the animals. Concomitantly, temperature (degC), relative humidity (RH), and rainfall (RNFL) were recorded. Animals in the RG45 group had the lowest count of R. microplus compared to the RG30 and CG00 groups; these results suggest that RG45 days of rest could be a potential tool to control R. microplus in cattle. Yet, we also observed the highest population of ticks on the animals under rotational grazing with a 30-day pasture rest. A low tick infestation characterized rotational grazing at 45 days of rest throughout the experiment. The association between the degree of tick infestation by R. microplus and the climatic variables was nil (p > 0.05). cattle ectoparasites control grasslands ticks DGAPA-UNAMIT229320 DGAPA-UNAM supported this work through the research PAPIIT Project IT229320. pmc1. Introduction Ticks are one of the main threats to cattle production, affecting about 80% of livestock worldwide. These parasites generate losses ranging from 13.9 to 18.7 billion US dollars annually , mainly by affecting productive and welfare parameters. Rhipicephalus microplus (Canestrini, 1887) (Acari; Ixodidae) is the main ectoparasite affecting cattle in tropical, subtropical, and temperate areas of the world where it is a transmitter of pathogens such as Babesia bovis, B. bigemina, and Anaplasma marginale . Worldwide control of this tick has mainly been based on therapeutic interventions using chemical treatments; however, these chemicals have developed acaricidal resistance in ticks and their ecological impact . Non-conventional methods to control tick populations have been proposed to mitigate the effects of this resistance . Among these are rotational grazing, in which the pasture manager maintains the grazing time cattle remains in the grazed section and determines the length of the recovery periods the animal stays off a pasture subdivision . It aims to reduce the parasite-host interaction and has been mentioned as an ecological, profitable way and would also help optimize forage resources . Rotational grazing as a means of tick control has received the attention of researchers for several years; nevertheless, pointed out that most studies have been based on mathematical models, and there is little information on the effect of rotational grazing in the field. The preceding agrees with , who mentioned that population dynamics and dispersal of ticks in rotational grazing systems are complex and relatively unstudied. In field studies, it is observed that the effect of rotational grazing on ticks may depend on the recovery time of the paddock. Those rotational grazing with short periods of rest (20 days) showed higher tick infestations on-host than did those in continuous grazing. Some studies with longer paddock recovery times report that rotational grazing is a promising non-conventional strategy to control ticks. On the other hand, generations of R. microplus are increasing throughout the year, a phenomenon linked with global warming , which justifies the need for current studies of the seasonal dynamics of this ectoparasite. A successful tick-control strategy will also depend on the interaction of biotic and abiotic factors that leads to seasonal population abundance that, in turn, determines tick behavior in each region. This knowledge plays a decisive role in an ectoparasite control program . There is no information on the effect of rotational grazing with different resting times of paddocks on the control of R. microplus in cattle. The objectives of this study were: (1) to evaluate the effect of three grazing modalities (continuous grazing and rotational grazing with 30- and 45-day pasture rest) on R. microplus infestation in cattle, (2) to determine population dynamics of R. microplus in cattle under the three grazing modalities mentioned in the environmental conditions of the humid tropics. 2. Material and Methods 2.1. Study Site The study took place in the Center for Teaching, Research and Extension in Tropical Livestock Production (CEIEGT) of the Faculty of Veterinary Medicine and Zootechnics of the National Autonomous University of Mexico (20deg02' N, 97deg06' W) , from April 2021 to March 2022. 2.2. Experimental Design We tested three grazing management strategies: CG00, continuous grazing, where the animals roamed free in a single paddock without internal divisions; RG30, rotational grazing with 3-day and 30-day grazing and recovery times, respectively, with 11 paddocks of 0.18 ha each; and RG45, rotational grazing with 3-day and 45-day grazing and recovering times, respectively, with 16 paddocks of 0.12 ha each. The three experimental areas share the same latitude, and land irregularities are similar. Each treatment consisted of 2 ha of pastures where African star grass (Cynodon nlemfuensis) predominated and with natural tick infestations. Grazing by cattle has been the only use received by the pastures over the last 30 years. We used the flag technique to verify the presence of larvae in the three experimental areas before the start of the experiment. The number of larvae was very low and similar among pastures. Paddocks did not receive any anti-tick treatment before the investigation. The experimental animals were thirty heifers between 8 and 12 months of age and had an average live weight of 182 +- 44 kg. We allocated ten heifers randomly to each treatment. Eight animals from each group were F1 (Holstein x Zebu) and two 5/8 x 3/8 (Zebu x Holstein). Another grouping criterion was coat color. The stocking rate for each experimental area at the beginning of the study was four animal units per hectare (au = 450 kg of live weight). Fifteen days before starting the experiment, all animals were treated against gastrointestinal parasites (albendazole), ticks, and flies (coumaphos) in order to start with similar tick loads. Report indicates that the local tick populations resist amitraz, synthetic pyrethroids, chlorpyrifos, diazinon, and ivermectin. However, no study has evaluated tick susceptibility to coumaphos, a chemical that has been nil for at least 15 years. 2.3. Animal Management Throughout the experiment, all animals received a feed supplement at a daily rate of 1 kg per head. We supplied water ad libitum to the animals. During the winter, the available grass decreased, so every other day, we supplemented the heifers with two bales of grass hay (C. nlemfuensis and Brachiaria sp., 22 kg each). There are no common areas between any of the three treatments. Each treatment had mobile and exclusive drinkers and feeders for the animals. The animals were not treated against ticks during the study; however, cattle were under medical supervision to monitor tick loads and clinical signs that might be present. 2.4. Tick Counts in Cattle The count was carried out throughout the year every 14 days from 7:00 to 9:00 h. In total, we did 26 counts, beginning in April 2021 and ending in March 2022. With the aid of a compression ramp, we counted R. microplus only if its length was > 4.5 mm in each heifer. Ticks remained in place after counting. A qualified veterinarian examined the total body surface area of the right side and the number of ticks multiplied by two . 2.5. Climate Data Collection Two weeks before and throughout the experiment, the environmental temperature (degC) and rainfall (mm) were recorded daily through the National Meteorological Service database. The Weather Channel (c) mobile application provided the relative humidity data (RH, %) . The climate is hot and humid with three climatic seasons: rainfall (June-September), winter (October-January), and dry (February-May). Our records (CEIEGT) and INEGI indicated that the rainy season has temperatures ranging from 15 degC to 27 degC, rainfall is 715 mm, and relative humidity is 90-95%. The winter season (also known as "norths") presents temperatures from 9 degC to 23 degC, with a total rainfall of 190 mm with a relative humidity of 30-90%. The dry season temperature varies from 11 degC to 29 degC, with rains of 150 mm and relative humidity of 20 to 80%. 2.6. Statistical Analysis The data were analyzed using D'Agostino & Pearson, Anderson-Darling, Shapiro-Wilk, and Kolmogorov-Smirnov tests to determine normality and homogeneity of variances using StatGraphics 19.1.3 (StatPoint, Inc., Herndon, VA, USA). Tests showed that our data were not normally distributed. Tick counts for each treatment were compared using the Kruskal-Wallis test with Statistica 10.0 (StatSoft, Inc., Tulsa, OK. USA). A 95% confidence interval and a p value of less than 0.05 were considered. Environmental temperature, relative humidity, and rainfall were correlated with tick load using the Spearman test with Software R version 2021 (R Core Team, Vienna, Austria). The tick count in the bovines of each treatment was analyzed by descriptive analysis (Software R version 2021). 3. Results Table 1 shows 26 counts of R. microplus teleogins (>4.5 mm). In the first three months (April, May, and June) after starting the experiment (six counts), the parasitic loads were very low and similar among treatments (p > 0.05) (Table 1). From count seven to count sixteen, corresponding to July to November, the animals in RG30 had the highest counts of R. microplus (p < 0.05) (Table 1). In the last ten samplings, corresponding to the November-March period, the tick count recorded in RG45 was significantly lower than RG30 and CG00 (p < 0.05). The animals in the RG30 group had a higher cumulative parasite load at the end of the experiment with 13,352 teleogins, followed by animals from CG00 and RG45 treatments with 1882 and 660 teleogins. The dispersion patterns of parasitic loads among animals were different within each treatment. In total, 30% of the animals in RG30 and RG45 treatments concentrated 55% (7344/13,352) and 57% (1073/1882) of parasite loads, while in the CG00 treatment, the pattern was 42% (277/660). None of the animals showed health problems during the experiment. The population dynamics of engorged ticks on each treatment showed variable patterns . Animals in the RG30 group presented the highest infestations of R. microplus ticks (>4.5 mm in length) throughout the year, and the population fluctuation showed five distribution peaks. The first peak of engorged females was in June and July, averaging 71.5 ticks per animal. The second and third peaks occurred during September and October, reaching an average of 188 ticks and 115 ticks per animal; fourth and fifth peaks occurred during January and February, with an average of 31 and 48 ticks per animal, respectively . The experiment's minimum and maximum environmental temperature fluctuated between 11.5 degC and 37.0 degC. Monthly relative humidity (RH) and precipitation were between 67 and 85% and 54.0 to 427.7 mm, respectively. There was no association between the degree of tick infestation by R. microplus and the climatic variables (p > 0.05). 4. Discussion Rotational grazing has been reported as a viable alternative to control R. microplus in cattle. However, there is limited information on the effects of this non-chemical alternative at the farm level. Ours is the first report about the impact of three grazing management variations on R. microplus infestation in cattle. The evaluations that exist have been carried out in regions with different climates, different stocking rates and different types of pastures. However, these studies have reported significant findings that could help to understand the results obtained in the present report. In this study, we observed that reducing the length of the recovery period from continuous grazing or 45 days to a 30-day recovery stimulated the tick loads on heifers. Animals in the RG30 group had the highest count of R. microplus compared to the RG45 and CG00 groups; indeed, the animals in treatment RG30 had a higher cumulative parasite load at the end of the experiment. Rotational grazing (with 20 days of rest) in Cynodon dactylon pastures was ineffective in reducing the parasitic loads of R. microplus on animals compared with continuous grazing . The duration of the non-parasitic phase (in pastures) depends directly on climate and vegetation, which determine the abundance of the populations . Under controlled field conditions, the average pre-hatching time is 42 days. Larvae show better activity to adhere to the potential host at 3 to 8 days post-hatching, indicating that the adequate time for the presence of viable and vigorous larvae in the pasture could be 45-50 days post detachment of engorged ticks . If we consider in this study that the animals return to the paddock after 30 days, there would still be no viable larvae to infest. Still, in the next round, the animals would return after 60 days, when the larvae have 15-20 more days of their best viable age, which could be an adequate time for a high level of infestation under the conditions of this study. Further research is needed to determine the duration of the biological parameters of ticks under this grazing system and to consider other factors inherent to animal behavior. On the other hand, short-term rotational grazing induces a high stocking density because the number of paddocks increases, but the number of animals remains more or less constant. As the available pasture and area per animal decrease, the probability of the larva-host encounter would increase, leading to augmented tick loads on animals . However, other factors can also influence infestations, such as animal behavior, cattle trampling, and the vegetation cover of pastures, among others. Animals in the RG45 group, with a greater-density grazing system, had the lowest count of R. microplus compared to the RG30 and CG00 groups; indeed, the animals also had the lowest cumulative parasite load at the end of the experiment. These results suggest that RG45 days of rest could be a potential tool to control R. microplus in cattle. There is no information about the effect of rotational grazing with 45-day pasture rest on R. microplus infestation compared with grazing modalities such as 30-day pasture rest and continuous grazing. Unlike the RG30 group, the animals return to the paddock after 45 days, which could still mean a low percentage of viable ticks to infest. Still, in the next round, the animals would return after 90 days, when the larvae have 40-45 more days of their best viable age. Such length could be enough time for environmental conditions to damage the larvae, reducing the chances of infesting cattle. The effect of rotational grazing on tick populations results from the impact that abiotic factors, type of pasture, and the recovery time can have on non-parasitic phases (pre-oviposition, oviposition, incubation, egg hatch, and larval maturation) of R. microplus. The vegetation architecture influences tick loads by protecting the larvae, as it climbs the pasture, from non-ideal environmental factors ; furthermore, it can also offer protection to other non-parasitic phases. In this study, and after each grazing period, the pasture in the RG45 could have been shorter and could expose larvae to harsh environmental conditions that increased the probability of dehydration. Although ticks have an essential capacity to resist prolonged starvation, the decrease in their energy reserves and overexposure to adverse climatic factors (such as high temperature and low humidity) are among the leading causes of mortality in field conditions . Some authors from different countries have repeatedly mentioned achieving completely tick-free pastures. For example, refs. report 98, 105, and 136 to 192 days, respectively, because the larvae of R. microplus lose water and energy without having a source of nutrition. This variation highlights the need for studies under different conditions of vegetation cover (grazing times) and other times of the year to better understand the behavior of ticks under different pasture management systems and geographical conditions. In addition, since there is a higher stocking density than group RG30, the soil, pastures, and ticks are exposed to greater trampling, which could affect their survival. Further studies are suggested to determine these factors' influence on ticks' survival at the pasture level. Remarkable data from the present study show that three out of ten animals in the RG30 and RG45 treatments maintained 55% and 57% of ticks, respectively. reported a similar pattern, where 25 out of 36 animals were responsible for 50% of the total ticks. These results further reinforce the idea that animals have different susceptibilities or responses to tick infestations . The above would help us to detect and treat only susceptible animals, as this would control around 55% of infestations and exert less selection pressure for resistance on ticks when treating animals. On the other hand, it is also essential to highlight behavior since some animals are leaders in the group and move ahead of others when the herd enters a new pasture; in this way, they can collect most of the tick larvae. Animal hierarchy can also explain why the tick load patterns are similar in the case of rotational grazing (55 and 57%) and lower (42%) for continuous grazing. The growth and establishment of tick populations are directly related to the availability of hosts and the climate, such as temperature, humidity, and precipitation . The observations during one year of the parasitic phase of R. microplus in bovines helped us to determine that the ectoparasite showed approximately five peaks in the RG30 and one in RG45 and group CG00. It is worth mentioning that these populations, being highly influenced by environmental conditions, can present various diapause times, which could manifest in the absence of marked peaks in some seasons and treatments. Previous studies in tropical and subtropical areas reported 3 to 4 peaks per year for this tick species in cattle . However, a recent experiment showed the occurrence of five annual peaks of R. microplus in cattle , attributing temperature as a possible factor in the increase in peaks. In this regard, the cattle tick population dynamics from 40 years ago until now showed a tick population growth, with different peaks in a year depending on the seasonality (i.e., rainfall and dry seasons) or associated with the increase of the environmental temperature over the years . The present study had no significant association between the parasite loads and the climatic variables as analyzed (p > 0.05). An aspect to highlight in the current experiment was the increase to a peak in group CG00 in winter, which is probably an effect of the cumulative number of larvae in the grasses that became adults in previous generations. This observation during the winter season in this experiment agrees with that reported by in Brazil, where they found high peaks of R. microplus in continuous grazing, attributing to the quality of pasture and the nutritional status of cattle, inducing a lower probability of susceptibility of animals to ticks. 5. Conclusions We observed the highest population of ticks on the animals under rotational grazing with a 30-day pasture rest. A low tick infestation characterized rotational grazing at 45 days of rest throughout the experiment. None of the climatic variables evaluated was related to tick loads in the experimental groups. Acknowledgments Gabriel Cruz-Gonzalez thanks the CONACyT for his doctoral scholarship 804128, Mexico. Author Contributions G.C.-G.: Executed the fieldwork (tick counts) and wrote the manuscript. M.A.A.-D.: Contributed to the founding acquisition, resources, and revision of the final manuscript. J.M.P.-R.: Contributed to the founding acquisition, resources, and revision of the final manuscript. J.J.-R.: Designed pasture treatments layout, implemented and executed the pasture sampling schedule. A.F.-S.: Counted ticks, checked daily cattle health, and collaborated in implementing pasture treatments. E.C.-G.: Designed the experiment, analyzed the data statistically, and reviewed the manuscript. D.R.-S.: Contributed to the founding acquisition, resources, and revision of the final manuscript. J.G.V.-M.: Contributed to the founding acquisition, resources, and revision of the final manuscript. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was approved by the Bioethics and Animal Welfare Commission (number 015/21) of the Faculty of Veterinary Medicine and Zootechnics, University of Veracruz (UV-FMVZ). Informed Consent Statement Not applicable. Data Availability Statement The database and the statistical analyses are available upon reasonable request. Conflicts of Interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced this research. Figure 1 Number of Rhipicephalus microplus (mean +- standard deviation) under continuous grazing (CG00), rotational grazing with 30 and 45 days of rest (RG30 and RG45, respectively) on cattle (n = 10 per treatment), for 1 year (April 2021 to March 2022). Tmax = maximum temperature in degC; Tmin = minimum temperature in degC (a), degC = degrees centigrade, RH = relative humidity (%) (b), PP = pluvial precipitation (mm) (c). Within dates, bars with the same upper script letter are statistically equal at p < 0.05. animals-13-00915-t001_Table 1 Table 1 Number of Rhipicephalus microplus in cattle (>4.5 mm) under three grazing systems over a year. Date Sampling Number CG00 RG30 RG45 Means 1 SD Means SD Means SD 02/04/2021 1 1.60 a 1.58 1.40 a 1.65 1.00 a 1.41 17/04/2021 2 1.40 a 1.65 1.40 a 2.12 1.60 a 2.07 01/05/2021 3 2.00 a 2.49 2.20 a 3.19 2.80 a 3.68 18/05/2021 4 0.80 a 1.69 6.20 a 10.73 2.60 a 4.01 01/06/2021 5 5.00 a 4.74 2.20 a 3.19 1.40 a 2.50 15/06/2021 6 0.00 a 0.00 8.60 b 10.50 2.00 a 5.66 29/06/2021 7 3.00 a 3.16 59.20 b 47.27 0.00 c 0.00 13/07/2021 8 3.40 a 3.53 83.80 b 59.45 11.20 a 14.58 27/07/2021 9 4.80 a 3.16 61.80 b 40.09 15.00 c 7.62 11/08/2021 10 0.40 a 0.84 90.80 b 77.00 1.00 a 1.94 24/08/2021 11 11.20 a 8.12 117.20 b 94.07 4.40 c 6.24 08/09/2021 12 0.20 a 0.63 188.40 b 159.84 0.60 a 1.35 20/09/2021 13 3.80 a 5.29 162.40 b 138.17 0.00 c 0.00 05/10/2021 14 3.40 a 3.41 70.80 b 63.01 2.20 a 2.90 18/10/2021 15 2.00 a 3.27 115.00 b 81.83 0.00 c 0.00 03/11/2021 16 6.00 a 6.18 88.00 b 82.84 3.20 a 4.54 16/11/2021 17 3.40 a 3.66 36.60 b 17.39 0.40 c 0.84 30/11/2021 18 0.60 a 1.90 29.00 b 15.03 0.00 a 0.00 14/12/2021 19 16.40 a 13.16 13.00 a 5.10 2.20 b 2.57 28/12/2021 20 17.00 a 5.52 25.00 a 29.55 1.60 b 2.27 11/01/2022 21 11.20 a 7.19 31.00 b 17.64 3.20 c 3.16 25/01/2022 22 19.20 a 14.37 23.00 a 11.21 0.80 b 1.93 08/02/2022 23 16.40 a 11.46 16.20 a 13.45 1.80 b 2.57 22/02/2022 24 16.80 a 9.53 48.00 b 36.49 0.80 c 1.40 09/03/2022 25 21.80 a 5.37 22.20 a 10.17 2.80 b 3.43 23/03/2022 26 16.40 a 8.04 31.80 b 21.63 3.40 c 2.32 1 Arithmetic mean of tick counts. a-c Values within a row with different superscripts differ significantly at p < 0.05. 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PMC10000024

An 8-week feeding trial was conducted to investigate the effects of dietary protein levels on growth performance, feed utilization, and energy retention of juvenile dotted gizzard shad Konosirus punctatus based on the variation of fish meal. Fish meal was used as the sole protein source; five semi-purified diets were formulated with varying crude protein (CP) levels of 22.52%, 28.69%, 34.85%, 38.84%, 45.78% (CP1-CP5 diets). A total of 300 uniform juveniles with initial body weight 3.61 +- 0.20 g fish-1 were randomly divided into five groups with three replicates in each group. The results showed that different CP levels did not significantly affect the survival of juvenile K. punctatus (p > 0.05). The values of weight gain (WG) and specific growth ratio (SGR) showed a general enhancing trend and then weakened with increasing dietary CP levels (p > 0.05). Feed utilization also improved with increasing dietary CP levels (p > 0.05), and the optimal feed conversion ratio (FCR) value was found in fish fed the diet with CP3 (p > 0.05). The rise of dietary CP from 22.52% to 45.78% enhanced the daily feed intake (DFI) and protein efficiency ratio (PER) values of K. punctatus (p < 0.05). With the increase of dietary CP levels, daily nitrogen intake (DNI), energy retention (ER), and lipid retention (LR) elevated, while retention (NR), daily energy intake (DEI), and daily lipid intake (DLI) reduced (p < 0.05). No statistical differences in the content of water, crude protein, and crude lipid were observed among different treatments (p > 0.05). The activity of lipase in CP3 and CP4 diets was significantly higher than that of the CP1 diet (p < 0.05). Fish fed CP2 and CP3 diets had significantly higher amylase activity than that of the CP5 diet (p < 0.05). The levels of alanine aminotransferase (GPT) first enhanced and then decreased as dietary CP levels raised. The second-order polynomial regression model analysis of the WG and FCR indicated that the optimal dietary protein level for K. punctatus is about 31.75-33.82% based on the variation of fish meal. Konosirus punctatus growth feed utilization body composition energy retention dietary protein levels International Cooperation Technology Research and DevelopmentDemonstration Promotion2021C04016 key research and development projects2021C02047 This work was supported by the International Cooperation Technology Research and Development, and Demonstration Promotion (2021C04016), as well as the key research and development projects in Zhejiang Province (2021C02047). pmc1. Introduction Protein, as the main organic matter in fish tissue, accounts for about 65-75% of the dry weight . Dietary protein is identified as a crucial source of amino acids for fish synthetic body protein and is an important component of many active substances such as enzymes and hormones with key functions . Moreover, despite the fact that dietary protein is the most expensive component of most aquafeeds, it is the essential nutrient to determine the growth rate of fish . It has been reported that inadequate dietary protein may hinder growth and even damage the health of fish . In contrast, excessive dietary protein not only increases feed costs but also results in nitrogen loss and water pollution . Fish meal (FM) has been recognized as the preferred protein source because of its well-balanced nutrition and reasonable palatability . It is feasible to use fish meal as the only source of protein in feed formulation to determine the optimal dietary protein requirement of fish. For instance, Zhang et al. observed that dietary protein level had a clear effect on growth performance, and the optimum protein level in diets for juvenile sparus macrocephalus, defined by the SGR, was 41.4% when white fish meal was the sole protein source and the dietary energy value was 15.9 kJ/g. The optimal dietary protein requirement based on the variation of fish meal for the growth of juvenile Bidyanus bidyanus was estimated to be 42.15% . Therefore, it is essential to determine the variation of fish meal that provides the optimum balance of protein requirements, nutrition, and low cost while being environmentally friendly. The dotted gizzard shad Konosirus punctatus is a small nearshore pelagic fish in the family Clupeidae (Clupeiformes), and is widely distributed in the coastal areas of China, South Korea, and Japan . It is a euryhalinous species and can even live in freshwater . With many advantages such as fast sexual maturity, delicious meat, high market value, and nice appearance, K. punctatus has become one of the important commercial aquaculture species in China . In recent years, due to the decline of marine ecological resources, small, excellent commercial fish are gradually replacing traditional commercial fish, and K. punctatus is a typical representative of small commercial fish . To date, the studies on the K. punctatus mainly focus on biological characteristics, genetics, and resources . In nutrition research on fish, ensuring the dietary protein requirement is the most crucial consideration. However, to our knowledge, limited information is utilized about the protein demand in diets of this species, which has brought difficulties to the culture and reproduction as well as artificial release. Therefore, five kinds of semi-purified feeds with different CP levels (ranging from 22.52% to 45.78%) were designed with fish meal as the only protein source in this study to evaluate the effects of different protein levels on the growth performance, feed utilization and energy retention based on the variation of fish meal. 2. Materials and Methods 2.1. Experimental Diets Fish meal was used as the only protein source, and fish oil and soybean oil were used as the main lipid sources. Five isoenergetic test diets were prepared to contain graded levels of crude protein (22.52%, 28.69%, 34.85%, 38.84%, and 45.78%, CP1-5 diets) based on the variation of fish meal. The ingredients and proximate composition of diets are shown in Table 1. All dry ingredients were weighed after passing through an ultra-fine pulverizer (120 mm), and fully mixed in a mixer for 20 min. Then, the weighed fish oil and soybean oil and a suitable amount of double-distilled water were added into the mixer until completely mixed. Five kinds of wet pellet feeds (1.5 mm) were individually obtained by a twin-screw extruder (G-250; Technology Machinery Factory of South China University, Guangzhou, China), and were dried at 45 degC. Then, these pellets were sieved and placed in a refrigerator at -20 degC. 2.2. Experimental Fish and Feeding Trial K. punctatus juveniles for the experiments were provided by the Key Laboratory of Mariculture and Improvement, Zhejiang Institute of Marine Fisheries, Zhoushan, China, and the feeding experiment was carried out in this place. Prior to starting the experiments, all fish were placed in concrete ponds (13.0 x 4.0 x 1.5 m; L x W x H) for 2 weeks and fed the same kind of commercial feed. At the beginning of this trial, 20 juvenile fish (initial body weight 3.61 +- 0.20 g fish-1) after being fasted for 24 h were randomly selected and put into 15 tanks (300 L). Three replicate tanks were used in order to test 5 kinds of diets for 8 weeks. During feeding, all the test fish were fed with experimental diets at 09:00 and 17:00 to apparent satiation in a natural photoperiod. Sand-filtered and flowing seawater was provided during the culture experiment, and the water flow was maintained at 1 L min-1. The roots blower was used for uninterrupted aeration to ensure that dissolved oxygen exceeded 6 mg L-1. The temperature, salinity, and pH value of water fluctuated around 27.7 +- 1.4 degC, 26.10 +- 0.9 g L-1, and 7.5 +- 0.1, respectively. Ammonia nitrogen concentration was controlled below 0.05 mg L-1. 2.3. Sample Collection and Analysis After the acclimatization period, 20 fish with body weight of 3.61 g fish-1 were randomly selected and euthanized, and then placed in pre-marked sealed bags and stored in a -80 degC refrigerator for subsequent analysis. After the mariculture experiment, all surviving fish were fasted for 24 h, and the number and weight of the fish in each tank were recorded, respectively. The surviving juveniles in each tank were anesthetized with 150 mg L-1 of MS-222. Then, 6 fish were randomly selected from each tank to calculate the morphological parameters including body weight and body length. The 6 fish were dissected to obtain the liver and placed in a -80 degC refrigerator for subsequent physiological index analysis. The other 5 fish were randomly collected from each tank and placed in marked, sealed bags, and stored in a -80 degC refrigerator for subsequent analysis. Proximate composition of samples (experimental diets, fish) was measured according to AOAC . Moisture was analyzed via a constant temperature drying oven (diets, 105 degC) or a lyophilizer (whole-body, -110 degC, LL1500, Thermo Fisher Scientific, Waltham, MA, USA). An automatic Kjeldahl system (K355/K437 Buchi, Flawil, Switzerland) was used to estimate the crude protein (N x 6.25) of samples based on the Kieldahl method. Soxhlet apparatus (E816, Buchi, Flawil, Switzerland) was used to estimate crude lipid via petroleum ether extraction. A Muffle furnace (550 degC) was used to measure the ash of samples. An adiabatic bomb calorimeter (HWR-15E, Shangli, Shanghai, China) was used to estimate the gross energy of samples. Lipase and amylase activities of liver were analyzed by the method of Li et al. . Glutamyl pyruvic transaminase (GPT) activity was determined as described by Reitman and Frankel . All parameters were assayed in triplicate by commercial kits (Jiancheng, Ltd., Nanjing, China), and performed by a microplate reader. 2.4. Calculation and Statistical Analysis Initial body weight = IBW; final body weight = FBW. Weight gain (WG, %) = 100 x (FBW - IBW)/IBW. Specific growth rate (SGR, % per day) = 100 x (Ln (FBW) - Ln (IBW)/days. Daily feed intake (g 100 g fish-1 day-1) = 100 x feed offered/average total weight/days. Feed conversion ratio (FCR, %) = dry feed consumed/wet weight gain, Protein efficiency ratio (PER) = wet weight gain/protein intake. Average body weight (ABW, g) = (IBW + FBW)/2. Daily nitrogen intake (DNI, g kg ABW-1 day-1) = feed intake nitrogen/ABW x days. Daily nitrogen gain (DNG, g kg ABW-1 day-1) = (FBW x final body nitrogen - IBW x initial body nitrogen)/ABW x days. Nitrogen retention (NR, %) = 100 x DNG/DNI. Daily energy intake (DEI, 102 kJ kg ABW-1 day-1) = feed intake energy/ABW x days. Daily energy gain (DEG, 102 kJ kg ABW-1 day-1) = (FBWx final body energy - IBW x initial body energy)/ABW x days. Energy retention (ER, %) = 100 x DNG/DNI. Daily lipid intake (DLI, g kg ABW-1 day-1) = feed intake lipid/ABW x days. Daily lipid gain (DLG, g kg ABW-1 day-1) = (FBW x final body lipid - IBW x initial body lipid)/ABW x days. Lipid retention (LR, %) = 100 x DLG/DLI. Data were expressed as an average value (n = 3) +- SD and analyzed statistically using SPSS 24.0 (IBM, Chicago, IL, USA). Data were analyzed using one-way ANOVA and deviating replicates through Duncan's multiple-range tests. p < 0.05 was regarded as a significant difference. The second-order polynomial regression model was applied to measure the dietary protein requirement for this species on the basis of weight gain (WG) and feed conversion ratio (FCR). 3. Results 3.1. Growth Performance and Feed Utilization No statistical differences were observed in the survival rate among different CP levels (p > 0.05; Table 2). With the increase of dietary CP levels, the values of weight gain (WG) and specific growth rate (SGR) showed a general enhancing trend and then reduced slightly (p > 0.05). Feed conversion efficiency (FCR) was improved by dietary CP levels up to 34.85% and then weakened beyond this level (p > 0.05). The values of daily feed intake (DFI) and protein efficiency ratio (PER) in the CP1 diet were the highest, which was significantly higher than those in CP3, CP4, and CP5 diets (p < 0.05). According to the second-order polynomial regression model analysis of WG and FCR, the maximal protein requirement for juvenile K. punctatus is 31.75-33.82% . 3.2. Retention and Deposition of Energy, Nitrogen, and Lipid As shown in Table 3, there were no significant differences in the values of daily nitrogen gain (DNG), daily energy gain (DEG), and daily lipid gain (DLG) among different treatments (p > 0.05). The values of daily nitrogen intake (DNI), energy retention (ER), and lipid retention (LR) values increased significantly with increasing dietary CP levels (p < 0.05). Increasing dietary CP levels from 22.52 to 45.75% reduced daily energy intake (DEI), daily lipid intake (DLI), and nitrogen retention (NR) values (p < 0.05). 3.3. The Whole-Body Composition In this study, there were no significant differences in the contents of moisture, crude protein, and crude lipid among dietary CP treatments (p > 0.05; Table 4). 3.4. Liver Enzyme Activities The activity of lipase in CP3 and CP4 diets was significantly higher than that in the CP1 diet . There was no significant difference in the amylase activity among CP1-CP4 diets, but the amylase activity in the CP2 and CP3 diets was significantly higher than that in the CP5 diet . Fish fed the diet with CP3 showed significantly higher glutamyl pyruvic transaminase (GPT) than that in CP1, CP2, and CP5 diets . 4. Discussion During the feeding trial, all experimental diets were properly accepted by juvenile dotted gizzard shad. The survival rate of this species ranged from 92.42% to 96.97%, and no mortalities were ascribed to dietary CP treatments. After 56 days of culture experimentation, although there were no statistical differences in WG and SGR of this species among treatments, WG and SGR showed an overall upward trend and then decreased slowly as dietary CP levels increased when fish meal was used as the single protein source . A similar phenomenon has been observed in the studies of Puntius gonionotus , Diplodus vulgaris , Takifugu rubripes , Ctenopharyngodon idella , and Epinephelus akaara . In addition, the feed utilization of fish fed the low-protein diet was obviously poor, and the highest FCR value appeared in the CP1 diet. Similar results were also observed in other fish species, such as T. rubripes , Nibea japonica , and Lepomis macrochirus . The feed utilization improved with increasing dietary CP levels, and the optimal FCR was found in fish fed the diet with CP3. However, the FCR in high-protein diets increased again with the further increase of dietary CP levels (CP4-CP5). Based on the second-order polynomial regression model analysis of WG and FCR, the maximum protein requirement for juvenile dotted gizzard shad is about 31.75-33.82% when fish meal was used as the sole protein . This level is similar to the protein requirement reported for other omnivorous fish, such as 30% Calrias batrachus , 32% Zacco barbata , 36% D. vulgaris ), 30% Hypostomus commersoni , 30-33% Betta splendens Regan , and 30 % Chanos chanos . Generally, fish can regulate feed intake to satisfy their energy metabolic requirements . Nonetheless, when diets lack an essential nutrient, fish may consume more feed to meet the specific nutrient requirement . In the present study, the DFI of juvenile dotted gizzard shad was significantly influenced by different CP levels. The highest DFI value was observed in fish fed the lowest dietary CP diet (CP1), which was significantly higher than that in CP3, CP4, and CP5 diets. A general enhancing trend in DFI with descending dietary protein levels has also been observed in L. macrochirus , E. akaara , and Eleginops maclovinus . It is well known that fish require adequate protein intake to obtain benign growth performance. However, when fish are fed diets with insufficient protein inclusion, a high proportion of protein in diets may be applied to nitrogen deposition . The results of this study support the hypothesis that the values of PER and NR increase significantly with the reduction of dietary CP levels. A similar result also appeared in the study of L. macrochirus . Although fish fed with the CP1 diet had excellent DFI, PER, and NR compared with those fed with higher levels of dietary protein (CP2, CP3, CP4), obvious reductions in WG, DNI, and DNG were also observed in the CP1 diet. These data suggest that the rise of DFI, NR, and PER of this species might not be enough to obtain a sufficient amount of protein in a low-protein diet. Similar results have been reported for Megalobrama terminalis . Therefore, the lack of dietary protein may be the main reason for the phenomenon of fish exhibiting poor growth and reduced feed utilization . Protein to energy (P/E) ratio plays a considerable role in establishing a formulated feed, which may affect growth performance and feed efficiency of fish . There is a strong view that P/E ratio is a more reasonable way of expressing protein requirements than dietary crude "protein requirements" . In this study, the addition of corn starch was used to balance the energy of the diets with P/E ratios ranging from 11.69 mg kJ-1 to 24.15 mg kJ-1. After the experiment, dotted gizzard shad fed 22.52% CP diet with P/E ratio of 11.69 mg kJ-1 showed poor growth performance and feed utilization, which may be attributed to the unbalanced P/E ratio of diets. Although the DFI value of the P1 diet is significantly higher than that of feed with higher levels of dietary protein (CP2, CP3, and CP4), nitrogen intake is still insufficient, and more lipid is ingested, which further exacerbates the imbalance of nutrients and affects the utilization of protein. On the other hand, fish cannot use excessive dietary protein for protein synthesis because a high content of dietary protein is usually used as energy fuel for metabolism . In this study, fish fed the diet with P5 obtained the lowest WG and SGR values. The result may be ascribed to the excessive amino acid metabolism in the high proportion of dietary CP , which causes additional energy consumption through deamination and amino acid metabolism . In this study, although the values of DEI and DLI in the CP1 diet were the highest, the lowest values of ER and LR were also observed in the CP1 diet. Sa et al. reported that fish fed the low-protein diets (isoenergetic) had poor digestibility, and the increase of feed intake was probably to overcome the digestibility of low-protein diets. In this study, corn starch was used to balance energy, and cellulose made up for the insufficiency of the diets, leading to the lowest protein group (CP1) having the highest content of corn starch and cellulose. Usually, high content of cellulose may result in poor digestibility for fish . In a sense, the imbalance of formula, or excessive intake of non-protein energy may be the important influencing factors of poor growth performance and feed utilization in fish fed low-protein diets. NR and PER could reflect the utilization of dietary protein . In this study, DNI values in CP4 and CP5 diets were significantly higher than those in the CP2 diet. Although the ER and LR values of CP3, CP4, and CP5 diets were not significantly different from those of the CP2 diet, the NR value of the CP5 diet was significantly lower than that of the CP2 diet. On the other hand, the FCR value gradually increased as dietary protein level increased from 34.85% to 45.78%. The studies of N. japonica and L. macrochirus also showed that the high-protein diets had poor feed utilization. Moreover, the lowest PER value was observed in the CP5 diet; and the PER value of the CP5 diet was significantly lower than that of CP2, CP3, and CP4 diets. Similarly, the research of Diplodus puntazzo and M. terminalis also found the PER value decreased gradually with the increase of dietary CP level. Generally, high level of dietary protein contains insufficient non-protein energy, and excessive dietary protein is considered metabolized for energy or converted into lipid . It may lead to a decrease in the efficiency of protein utilization, increase the metabolic load of the fish body, and even cause ammonia toxicity . Therefore, it is necessary to avoid the high FCR and low PER caused by high protein content in fish diets, which leads to high feed cost, excessive nitrogen excretion, and even harm to fish. For some fish, excessive dietary protein may be used as lipid deposits or energy metabolism . Compared with the CP3 diet, the dotted gizzard shad fed with the CP4 diet showed relatively low PER and NR values, whereas the LR value of the CP3 diet was higher than that of the P4 diet. Moreover, the crude lipid of the whole body in the CP4 diet is higher than that in the CP3 diet, which indicates that excessive dietary protein may not be used for protein deposition, but may be deposited as lipid. This is consistent with the studies of T. rubripes and Brachymystax lenok . However, some other studies also showed that the crude lipid of the whole body for L. macrochirus and E. akaara decreased significantly with the increase of dietary CP levels. The difference may be related to fish species or culture environment. There were no significant differences in the crude protein of the whole body among treatments, suggesting the deposition of body protein of juvenile dotted gizzard shad was not affected by the level of dietary CP. This is consistent with the study of E. akaara . These observations may be attributed to the fact that the protein biological titer of fish with low-protein diets is apparently higher than that of fish with high-protein diets, so as to make up for the deficiency of dietary protein level and ensure the accumulation of body protein . This hypothesis is consistent with the highest NR value of the lowest dietary protein level. In addition, there was no significant difference in the moisture of the whole body among CP treatments. This is similar to the results of E. akaara and T. rubripes . Digestive enzyme activity (including amylase and lipase) is considered as a predictor of potential feed utilization and growth differences of some fish . Wang et al. reported that Nibea albiflora fed the diet with 47% CP has significantly higher intestinal lipase activity than those fed 40% and 54% CP diets. Moreover, Ping et al. reported that the activity of hepatopancreas amylase was increased to some extent, and then reduced as dietary CP levels continued to increase. These observations showed that the increase of dietary protein level in a certain range may promote the secretion of lipase and amylase, enhance the absorption of the digestive function of fish, and be beneficial to fish growth . However, excessive or too low dietary protein may lead to the decrease of enzyme activity, which is not conducive to growth and health . Similar results also appeared in our study, and the trend of lipase and amylase activities was generally consistent with the growth and feed utilization of juvenile K. punctatus. The content of dietary protein can affect the activities of enzymes involved in amino acid catabolism . Aminotransferase such as GPT could decompose amino acids and transfer amino groups to alpha-keto acids (reversible catalysis) . The activity of GPT reflects the metabolic intensity of amino acids and can be used as a reference index to evaluate the nutrition and health of fish . Zhang et al. reported that Channa maculata x Channa argus fed the diet with 46% CP showed significantly higher plasma GPT activity than those fed 34% CP diet. Moreover, the activity of GPT is considered an indicator of proper liver function, and high GPT activity generally indicates a weakening or impairment of normal liver function . In this study, GPT activity increased significantly as dietary CP levels increased from 22.52% to 34.85%, which may indicate the amino acid metabolism of K. punctatus was affected. The mechanism of dietary CP level on amino acid metabolism of different fish species still needs further study. In conclusion, this study found that the suitable levels of dietary protein for juvenile K. punctatus is recommended to be 31.75-33.82%, based on WG and FCR when fish meal was used as the sole protein. Inadequate or excessive dietary protein levels are unfavorable for fish growth and feed utilization. In addition, higher dietary protein levels also alter the digestive enzyme activities and metabolism of amino acids. Author Contributions J.W., X.W. and T.L. conceived the idea of the study; T.L., J.W., T.H., Y.W. and X.C. performed the research; X.W. and T.H. analyzed data; T.L. and J.W. interpreted the results; T.L. and J.W. wrote the paper. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The present study was conducted under the permission and supervision of the Institutional Animal Care and Use Committee of Zhejiang Ocean University. All experimental procedures followed established guidelines for the care and handling of laboratory animals. The present study is approved by Animal Experimental Ethical Inspection, Institutional Animal Care and Use Committee of Zhejiang Ocean University on 13 January 2023 (The approval code is 2023001). Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Relationship of weight gain (WG) with dietary crude protein (CP) levels of juvenile K. punctatus. Figure 2 Relationship of feed conversion rate (FCR) with dietary CP levels of juvenile K. punctatus. Figure 3 Lipase activity of juvenile K. punctatus fed diets containing different CP levels. Value columns with different letters mean significant difference (p < 0.05). Figure 4 Amylase activity of juvenile K. punctatus fed diets containing different CP levels. Value columns with different letters mean significant difference (p < 0.05). Figure 5 Glutamic-pyruvic transaminase (GPT) activity of juvenile K. punctatus fed diets containing different CP levels. Value columns with different letters mean significant difference (p < 0.05). animals-13-00788-t001_Table 1 Table 1 Formulation and composition of experimental diets (% as dry matter basis). Ingredients (g 100 g-1) Dietary Crude Protein (CP) Levels (%) 22.52 (CP1) 28.69 (CP2) 34.85 (CP3) 38.84 (CP4) 45.78 (CP5) Fish meal 1 29.43 38.25 47.08 55.91 64.74 Corn starch 2 34.02 28.02 22.02 16.02 10.02 Fish oil 3 3.85 3.24 2.63 2.02 1.41 Soybean oil 4 2.94 2.94 2.94 2.94 2.94 Vitamin mix 5 2.50 2.50 2.50 2.50 2.50 Mineral mix 6 1.50 1.50 1.50 1.50 1.50 Vitamin C 0.50 0.50 0.50 0.50 0.50 Choline chloride 0.30 0.30 0.30 0.30 0.30 Sodium alginate 4.00 4.00 4.00 4.00 4.00 Cellulose 20.96 18.74 16.53 14.31 12.09 Total 100.00 100.00 100.00 100.00 100.00 Proximate composition (g 100 g-1 dry matter) Moisture 4.10 4.18 3.99 5.04 4.67 Crude protein 22.52 28.69 34.85 38.84 45.78 Crude lipid 9.03 8.38 8.37 8.55 8.88 Gross energy (kJ g-1) 19.26 18.77 18.91 18.91 18.96 Protein to energy ratio (mg kJ-1) 11.69 15.29 18.43 20.54 24.15 1 Imported from Trident Seafoods Corporation, Seattle, USA. 2 Obtained from Sanzhenzhai Foodstuff Co., Ltd., China. 3 Obtained from Zhejiang Industrial Group Co., Ltd., China. 4 Obtained from Jia Li Food Co., Ltd., China. 5 Vitamin (g kg-1 premix) premix consisted of the following ingredients: menadione (4.00); riboflavin (5.00); inositol (200.00); biotin (0.60); folic acid (1.50); cyanocobalamin (0.01); D-Ca pantothenate (10.00); nicotinic acid (20.00); pyridoxine hydrochloride (4.00); thiamin hydrochloride (5.00); tocopherol (40.00); axerophthol (5.00); Vitamin D (4.80); a-cellulose (700.09). 6 Mineral (g kg-1 premix) premix consisted the following ingredients: MgSO4*7H2O (90.43); Ca(H2PO4)2 (122.87); KI (0.02); KH2PO4 (42.03); FeSO4*7H20 (19.73); CuSO4*5H2O (0.34); NaCl (32.33); KCl (65.75); CoCl2*6H2O (0.79); ZnSO4*7H2O (8.44); ferric citrate (38.26); K2SO4 (163.83); C6H10CaO6*5H2O (683.62); MnSO4*H2O (0.37). animals-13-00788-t002_Table 2 Table 2 Growth performance and feed utilization of juvenile K. punctatus fed diets containing different CP levels. Items Dietary CP Levels (%) 22.52 (CP1) 28.69 (CP2) 34.85 (CP3) 38.84 (CP4) 45.78 (CP5) IBW (g fish-1) 1 3.51 +- 0.06 3.46 +- 0.13 3.48 +- 0.11 3.68 +- 0.16 3.91 +- 0.15 FBW (g fish-1) 2 9.77 +- 0.57 9.96 +- 0.64 9.94 +- 0.66 10.70 +- 0.82 10.70 +- 0.39 Survival (%) 96.97 +- 5.25 95.45 +- 4.55 93.94 +- 6.94 92.42 +- 6.94 92.42 +- 13.12 WG (%) 3 177.96 +- 11.84 185.41 +- 7.94 187.92 +- 25.74 191.12 +- 21.95 173.93 +- 10.64 SGR (% day-1) 4 1.82 +- 0.08 1.88 +- 0.05 1.87 +- 0.16 1.90 +- 0.14 1.80 +- 0.07 FCR 5 1.97 +- 0.27 1.70 +- 0.04 1.54 +- 0.21 1.54 +- 0.20 1.73 +- 0.12 DFI (g 100 g fish-1 day-1) 6 4.92 +- 0.71 b 4.42 +- 0.96 ab 3.92 +- 0.14 a 3.83 +- 0.19 a 3.85 +- 0.15 a PER 7 2.18 +- 0.19 c 1.97 +- 0.44 bc 1.81 +- 0.24 b 1.60 +- 0.20 b 1.20 +- 0.81 a 1 Initial body weight; 2 final body weight; 3 weight gain; 4 specific growth rate; 5 feed conversion rate; 6 daily feed intake; 7 protein efficiency ratio. Values are the means of three replicates; Means in the same row with different superscripts are significantly different (p < 0.05). animals-13-00788-t003_Table 3 Table 3 Nitrogen, energy, and lipid utilization by juvenile K. punctatus fed diets containing different CP levels. Items Dietary CP Levels (%) 22.52 (CP1) 28.69 (CP2) 34.85 (CP3) 38.84 (CP4) 45.78 (CP5) Nitrogen DNI (g kg-1 ABW-1 day-1) 1 1.51 +- 0.19 a 1.73 +- 0.01 b 1.89 +- 0.02 bc 2.03 +- 0.05 c 2.43 +- 0.07 d DNG (g kg-1 ABW-1 day-1) 2 0.39 +- 0.05 0.44 +- 0.01 0.44 +- 0.03 0.40 +- 0.09 0.41 +- 0.04 NR (%) 3 26.60 +- 6.24 b 25.50 +- 0.40 b 23.08 +- 1.71 ab 19.93 +- 4.92 ab 17.09 +- 1.92 a Energy DEI (kJ kg-1 ABW-1 day-1) 4 8.03 +- 1.041 b 7.08 +- 0.02 a 6.41 +- 0.08 a 6.16 +- 0.15 a 6.29 +- 0.19 a DEG (kJ kg-1 ABW-1 day-1) 5 3.89 +- 0.24 4.03 +- 0.18 3.80 +- 0.69 4.11 +- 0.17 3.91 +- 0.10 ER (%) 6 48.82 +- 5.87 a 56.86 +- 2.80 ab 59.36 +- 10.88 ab 66.80 +- 4.31 b 62.15 +- 0.30 b Lipid DLI (g kg-1 ABW-1 day-1) 7 3.77 +- 0.49 b 3.16 +- 0.01 a 2.84 +- 0.03 a 2.79 +- 0.07 a 2.95 +- 0.09 a DLG (g kg-1 ABW-1 day-1) 8 1.75 +- 0.38 2.03 +- 0.12 1.71 +- 0.21 1.96 +- 0.44 1.83 +- 0.07 LR (%) 9 47.82 +- 5.06 a 64.08 +- 4.03 ab 60.15 +- 7.15 ab 70.52 +- 7.46 b 62.03 +- 1.64 ab 1 Daily nitrogen intake; 2 daily nitrogen gain; 3 nitrogen retention; 4 daily energy intake; 5 daily energy gain; 6 energy retention; 7 daily lipid intake; 8 daily lipid gain; 9 lipid retention. Values are the means of three replicates; Means in the same row with different superscripts are significantly different (p < 0.05). animals-13-00788-t004_Table 4 Table 4 Whole body composition of juvenile K. punctatus fed diets containing different CP levels. Items (%) Dietary CP Levels (%) 22.52 (CP1) 28.69 (CP2) 34.85 (CP3) 38.84 (CP4) 45.78 (CP5) Moisture 71.93 +- 0.85 70.54 +- 0.56 71.25 +- 1.44 70.86 +- 1.07 71.04 +- 0.97 Crude protein 14.10 +- 1.21 15.03 +- 0.39 14.97 +- 0.12 14.01 +- 1.70 14.75 +- 0.78 Crude lipid 9.76 +- 1.49 10.63 +- 0.36 9.46 +- 0.37 10.30 +- 1.30 10.11 +- 0.14 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000025

Efficient nutritional assimilation and energy metabolism in the stomachs of yaks contribute to their adaption to harsh environments. Accurate gene expression profile analysis will help further reveal the molecular mechanism of nutrient and energy metabolism in the yak stomach. RT-qPCR is regarded as an accurate and dependable method for analyzing gene expression. The selection of reference genes is essential to obtain meaningful RT-qPCR results, especially in longitudinal gene expression studies of tissues and organs. Our objective was to select and validate optimal reference genes from across the transcriptome as internal controls for longitudinal gene expression studies in the yak stomach. In this study, 15 candidate reference genes (CRGs) were determined according to transcriptome sequencing (RNA-seq) results and the previous literature. The expression levels of these 15 CRGs were quantified using RT-qPCR in the yak stomach, including the rumen, reticulum, omasum and abomasum at five stages: 0 days, 20 days, 60 days, 15 months and three years old (adult). Subsequently, the expression stabilities of these 15 CRGs were evaluated via four algorithms: geNorm, NormFinder, BestKeeper and the comparative CT method. Furthermore, RefFinder was employed to obtain a comprehensive ranking of the stability of CRGs. The analysis results indicate that RPS15, MRPL39 and RPS23 are the most stable genes in the yak stomach throughout the growth cycle. In addition, to verify the reliability of the selected CRGs, the relative expression levels of HMGCS2 were quantified via RT-qPCR using the three most stable or the three least stable CRGs. Overall, we recommend combining RPS15, MRPL39 and RPS23 as reference genes for the normalization of RT-qPCR data in the yak stomach throughout the growth cycle. yak stomach transcriptome-wide reference gene RT-qPCR Double Word-ClassNatural Science Foundation of Sichuan Province2022NSFSC1665 Sichuan Science and Technology Program, China2021YFYZ0001 2021YFN0001 This research was funded by "Double Word-Class" projects of Southwest Minzu University; the Natural Science Foundation of Sichuan Province (2022NSFSC1665); and the Sichuan Science and Technology Program, China (2021YFYZ0001 and 2021YFN0001). pmc1. Introduction The yak (Bos grunniens), a precious domesticated ruminant, also known as the "boat on the plateau", is mostly found on the Qinghai-Tibetan Plateau and nearby areas at an altitude above 3000 m. As the most significant livestock in this region, yaks are capable of surviving and providing milk, meat, hair and cheese for local herders in a hostile environment . A previous study found that efficient nutritional assimilation and energy metabolism in the yak stomach contributes to their adaption to a harsh environment . In ruminants, the stomach and small intestine are mostly where nutrients are digested and absorbed . Additionally, the development of the yak stomach at different stages plays a vital role in digestive ability and nutrient supply . Thus, accurate analysis of gene expression profiles in the yak stomach are of major priority to further reveal the molecular mechanisms of nutrient and energy metabolism. As a typical ruminant, a remarkable feature of the yak is that it has a complex stomach consisting of four gastric compartments: rumen, reticulum, omasum and abomasum . The first three compartments of the compound stomach (i.e., rumen, reticulum and omasum) are commonly referred to as the "forestomach" and perform cooperative functions . They serve as fermentative chambers where bacteria break down the ingested cellulose, producing enormous amounts of gas . By contrast, only the abomasum can generate digestive juices and gastric enzymes . Hence, the abomasum is also called the true stomach. In newborn ruminants, dietary requirements are fulfilled by the uptake of colostrum, which is digested in the abomasum to provide energy and essential nutrients, as well as immunity molecules . In comparison, the rumen acts as the primary location of digestion and absorption in grown ruminants, and microorganisms decompose ingested feed in the rumen to produce volatile fatty acids (VFA) that serve as the main source of energy . Understanding which genes in the stomach are crucial for nutrient absorption and digestion and how they might be regulated to contribute to growth and maintenance is a major concern in the field of yak research. RT-qPCR is extensively used for the analysis of gene expression patterns due to its sensitivity, accuracy and specificity, as well as practical simplicity . However, several drawbacks such as nucleic acid quality, poor choice of primers or probes and inappropriate data and statistical analyses encumber the authenticity of RT-qPCR results . Therefore, various strategies have been applied to normalize RT-qPCR results. The use of reference genes that are not affected by study conditions is a generally accepted strategy for normalizing RT-qPCR data . Despite the fact that several genes such as ACTB and GAPDH are commonly employed as reference genes in a wide range of studies, it is unlikely that any genes have enough overall expression stability to be appropriate for any kind of experiment . Therefore, it is essential to select reliable reference genes for the specific experimental context under study. To date, no studies have shown suitable reference genes for the normalization of RT-qPCR data in the yak stomach. Many studies have concentrated on verifying subsets of frequently used reference genes for specific experimental contexts . However, it is biased to select the CRGs from a minority of genes and assume that at least a few of those genes are appropriate for the certain experimental context. The emergence of high-throughput RNA-seq technology provides a novel strategy for identifying reference genes . Based on the RNA-seq dataset, CRGs with stable expression and high abundance were preliminarily selected. Subsequently, the expression levels of CRGs were quantified using RT-qPCR and their stabilities were evaluated using geNorm , NormFinder , BestKeeper and the comparative CT method . This strategy has been successful for identifying reference genes for fish , Holstein cows , goats , and so on. The purpose of this study was to select and validate reliable reference genes from across the transcriptome that can serve as internal controls for longitudinal gene expression studies in the yak stomach throughout the growth cycle. 2. Materials and Methods 2.1. Animals and Sample Collection All the experimental protocols were approved by the Institutional Animal Care and Use Committee of Southwest Minzu University (permit number: 2020-07-02-11). All Maiwa yaks were raised in Hongyuan County of Sichuan Province and fed with natural lactation and pasture. A total of 15 Maiwa yaks (7 males and 8 females) were selected from the same herd at 5 different growth stages: 0 days (lactating stage), 20 days (lactating stage and starting to graze), 60 days (lactating stage and graze stage), 15 months (graze stage but still lactating) and 3 years old (natural graze stage). For sample collection, three separate yaks of each age were slaughtered. The stomach tissues of the yaks, including rumen, reticulum, omasum and abomasum, were rinsed immediately in 0.1% DEPC water after slaughter and frozen in liquid nitrogen until processing for total RNA extraction. 2.2. RNA Extraction and cDNA Synthesis The total RNA of the rumen, reticulum, omasum and abomasum tissues were extracted using the mirVana miRNA Isolation Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. The purity and concentration of total RNA were confirmed using the NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The integrity of total RNA was assessed using 1% agarose gel electrophoresis. The cDNA was generated from 1000 ng total RNA using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) in a reaction mixture of 20 mL. The cDNA was stored at -80 degC until required. 2.3. Selection of CRGs Based on our previous RNA-seq results of the compound stomach at five stages in fifteen yaks (unpublished data), 7 CRGs, ribosomal protein S15 (RPS15), ribosomal protein S23 (RPS23), 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), ribosomal protein L13a (RPL13A), b-actin (ACTB), ribosomal protein S9 (RPS9) and glyceraldehyde-3-phos-phate dehydrogenase (GAPDH), were selected according to the fragments per kilobase of exon model per million mapped reads (FPKM) and the coefficient of variation (CV, %). The value of FPKM was higher than 100 and the CV value was less than 20%. FPKM = cDNA fragments/[mapped fragments (millions) x transcript length (kb)] and CV = standard deviation (SD) FPKM/MeanFPKM x 100%. Subsequently, based on the previous literature, eight genes were selected as CRGs: ubiquitously expressed prefoldin-like chaperone (UXT), dystrobrevin binding protein (DBNDD2), DEAD box polypeptide 54 (DDX54), hydroxymethylbilane synthase (HMBS), protein phosphatase 1 regulatory inhibitor subunit 11 (PPP1R11), mitochondrial ribosomal protein S15 (MRPS15), mitochondrial ribosomal protein L39 (MRPL39) and TATA box binding protein (TBP). 2.4. Primer Pairs Design Primers for RT-qPCR were designed using Primer-BlAST with a length of 20 +- 3 bases and amplicon sizes ranging from 100 to 150 bp. The sequences of the CRGs were obtained from NCBI accessed on 25 June 2022). The primer specificity of each CRG was verified using 2% agarose gel electrophoresis and melting curve analysis. To validate the specificity of each primer pair, the products of PCR were purified and sequenced using a 3730 DNA analyzer (ABI, Carlsbad, CA, USA), and the sequencing results were compared with all potential transcript sequences in NCBI using BLAST. 2.5. RT-qPCR Assay All RT-qPCR assays were carried out in triplicate for each sample using the LightCycler 96 System (Roche Diagnostics, Indianapolis, IN, USA). The total volume of each reaction mixture was 20 mL, including 10 mL of TB Green Premix Ex Taq II (TaKaRa, Dalian, China), 2 mL of diluted cDNA, 0.5 mL of each of 10 mM forward and reverse primers and 7 mL of RNase Free dH2O. The PCR program was conducted as follows: 95 degC for 30 s (pre-denaturation), 40 cycles of 95 degC for 5 s and 60 degC for 30 s (quantitative analysis), 95 degC for 5 s and 60 degC for 1 min (melting curves analysis). To determine the correlation coefficient (R2) and amplification efficiency (E) for each primer pair, a five-point standard curve was generated using a five-fold dilution of cDNA. The correlation coefficient (R2) and amplification efficiency (E) of each primer pair were calculated using the LightCycler 96 System. A modified Pfaffl equation was used to determine the relative quantity (RQ) of each gene :RQsample= E (Cq (Calibrator)- Cq (Sample) ) Cq (calibrator) = Cq for the arithmetic mean of all samples at 5 stages, Cq (sample) = Cq for the sample. The formula for calculating the relative expression level of a target gene is as follows:Relative gene expression=RQGOIGeomean[RQREFs] RQGOI: the RQ value of the target gene, Geomean[RQREFS]: the geometric mean of the RQ value of selected reference genes. The normalization factor (NF) was calculated using the geometric mean of the RQ value of the selected reference genes . 2.6. Stability Analysis of CRGs The expression stability of 15 CRGs was evaluated using 4 algorithms: geNorm, NormFinder, BestKeeper and the comparative CT method. In addition, RefFinder accessed on 10 October 2022) was used to synthesize the evaluation results of the above four algorithms to give an overall ranking. 2.7. Validation of Optimal Reference Gene Combinations HMGCS2 is the key rate-limiting enzyme in the ketogenic pathway and plays an important role in the digestion and absorption of nutrients in the stomach. The expression levels of HMGCS2 were quantified using RT-qPCR to validate the selected reference genes. The expression levels of HMGCS2 in the stomach at 5 stages were normalized using the three most stable gene combinations and the three most unstable gene combinations identified from this study. The relative mRNA expression of HMGCS2 was calculated using the 2-Ct method. In addition, statistical significance was analyzed using one-way analysis of variance via SPSS 25.0 software (IBM, Armonk, NY, USA). A p value below 0.05 was regarded as statistically significant. 3. Results 3.1. Quality Control of Total RNA The 260/280 ratio of total RNA for each sample ranged from 1.8 to 2.2, and the purity and concentration were qualified for subsequent experiments (Table S1). The RNA of all samples clearly displayed two prospective bands at 18 s and 28 s without any signs that the products were degraded . The above results indicate that the RNAs of all samples were equipped for cDNA synthesis. 3.2. Selection of CRGs Based on RNA-seq Data and Previous Literature The criteria for preliminary selection of reference genes were relatively high transcriptome abundance and low expression variation . As a result, preliminary selection comprised genes with relatively high transcriptome abundance (FPKM > 100) as identified by the mean FPKM value and low variability as identified by the coefficient of variation (CV < 20%). A total of 80 CRGs were preliminarily selected using our previous RNA-seq results of the stomach at five stages in fifteen yaks (Table S2). Furthermore, 7 genes (RPS15, RPS23, YWHAZ, RPL13A, ACTB, GAPDH, and RPS9) were considered as CRGs due to their lower CV values, higher FPKM values, and easier primer designs. In addition, eight CRGs were selected based on previous studies. Among these CRGs: UXT, HMBS, MRPS15, PPP1R11, MRPL39 and TBP were validated to be appropriate reference genes for RT-qPCR in yak . Additionally, DBNDD2 and DDX54 were verified as suitable reference genes for RT-qPCR in the rumen epithelium of cows . In conclusion, 15 genes were chosen as CRGs for further evaluation. 3.3. Characteristics of Primer Pairs The details of primer pairs of 15 CRGs are displayed in Table 1. Primer pairs of amplification efficiency (%) ranged from 91 to 109%, the amplicon's size lay in 100-286 bp and the R2 of each primer pair was not less than 0.99. The specificity of primer pairs for each gene was verified via 2% agarose gel electrophoresis and melting curve analysis . To further validate the specificity of each primer pair, the products of PCR were purified and sequenced. Then, the sequencing results were compared with all potential transcript sequences in NCBI using BLAST (Table S3). 3.4. RT-qPCR Analysis for CRGs The mean Cq values of all tested samples calculated to determine the expression levels of the 15 CRGs are illustrated in Figure 1. Cq value had a negative correlation with gene expression level. In other words, higher gene expression levels are associated with lower Cq values and vice versa. The Cq values of all CRGs ranged from 18.49 to 32.67. For each CRG, the mean and median Cq values were relatively close. Among all the CRGs, RPS23 demonstrated the highest expression level, with Cq = 20.18 +- 0.76, while PPP1R11 had the lowest expression level, with Cq = 30.59 +- 0.89. 3.5. Evaluation of Expression Stability for CRGs In this study, four algorithms: geNorm, NormFinder, BestKeeper and the comparative CT method were used to evaluate CRGs for stability ranking. The stability rankings obtained from the four algorithms were different. Thus, RefFinder was employed to obtain a total score that was used to rank the stability of the 15 CRGs (Table 2). The M-value was calculated via geNorm analysis to identify gene expression stability. Then, the M-value was used to rank the stability of expression for the 15 CRGs, and the M-value was negatively correlated with the stability of gene expression. According to the geNorm method, the results show that RPS15 and DBNDD2 were the most stable CRGs with the lowest M-value of 0.48, while RPL13A was the least stable gene with the highest M-value of 0.74 in the yak stomach throughout the growth cycle. The NormFinder algorithm was used to calculate the stability value (SV) to identify the ranking of the CRGs, with the most stable gene showing the lowest SV. For the yak stomach throughout the growth cycle, the most stable gene was RPS15 with the lowest SV of 0.35, and RPL13A was the most unstable gene with the highest SV of 0.62. The BestKeeper and the comparative CT method regard standard deviation (SD) as one of the criteria to evaluate the stability of gene expression.. The lower the SD value, the more stable the gene expression. Based on BestKeeper analysis, RPS15 was the most stable gene, whereas YWHAZ was the least stable gene with the highest SD value. By contrast, according to the comparative CT method, MRPL39 had the highest stability, and the YWHAZ was the most unstable gene. Based on the results obtained using these four algorithms, RefFinder was used for comprehensive ranking. As a result, the comprehensive rankings according to stability from the highest to the lowest are RPS15 > MRPL39 > RPS23 > DDX54 > DBNDD2 > GAPDH > TBP > RPL13A > MRPS15 > PPP1R11 > ACTB > HMBS > RPS9 > UXT > YWHAZ. 3.6. Optimal Number of Reference Genes The pairwise variation values (V) were calculated using geNorm software, which is a valid tool to identify the optimal number of reference genes for RT-qPCR. Vandesompele et al. proposed taking 0.15 as a cut-off value below which the inclusion of additional reference genes is not necessary. Thus, according to the cut-off value (V = 0.15), the results indicate that the combination of three genes was the optimal number for normalization of RT-qPCR data in the yak stomach throughout the growth cycle . Furthermore, low pairwise variation values correspond to a high correlation coefficient . Clearly, there is no need to include an additional gene when using the three most stable reference genes for calculating the NF . In contrast, it is essential to have more than an additional gene when using the two most stable reference genes for calculation of NF . Thus, we recommend the combination of the three most stable genes (RPS15, MRPL39, and RPS23) to normalize RT-qPCR data in the yak stomach throughout the growth cycle. 3.7. Validation of the Combination of CRGs To verify the effect of the combination of RPS15, MRPL39 and RPS23 for the normalization of RT-qPCR data, the expression of HMGCS2 was quantified via RT-qPCR in yak stomach at 5 stages (0 d, 20 d, 60 d, 15 m and adult). Moreover, the expression patterns of HMGCS2 in yak stomach at 5 stages were also identified using the FPKM of RNA-seq results. The results show a correspondence between the RT-qPCR and RNA-seq, indicating the RT-qPCR data of HMGCS2 using the RPS15, MRPL39 and RPS23 for normalization were reliable . To further validate the selection of CRGs, the three most stable CRGs (RPS15, MRPL39, and RPS23) and the three least stable CRGs (RPS9, UXT and YWHAZ) were used to normalize the expression of HMGCS2. As shown in Figure 4A,B, the expression patterns of HMGCS2 in the rumen, reticulum, omasum and abomasum at five stages (0 d, 20 d, 60 d, 15 m and adult) were similarly obtained using FPKM based on RNA-seq results and the combination of three most stable CRGs (RPS15, MRPL39, and RPS23) for normalization. Furthermore, the expression of HMGCS2 in the rumen, reticulum and omasum were the lowest at 0 d and the highest at adulthood, while the opposite was true in the abomasum. However, compared with the expression of HMGCS2 based on RNA-seq results , normalization of HMGCS2 expression using the three least stable CRGs (RPS9, UXT and YWHAZ) demonstrated significant differences . Hence, it is essential to select suitable reference gene combinations to normalize the expression of target genes. 4. Discussion Gene expression analysis via RT-qPCR is a dependable and extensively used method to reveal the molecular mechanism of digestion and absorption of nutrients in the stomach. The use of reference genes is the most credible strategy for taking into account the initial concentration of RNA, sample loss during experimentation, the efficiency of cDNA synthesis, and so on . However, the selection of inappropriate reference genes also affects the authenticity of RT-qPCR data . Therefore, selecting suitable reference genes is essential to obtain meaningful RT-qPCR results. Until now, strategies for identifying reference genes from the transcriptome have been widely used. Reference genes selected from the transcriptome increase the reproducibility and sensitivity of results, give a stronger correlation between protein expression levels, and have better detection and coverage . Although RNA-seq screening has many merits in predicting reference genes, this strategy is not absolutely trustworthy and needs further validation via RT-qPCR . In this study, 15 CRGs were determined via RNA-seq and the previous literature, and further verified using RT-qPCR. In this study, geNorm, NormFinder, BestKeeper and the comparative CT method were employed to assess the stability of 15 CRGs. Ribosomal protein S15 (RPS15) is a component of the 40S ribosomal subunit and functions as a nuclear export factor . Although different algorithms were used for the stability ranking of 15CRGs, RPS15 had the best stability in all the algorithms except the comparative CT method (geNorm, NormFinder and BestKeeper) (Table 2). Furthermore, RPS15 was also the most stable gene in the comprehensive ranking of the results of the four algorithms using RefFinder. This is consistent with Bionaz et al. finding that RPS15 is one of the best reference genes used for the normalization of gene expression data in the bovine mammary gland during the lactation cycle. Tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), belonging to the 14-3-3 protein family, participates in various cell activities including cell growth, cell cycle, apoptosis, and so on . Some studies have demonstrated the consideration of YWHAZ as an appropriate reference gene due to its high stability in cattle , buffaloes and yak . By comparison, it seems that YWHAZ was the least stable gene in our study (Table 2). Even so, the M value of YWHAZ (0.73) derived from the geNorm analysis is well below the threshold (M = 1.5) proposed by Vandesompele et al. , suggesting that it is also a relatively stable gene in the yak stomach. These results indicate that the stability of reference genes is highly specific and should be evaluated for a given experimental context. Although RPS15 had the highest stability in our evaluation, we still do not recommend using it alone as the reference gene for the normalization of RT-qPCR data in the yak stomach. Many studies show that using a single gene as the reference gene should be avoided . It has been reported that using a single reference gene results in significant bias . To date, no specific theory prescribes a certain number of reference genes to be used. Use of geNorm can provide the optimal number of reference genes needed to eliminate the majority of technical variation . Accuracy and practicality are trade-offs when determining the optimal number of reference genes. It is an unnecessary waste of resources to use more reference genes if the inclusion of additional genes has no significant effect on NF . In our study, there was no significant change between the NF calculated with the three most stable CRGs and that calculated with the four most stable CRGs, indicating that it was superfluous to add a gene for normalization . In addition, the digestive tract of the yak has three developmental stages: pre-rumination (0-20 days), transition from pre-rumination to rumination (20-60 days), and rumination (after 60 days). The diets of yaks are different at different developmental stages. Therefore, we evaluated the stability of these genes in yak stomach tissue over five developmental stages. Our results support the use of these reference genes in the normalization of RT-qPCR data under different dietary conditions. As a result, we recommend using the combination of three most stable genes (RPS15, MRPL39, and RPS23) to calculate the NF for normalization of RT-qPCR data in the yak stomach throughout the growth cycle. The expression profiles of HMGCS2 were quantified via RT-qPCR in the yak stomach at five developmental stages, and its expression levels were normalized by the selected combination of reference genes. HMGCS2 is the key rate-limiting enzyme in the ketogenic pathway and induces the biosynthesis of HMG-CoA, which is the central metabolite of rumen epithelial cells . The ketogenic capacity of ruminal epithelium in ruminants increases with age, and newborn ruminants have no ketogenic capacity . Thus, we hypothesized that the expression level of HMGCS2 in the rumen should increase in terms of age, as well as those in the reticulum and omasum because they serve analogous functions to the rumen. In this paper, the expression level of HMGCS2 did increase with age, as determined using RNA-seq and RT-qPCR in the rumen, reticulum and omasum . For newborn ruminants, the rumen was not fully developed and ingested colostrum is instead digested in the abomasum . Consequently, the expression level of HMGCS2 in the abomasum should be highest at birth and lowest in adulthood . In addition, although target genes with significant expression changes can be identified using less stable reference genes, target genes with imperceptible expression changes can only be detected using the best reference genes . Our results confirm that significant changes in the expression of HMGCS2 between birth and adulthood could be identified using either the three most stable CRGs (RPS15, MRPL39, and RPS23) or the three least stable CRGs (RPS9, UXT and YWHAZ). However, when the change in HMGCS2 expression is slight, errors may occur when using RPS9, UXT and YWHAZ for normalization. For example, in the rumen, there were no significant differences in HMGCS2 expression levels between 60 days and 15 months either based on the results of RNA-seq or using RPS15, MRPL39, and RPS23 for normalization, whereas its expression levels normalized using RPS9, UXT and YWHAZ had significant differences (p < 0.05) between 60 days and 15 months . These results imply that using suitable reference genes is essential for accurate normalization of target gene expression. 5. Conclusions In this study, 15 CRGs were selected using transcriptome sequencing results and the previous literature, and their expression stability was evaluated using five algorithms. Therefore, we recommend the combination of the three most stable genes RPS15, MRPL39, and RPS23 as reference genes for the normalization of RT-qPCR data in the yak stomach throughout the growth cycle. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Agarose gel electrophoresis for all tested RNA samples; Figure S2: Agarose gel electrophoresis for 15 candidate reference genes; Figure S3: Melting curves of 15 candidate reference gen; Table S1: The purity and concentration of RNA samples used in this study; Table S2: The stably expressed genes identified in the yak stomach via RNA-sequencing; Table S3: Sequence alignment results in NCBI for 15 candidate reference genes. Click here for additional data file. Author Contributions Conceptualization, M.J. and Q.M.; methodology, Q.M. and L.Y.; validation, Q.M. and L.Y.; data analysis, Q.M. and Y.W.; investigation, Y.L.; resources, M.J.; data curation, M.J.; writing--original draft preparation, Q.M.; writing--review and editing, Q.M.; visualization, Q.M. and Y.W.; supervision, M.J.; project administration, M.J.; funding acquisition, M.J. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The protocol for Animal Care and Use was approved by the Animal Ethics and Welfare Association of the Southwest Minzu University (date of approval: 2020-07-02; permit number: 2020-07-02-11), and experiments were conducted in accordance with the regulations and guidelines established by this committee. Informed Consent Statement Not applicable. Data Availability Statement Data sharing is not applicable to this article. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The mean Cq values of 15 CRGs in a total of 60 samples of yak stomach including rumen, reticulum, omasum and abomasum at 5 developmental stages. The Cq values indicate quantification cycle and are also known as the threshold cycle (Ct). The 75th and 25th percentiles are shown at the top and bottom of each box, respectively. The black line within the box depicts the median. The upper whisker caps represent Q3 + 1.5 x IQR (where Q3 is the third quartile and IQR is the inter-quartile range, or distance between the first and third quartiles) and the lower whisker caps represent Q1 - 1.5x IQR (where Q1 is the first quartile). The black dots indicate outliers. Figure 2 The best number of reference genes for RT-qPCR. (A) Pairwise variation (Vn/n + 1) analyses of 15 CRGs. The y-axis indicates Vn/Vn + 1 between the calculation of the NFn using the most stable reference genes and the NFn + 1 using an addition of the next most stable reference genes. The gray line depicts the cut-off value (V = 0.15). (B,C) NFn/n+1 scatterplots before and after the addition of (n + 1) reference gene (x-axis and y-axis). The NFn was calculated using the geometric mean of the RQ data of n CRGs. r: Spearman rank correlation coefficient. Figure 3 Expression profiles of HMGCS2 in (A) rumen, (B) reticulum, (C) omasum and (D) abomasum at five developmental stages (0 d, 20 d, 60 d, 15 m and adult). Relative expression level: the RT-qPCR data of HMGCS2 were normalized using RPS15, MRPL39 and RPS23. RNA-seq: the arithmetic mean values of FPKM from samples in triplicate at each developmental stage in transcriptome sequencing results. Figure 4 The expression levels of HMGCS2 (A) based on FPKM, (B) normalized using the 3 most stable CRGs (RPS15, MRPL39 and RPS23), and (C) normalized using the 3 least stable CRGs (RPS9, UXT and YWHAZ). The values are means +- SE. Each histogram is divided into four groups (rumen, reticulum, omasum and abomasum). Within each group, the developmental stages with different superscripts indicate significant differences (p < 0.05) in HMGCS2 expression between them. (For example, d is significantly lower than a, b and c). animals-13-00925-t001_Table 1 Table 1 Candidate reference genes and primer pairs characteristics. Gene Accession No. Primer Sequence (5'-3') 1 Size (bp) 2 E (%) 3 R2 GAPDH XM_014482068.1 F: TGGGTGTGAACCACGAGAAG R: CGTGGACGGTGGTCATAAGT 141 95 0.9970 ACTB XM_005887322.2 F: GAGCTACGAGCTTCCTGACG R: CGCAGGATTCCATGCCCAG 104 99 0.9961 UXT XM_005899362.2 F: TGAGCGACTCCAGGAAGCTA R: CCAAGGGCCACATAGATCCG 114 97 0.9955 DBNDD2 XM_014477527.1 F: TTCTTGCCTTGTGAAGACCCTC R: AGGACAAGGAGGAAGTACGAGAC 124 106 0.9996 RPS9 XM_014483477.1 F: CTGAAGCTGATCGGCGAGTA R: GGGTCTTTCTCATCCAGCGT 119 101 0.9940 DDX54 XM_005904734.2 F: CCTTGCACGAAAATCCCGAC R: AGCCCATTTCAAAGAGCCTGT 135 97 0.9937 HMBS XM_005897125.2 F: TTGGATCTGGTGGGTGTGTT R: CTCCAGTCAGGTACAGTTGCC 148 100 0.9949 RPS15 XM_005890466.2 F: GCGGAAGTGGAACAGAAGAA R: GCATCAGTTGCTCATAGGACAT 100 91 0.9979 MRPS15 XM_014477429.1 F: CTCAAGTCCTGGAGGTCTCAT R: CTGGTAGTCCTTCAGCAGCAT 115 99 0.9967 RPS23 XM_005903762.2 F: TGTGCTGGAAAAAGTAGGAGTT R: AGCAACCATCATTGGGTACAA 122 109 0.9999 PPP1R11 XM_014483599.1 F: AGTGGGTTTGGGAGAATCGC R: GTTAGGCTCCGGTTCTCAGAC 143 92 0.9965 MRPL39 XM_005898618.2 F: AGAGCCCCAGAAGTTCCAGT R: AGAACGCAGGTTCTCTTTTGTTG 102 92 0.9948 TBP XM_005908678.2 F: AAGATAACCCACAGAGCCGAG R: GCTCCTCCAGAATAGACAGACTGTT 286 97 0.9958 YWHAZ XM_005887010.2 F: CCTACTCCGGACACAGAACAT R: CAGGCTGCCATGTCATCATATC 101 99 0.9985 RPL13A XM_005904989.2 F: GGTTCCTTCTTTCCCAGGCA R: CAACCTTGCGGCCCAGAA 130 107 0.9984 1 F: forward primer, R: reverse primer. 2 size: amplicon size. 3 E (%): amplification efficiency (%) = (10(-1/slope) - 1) x 100%. animals-13-00925-t002_Table 2 Table 2 Stability of CRGs in yak stomach throughout the growth cycle. CRGs GeNorm NormFinder BestKeeper Delta Ct Comprehensive Ranking R-Based R-Based Excel Plug-in Excel Plug-in RefFinder Rank Value Rank Value Rank Value Rank Value Rank Value RPS15 1 0.48 1 0.35 1 0.51 2 0.78 1 1.41 MRPL39 3 0.53 2 0.38 8 0.67 1 0.77 2 2.51 RPS23 4 0.56 8 0.55 4 0.55 7 0.85 3 3.74 DDX54 6 0.61 5 0.49 6 0.58 3 0.81 4 3.83 DBNDD2 1 0.48 3 0.44 3 0.54 6 0.85 5 5.05 GAPDH 13 0.72 11 0.58 5 0.57 9 0.87 6 5.90 TBP 7 0.63 6 0.50 12 0.70 4 0.83 7 6.31 RPL13A 15 0.74 15 0.62 2 0.52 11 0.88 8 6.42 MRPS15 10 0.69 10 0.57 9 0.68 5 0.85 9 6.89 PPP1R11 5 0.59 4 0.48 11 0.70 8 0.86 10 8.85 ACTB 12 0.71 12 0.59 7 0.63 10 0.87 11 9.37 HMBS 9 0.67 9 0.56 10 0.69 12 0.91 12 11.92 RPS9 8 0.65 7 0.53 14 0.76 13 0.93 13 12.98 UXT 11 0.70 13 0.61 13 0.73 14 0.93 14 13.49 YWHAZ 14 0.73 14 0.62 15 0.80 15 0.93 15 15.00 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000026

Automated monitoring systems have become increasingly important for zoological institutions in the study of their animals' behavior. One crucial processing step for such a system is the re-identification of individuals when using multiple cameras. Deep learning approaches have become the standard methodology for this task. Especially video-based methods promise to achieve a good performance in re-identification, as they can leverage the movement of an animal as an additional feature. This is especially important for applications in zoos, where one has to overcome specific challenges such as changing lighting conditions, occlusions or low image resolutions. However, large amounts of labeled data are needed to train such a deep learning model. We provide an extensively annotated dataset including 13 individual polar bears shown in 1431 sequences, which is an equivalent of 138,363 images. PolarBearVidID is the first video-based re-identification dataset for a non-human species to date. Unlike typical human benchmark re-identification datasets, the polar bears were filmed in a range of unconstrained poses and lighting conditions. Additionally, a video-based re-identification approach is trained and tested on this dataset. The results show that the animals can be identified with a rank-1 accuracy of 96.6%. We thereby show that the movement of individual animals is a characteristic feature and it can be utilized for re-identification. deep learning re-identification computer vision animal identification animal welfare automated behavior analysis motion features video-based method dataset This research received no external funding. pmc1. Introduction Assessing animal welfare is a major challenge for every animal-keeping institution and thus focus of biological research. It is not trivial to obtain a comprehensive picture of an animal's physical and psychological condition . One crucial tool for investigating animal welfare is the direct observation of the animals under care. Compared to other approaches, such as hormone level evaluation or blood analysis, behavioral observation offers the advantage that it is an inexpensive and non-invasive method . Nevertheless, it is common for many biologists, veterinarians, and animal caretakers to observe the animals manually, which comes with severe limitations. It is very labor-intensive, time-consuming and cannot be carried out continuously. Moreover, it is prone to human mistakes. Automated camera-based observation systems help to solve these issues and allow for continuous recording and analysis. Unlike a human observer, such systems can record multiple animals simultaneously on a 24/7 scale. The development of camera-based observation systems has increasingly been carried out in the last few years as advances in deep learning continue to offer new tools to study animal ecology and behavior . The most crucial and challenging step for such a system is the identification of individual animals in the recorded data. Only by performing this stage can the behavior of individuals be analyzed. This identification step helps biologists and animal keepers to tailor possible measures or treatments to individual needs. The problem of identifying animals in different camera views is called re-identification (short re-ID). Re-ID refers to the task of ranking a list of known individuals (the gallery) when confronted with a new image or video (the query sample). The generated list contains the best-matching identities in descending order. Ideally, the first listed individual corresponds to the animal shown in the query sample . Researchers initially focused on re-identification in humans. Large benchmark datasets such as Mars or Market-1501 enabled the development of various methods for this task. In recent years, some of these approaches could be utilized for re-ID in animals. However, there are still numerous gaps in this area of research, which we are helping to fill through our work. Deep learning approaches to identify animals were first introduced in 2014 and since then, the number of methods used for animal re-ID is increasing . Many of them tackle re-ID for specific species with unique visual features such as salamanders , manta rays , cows or Amur tigers . In recent years, species-unspecific approaches have been introduced more and more frequently. These methods offer the advantage of not needing unique visual markers such as fur stripes or skin patterns. Most well-performing approaches are based on convolutional neural networks (CNNs). Freytag et al. showed that the CNN architecture AlexNET outperformed all previous re-ID approaches on two groups of chimpanzees. Brust et al. followed the same approach and used AlexNET for the re-ID of gorillas. Schneider et al. took a different path and showed that similarity learning networks could be utilised to re-identify animals across species without handcrafted feature extraction. This approach is promising as it performed well on five different species: humans, chimpanzees, whales, fruit flies and tigers. In a few projects, animal re-ID methods are already embedded in frameworks to collect information about single individuals' behavior automatically. Marks et al. present an end-to-end pipeline to extract specific behaviors (e.g., social grooming or object interaction) as well as the pose of the animals. Their approach is species-unspecific and was tested on primates and mice, showing promising results. In our previous work , we proposed a similar end-to-end framework with a focus on fast inference times to analyse individual polar bears' trajectories over long time periods. In automated observation of animal behavior, videos are the primary data source. However, all previously listed methods for re-ID in animals are image-based approaches. Nevertheless, the movement and gait of humans are individual characteristic features used for re-ID in several methods . Video-based approaches aim at incorporating these individual movement characteristics by embedding not only spatial, but also temporal information into their feature representation. When conducting observations of animals in zoological facilities, it is common to encounter low-resolution videos with occlusions. In such cases, utilizing all available information from the video, rather than relying solely on a single image, is essential for successful animal re-identification. In the future, we expect video-based approaches to be used more and more extensively for automated observation of animals in zoos. At this point, however, there are no video-based approaches for re-ID in animals. Developing deep learning-based approaches for re-ID in animals requires large amounts of labeled data. Unlike in humans, it is difficult to record a large number of individual animals. In particular, publicly available datasets are very limited . Table 1 shows a list of publicly available datasets for re-ID in animals. However, all available datasets are image-based, meaning that each sample is a single image showing a specific animal. To be able to develop a video-based re-ID method, the need for a video-based dataset is a matter of urgency. To the best of our knowledge, we introduce the first video-based dataset for animal re-ID to date: the PolarBearVidID dataset. It includes 13 individual polar bears, each with at least 100 annotated sequences incorporating the movement of the animals. The main concept of the dataset is depicted in Figure 1. Polar bears are particularly challenging as individuals lack prominent distinct visual features. Two biologists independently labeled the data in a competitive procedure aimed at ensuring a high quality of the annotations. Furthermore, we present a novel approach to extend labels to multiple adjacent frames and reduce the labeling effort for video-based datasets. The PolarBearVidID dataset can be used for research and development of algorithms and systems for video-based animal re-identification. It is the first dataset that allows for the investigation of using the movement of individual animals as a feature for the task of re-ID. As this dataset directly contributes to the improvement of automated animal behavior analysis systems, it may have applications in fields such as wildlife conservation and animal behavior research. To benchmark the PolarBearVidID dataset, we chose a state-of-the-art video-based method introduced by Li et al. for human re-ID. It is sensitive to the movement characteristics of the individual being classified, making it ideal for benchmarking the dataset. We compare the performance to an image-based method to estimate the advantages of a video-based re-ID approach. In summary, with PolarBearVidID, we contribute the first-ever video-based dataset for animal re-identification similar to the benchmark datasets for humans. This dataset allows us to test a state-of-the-art video-based approach for re-ID in animals for the very first time. Finally, we compare this method to an image-based baseline. This allows us to investigate whether the use of temporal information of the movement yields performance improvements. 2. The PolarBearVidID Dataset With a sequence length of up to eight seconds, a frame rate of 12.5 fps and at least 100 sequences per animal, the PolarBearVidID dataset contains 138,363 identity-annotated images. As it is not feasible to manually annotate this many images, we developed a pipeline for the generation of sequences for each annotated image. This reduces labeling effort to 1%, as only one frame per sequence (consisting of up to 100 frames) needs to be annotated. In the following, we describe the data collection phase, the labeling process and how a video sequence is generated for each of the labeled images. 2.1. Data Collection We collected videos in six different zoos, each housing at least two polar bears. An overview of the participating animals and institutions is depicted in Table 2. One challenge in our setting is that the animals' enclosures are designed to be very diverse. In order to ensure consistent data recordings, we filmed the animals in up to three different suitable camera angles. The cameras cover as large an area of the enclosure as possible, which is also the natural setup commonly used in automated animal observation. We conducted the recordings using a tripod and consistently filmed with at least 12.5 frames per second. 2.2. Labeling Process In order to train and evaluate a deep learning model, the identity of the animals in every single image must be determined first. Unfortunately, manual labeling is very time and labor-intensive and unfeasible in our case, as the dataset includes over 130,000 images. Furthermore, it is especially challenging for polar bears because only experts can distinguish this species reliably. In the first step, single frames are extracted from the videos, and two biologists create a bounding box for each visible animal. In the second step, they assign them to a polar bear identity. An overview of this process is depicted in Figure 2. This process is performed competitively, which means that the two experts do not know what the other person has decided. The instances of the dataset for which the identity was not consistently determined are discussed with both experts. Depending on whether they were able to agree on a common statement, the image was either kept or removed. This procedure excluded 10 images. 2.3. Sequence Generation The annotated frames form the basis for further processing. We generated an eight-second video sequence for each individual frame, starting four seconds prior to the annotated frame and ending four seconds after the selected frame. The resulting sequences feature a frame rate of 12.5 fps, including a total of 100 frames. We label the remaining images in this short video sequence using the following procedure. First, we apply a state-of-the-art object detection algorithm , which has been trained on the class polar bear to each frame of an unlabeled video sequence. The output of this process should be the identification and localization of any polar bear instances present in the video frames. The object detection algorithm will analyze each frame and output the locations and bounding boxes of any polar bear instances detected within the frame. Of course, it is possible that more than one bear would be found in an image. To ensure that the correct individual is being tracked, we calculate the distance of the bounding boxes between two consecutive images. We map the boxes with the smallest distance to the same individual, as we assume this to be a correct match. The procedure is shown in Figure 3. Each individual track of one individual found during this process is cropped from the original video and saved. In order to exclude errors, we checked all sequences manually and shortened them if necessary. 2.4. Dataset Statistics PolarBearVidID includes 13 individual polar bears housed in six institutions. Each identity has 110 sequences on average. The maximum length of the sequences is 8 s, correspondingly, 100 frames at a frame rate of 12.5 frames per second. The average length of the sequences is 96.69 images. In total, the dataset includes 1431 sequences. The resolution of the images is set to 256 x 128 pixels. Finally, PolarBearVidID is the first dataset to enable utilizing the movement of a non-human species as a feature for the task of re-identification. We provide the data including all relevant annotations under public license (see Data Availability Statement). 3. Methodology This section provides a detailed description of both the image- and video-based re-ID methods. Furthermore, we present the training and testing procedures as well as the metrics used for evaluation. Both methods have been trained and evaluated as comparable as possible. However, there is an important distinction regarding the terminology: for the image-based approach, a data sample is a single frame, whereas for the video-based approach, a data sample means an entire sequence. Both models follow the same processing pipeline. The input is one data sample depicting an individual polar bear to be re-identified (the query). The model outputs a feature vector (i.e., a mathematical representation) corresponding to that query sample. Meanwhile, both models need to have a gallery including examples of all individuals. All entries in the gallery are embedded into the feature space using the same procedure used for the query sample processing. Now, we compare the feature vector of the query sample using the Euclidean distance metric to every feature embedding in the gallery to receive the ranked list of individuals. The closer an individual from the gallery is to the query, the more likely it is that it resembles the same identity. This ranked list is the output of both re-ID approaches. When used in an automated observation system, the first rank in this list would be determined to be the identity. 3.1. Video-Based Benchmark To create a video-based re-ID benchmark, we chose the model Global-Local Temporal Representations For Video Person Re-Identification (GLTR) introduced by Li et al. . This approach was designed to utilize both spatial as well as temporal information embedded in video-based datasets. The model's compounds are two sub-networks. The first is a backbone ResNet50 that extracts a feature vector for each frame of a video sequence analogue to the image-based baseline approach. The second sub-network inputs the frame feature vectors from the backbone and combines them into a single feature vector for the entire video sequence. That same second network is designed to model the short-term temporal cues between adjacent frames and capture the long-term relationships between frames that are further apart. The short- and long-term temporal features are aggregated with a final simple single-stream CNN . 3.2. Image-Based Baseline We require an image-based baseline to estimate the improvement video-based approaches can provide for re-ID in animals. For this, we trained and tested a straightforward image-based method. Basically, we kept the approach as similar as possible to the video-based method, leaving out the temporal cue utilization for re-identifying. Comparable to the video-based approach, which utilizes a ResNet50 as its backbone, we also use this CNN architecture for the image-based implementation. Furthermore, we developed and evaluated an alternative version of the image-based method to address the limitation that, unlike GLTR, the approach defined here inputs a single input frame. For the sake of the readability of this work, we present this in Appendix B. 3.3. Normalization As we acquired the data with different cameras and at different enclosures, the videos show varying lighting conditions and color calibrations. Therefore, we normalized the images before training and testing to reduce a possible bias. For normalization, we calculated the mean and standard deviation for each color channel throughout the whole data. 3.4. Training Procedure We kept the training procedure the same for both the image- and the video-based approach. This ensures the comparability of both methods. First, each training sample is processed into a feature vector. This feature vector is mapped to a single class by an additional classification layer. This single class corresponds to the individual's ID shown in the dataset sample. Figure 4 shows the training procedure. Note that, for the image-based method, the instances of the dataset are single images, whereas, for the video-based approach, one instance corresponds to one sequence. 3.5. Evaluation Procedure For the evaluation procedure, we compute the embedding vector for every instance of the test set. All test samples end up in the gallery. For the evaluation, we take out one entry of the gallery and use it as the query. Then, we compare the query to the embeddings of the gallery embedding using the Euclidean distance. This procedure results in an ascending ordered list, meaning entries in the gallery, which are deemed similar to the query sample, are at the top of the list. This ranked list of individuals is further used in the evaluation to calculate all relevant metrics (see Section 3.6). The overall procedure is depicted in Figure 4. Note that we only used one image per sequence for the image-based dataset. 3.6. Metrics The use case in zoological institutions is equivalent to a closed-world setting, meaning that all individuals to be identified are known. Hence, the re-ID approaches compare a query image or video of an individual to be identified with the instances of known animals stored in the gallery to retrieve an ascending list of gallery images or videos with the most similarities to the given query. A commonly used metric to measure the performance of re-ID methods is the rank-k accuracy. It describes the probability that a correct example from the gallery with the same ID as the query is in the first k elements of the resulting list . Naturally, in many applications, especially in zoos, only the rank-1 metric is of interest, as we need to identify the correct individual to conduct further behavioral analysis. Another helpful metric is the mean average precision (mAP) . It describes the average recognition performance compared to the best theoretically possible re-ID method. The metric calculation is based on the precision value for each k-rank, and a subsequent averaging step. The mAP is a good measure to determine how well a re-ID solution can re-identify people in a given database. 4. Experiments In this section, we describe the evaluation of both the image-based and the video-based re-ID model for the PolarBearVidID dataset. We performed the same experiment for each approach, keeping all parameters and procedures as similar as possible. 4.1. Cross-Validation As the polar bear dataset is rather small, variations in the train and test data can have a larger impact on the performance of the system. We address this issue by performing a five-fold cross-validation when training the models. First, we split the dataset into five equally sized parts. Then, we train the algorithms on four parts and evaluate the performance on the unseen fifth part. This allows us to make a more general statement about the generalization capabilities of the models. For all described experiments, we used the same data splitting according to Table A1. 4.2. Results We assessed the performance of two approaches to re-identify the individuals included in the PolarBearVidID. Table 3 shows the results for each the image- and video-based method. The rank-1 score, as well as the mean average precision (mAP), are given as a mean result of all runs of the five-fold cross-validation, including the overall standard deviation. The data distribution over the five folds was kept the same for both approaches. To determine which misidentifications occurred, we show the confusion matrix for both approaches in Figure 5. We display the sum over all folds. With the predicted labels (equivalent to the rank-1 instances) compared to the ground truth, the matrices display how many sequences from each individual polar bear are correctly or falsely identified. Ideally, the model outputs the correct identity, which is shown as the entries on the diagonal of the matrix. We sorted the identities according to the corresponding zoos, also denoted in the matrices. Therefore, one can assess whether misidentifications occur inter- or intra-zoos. To assess the final use case, we evaluated the trained video-based model on the individual zoos. Thus, only the animals from the respective institution are in the Gallery. The distribution of the 5 folds remains the same as in the previous experiments. The results are shown in Table 4. 5. Discussion The PolarBearVidID dataset is the first video-based re-ID dataset for animals. It offers many possibilities and comes with some limitations, which we will discuss in the following. Furthermore, we evaluated the performance of a state-of-the-art video-based re-ID approach and compared it to an image-based method. 5.1. Dataset With the PolarBearVidID dataset, we provide the first-ever video-based dataset for the task of re-ID in animals. For this dataset, we present the first implementation of a video-based model utilizing state-of-the-art re-identification techniques developed for human subjects. While this evaluation is limited in scope, it serves as an initial exploration of the feasibility of transferring re-identification techniques developed for human subjects to the animal domain. By choosing polar bears, we introduced a particular challenge to the dataset, as this species lacks prominent distinct visual features and is, therefore, more difficult when it comes to re-ID. Another specific challenge is the relatively unconstrained zoo setting. In a laboratory setting, data acquisition is performed in a controlled environment with a fixed camera angle, high camera resolution, consistent lighting conditions, a small enclosure, a uniform background and minimal occlusion of the animals. These constraints allow for more uniform data collection. However, in the zoo setting, all these parameters may vary and be less controlled. This applies intra-zoo when, for example, the weather changes or the topology of the enclosure requires cameras at different heights. Inter-zoos, of course, the parameters change even more drastically due to different enclosure designs. Therefore, data acquisition in different zoos results in a rather unconstrained re-ID dataset compared to one created in a lab. However, a re-ID method developed on a dataset recorded under laboratory conditions is not necessarily suitable for the more open zoo setting. Therefore, the PolarBearVidID dataset more closely reflects real-world conditions and is more representative of realistic re-ID scenarios. One reason why all animal re-ID datasets to date are only image-based might be that the annotation of videos is a big challenge. In the case of our PolarBearVidID dataset, each sequence contains up to 100 individual images. Only the method of extrapolation presented by us using an object detection algorithm allows annotating this amount of images. With this method, it will be easier to create more video-based datasets for animal re-ID in the future. Note that for other species that live in herds or move close together, one might have to use a more advanced tracking method (e.g., ). In the end, although the experts only had to annotate 1431 images, PolarBearVidID offers a large number of images with almost 140,000 images compared to other publicly available datasets (see Table 1). Finally, many authors of public datasets do not provide information about the quality of ground truth annotations or details about how they were created (e.g., ). Our scope was to ensure that the annotations of the PolarBearVidID dataset provide the highest possible quality. Especially the identification of individuals of a species such as polar bears lacking distinct coat features is very challenging and can only be performed by experts. Therefore, each data sample was annotated independently by two biologists. Unclear identities were discussed in a second step. The samples for which no agreement could be reached were excluded from the dataset. The fact that only 10 sequences had to be removed during this process shows that the level of agreement between the experts was very high. Overall, the PolarBearVidID dataset can be assumed to comprise labels of excellent quality. The main limitation of our dataset is the rather small number of individual animals. In contrast, video-based benchmark datasets for humans usually include hundreds of individuals (e.g., MARS ), recorded only by a few cameras that do not change position (for example, at an intersection). However, the recording of animals in zoos is restricted to a significantly smaller number of individuals in a single enclosure. This is due to species-specific requirements and is usually limited to a maximum of three animals in the case of polar bears. This key difference limits a fair comparison between the domain of human re-ID and the domain of polar bear re-ID. In particular, the setting for human benchmark datasets cannot be replicated, and, therefore, all future video-based re-ID datasets for animals will differ substantially from those for humans, including the PolarBearVidID dataset. 5.2. Re-Identification Performance We find that the video-based approach outperforms the image-based method in both scorings. While the image-based model attains a rank-1 score of 85.3+-1.1%, the video-based one achieves an impressive score of 96.6+-1.6%. Since the rank-k metric for k = 1 indicates how often the exact identity is determined for a query sample, the video-based model is perfectly suited for application in the zoo setting. The small standard deviation of the scores over the five folds indicates that both models perform very robustly on the dataset and that the train/test distribution does not influence the scores. Li et al., who introduced the video-based method (GLTR) in 2019, reported a performance of the approach on the benchmark dataset MARS with a rank-1 score of 87.02%. Thus, GLTR performs slightly better on the PolarBearVidID dataset. However, this statement is subject to the limitation that with 1261 individuals and over 20,000 sequences, the quantity of MARS is not comparable to our dataset. The mean average precision of the two models differs significantly. While the video-based approach shows an excellent mAP of 88.2+-9.0%, the image-based approach only achieves an mAP of 45.4+-2.4%. This metric allows a good insight into the robustness of a method since it not only considers the first entry of the ranked list but also considers the remaining entries with descending weighting. The image-based method outputs the correct identity on the first rank relatively often. However, at the same time, it ranks other gallery samples of the correct identity quite often far down in the list. This means that this approach only attains a satisfactory rank-1 score because in the PolarBearVidID dataset for each query sample, there is often a very similar sample in the gallery. If we were to shrink the gallery size or record a more diverse dataset in the wild, the image-based method would no longer work properly. The video-based method promises much better robustness here. The confusion matrices depicted in Figure 5 provide insight into the mismatches between predicted ID and actual ID. The matrices show that the image-based approach determines the wrong identity much more frequently. In total, the prediction does not match the actual label 211 times over all 5 folds of the test set, whereas this number drops to only 49 false identities for the video-based approach. It shows that the inter-zoo confusions almost vanish, from 81 false predictions to only 6 for the video-based model. Here, 89.58% of all misclassifications occurred within the same zoo, while only 10.42% identities were mistakenly matched to one of another zoo. This observation is to be expected, as the recordings of the animals in the same institution are identical in camera perspective and background. The zoos differ slightly in the occurrence of this issue, with inter-zoo misclassifications occurring more frequently in Berlin and Vienna. Overall, however, this finding is considered positive since, within one zoo, the animals can still be distinguished with sufficient accuracy. The influence of camera position and background is, therefore, not problematic. This work's use case is the re-ID method's application in zoological institutions. When the model is deployed, the gallery includes only the animals kept in the respective enclosure. For the participating zoos, this means two or three animals, respectively. The results of this experiment are shown in Table 4. The important rank-1 score is above 90% for all zoos, meaning that wrongly classified identities occur in less than 10% of all attempts. The animals in Berlin are most frequently misidentified. The rank-1 score is 92.0%. One possible explanation is that the two polar bears in Berlin are mother and daughter. Biologists also reported that distinguishing between the two animals was particularly challenging. For this zoo, the mAP score is also the smallest, at 84.7%. This is due to the fact that in the ranked list, a false identity is more often listed in the top ranks. However, the performance for all zoos is very promising and within the scope of this project. With a re-ID performance of >90%, false detections can easily be corrected by common interpolation or filtering methods. One limitation in evaluating both methods is the composition of the gallery. Due to the limited data set size, we used each sequence from the test set once as a query, while all remaining sequences constitute the gallery set. This procedure has a positive influence on the rank-1 scores. For the models to place the correct identity at the top of the list, there needs to be a proximate identity in the feature embedding space. A more extensive gallery helps in this regard. For the image-based approach, a smaller gallery will quickly become a problem, as suggested by the poor mAP. The video-based approach, however, has a very high mAP score, so the limitation here is less severe. Finally, it can be concluded that the video-based approach, which utilizes the movement of the animals as a feature for re-ID, achieves a significant improvement for this task. As a result, these methods will become the go-to solution for open settings such as zoos or the wild in the coming years. 6. Conclusions The greatest challenge and limitation within the animal re-ID research area is the availability of data . With the PolarBearVidID dataset, we contribute not only a novel re-ID dataset for a species not published before but also the first-ever dataset to include fully labeled sequences of the individual animals. Furthermore, with our novel sequence generation procedure that reduces annotation effort drastically, we hope to encourage other researchers to contribute further datasets for the task of video-based re-ID. Only if the number of datasets for other species keeps increasing can the previous limitations be overcome and the field of re-ID for animals be further developed. Developing re-ID models will greatly facilitate the work of biologists and animal caretakers in the future. Progress in this area of research enables the deployment of automated behavior observation systems and therefore contributes to the evaluation of animal welfare. With improved observation methods, the focus of biologists and veterinarians can be shifted to early recognition of behavioral changes as signs of disease, stress situations within groups, as well as the effectiveness of changed management procedures or the use of enrichment items . While the PolarBearVidID is still limited in size, the success of our dataset and the video-based model shows that utilizing an animal's movement as an indicator of its identity will become even more critical when we shift the scenario to the wild. In order to study animal welfare in the wild, conserve animal habitats, protect animals and preserve biodiversity, animal populations and movement patterns have to be detected and evaluated. This is performed with the help of photo traps . However, with classical image-based approaches, it is not yet possible to determine single individuals on images of low quality. Therefore, animal observation and welfare investigations will significantly benefit from future developments in video-based approaches for animal re-ID. Therefore, we want to encourage the research community focused on re-ID in animals to use the PolarBearVidID dataset to drive further investigation in video-based methods for this task. Acknowledgments We sincerely thank all zoos which contributed to this project by providing data: Tiergarten Nurnberg, Tiergarten Schonbrunn, Tierpark Berlin-Friedrichsfelde, Tierpark Neumunster Zoo Hannover and Zoo Karlsruhe. Furthermore, we would like to express our sincere gratitude to Marie Krausse for her efforts in labeling the data. Bjoern Eskofier gratefully acknowledges the support of the German Research Foundation (DFG) within the framework of the Heisenberg professorship program (grant number ES 434/8-1). We acknowledge financial support by Deutsche Forschungsgemeinschaft and Friedrich-Alexander-Universitat Erlangen-Nurnberg within the funding programme "Open Access Publication Funding". Author Contributions Conceptualization, M.Z.; methodology, M.Z., R.D. and J.S.; software, M.Z., R.D., N.S. and J.S.; validation, M.Z., R.D. and J.S.; formal analysis, M.Z.; investigation, M.Z., R.D., N.S. and J.S.; resources, M.Z., N.S., I.B. and L.v.F.; data curation, M.Z., R.D., N.S. and I.B.; writing--original draft preparation, M.Z.; writing--review and editing, M.Z., R.D., F.K., N.S., J.S., D.Z., I.B., L.v.F. and B.E.; visualization, M.Z., R.D. and J.S.; supervision, D.Z. and B.E.; project administration, M.Z.; All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethical review and approval were waived for this study due to a camera-based non-invasive approach without using flashes or spotlights, carried out from the visitor area during standard opening times of the respective institution or using a CCTV system for permanent monitoring of the enclosure. Data acquisition was exclusively carried out in zoological institutions without altering any management procedures of the animals. Informed Consent Statement Not applicable. Data Availability Statement We provide the data presented in this study openly available as the PolarBearVidID Dataset at Conflicts of Interest The authors declare no conflict of interest. Abbreviations The following abbreviations are used in this manuscript: CNN convolutional neural network GLTR global-local temporal representations mAP mean average precision re-ID re-identification Appendix A animals-13-00801-t0A1_Table A1 Table A1 Number of sequences for each polar bear and the distribution of data over the folds. ID Name Sequences All Fold 1 Fold 2 Fold 3 Fold 4 Fold 5 0 Vera 123 25 25 25 24 24 1 Nanuq 105 21 21 21 21 21 2 Tonja 108 22 21 21 22 22 3 Hertha 118 23 24 24 24 23 4 Nora 104 21 21 21 20 21 5 Finja 107 21 21 21 22 22 6 Larissa 107 22 22 21 21 21 7 Vitus 104 21 20 21 21 21 8 Lloyd 120 24 24 24 24 24 9 Charlotte 112 22 23 23 22 22 10 Nana 119 24 24 23 24 24 11 Milana 104 21 20 21 21 21 12 Sprinter 100 20 20 20 20 20 Sum 1431 287 286 286 286 286 Appendix B The video-based re-ID method GLTR outperforms the image-based method significantly. Thus, using video clips instead of single images brings a performance advantage for the re-identification task in our dataset. This analysis, however, does not allow us to understand to what extent the advantage is due solely to the greater amount of input data, or to the ability of the video-based model to capture dynamic features and movements characteristic of individual animals. We developed an improved image-based approach to investigate whether only the larger number of images for each query sample is the reason for the better performance of the video-based method. In this alternative method, we not only calculated a feature vector for one image per sequence but for every frame, resulting in up to 100 feature vectors per sequence. Since there is still the need to select a single feature vector as the representative, we calculate the element-wise median vector. Thus, the information of all frames of a sequence contribute to the final feature vector. The result of this third approach is listed in Table A2. The improved version performs better than the basic image-based model, but the rank-1 score is still 5% worse than for the video-based approach.The mAP is also slightly improved with 52.8%, but is much worse compared with the video-based approach (88.2% mAP). animals-13-00801-t0A2_Table A2 Table A2 Performance of the image-based, improved image-based and video-based re-ID approach. The rank-1 score, as well as the mean average precision (mAP), is given as a mean result of all runs of the five-fold cross-validation, including the overall standard deviation. Method Rank-1 mAP Image-based (single-image) 0.853 +- 0.011 0.454 +- 0.024 Image-based (multi-image) 0.913 +- 0.016 0.528 +- 0.033 Video-based 0.966 +- 0.016 0.882 +- 0.090 Figure 1 The PolarBearVidID dataset. It is the first dataset to date to include the movement of non-human animals to utilize this as a feature for re-ID. PolarBearVidID includes 13 individual polar bears housed in six institutions. Each identity has at least 100 sequences. The maximum length of the sequences is 8 s, respectively 100 frames at a frame rate of 12.5 frames per second. In total, the dataset includes 1431 sequences. Figure 2 First, single frames are captured from the videos recorded in the zoos. Next, two biologists create bounding boxes for the animals and label their identities. They do not know the other person's decision during the labeling process. The agreement between the two experts is checked across the whole dataset. Those instances in which they disagreed regarding the identity of the shown animals were collaboratively discussed. Only ten instances remained unclear. We excluded those from the dataset to ensure the highest quality of labels. Figure 3 Approach to creating the sequences. For each image labeled by the experts, there is a bounding box including information about the identity of the animal (step 1). The adjacent images are then searched for polar bears in the second step, using an object detection algorithm. To ensure that the correct animal is tracked, we compute the bounding box distances between two consecutive images. If this is below a threshold, we can assign the same ID to the found animal (step 3). In step 4, the bounding boxes are then cropped and saved as a sequence. Figure 4 Schematic depiction of the training and testing procedure for each approach. We trained each model using a simple classifying layer. To evaluate the performance, one data sample from the test set is used as the input for the model. We calculate the Euclidean distance of the resulting feature vector to all vectors included in the gallery. This results in a ranked list of the individuals. With this list, the rank-k accuracy and the mean average precision (mAP) can be calculated. Figure 5 Confusion matrix of the two re-identification approaches. When confronted with a data sample of identity X, the model outputs a ranked list of all known identities with descending probability. Therefore, the identity Y listed first is the prediction of the model. In the case of X=Y, the model outputs the correct identity, which is shown as the entries on the diagonal of the matrix. The identities are sorted considering the respective zoos: Nuremberg (0 and 1), Berlin (2 and 3), Vienna (4 and 5), Neumunster (6 and 7), Karlsruhe (8 and 9) and Hannover (10, 11 and 12). animals-13-00801-t001_Table 1 Table 1 Publicly available datasets for the task of re-ID in animals. The PolarBearVidID dataset is the only one providing video sequences of the animals. Therefore, this dataset is the only one to make is possible to utilize the individuals' movement as a unique feature for re-ID. Dataset Species # Individuals # Images Video-Based ATWR Amur Tigers 92 4434 Chimpface Chimpanzees 90 5559 ELPephants Elephants 276 2078 GoldenMonkeyFace Golden Monkeys 49 1450 Howard et al. Humpback Whales 4251 9850 LemurFace Lemurs 129 3000 Schneider et al. Fruit Flies 20 16,320 SealID Saimaa Ringed seals 57 2080 SeaTurtleID Sea Turtles 400 7774 PolarBearVidID Polar Bears 13 138,363 animals-13-00801-t002_Table 2 Table 2 Full list of the individual animals in the PolarBearVidID dataset. ID Name Gender Birth Zoo Website 0 Vera Female 2002 Nuremberg 1 Nanuq Male 2007 Nuremberg 2 Tonja Female 2009 Berlin 3 Hertha Female 2018 Berlin 4 Nora Female 2013 Vienna 5 Finja Female 2019 Vienna 6 Larissa Female 1990 Neumunster 7 Vitus Male 2000 Neumunster 8 Lloyd Male 2000 Karlsruhe 9 Charlotte Female 2014 Karlsruhe 10 Nana Female 2019 Hannover 11 Milana Female 2009 Hannover 12 Sprinter Male 2007 Hannover animals-13-00801-t003_Table 3 Table 3 Performance of the image-based and video-based re-ID approach. The rank-1 score, as well as the mean average precision (mAP), is given as a mean result of all runs of the five-fold cross-validation, including the overall standard deviation. Method Rank-1 mAP Image-based 0.853 +- 0.011 0.454 +- 0.024 Video-based 0.966 +- 0.016 0.882 +- 0.090 animals-13-00801-t004_Table 4 Table 4 Performance of the video-based re-ID approach on each zoo. The rank-1 score, as well as the mean average precision (mAP), is given as a mean result of all runs of the five-fold cross-validation, including the overall standard deviation. Zoo Rank-1 mAP Nuremberg 0.991 +- 0.011 0.978 +- 0.013 Berlin 0.920 +- 0.038 0.847 +- 0.070 Vienna 0.948 +- 0.041 0.854 +- 0.060 Neumunster 0.995 +- 0.010 0.976 +- 0.022 Karlsruhe 0.983 +- 0.025 0.937 +- 0.100 Hannover 0.972 +- 0.034 0.856 +- 0.159 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000027

Feeding increased volumes of milk in the preweaning phase has been shown to improve growth, morbidity and mortality rates in calves (Bos Taurus). This experiment enlisted 20 Holstein-Friesian dairy replacement calves from birth until weaning (at 10 weeks of age) and assessed the effect of feeding either 4 L (Low) or 8 L (High) of milk per calf per day on their growth, immune competence and metabolic characteristics. The responsiveness of these systems was compared through a vaccination immune challenge. Calves in the High treatment group were significantly heavier from two weeks of age and were 19 kg heavier than calves in the Low treatment group at weaning. Calves in the High treatment group also exhibited greater immune responses, with significantly higher white cell counts and neutrophil counts than calves in the Low treatment group post-vaccination. Calves in the High treatment group also had lower beta-hydroxybutyrate both pre- and post-vaccination, and higher glucose and insulin levels post-vaccination, indicating superior metabolic characteristics. Calves had ad libitum access to lucerne hay (Medicago sativa) and a commercial concentrate. Solid feed intakes were mostly the same between treatments, with differences in hay intake only detected at 7 and 8 weeks of age. Results from this experiment are indicative of a positive influence of accelerated preweaning nutrition on growth, immune response and metabolic characteristics. immune challenge accelerated milk feeding heifer calf development Agriculture Victoria (Australia)Dairy Australia (Australia)University of Melbourne (Australia)This research was funded by Agriculture Victoria (Australia), Dairy Australia (Australia) and the University of Melbourne (Australia). pmc1. Introduction In Australia, dairy calf morbidity and mortality rates are around twice industry targets (morbidity 23.8% with <10% target; and mortality 5.6% with <3% target) . Recent research has called for a re-evaluation of management strategies for rearing replacement heifers to reduce calf morbidity and mortality rates . Current recommendations prioritise keeping labour and feed costs to a minimum by limiting milk consumption to 10% birth bodyweight daily with the aim of increasing concentrate intake to ultimately promote early weaning . It has been widely demonstrated that calves with access to milk ad libitum consume 50-90% more milk than recommended feeding levels, while those rationed to current recommendations show behaviours associated with hunger . Early life nutrition is widely understood to affect the development of physiological systems in all species . Optimising early life development in dairy calves has been shown to have both short- and long-term benefits on health, welfare and production. Improved nutrition in the preweaning phase has been shown to increase growth rates, decrease disease susceptibility and mortality, and in turn increase economic benefits with reduced veterinary costs and fewer animal losses . Long-term, these benefits are also evident through increased milk production . The mechanisms behind the health and production benefits of improved preweaning nutrition are not well understood. It has been widely cited that increased nutrition gives rise to greater growth rates in calves, and malnutrition is well known to negatively impact immunity in all species . Yet, as is the case with much calf-related research, the effect of increased milk feeding on pre-weaned calves and their immunity is mixed (either having a positive influence or a negative influence or as reviewed by Verdon and Palczynski et al. for greater detail). It has been previously demonstrated that weaned calves with greater average daily gains (ADG) were categorised as having greater immune responsiveness when faced with an immune challenge . In this case, it would be reasonable to assume that manipulating nutrition in pre-weaned calves would therefore result in a variation in immune function. However, this is yet to be determined. Furthermore, given the suspected differences in metabolic reserves available to calves with varying nutrition, their ability to respond to an immune challenge could be impacted. When evaluating the influence of nutrition on the immune capabilities of an animal, immune challenges, such as those previously mentioned, are considered the gold standard , and are generally lacking in calf studies. Further, a considerable number of these experiments are conducted using male calves, when it has been demonstrated that the immune response differs with sex . This experiment examined the effects of two milk feeding strategies (High and Low milk volume) on the growth, immune competence and metabolic characteristics of pre-weaned calves. These physiological parameters were also compared in response to an immune challenge via routine vaccination at 6 weeks of age. The hypotheses tested were: (1) that calves in the High treatment group would have greater body weights at weaning than calves in the Low treatment group; (2) that calves in the High treatment group would have superior immune and metabolic responses to the immune challenge than calves in the Low treatment group. 2. Materials and Methods This experiment was undertaken at the Agriculture Victoria Dairy Research Centre, Ellinbank, Victoria, Australia (38deg14' S, 145deg56' E) from July-October 2020 (Australian Spring). Approval to proceed was granted by the Agricultural Research and Extension Animal Ethics Committee, application number 2020-06. 2.1. Experimental Design and Treatments This experiment used 20 spring-born Holstein-Friesian heifer calves from birth until 10 weeks of age. Calves were all born from heifer dams, within eight days of their expected due date. At birth, one of two treatments was randomly assigned to calves:High: 8 L of milk daily Low: 4 L of milk daily Treatments were balanced for calves' birth weight and estimated Balance Performance Index (BPI) (Australia's national selection index). Estimated BPIs were calculated prior to birth using the average of the parents' BPI. If necessary, randomised allocation of treatments was overruled in the final stages of the recruitment process to ensure these parameters remained balanced between treatments. 2.2. Animal Management At birth each calf is fitted with an individual ear tag to allow identification within the herd as part of normal farm practice. All calves were group-housed within a single pen inside a three-sided shed. The pen dimensions allowed for 4.5 m2 floor space for each calf. Calves were separated from their dam and tube fed 4 L of high quality (>22% Brix) pooled colostrum within 8 h of birth. A second 2 L feed of colostrum was tube fed 12 h after the first colostrum feeding. At 24-48 h of age, passive transfer of immunity was initially screened onsite using serum Brix refractometer methods before enrolment. Passive transfer of immunity was subsequently confirmed through blood serum Immunoglobin G (IgG) concentrations using ELISA methods at a commercial laboratory (Gribbles Veterinary Pathology, Clayton, Victoria, Australia). Following colostrum tube feeding, the first four milk feeds given to all calves was 2 L of colostrum milk (milk from the 2nd-4th milking post birth), before transition to whole milk from the main vat. Daily milk allocation was divided into two equal feeds and supplied at approximately 0600 and 1630 h. Calves were separated while being fed milk via single-teat feeders to accurately measure each calf's individual intake. Calves in the High treatment group were gradually accustomed to the higher level of feed to reduce the risk of scours. This was done by increasing the amount of milk offered by 2 L each day (1 L per feed) until they reached the full treatment volume after 2 days. Any refusals were measured, and individual intake was recorded throughout the experiment. A commercial calf concentrate (Reid Stockfeeds- Gippsland, Trafalgar, Victoria, Australia) and lucerne hay were available ad libitum within the pen, and individual feed intakes recorded via automatic feeders (Gallagher Animal Management Systems, Hamilton, New Zealand). The automatic feeder system identified calves via an electronic ear tag. The system constantly recorded the weight of the feed bin, enabling feed intake to be determined each time a calf visited the feeder. The feed intake of hay and concentrate was reported on a dry matter basis following correction for dry matter concentration. Calves were weighed using electronic scales at 24-48 h of age to establish birth bodyweight. Bodyweights were then measured weekly for the remainder of the experiment prior to AM milk feeding. Calves completed the experiment at the commencement of weaning at 10 weeks of age. 2.3. Nutritional Measurements 2.3.1. Milk A representative sample of approximately 20 mL of milk fed to calves was taken twice weekly and sent to a commercial laboratory (HICO laboratories, Korumburra, Victoria, Australia) for milk composition analysis (fat, protein, and lactose concentration). The mean nutritive content of whole milk fed to calves throughout the preweaning period was 3.6 +- 0.58% (mean +- SD) fat, 3.2 +- 0.16% protein, and 4.9 +- 0.09% lactose. 2.3.2. Concentrate & Hay Representative samples of concentrate and hay were taken each fortnight throughout the duration of the experiment. Half of each sample was dried immediately at 105 degC for 48 h to determine the percentage dry matter (DM). The remaining half was stored at -18 degC until the completion of the experiment. At the time of processing the remaining sample was oven-dried at 60 degC for 72 h before being ground through a 1 mm sieve and sent to a commercial laboratory for wet chemistry analysis (Dairy One Forage Laboratory, Ithaca, NY, USA) . The nutritive characteristics are presented in Table 1. 2.4. Immune Challenge At 42 days of age, each calf commenced an immune challenge using a protocol combined and modified from Aleri et al. . An interruption of homeostasis occurred via the use of a commercial vaccination for Bovine Respiratory Disease (BRD) (Bovilis MH + IBR; Coopers Animal Health, Macquarie Park, NSW, Australia), and subsequent responses between treatments were compared. 2.4.1. Acquired Immune Response At day 42, a blood sample was taken from each calf. Immediately following the blood sample, the vaccination was administered according to the manufacturer's protocol. Additional blood samples were taken at day 50 and day 52 of age to determine the responses. Samples taken on day 42 and 52 involved the collection of 35 mL of blood via jugular venepuncture into four vacutainers (1 x 10 mL lithium heparin, 1 x 10 mL plain, 1 x 10 mL EDTA and 1 x 5 mL fluoride/oxalate tubes). The sample taken on day 50 was a smaller sample and involved the collection of 10 mL of blood into a 1 x 10 mL plain tube. Lithium heparin, EDTA and fluoride/oxalate blood samples were stored on ice prior to centrifugation at 1578 g for 15 min at 4 degC. Prior to centrifugation, a sample of whole blood was extracted from the EDTA tube and sent for immediate haematology analyses (Gribbles Veterinary Pathology, Clayton, Victoria, Australia). Plain tube samples were incubated at 24 degC for 2 h prior to centrifugation at 1258 g for 15 min at 24 degC. Plasma and serum samples were then stored at -20 degC prior to analyses. 2.4.2. Cell Mediated Immune Response On day 50, hypersensitivity skin fold testing also commenced as per the protocols described in Aleri et al. , Aleri et al. and Aleri et al. . On day 50, 0.1 mL of the vaccine was injected intradermally into the right caudal fold of the tail, and 0.1 mL of saline into the left. The caudal skin fold thickness was measured in triplicate prior to injection and repeated 24 and 48 h post-injection using Harpenden skin fold callipers (Creative Health Products Inc., Ann Arbor, MI, USA). The delayed-type hypersensitivity response was corrected using the following formula from Aleri et al. , Aleri et al. and Aleri et al. :Increase (mm) = (A - B) - (C - D), whereby A = mean test site thickness at 48 h post injection, B = mean test site thickness prior to injection, C = mean control site thickness at 48 h post injection, and D = mean control site thickness prior to injection. 2.5. Biomarker Analysis The first blood sample was taken 24-48 h post birth and was used to confirm passive transfer of immunity, while also providing an experimental baseline of all the biomarkers. The subsequent samples were taken at 42, 50 and 52 days for the immune challenge as previously described. Analyses for the first, second and final samples included the metabolic biomarkers beta-hydroxybutyrate (BHB), non-esterified fatty acid (NEFA) glucose and insulin, and immune biomarkers including a white blood cell (WBC) count and differential and infectious bovine rhinotracheitis (IBR) antibodies. The third blood sample, taken on day 50, measured the IBR antibody titres only. BHB, NEFA and glucose concentrations were analysed by a commercial laboratory (Regional Lab Services, Benalla, Victoria, Australia) using a Konelab 20XTi autoanalyzer (Thermo Fisher Scientific, Waltham, MA, USA) with assays as follows: BHB assay as per McMurray et al. ; NEFA assay kit: Randox Laboratories Ltd., Crumlin, UK; Glucose assay kit: Beckman Coulter Inc., Brea, CA, USA. Insulin assays were developed and conducted at the Assay Centre, Department of Agriculture and Food, Melbourne University. Insulin concentrations were measured in a homologous double antibody radioimmunoassay (RIA). The RIA was performed using purified insulin antiserum raised in guinea pig (Antibodies Australian) and purified bovine insulin for iodination and standard (Sigma-Aldrich, cat#I5500). Briefly, 200 mL or 100 mL assay buffer (0.5% BSA/0.03M sodium phosphate monobasic/0.2M sodium phosphate dibasic/0.1% sodium azide/0.1% triton X) and 200 mL first antibody (1:60,000) diluted in 1:500 normal guinea pig serum (NGPS) was added to duplicate plastic tubes containing either 100 mL standard (1.211-620 mIU/mL) or 200 mL ovine plasma. After preincubation at 4 degC for 72 h, 125I iodinated bovine insulin was added (15,000 cpm/100 mL) and the incubation continued for a further 24 h at 4 degC. The antibody-bound hormone was separated from the free hormone by the addition of 200 mL goat-anti-guinea pig serum (GaGPS) (1:200). The tubes were incubated with the second antibody overnight at 4 degC before centrifugation (3500 rpm, 30 min, 4 degC), after which the supernatant was aspirated and the precipitate counted in a gamma counter. Immune biomarkers were also analysed in commercial laboratories. The white cell count and differential were determined using a Cell-Dyn 3700 autoanalyzer (Abbott Diagnostics, IL, USA) at Gribbles Veterinary Pathology (Clayton, Victoria, Australia). IBR antibodies were analysed at Veterinary Diagnostic Services-Agriculture Victoria (Bundoora, Victoria, Australia) using IBRGBC ELISA kit (IDvet, Grabels, France). 2.6. Statistical Analysis Weekly bodyweight, nutritive intakes and physiological biomarkers were analysed using linear mixed models (LMMs) which were fitted using restricted maximum likelihood, with individual calf as the unit of analysis. In LMMs, the effect of the covariates birth body weight and estimated BPI were fitted as fixed effects, as was the effect of treatments. The effect of calf was fitted as a random effect and used as a residual term, and was assumed to follow a normal distribution with zero mean and constant variance. Histograms of residuals and plots of residuals versus fitted values were examined for normality of distribution with constant variance. Prior to the final analyses, blood insulin measurements were logarithmically transformed to satisfy the assumption of normality with constant variance. The treatment means of blood insulin measurements were back transformed using the bias correction factor expm^+s^22 where m^ and s^2 is the estimated treatment mean and residual variance, respectively, in the logarithmic scale. All statistical analyses were performed using GenStat 22nd Edition (VSN International, Hemel Hempstead, UK). Haematology data from one calf in the High treatment group at birth was unable to be used in the analysis due to haemolysis in the sample. 3. Results There was no difference in birth weight between the treatments . From 2 weeks of age onwards, calves in the High treatment group were significantly heavier than calves in the Low treatment group (p-value < 0.05). By 10 weeks of age, the calves in the High treatment group were, on average, 19.0 kg heavier than those in the Low treatment group (96.7 kg and 77.7 kg, respectively). From birth, the average daily gain (ADG) for calves in the High treatment group was 0.83 kg/day, compared to 0.56 kg/day for calves in the Low treatment group. By design, daily milk consumption was higher for calves in the High treatment group than the Low treatment group throughout the experimental period . Apart from the first week, when they consumed 3.9 L, all calves in the Low treatment group consumed the total 4 L of milk offered daily. Calves in the High treatment group consumed almost twice that, drinking an average of 7.9 +- 0.1 L of milk daily from two weeks of age onwards. Prior to this, the High treatment group consumed an average of 5.4 +- 0.1 L and 7.6 +- 0.2 L of milk daily during week 1 and week2, respectively. Over the entire experimental period, the mean amount of milk consumed by the Low treatment group was 279 L, which contained a total ME of 61.5 MJ and 71.6 kg of crude protein (CP). The High treatment group consumed 533 L, containing an ME of 117.3 MJ and 136.5 kg CP throughout the experimental period. Daily concentrate intake did not differ between the High and Low treatment groups throughout the experiment . Concentrate intakes for the Low treatment group started to increase steadily at 5 weeks of age and the High treatment group a week later at 6 weeks of age. Concentrate intake increased by an average of 0.12 kg DM/calf/day each week from week 5 onwards for calves in the Low treatment group, and 0.10 kg DM/calf/day for the High treatment group. The average intake for both groups remained under 1 kg DM/calf/day throughout the experimental period. The mean amount of concentrate consumed by the Low treatment group was 25.3 kg DM (totaling 3.4 MJ of ME, and 5.5 kg CP) compared to the High treatment group 22.5 kg DM (totaling 2.3 MJ of ME and 4.8 CP) throughout the experimental period. Hay intakes were significantly higher for the Low treatment group during weeks 7 (p-value = 0.034) and 8 (p-value = 0.033) only. They were not different between treatments throughout the rest of the preweaning period. Hay intakes were lower than concentrate intakes, with the average daily intake increasing by 0.04 kg DM/calf/day for the Low treatment group and 0.03 kg DM/calf/day each week for the High treatment group. Daily intakes remained below 0.40 kg DM/calf/day throughout the experimental period. Mean hay intake for the Low treatment group was 10.9 kg DM (totaling 0.9 MJ of ME and 1.8 kg of CP) compared to the High treatment group's 7.8 kg DM (totaling 0.6 MJ of ME and 1.3 kg of CP) throughout the experimental period. All calves received successful passive transfer of immunity, with IgG levels all above the minimal target level of 10,000 mg/mL between 24-48 h of age. The WCC and neutrophil count were not significantly different between treatments prior to vaccination (Table 2). However, 10 days post-vaccination, the WCC was significantly higher (p-value = 0.048) and the neutrophil count was trending towards significantly higher (p-value = 0.054) in the High treatment group. No significant differences were found for monocytes, eosinophils, basophils or the IBR titre between the treatments at birth, pre- or post-vaccination. This is excluding the lymphocytes, where the High treatment group recorded a significantly higher lymphocyte count at birth (p-value = 0.050), and the Low treatment group significantly higher pre-vaccination (p-value = 0.028). The High treatment group had significantly lower BHB levels both pre- (p-value < 0.001) and 10 days post-vaccintion (p-value = 0.012) (Table 2). The High treatment group also had significantly higher glucose (p-value < 0.001) and insulin (p-value < 0.001) levels 10 days post-vaccination than the Low treatment group, yet not at the sample times prior. There was no difference in NEFA levels detected between treatments at any sample time point. There was no significant difference in the increase in test site skin fold thickness post-delayed-type hypersensitivity skin fold testing between treatments. Twenty-four hours post-intradermal injections, the Low treatment group measured a corrected increase in skin fold thickness of 5.9 mm, in comparison to the High treatment group who measured a corrected increase of 5.4 mm prior to injections (SED = 0.36; p-value = 0.175). At 48 h, the test site skin fold thickness corrected increase was still the same between treatments, with the Low treatment group recording a corrected increase of 6.2 mm versus High treatment with 6.6 mm prior to injections (SED = 0.63; p-value = 0.627). 4. Discussion Feeding dairy calves increased volumes of milk in the preweaning phase increased growth rates and improved immune competence and metabolic characteristics without effecting solid feed intake. There are many management factors in calf rearing that contribute to the overall health and development of calves , including but not limited to: milk type and volume, forage source and particle size, concentrate texture and ingredients, weaning age and methods. These variations between studies can considerably impact or confound results, and may be responsible for the discrepancies throughout the calf nutritional studies discussed in this paper. Unlike the current experiment, many calf experiments lack immune challenge protocols that are considered the gold standard when evaluating the influence of nutrition on the immune capabilities of an animal . Additionally, many of these experiments are conducted using male calves, and it has been reported in many human and animal studies, including prepubertal calves, that the innate and adaptive immune response differs between males and females . The current experiment implemented an immune challenge on female replacement calves as these are the animals that remain within the herd and therefore more accurately reflect the industry. However, given the lack of data for female calves within the literature, studies using male calves have been cited throughout this report; therefore, discretion needs to be used in such instances. 4.1. Growth Rates Feeding increased volumes of milk resulted in heavier calves from two weeks of age until weaning. Calves in the High treatment group demonstrated similar growth paths to other accelerated milk-fed calves . However, calves in the Low treatment group were slightly lighter than restricted milk fed calves reported elsewhere . The smaller average body weight recorded for the Low treatment group exacerbated the difference between treatment bodyweights at weaning. This is thought to be due to the lower concentrate intake from these calves than expected. Given the lack of differences in solid feed intakes recorded in this experiment, all body weight gains in the High treatment group can be attributed directly to increased milk consumption. 4.2. Feed Intake By design, milk intakes for the High treatment group were significantly higher than the Low treatment group throughout the experiment. Calves in the Low treatment group always consumed the 4 L of milk offered daily. Most calves in the High treatment group consumed the increased volume of milk daily. However, two individual calves in the High treatment group would often record refusals bringing the group average below 8 L daily. However, the amount of milk consumed by these High group calves was always more than that of the Low group. Studies offering calves milk ad libitum have recorded daily intakes twice that of current industry practice and greater, with intakes of 10 L , and even up to 16 L reported from two weeks of age. These ad libitum intakes suggest that even the High treatment group underwent restricted feeding in the current experiment. In which case, the question is posed whether the biological differences observed thus far could be further enhanced with the provision of even greater volumes of milk. The milk intake results for this experiment support the idea that calves can consume far greater amounts of milk daily than the current Australian dairy industry standard . Concentrate intakes were not significantly different and were considered low for both treatment groups. Typically, restricted milk-fed calves are shown to have higher solid feed intake as compensation for being on limited milk . It is this common principle that forms the basis of the current calf feeding recommendations in Australia. However, given the differences in energy availability between milk and concentrates, calves cannot consume enough concentrate to match the energy source from milk, and the recommendations are now being revised . Both treatment groups recorded low solid feed intakes; while the reduced solid feed intake was expected in the High treatment group, the reason for the reduced solid feed intake in the Low group is unclear. Due to the lack of compensatory concentrate intake by the Low treatment group, all differences in ADG, immune and metabolic biomarkers found in this experiment can be attributed to the greater overall increased energy intake from the higher volume of milk fed to calves in the High treatment group. Hay intake increased from two weeks of age yet remained the lowest contributor to overall daily feed intake. Intakes of hay in this study were slightly higher for both treatment groups than previously reported , this could be due to the type of hay offered. The current study provided lucerne hay as the forage source, which has been noted as highly palatable leading to increased intakes than other forage sources . The suitable provision of hay and its effect on the development of young calves is contrary . Like milk, previous recommendations have advised limiting forage supplementation to avoid non-nutritive gut fill and promote more energy-rich concentrate intake . On the other hand, forage intake early in the preweaning phase is considered vital for both physical and chemical rumen development . While the current intakes of hay were only slightly higher than those reported by others , it would not be unreasonable to question its contribution to the current experiment's low concentrate intakes. While hay intakes were significantly higher for the Low treatment group during weeks 7 and 8 alone (week 7: p-value = 0.034; week 8: p-value = 0.033), it is unlikely that these two weeks of increased intakes had much influence on overall results. Furthermore, like concentrate intakes, the differences in ADG, immune and metabolic biomarkers detected in this experiment were not influenced by hay intake alone, but most likely by the overall increase in energy provided to calves in the High treatment group. 4.3. Immune Biomarkers Immune biomarkers analysed were not significantly different between treatments at birth or prior to vaccination. However, there was a small difference in lymphocyte count at birth and pre-vaccination between the High and Low treatments. While significantly different, all results remain within the normal biological range , so the biological significance of this result is minimal. Post vaccination, the WCC and neutrophil count were both significantly higher in the High treatment group when compared to the Low treatment group (p-value = 0.048 and p-value = 0.054 respectively). This result indicates that in this experiment calves in the High treatment group have an improved immune response due to the increased level of nutrition. In contrast, Foote et al. found no difference in the WCC differential of calves at different preweaning growth rates after a vaccination. Furthermore, Obeidat et al. , reported higher neutrophil activity in calves fed a lower plane of nutrition. While these two experiments are some of the few to include a form of immune challenge, they differed from the current experiment as they were both conducted using male calves. However, like both of these studies we conclude that further research is required to understand the mechanisms behind our findings, and what implication they have on the overall immune function of calves. The IBR (vaccine specific antibody) titre was not different between the two treatments. Previous experiments undertaking vaccine immune challenges have found relationships between vaccine titres and greater ADG using similar challenge protocols with a different vaccine in older calves . The IBR vaccine was selected for use in this experiment because it was a novel vaccine that was not routinely used as part of the farm's vaccine protocol. However, at birth, positive IBR titres were evident in the blood of some calves, suggesting pre-exposure to IBR. Further, post-vaccination, one calf returned a negative titre and two others doubtful S/N% titres. Given the positive immune responses found in the WCC and neutrophil count post-vaccination we are confident an adequate immune response was generated from the vaccine. The lack of response from some of the IBR titre results potentially indicate the need for further consideration of protocols including vaccine type and duration between blood tests in future research. In more general terms, the effect of increased nutrition on the immune status of calves is mixed. Supportive of the previously discussed immune biomarker results, calves on an increased plane of nutrition have reported improved immune status with fewer incidences or faster resolution of diarrhea . On the other hand, Borderas et al. and Gerbert et al. reported no difference in health or immune status. Quigley et al. and Curtis et al. reported that calves fed greater volumes of milk recorded more incidences of diarrhea requiring veterinary treatment. While our increased WCC and neutrophil count results indicate superior immune competence in calves fed high volumes of milk, the lack of difference from the IBR titre results are potentially contrary. Further research with greater sample size and vaccine protocol consideration would assist in confirmation of the effect of feeding a greater volume of milk on the immune status of calves. 4.4. Metabolic Biomarkers Metabolic biomarkers did not differ between treatments at birth. However, at 42 days of age, prior to the immune challenge, calves in the Low treatment group already had significantly higher levels of BHB (p-value =< 0.001), which was maintained post vaccination when both groups reported increased levels of BHB (p-value = 0.012). NEFA levels, however, did not differ at any point between the treatments. Increased circulating levels of NEFA and BHB are a result of the increased mobilisation of body fat reserves in response to a negative energy balance. In mature cows this can be an indication of metabolic disease . In well-fed pre-weaned calves, the mobilization of body reserves is not likely, and so elevated levels of BHB and NEFA are not typically reported in the literature. While the BHB levels did differ between treatments, all results in the experiment were within normal diagnostic limits . The level of nutrition is commonly reported as having no influence on the level of NEFA in pre-weaned calves . However, attempts have been made to use higher levels of BHB as a proxy indicator for increased concentrate intake and rumen development with mixed results. Khan et al. (2011) and Byrne et al. (2017) both found it a suitable indicator, whereas Suarez-Mena et al. (2017) did not. Calves' BHB levels are also understood to increase with age . BHB levels in the Low treatment group did increase with age and concentrate intake, supportive of previous trends in the literature. However, the results for the High treatment group seem to more closely resemble metabolic trends in the adult cow, and therefore may query the appropriateness of the current theories of calf BHB trends. BHB levels were lowest pre-vaccination, when obviously age and concentrate intakes were higher than at birth. Further, BHB levels were highest at times of increased metabolic stress imposed by the immune challenge and birth, and lowest at a time when metabolic stress on the animal would be considered lowest for the experiment. Additionally, age and concentrate intakes were not different between treatments, and therefore it is unlikely that these factors influenced the differences detected, indicating that the differences in BHB levels observed between treatments were potentially a direct result of the level of nutrition provided by milk. Glucose and insulin levels were significantly lower for the Low treatment group post-vaccination (p-value =< 0.001 and p-value =< 0.001 respectively). Pre-vaccination, Low treatment group calves also reported a trend towards lower glucose and insulin levels, although not a significant one (p-value = 0.056 and p-value = 0.063 respectfully). Similar nutritional effects were observed by Byrne et al. , where glucose levels were higher in calves fed greater volumes of milk. Glucose levels remained constant for the High treatment group both pre- and post-vaccination, whereas glucose levels continually reduced throughout the experiment for the Low treatment group. In calves, glucose levels are understood to decrease with age, as they transition from a monogastric animal into a ruminant and energy sources shift . Like BHB, glucose levels in the restricted fed calves support this theory, but those in the High treatment group do not. Glucose levels were not affected by the immune challenge in the High treatment group like the Low group. Considering the differences in immune responses previously discussed, this could indicate greater metabolic pressure for an inferior response from the Low milk-fed calves, as well as fewer energy reserves available for maintenance and growth. Unlike glucose, the difference in insulin levels between treatments was not solely due to the reduction in insulin levels from the Low treatment group, but mostly due to a simultaneous increase in insulin levels from the High treatment group. Insulin levels increased post-vaccination for the High treatment group, with little change (slight reduction) in circulating glucose levels. While its exact role is not completely understood, insulin is known to play a modulatory role in immunity and the inflammatory response . Higher levels of insulin have been associated with lower disease severity in human patients suffering various inflammatory diseases . Despite insulin initially being examined as a metabolic biomarker, these results may have further contributed to the evidence supporting the superior immune response generated by calves fed a high volume of milk. From these results it would not be unreasonable to question the existing theories of biomarkers as indicators of calf development. Calves' energy demands for maintenance and growth rise with age and increasing size. As the energy supply from milk is restricted and compensation of energy from solid feed intake is increasingly insufficient, it may be plausible that they become more metabolically strained with age. More research is needed to re-evaluate the various biomarkers and the effect increasing preweaning nutrition has on their trends. 4.5. Delayed-Type Hypersensitivity Skin Fold Testing Reactions to the delayed-type hypersensitivity skin fold tests were not different between treatments. Like the current experiment, Aleri et al. and Aleri et al. also did not find significant correlations between ADG and hypersensitivity skin fold testing responses in older calves or cows. Foote et al. did find differences in skin welt size in calves at various growth rates; however, it did not correlate with ADG. While nutrition seems to affect some areas of immunity, these results further support that cell-mediated immunity is not influenced by nutrition. 5. Conclusions The findings from this experiment stipulate that feeding calves increased volumes of milk in the preweaning phase has a positive influence on growth, immune competence and metabolic characteristics. An improved immune response to the vaccination was observed in the High treatment, where there were higher WCC, neutrophil count and insulin levels compared to the Low treatment. The greater metabolic reserves available for these calves allows greater immune responses with additional energy available for maintenance and growth. The results from this experiment do not support the common industry practice of restricted milk feeding of calves. How to best balance the supply of energy from liquid and solid feed is yet to be determined, alongside an understanding of their interactions on developing systems to improve calf nutrition. Future work is needed to confirm these uncertainties in addition to the preservation of such traits (i.e., growth, immunity and metabolism), and the ongoing effect this has on future milk production. Furthermore, information on the contribution of the reported improved immune response on reducing morbidity and mortality rates in dairy calves would benefit the industry and recommendations moving forward. Acknowledgments The authors acknowledge the technical input of T. Luke, K. Hoffman, C. Goode C. Parker, G. Morris, W. Ryan, M. Jenkin, L. Burns, M. Summerfield, L. Dorling, M. Douglas, T. Hookey, A. McDonald, D. Mapleson, M. Hannah, and all the Science, Dairy and Technical staff of Agriculture Victoria in Ellinbank. Author Contributions Conceptualization, E.M.O., V.M.R., B.J.L. and W.J.W.; Data curation, E.M.O., V.M.R. and K.G.; Formal analysis, E.M.O., V.M.R. and K.G.; Funding acquisition, W.J.W.; Investigation, E.M.O. and V.M.R.; Methodology, E.M.O., V.M.R., B.J.L. and W.J.W.; Project administration, V.M.R. and W.J.W.; Supervision, V.M.R., B.J.L. and W.J.W.; Validation, E.M.O.; Writing-original draft, E.M.O.; Writing-review & editing, V.M.R., B.J.L. and W.J.W. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All procedures were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (National Health and Medical Research Council 2004). Approval to proceed was obtained from the Agricultural Research and Extension Animal Ethics Committee, application number 2020-06. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Figure 1 Body weight (kg) of calves offered 4 L (Low) vs. 8 L (High) of milk daily. Asterisks * indicate significant difference (p-value < 0.05). The least significant difference (LSD) is given with vertical bars at each week. Figure 2 Average daily concentrate (kg DMI/calf/day) and hay (kg DM/calf/day) consumption of calves offered 4 L (Low) vs. 8 L (High) of milk daily. Asterisks * indicate significant difference (p-value < 0.05). The least significant difference (LSD) is given with vertical bars at each week (concentrate LSD lines appear above hay). animals-13-00829-t001_Table 1 Table 1 Mean nutritive characteristics of feed offered during the preweaning phase 1 (Crude protein (CP), acid detergent fibre (ADF), neutral detergent fibre (NDF), lignin, non-fibre carbohydrates (NFC), starch, crude fat (CF) and metabolizable energy (ME)). CP ADF NDF Lignin NFC Starch CF Ash ME 2 Concentrate 21.7 8.0 14.1 3.2 52.5 33.5 3.7 8.1 13.2 Lucerne hay 16.5 38.7 47.9 9.8 22.9 0.4 1.8 11.0 8.1 1 All values are % DM unless otherwise stated; 2 MJ/kg DM. animals-13-00829-t002_Table 2 Table 2 Immune and metabolic biomarkers of calves offered 4 L (Low) vs. 8 L (High) of milk daily at birth, pre-vaccination, 8 days post-vaccination and 10 days post-vaccination (WCC, white cell count; IBR, infectious bovine rhinotracheitis; BHB, beta-hydroxybutyrate; NEFA, non-esterified fatty acid). Time Biomarker Low High SED p-Value Birth WCC 1 9.4 12.4 2.14 0.170 Neutrophils 1 6.9 9.1 1.85 0.246 Lymphocytes 1 1.9 2.6 0.33 0.050 * Monocytes 1 0.3 0.5 0.12 0.108 Eosinophils 1 0.1 0.1 0.06 0.443 Basophils 1 0 0 - - BHB 2 0.04 0.04 0.013 0.733 NEFA 2 0.30 0.27 0.059 0.586 Glucose 2 8.12 8.02 0.493 0.852 Insulin (mIU/mL) 15.72 22.61 - loge Insulin 2.66 3.02 0.198 0.085 Pre-vaccination WCC 1 7.3 7.7 0.75 0.574 Neutrophils 1 2.6 3.8 0.62 0.081 Lymphocytes 1 4.3 3.6 0.30 0.028 * Monocytes 1 0.4 0.4 0.09 0.728 Eosinophils 1 0.2 0.0 0.09 0.088 Basophils 1 0 0 - - IBR Titer (S/N%) 17.0 12.6 8.14 0.593 BHB 2 0.07 0.02 0.010 <0.001 * NEFA 2 0.16 0.15 0.027 0.740 Glucose 2 6.01 6.80 0.383 0.056 Insulin (mIU/mL) 12.23 32.62 - loge Insulin 1.92 2.90 0.490 0.063 8 days post-vaccination IBR Titer (S/N%) 18.5 14.5 8.45 0.642 10 days post-vaccination WCC 1 9.3 11.9 1.21 0.048 * Neutrophils 1 5.2 7.6 1.18 0.054 Lymphocytes 1 3.6 3.5 0.49 0.783 Monocytes 1 0.4 0.6 0.16 0.252 Eosinophils 1 0.1 0.1 0.04 0.460 Basophils 1 0 0 - - IBR Titer (S/N%) 18.6 16.2 8.59 0.785 BHB 2 0.11 0.06 0.018 0.012 * NEFA 2 0.20 0.15 0.032 0.095 Glucose 2 5.46 6.72 0.183 <0.001 * Insulin (mIU/mL) 11.08 40.18 - loge Insulin 2.25 3.54 0.252 <0.001 * 1 Values are x109/L, 2 Values are in mmol/L, Asterisks * indicate significant difference (p-value < 0.05). Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000028

The purpose of this experiment was to examine the effects of MFL supplementation on feed intake, nutrient digestibility, milk yield, and milk composition in early lactating dairy cows. Twelve, Thai crossbred Holstein Friesian cows in early lactation, 500 +- 30 kg of body weight (BW), were randomly assigned according to a completely randomized design (CRD). MFL supplementation levels of 0, 100, 200, and 300 mL/d were used as treatments. Experimental animals were fed a total mixed ration (TMR) with a roughage to concentrate ratio (R:C ratio) of 40:60, which contains 12% crude protein (CP) and 70% total digestible nutrient (TDN). Rice straw was a roughage source. MFL supplementation levels had no effect (p > 0.05) on body weight change and dry matter intake (DMI) expressed as %BW, whereas DMI expressed as metabolic body weight (BW0.75) was linearly (p < 0.05) increased, with the highest at 200 mL/d in the YFL supplementation group (147.5 g/kg BW0.75), whereas feed intake of organic matter (OM), CP, ether extract (EE), neutral detergent fiber (NDF) and acid detergent fiber (ADF) did not significant (p > 0.05) difference among treatments. Related to apparent digestibility, MFL levels did not affect (p > 0.05) on DM, OM, and EE digestibility, while apparent digestibility of CP, NDF, and ADF were linearly increased (p < 0.05) when increasing MFL supplementation levels, and the highest (p < 0.05) were the 200 and 300 mL/d FML supplemented groups. BUN at 0 h post feeding did not show a significant difference (p > 0.05) between treatments, while at 4 h after feeding, BUN was linearly (p < 0.05) increased from 0, 100, 200, and 300 (mL/day) MFL supplementation, the values were 12.9, 13.1, 19.7, and 18.4 mg/dL, respectively and the highest was 200 mL/head/day for the MFL supplemented group. MFL supplementation did not affect (p > 0.05) milk fat, lactose, solid not fat (SNF), and specific gravity of milk, while MFL supplementation at 200 mL/day caused a linear increase (p < 0.01) in BUN, MUN, milk yield, milk protein, total solids (TS) and 3.5% FCM when supplement levels were increased. In conclusion, MFL supplementation in early lactating dairy cows could improve feed intake, nutrient digestibility, milk yield, and milk composition. microbial fermented liquid nutrient digestibility lactating dairy cows milk production efficiency Saccharomyces cerevisiae The Research Fund (TRF) via the Research and Researchers for Industries (RRI)MSD60I0059 This work received financial support from The Research Fund (TRF) via the Research and Researchers for Industries (RRI) (Contract no. MSD60I0059). pmc1. Introduction Recently, numerous researchers have tested the use of microorganisms in ruminants' feed, either directly or as a supplement. Ruminant direct-fed microorganisms (DFM) that are commonly used. The main types of DFM used in ruminants studies include rumen-derived bacteria that can utilize lactic acid (LUB), such as Megasphaera elsdenii, Propionibacterium freudenreichii, and Selenomonas ruminantium, they are modulation of lactic acid metabolism and by promoting the growth of fiber-degrading bacteria or that can convert starch into end products other than lactic acid such as Prevotella bryantii 25A, lactic-acid producing bacteria (LAB) of intestinal origin such as bifidobacteria, lactobacilli, and enterococci; They are hypothesized to stimulate the increase of LUB by administering lactic acid as a substrate, thereby improving ruminal pH active yeast such as Saccharomyces cerevisiae . Active yeast potentially scavenges traces of dissolved oxygen within the rumen, thereby creating an optimal anaerobic growth condition for fibrolytic microorganisms . By limiting the buildup of lactic acid in the rumen, it was also proposed that live yeast would promote optimal growth conditions for bacteria . Polyorach et al. reported that yeast fermented liquid (YFL) consisted of 29.0%DM, 91.7% OM, 8.3% ash, 15.0% CP and 3.2% EE, and that supplementation of YFL with coconut oil in diets that include cassava hay could improve rumen ecology by lowering protozoal populations and increasing bacterial and fungal populations, which improved CP and ADF digestibility in beef cattle. However, the interaction and subsequent results of mixed microbes such as yeast, lactic acid bacteria, and other microbes in ruminants have not been studied. Recently, Polyorach et al. found that yeast (Saccharomyces cerevisiae) and lactic-acid producing bacteria co-cultured in raw milk for 72 h which was the optimal cultivation time for yeast (6.90 log cells/mL) and LAB (9.67 log cells/mL). Moreover, Nampukdee et al. investigated the application of MFL, which was made from effective microorganisms (EM), a liquid mixed culture consisting of lactic acid bacteria, photosynthesis bacteria, and yeast supplementation in concentrate, by using the in vitro gas production technique. It was discovered that 20% MFL supplementation in concentrate improved the kinetics of gas production and increased in vitro digestibility . However, the use of MFL in in vivo trials has not been studied. It was hypothesized that MFL supplementation could enhance nutrient digestibility, feed intake, and milk production in lactating dairy cows fed a total mixed ration. Therefore, this study aims to examine the effects of MFL supplementation on feed intake and nutrient digestibility in early lactating dairy cows. 2. Materials and Methods The Animal Ethics Committee, in accordance with the Animal Care and Use Committee, King Mongkut's Institute of Technology Ladkrabang (Approval no. ACUC-KMITL-RES/2023/001) approved all of the experimental animals and methods used in this research. 2.1. Microbial Fermented Liquid (MFL) Preparation The MFL was prepared using the method described by Nampukdee et al. . Effective microorganisms (EM) were obtained from the manufacturer (Bionova Hygiene GmbH, Stans, Switzerland), with each mL containing 1.3 x 107 lactic acid bacteria, 3.3 x 104 photosynthetic bacteria, and 1.3 x 104 yeasts. EM were added to a flask along with 20 g of cane sugar and 100 milliliters of distilled water. The mixture was thoroughly stirred and kept at room temperature (22-33 degC) for one hour. Molasses was weighed at 48 g and dissolved in 100 mL of distilled water, followed by the addition of 48 g of urea to create a liquid culture medium (B). After mixing (A) and (B) in a 1:1 ratio and incubating them for 48 h at room temperature, they can be used in this experiment. 2.2. Animals, Diets, and Experimental Design Sakonnakhon Livestock Research and Breeding Center, Na Kham Road, Phang Khwang Subdistrict, Mueang Sakon Nakhon District, Sakon Nakhon, Thailand, was the location of the experiment. Prior to the commencement of the experiment, cows were of the same age and duration of lactation. Twelve early-lactation crossbred dairy cows (75% HF, 25% Thai native breed) were assigned to 4 levels of MFL supplementation with 3 replications in a completely randomized design (CRD). Control (no supplementation; T1), 100 g/head/day MFL supplementation (T2), 200 g/head/day MFL supplementation (T3), and 300 g/head/day MFL supplementation (T4), respectively, were the treatments. The total mixed ration (TMR) was composed of 40% rice straw and 60% concentrate mixtures (Table 1). The MFL was added to the TMR diets twice a day, at 7.30 a.m. and 4.30 p.m. The microbial fermented liquid was divided into two feedings. Each cow was housed in an individual pen with access to fresh water and mineral blocks. The experiment lasted 30 days, with 23 days dedicated to assessing treatment adaptability and feed intake and the remaining 7 days dedicated to sample collection. 2.3. Data Collection and Sampling Procedures At the start and finish of the experiment, the BW of each animal was determined. During the final week of the experiment, daily samples of feed, refusal, and feces were collected and divided into two equal groups. Half of the samples were analyzed daily for their DM content, while the remaining half were grouped by cow and frozen at -20 degC for chemical analysis. Spot sampling was used to collect the feces, which had a total fresh weight of 50 g/kg. Feed, refusal, and fecal samples were defrosted and oven-dried at 60 degC for 72 h in order to determine the chemical composition. The feed, refusal, and feces samples were then ground through a 1-mm screen and analyzed for dry matter (DM), ash, CP , NDF, and ADF . Acid insoluble ash (AIA) was used to evaluate nutrient digestibility . On the final day of the trial, 10 mL of blood was sampled from the jugular vein of each cow at 0 and 4 h after feeding. Each blood sample was stored in EDTA-containing tubes for BUN measurements . Daily milk yield was observed, and milk samples were obtained in the morning (05.00 h) and afternoon (16.00 h) milking times and kept at 4 degC. Measurements of milk were tested for fat, protein, lactose, total solids, and solids-not-fat using infrared methods. The FCM was determined using the following formula: 3.5% FCM = 0.35 milk yield (kg) + 15 fat yield (kg). 2.4. Statistical Analysis The data for a completely randomized design were analyzed using ANOVA in SAS software and the following test model: Yij = m + ti + eij where Yijk is the observation, m is the overall mean, ti is effects of treatments 1-4, eij is the residual effect. Using Duncan's new multiple range test, the treatment means were statistically compared . Orthogonal polynomials were used to compare treatment trends. At p < 0.05, significant treatment means were acceptable. 3. Results 3.1. Chemical Composition of Feeds Cassava chips were the primary energy source, while rice straw was used as the primary source of roughage. The TMR diets were carried out with 12% CP, which satisfied nutrient requirements for dairy cows to produce milk at a rate of 12-15 kg/day. In order to ensure that the animals ingested all of the MFL (20.6% CP), it was top-added to TMR diets at each feeding time. 3.2. Changes in Body Weight, Intake and Apparent Digestibility Effects of MFL supplementation levels on changes in body weight, dry matter intake (DMI), and nutrient intake are shown in Table 2. Animals supplemented with MFL at various levels did not change their final BW or nutrient intakes. However, compared to the group that did not receive MFL (p < 0.05), dry matter intake expressed as g/kg BW0.75 improved linearly with MFL supplementation. The addition of 200 mL of MFL to the TMR diet significantly improved the digestion of CP, NDF, and ADF (p < 0.05). 3.3. Blood Urea Nitrogen (BUN) The effects of MFL supplementation levels on blood urea nitrogen (BUN) are presented in Table 3. BUN at 0 h post-feeding showed no significant difference (p > 0.05) between treatments, while at 4 h after feeding, BUN was linearly (p < 0.05) increased from 0, 100, 200, and 300 (mL/head/day) MFL supplementation, the values were 12.9, 13.1, 19.7, and 18.4 mg/dL, respectively, and the highest was 200 mL/head/day MFL supplementation. 3.4. Milk Yield and Milk Composition The effects of MFL supplementation on milk yield and milk composition of lactating dairy cows (Table 4). It was found that milk yield was linearly increased (p < 0.01) and 3.5% FCM was quadratically increased (p < 0.05) when increasing MFL supplement levels, with the highest (p < 0.05) at 200 mL supplementation group. Milk protein, total solids, and milk urea-nitrogen levels significantly increased with the addition of MFL at 200 g/d, but milk fat and lactose levels remained unchanged (p > 0.05). 4. Discussion Microbial fermented liquid (MFL), a liquid mixed culture made up of yeast, bacteria that participate in photosynthesis, and lactic acid bacteria, should help ruminants better digest their feed. The current findings showed that the apparent digestibility of CP, NDF, and ADF appeared to increase linearly with increasing MFL supplementation. This may be because MFL contains a variety of microorganisms, including yeast and lactic acid bacteria (LAB), which are crucial for boosting the population of rumen microbial digestion activity . Polyorach et al. reported that yeast fermented liquid (YFL) could improve bacterial and fungal populations, which improved CP and ADF digestibility in beef cattle. Furthermore, yeast offers growth-promoting substrates for microbial growth in dairy cows, such as organic acids, amino acids, peptides, and vitamins . Moreover, yeast provides an anaerobic condition in the rumen, which is a good environment for rumen microbes, especially cellulolytic bacteria, and increases the binding affinity of anaerobic microbes to feed particles . Also, yeast cultures encouraged the growth of Fibrobacter succinogenes S85, Ruminococcus albus, Butyrivibrio fibrisolvens, and Ruminococcus albus . Dry matter intake (DMI), total milk yield, ruminal pH, total volatile fatty acid (VFA) concentrations, and OM digestibility of ruminants are supported by Saccharomyces cerevisiae supplementation . This is related to what Poppy et al. reported: improved DMI, total milk, milk composition, and milk yield by Saccharomyces cerevisiae supplementation. According to Ferraretto et al. , yeast supplementation can increase digestibility, the addition of live Saccharomyces cerevisiae increased DM digestibility (4.3 vs. 2.4%) and OM digestibility (3.9 vs. 2.4%). The addition of 4 g/d of yeast increased the digestibility of NDF by 7.5%. Erasmus et al. examined the effect of providing a yeast culture (10 g/d of Yea-Sacc) to lactating Holstein cows fed a high concentrate diet. They found that CP and ADF digestibility increased in comparison to the negative control. Ultimately, an increase in the digestibility of feed is essential for animal production and performance because it increases the passage rate and, consequently, DMI in ruminants. Polyorach et al. reported that yeast fermented liquid (YFL) contained 29.0% DM, 91.7% OM, 8.3% ash, 15.0% CP, and 3.2% EE and that supplementing with coconut oil with cassava hay diets could improve rumen ecology by decreasing protozoal populations and increasing bacterial and protozoal populations, improving CP and ADF digestibility compared to diets containing soybean meal. Determining the application of yeast in ruminants necessitates further examination of the confounding variables (i.e., yeast type, dose, and processing) that influence its efficacy. In Nampukdee et al. comparison between supplementation of yeast and MFL in a concentrate diet by using an in vitro gas production technique, it was found that the in vitro dry matter degradability (IVDMD) of the 20% yeast supplemented group was the highest (p < 0.01) (79.52%), and the 0% yeast supplemented group was the lowest (68.78%). Furthermore, the in vitro organic matter degradability (IVOMD) of the 20% yeast supplemented group was the highest (p < 0.01) (96.75%), and the 0% yeast supplemented group was the lowest (92.96%). The primary components of MFL are lactic acid-producing bacteria (LAB) (including Lactobacillus spp., Streptococcus spp., Pediococcus spp., and Enterococcus spp.), yeast, and other microorganisms . MFL also helps in the manipulation and efficiency of the rumen in ruminants. MFL is capable of balancing the intestinal microflora of animals, which increases nutrient absorption and reduces methane production. Additionally, lactic acid bacteria in MFL support fermentation and cellulose and lignin degradation . McAllister et al. demonstrated that LAB are desirable as directly fed microorganisms (DFM) because they are amenable to industrial culture, are environmentally reliable, and possess a variety of mechanisms by which they are able to modify or affect microbial communities. The mechanism of LAB is to change the intracellular pH of bacterial competitors and product antimicrobial peptides (bacteriocins) . LAB can inhibit Salmonella activity in vitro by producing hydrogen peroxide in an aerobic condition . It is unknown, however, what function these antimicrobials play in the intestinal tract, where oxygen levels are low. To achieve a multifactorial effect, many commercial DFM products contain LAB in addition to other bacteria or yeast. Although this strategy makes production sense, it makes it extremely difficult to attribute performance or pathogen exclusion responses to a particular microbial component within the DFM. Additionally, it is not possible to rule out the possibility that mixed DFM may have interactive effects on performance characteristics. Polyorach et al. discovered that 72 h post-cultivation was the optimal cultivation time for yeast (6.90 log cells/mL) and LAB (9.67 log cells/mL) populations co-cultured in fermented milk. It has been hypothesized that interactions between LAB and yeasts could affect the product's characteristics and quality due to their frequent co-occurrence . Moreover, yeast and lactic acid bacteria co-cultured in fermented milk could improve the nutritional value of soybean meal in terms of CP, EE, NDF, and ADF from 46.8, 3.2, 12.8, and 10.2, respectively, to 58.9, 6.2, 11.3, and 6.5, respectively. Soybean meal fermented milk (SBMFM) products are a high-quality source of protein that could improve degradability in the rumen, especially when added at 5% of the total concentrate substrate by using the nylon bag technique . Blood urea nitrogen (BUN) was linearly increased by MFL supplementation. The BUN concentrations corresponded to the digestibility of protein nutrients and were related to the concentration of ammonia-nitrogen (NH3-N) in the rumen . This explains that the increase in BUN is a result of the animal's diet containing high levels of protein or non-protein nitrogenous compounds . When it enters the rumen, microorganisms there, especially the proteolytic bacteria group, can digest more NH3-N , which is then absorbed through the rumen wall into the bloodstream and transported into the liver to be converted into more urea, resulting in increased BUN levels . In addition, an increase in BUN resulted in an increase in the concentration of MUN . Milk urea nitrogen (MUN) of 200 mL/head/day from MFL supplementation was the highest (15.4 mg/100 mL). MUN concentrations under the current study were similar to those reported by Jonker et al. , who reported that the optimum value of MUN should be between 10 and 16 mg/100 mL. The MUN concentration indicates adequate protein levels in the diet and a balance of protein and energy in the feed . Furthermore, the increase in milk protein percentage was consistent with higher digestibility of protein nutrients in feed intake and an increasing BUN concentration. Polyorach et al. reported that supplementation of mangosteen peel powder at 300 g/hd/d with yeast-fermented cassava chips (YEFFCAP) as a protein source could increase the milk protein percentage in dairy cows. Similar to the report by Dias et al. , yeast culture was studied in dairy cows fed a high starch diet. It was found that the percentage of protein intake was increased (1.18 kg/day). The increased milk protein under the current study might be due to improved rumen fermentation, which affects protein degradation and amino acid content . In addition, yeast, when decomposed, is also a nutrient for bacteria in the rumen. In particular, the proteolytic bacteria can produce amino acids and ammonia that can be absorbed by dairy cows through the rumen wall and absorbed through the intestine for use in increasing the percentage of protein in milk . 5. Conclusions Based on this study, it could be concluded that MFL supplementation levels in early lactating dairy cows did not affect DM, OM, and EE digestibility, milk fat, milk lactose, solid not fat (SNF), or the specific gravity of milk. However, DMI, digestibility of CP, NDF, and ADF, BUN, MUN, milk yield, milk protein, total solids (TS), 3.5% FCM were improved when 200 mL/day of MFL was supplemented. Supplementation in early lactation dairy cows may therefore improve feed intake, nutrient digestibility, milk output, and milk composition. Further research should be done on the supplementing impact of MFL on rumen parameters, rumen microorganisms, and higher animal numbers. Acknowledgments The authors are thankful the Phu Phan Sakon Nakhon Dairy Cooperative Limited, and the Sakon Nakhon Livestock Research and Breeding Center, Thailand. The experimental facilities were provided by the Department of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkut's Institute of Technology Ladkrabang, Bangkok, Thailand on use of their research facilities. Author Contributions Conceptualization, S.P.; Data curation, S.P., N.G. and A.L.; Formal analysis, S.P. and R.N.; Funding acquisition, S.P.; Investigation, S.P. and R.N.; Methodology, R.N.; Project administration, S.P.; Resources, S.P., A.T. and Y.J.; Supervision, S.P.; Validation, T.N.; Writing--original draft, S.P. and P.G.; Writing--review & editing, S.P., M.W., S.K., A.C., O.P., P.G. and S.F. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All animal procedures were approved by the Animal Care and Use Committee, King Mongkut's Institute of Technology Ladkrabang (Approval no. ACUC-KMITL-RES/2023/001). Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest Anuwat Lapmee works as a member of the Phuphan Sakon Nakhon Dairy Cooperative Ltd. The authors declare no conflict of interest. animals-13-00933-t001_Table 1 Table 1 Chemical composition of total mixed ration (TMR) and microbial fermented liquid (MFL). Item TMR MFL Rice Straw Ingredient, % of DM Rice straw 40.0 Cassava chip 39.6 Rice bran 5.0 Soybean meal 8.5 Urea 2.0 Molasses 2.0 Tallow 2.0 Salt 0.3 Sulfur 0.3 Mineral mixture 1 0.3 Chemical composition Dry matter (DM), % 89.6 22.2 90.2 % of dry matter Organic matter (OM) 90.7 98.9 83.0 Crude protein (CP) 12.3 20.6 2.7 Ether extract (EE) 3.5 1.2 2.0 Neutral detergent fiber (NDF) 56.4 - 80.4 Acid detergent fiber (ADF) 28.3 - 54.0 1 Minerals and vitamins (each kg contained): IU: vit. A 10,000,000, vit. E 70,000, vit. D 1,600,000; g: Fe 50, Zn 40, Mn 40, Co 0.1, Cu 10, Se 0.1, I 0.5. animals-13-00933-t002_Table 2 Table 2 Effects of MFL supplementation levels on body weight change, dry matter intake (DMI) and nutrient intake. Item Supplement Levels (mL/hd/d) 1 SEM Contrasts 2 0 100 200 300 L Q Body weight Initial body weight (kg) 512.0 505.3 505.7 517.3 19.32 0.22 0.66 Final body weight (kg) 531.0 526.3 529.0 539.5 20.35 0.32 0.45 Dry matter intake (DMI) Kg/d 13.3 15.3 15.7 15.5 0.34 0.27 0.22 %BW 2.6 3.0 3.1 3.1 0.09 0.78 0.77 g/kg BW 0.75 123.9 b 143.8 a 147.5 a 145.5 a 3.33 0.03 0.43 Nutrient intake (kg/hd/d) Organic matter (OM) 12.1 13.9 14.3 14.1 0.32 0.66 0.83 Crude protein (CP) 1.6 1.8 1.9 1.9 0.04 0.54 0.69 Ether extract (EE) 0.4 0.5 0.6 0.5 0.01 0.45 0.92 Neutral detergent fiber (NDF) 7.5 8.6 8.9 8.7 0.20 0.67 0.18 Acid detergent fiber (ADF) 3.8 4.3 4.5 4.4 0.10 0.97 0.65 Apparent digestibility (%) Dry matter (DM) 65.0 64.6 68.4 66.9 2.22 0.23 0.55 Organic matter (OM) 65.4 67.1 70.7 68.45 1.65 0.22 0.34 Crude protein (CP) 50.5 b 53.2 b 69.1 a 67.3 a 1.07 0.01 0.43 Ether extract (EE) 66.7 71.4 73.2 72.6 0.96 0.45 0.89 Neutral detergent fiber (NDF) 49.6 b 55.1 ab 63.2 a 61.9 a 1.52 0.02 0.65 Acid detergent fiber (ADF) 49.1 b 55.0 ab 64.3 a 59.7 ab 1.69 0.05 0.44 a,b Means in the same row with different superscripts, SEM = standard error of the means, 1 Levels of microbial fermented liquid supplementation, 2 L = Linear, Q = Quadratic. animals-13-00933-t003_Table 3 Table 3 Effects of MFL supplementation levels on blood urea nitrogen (BUN). Item Supplement Levels (mL/hd/d) SEM Contrasts 0 100 200 300 L Q BUN, mg/dL 0 h post feeding 11.1 11.7 12.4 12.7 0.29 0.44 0.98 4 h post feeding 12.9 b 13.1 b 19.7 a 18.4 a 0.25 0.01 0.78 Average 12.1 b 12.4 b 16.1 a 15.6 a 0.08 0.01 0.04 a,b Means in the same row with different superscripts. animals-13-00933-t004_Table 4 Table 4 Effects of MFL supplementation levels on milk yield, and milk composition in lactating dairy cows. Item Supplement Levels (mL/hd/d) SEM Contrasts 0 100 200 300 L Q Production Milk yield (kg/hd/d) 12.6 b 14.0 ab 15.4 a 14.8 a 0.22 0.01 0.23 3.5% Fat corrected-milk (kg/hd/d) 12.7 c 14.9 b 17.7 a 16.3 ab 0.32 0.01 0.04 Milk composition, % Fat 3.4 3.9 4.5 4.2 0.17 0.23 0.15 Protein 3.2 b 3.7 ab 4.0 a 3.7 ab 0.09 0.03 0.23 Lactose 4.2 4.4 4.8 4.6 0.15 0.33 0.27 Solid not fat (SNF) 8.1 8.5 8.8 8.6 0.12 0.44 0.34 Total solid (TS) 10.8 b 12.2 ab 13.0 a 12.6 a 0.23 0.04 0.66 Specific gravity 1.03 1.03 1.03 1.02 0.0003 0.54 0.45 Milk urea-nitrogen (MUN), mg/100 mL 11.9 b 12.2 bc 15.4 a 14.4 ab 0.39 0.03 0.43 a,b,c Means in the same row with different superscripts. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000029

A 13-year-old neutered, blue-eyed female Siamese cat with a bodyweight of 4.8 kg was admitted for enucleation of the right eye. An ultrasound guided retrobulbar block with 1 mL of ropivacaine was performed under general anaesthesia. When the tip of the needle was visualised inside the intraconal space, negative aspiration of the syringe before injection and no obvious resistance during injection were confirmed. Instantly, after ropivacaine was administered, the cat became apnoeic, and its heart rate and the blood pressure increased significantly for a short period of time. During surgery, the cat needed cardiovascular support to maintain blood pressure and was under continuous mechanical ventilation. Spontaneous breathing returned 20 min after the end of anaesthesia. Brainstem anaesthesia was suspected, and after recovery, the contralateral eye was examined. A reduced menace response, horizontal nystagmus, mydriasis, and absence of the pupillary light reflex were present. The following day, mydriasis was still present, but the cat was visual and was discharged. The inadvertent intra-arterial injection of ropivacaine was suspected to be the cause of the spread into the brainstem. To the current authors' knowledge, possible brainstem anaesthesia has only been reported in a cat 5 min after a retrobulbar block but never instantly. brainstem anaesthesia cat retrobulbar block intraarticular injection This research received no external funding. pmc1. Case Presentation A 13-year-old neutered, blue-eyed female Siamese cat with a bodyweight of 4.8 kg was scheduled for enucleation of the right eye due to panuveitis and secondary glaucoma. The menace response and dazzle reflex were normal on the left eye. Apart from being geriatric, the cat had no other comorbidities in her clinical history. Her preoperative clinical examination was normal without any nystagmus noticed or reported during her ophthalmological examination, and her biochemistry and haematological results were unremarkable. The cat's current medications included meloxicam and chloramphenicol ointment. For preanaesthetic medication, a combination of methadone (0.2 mg/kg) (Methadyne, Jurox, Leatherhead, UK), midazolam (0.3 mg/kg) (Hameln pharma Ltd., Gloucester, UK), and alfaxalone (1.5 mg/kg) (Alfaxan/Multidose, Jurox, Dublin, Ireland) was mixed in the same syringe and administered intramuscularly (IM). Anaesthesia was induced with a total of 1.8 mg/kg of alfaxalone administered through a right cephalic intravenous cannula. Intravenous fluid therapy was administered during anaesthesia at a rate of 3 mL/kg/h (Hartmann's solution; Aquapharm No. 11; Animalcare LTD; York, UK). Anaesthesia was maintained with sevoflurane (Sevohale, Chanelle, Galway, Ireland) in 100% oxygen administered through a Mapleson-D breathing system. Throughout anaesthesia, heart rate and rhythm with electrocardiography, haemoglobin oxygen saturation (SpO2), oscillometric blood pressure, oesophageal temperature, capnography, and spirometry were continuously monitored and recorded every 5 min using a multiparameter monitor (Datex-Ohmeda, GE, Helsinki, Finland). A retrobulbar block (RBB) was used as part of a multimodal analgesic plan. Following aseptic preparation of the periocular area and the area dorsal to the zygomatic process, an ultrasound (US)-guided RBB was performed using a 14 MHz linear transducer connected to a portable ultrasound device (Mindray bio-medical electronics Ltd., Shenzhen, China). Sterile ocular gel was applied to the globe, and the US transducer was placed over the cornea with the marker positioned laterally. The orientation was initially perpendicular to the transverse plane and parallel to the dorsal plane of the globe, and then it was oriented with an angle of 30deg approximately relative to the dorsal plane in order to position the US marker towards the junction between the zygomatic arch and the orbital ligament laterally. The depth and focus of the US were adjusted to achieve a good view of the retrobulbar space, the cone, and the optic nerve. The retrobulbar compartment was identified as a conic shape formed by the extraocular muscles positioned caudally to the eyeball. A 22 gauge, 50-mm ultrasound needle with extension tubing (USB 50 EVOLUTION, Temena GmbH) was introduced using a supra-temporal approach . The needle was inserted perpendicularly to the skin and oriented latero-medially behind the orbital ligament, dorsally to the zygomatic process of the temporal bone and ventral to the frontal bone, through the temporal muscle. When the tip and part of the needle were identified ultrasonographically using an oblique in-plane approach, the needle was advanced latero-medially to reach the intraconal space by crossing the extraocular muscles. In this case, the needle was visualised to go through the cone and appear in the medial aspect of it; therefore, it was slightly retracted until it was inside the cone again. At this level, negative pressure was applied to the syringe to exclude blood vessel penetration, and 1 mL of ropivacaine 0.75% (Naropin, Apen Pharma Trading Limited 0.75%, Dublin, Ireland) was injected without obvious resistance. The maximum recommended dose of ropivacaine of 2 mg/kg was calculated to be 1.28 mL . Immediately after the injection and before the needle had been removed, the cat became apnoeic. After a minute of apnoea, manual ventilation was initiated. At the same time, there was an immediate rise in the cat's heart rate (HR) from 102 to 140 bpm with a sinus rhythm. Unfortunately, there is no record of the concurrent blood pressure (BP), but we recall that it increased in parallel with the heart rate. Twenty minutes later, the cat was transferred to the operating theatre, and continuous mechanical ventilation (CMV) on a volume control mode was initiated using a veterinary ventilator (Merlin, Vetronic Services LTD, Devon, UK). During this time, no parameters were recorded on the monitoring sheet as the anaesthetist was involved in providing manual ventilation, but when the cat was moved to theatre, the blood pressure could not be measured with oscillometry or the flow Doppler technique. By that time, it was suspected that the administration of the local anaesthetic (LA) in the retrobulbar space could have been responsible for the occurrence of apnoea and the instability of the HR and BP. When the surgery started, the mean BP (MBP) was 50 mmHg, and the HR was 75 bpm. A dose of 0.01 mg/kg of glycopyronium (Accord Healthcare Limited, Durham, UK) was administered intravenously (IV) and was repeated after 5 min as there was no effect. Due to the lack of response to the anticholinergic, a dose of 0.1 mg/kg of ephedrine (MaCarthys Laboratories, Martindale Pharma, Brentwood, UK) was administered IV, and this was repeated again after 10 min, after which the MBP increased to 80 mmHg but only temporarily. The HR remained at levels >100 bpm after the above interventions, while the MBP increased again only after a dopamine (Martindale Pharma, UK) IV infusion at a dose of 5 mg/kg/min was initiated. During the whole anaesthetic, the SpO2 and the EtCO2 were maintained within the acceptable limits, while the oesophageal temperature was 34-36 degC. Anaesthesia ended at 75 min after induction, and the cat remained apnoeic. Twenty minutes following inhalant discontinuation, the cat was still apnoeic, and a decision was made to reverse the midazolam with 0.01 mg/kg of flumazenil IV (Hamlen Pharma gmbh, Gloucester, UK). The cat responded immediately and started breathing spontaneously following the administration of flumazenil. Dopamine was discontinued after extubation, as the cat was able to maintain MBP > 80 mmHg. During surgery, the cat lost approximately 29 mL of blood, which was 10% of her estimated total blood volume (4.8 kg x 60 mL/kg = 288 mL). On recovery, the cat remained on Hartman's solution at a rate of 3 mL/kg/h until the volume lost was replaced. After the cat had fully recovered, an ophthalmological examination of the left eye revealed a reduced menace response, horizontal nystagmus, mydriasis, and absence of the pupillary light reflex (PLR). The rest of the clinical examination was normal. She was responsive to stimulation and had a good appetite. The Glasgow Feline Composite Measure Pain Scale scores were between 0 and 2 postoperatively. The following day, she had a normal menace response, subtle horizontal nystagmus, mydriasis, and the PLR was present. The cat returned home on the day after surgery. One week later, at the follow-up examination, the left eye had a normal menace response, no nystagmus, a slightly larger than normal resting pupil size but was less mydriatic than postoperatively and within the normal limits for a blue-eyed Siamese cat, and with the PLR present. The owner reported no concerns, and the patient was discharged. 2. Discussion Regional anaesthesia of the eye is commonly applied in veterinary patients to provide analgesia during painful procedures, such as enucleation, or for akinesia of the extraocular muscles, which results in the globe maintaining a central position . Additionally, it reduces the incidence of the oculocardiac reflex during enucleation surgery by blocking the ophthalmic branch of the trigeminal nerve and the ciliary ganglion, as has been described in horses . The RBB and peribulbar blocks (PBB) are two different types of regional anaesthesia techniques in which the LA is either deposited in the retrobulbar or in the peribulbar space, respectively, with the final aim being for the LA to reach the intraconal space. The intraconal space is within the retrobulbar cone, and in the dog, the latter is formed by the ocular extrinsic muscles: the dorsal, ventral, lateral, and medial rectus muscles, the retractor bulbi with its four fascicles, and more superficially and medially, the dorsal oblique muscle . There is no fascia surrounding the muscles that form the cone. Within the retrobulbar cone are the ophthalmic nerve, the ciliary nerves, the optic nerve, the oculomotor nerve, and the abducens nerves. Additionally, the internal ophthalmic artery and the ciliary arteries run in the same space . Different techniques have been described for both blocks . In dogs, among other blind approaches to the RBB, the inferior-temporal-palpebral and the supra-temporal approaches have been described in cadavers. In one study with sedated dogs, the peribulbar technique was shown to be more reliable for producing anaesthesia than the RBB when using a bent needle inserted through the inferior eyelid . However, both methods produced adverse effects such as exophthalmos, chemosis, and anterior uveitis, which had resolved 14 h later . In cats, Shilo-Benzamini et al. described a superior-nasal approach for the retrobulbar block through the upper eyelid at the dorsomedial orbit using a curved needle. This was found to have a 70% success rate in cadavers, but this was only 50% when tested in sedated cats . Ultrasound-guided ophthalmic regional anaesthesia has been used recently in human ophthalmology, as it has the advantage of allowing visualisation of the retrobulbar structures and the needle at the same time as well as confirmation that the LA has been administered in the correct space. According to our knowledge, thus far, only extraconal deposition of the LA has been described in dog cadavers with US guidance . An US-guided PBB was shown to be as effective as a blind technique in anaesthetised dogs, although the dogs receiving the blind technique had a higher incidence of increased intraocular pressure (IOP) due to the possible wrong positioning of the needle, as the authors hypothesised . In cats, an US-guided RBB with a trans-palpebral approach, as mentioned above, was effective in only 50% of the animals , possibly due to "the difficulty in identifying the curved needle tip", as commented by the author in a review article on regional ophthalmic anaesthesia . More recently, the authors of a case report on a cat with microphthalmia described the use of an US-guided RBB using a modified technique similar to the supra-temporal approach, but in this case, the needle was introduced into the retrobulbar space from the subzygomatic area . Advocates of the PBB highlight the reduced risk of complications, such as the distribution of the LA in the central nervous system (CNS), retrobulbar haemorrhage, and globe penetration, compared to the RBB, as the needle is inserted further away from these structures . Indeed, the reported incidences of CNS complications in humans after a RBB are 0.09%, 0.27%, and 0.9% , whereas a much lower incidence of 0.007% has been reported with the PBB; however, the authors concluded that this incidence is probably underestimated . In veterinary patients, the incidence of complications is unknown. Only one case has been reported in a cat with suspected brainstem anaesthesia that occurred 5 min following the injection of LA into the retrobulbar space using a blind technique . The technique routinely performed at our hospital is the US-guided RBB using a supra-temporal approach with intraconal deposition of the LA. This preference is due to the perceived greater success rates when an US-guided technique with an in-plane approach is used and the correct deposition of the LA can be visualised. Additionally, a smaller volume of LA is necessary, as it is injected inside the cone. Humans receive regional ophthalmic blocks while awake, and the side effects vary from contralateral pupil dilation to amaurosis, convulsions, grand-mal seizures, hypertension/hypotension, tachycardia/bradycardia, and cardiopulmonary arrest . In veterinary medicine, the side effects reported in one cat following RBB were apnoea, increased HR and systolic blood pressure initially, and a reduction in blood pressure below normal values during surgery . A delay in recovery was also noticed, and after extubation, the cat presented with nystagmus, absence of the menace response, mydriasis, a lack of dazzle, and a negative PLR in the contralateral eye. There are three main potential mechanisms whereby a RBB or PBB may lead to CNS complications. First, these can occur due to penetration of the sheath of the optic nerve and the entry of LA into the subarachnoid space of the brainstem . If this occurs, symptoms appear 5 to 10 min after injection, depending on the onset of action of the LA. The second mechanism is through the inadvertent injection of the LA into the internal ophthalmic artery. The pressure of the injectate forces the LA to flow back into the internal carotid artery and into the brain. In such cases, clinical signs appear rapidly . Thirdly, complications can occur due to the systemic absorption of the LA . In the case presented here, tachycardia, hypertension, and apnoea appeared instantly before the removal of the needle, and the cat remained mechanically ventilated until recovery. No obvious resistance was noticed during the injection, and the needle did not seem to have penetrated the optic nerve on the US image. Given the rapid onset of the clinical signs, CNS spread of the local anaesthetic exclusively through penetration of the sheath of the optic nerve is less likely. Unfortunately, there are no saved images to corroborate this, as it is not common practice to save images for every patient, especially in routine cases. Similar to our case and to the other case report from a cat, the first manifestations of CNS spread in humans are often hypertension and tachycardia. This can be explained as a parasympathetic blockade through a combined vagal and carotid sinus reflex block . Although the immediate onset of symptoms in our case is in favour of inadvertent intra-articular injection, no blood appeared during the application of negative pressure to the syringe. Additionally, one would expect the duration of the symptoms to be transient due to the rapid redistribution of blood out of the brain, but this was only the case for hypertension and tachycardia. However, the cat remained apnoeic during the whole anaesthetic procedure and for 20 min after the end of anaesthesia. In dogs, the internal ophthalmic artery runs on the dorsal surface of the optic nerve . To the best of our knowledge, there is no published description of the ophthalmic vasculature in cats that mentions the exact pathway of the internal ophthalmic artery. In humans, because of an anatomical variation in the inferior ophthalmic artery in 15% of the population, there is a higher risk of inadvertent intra-arterial injection . The use of colour-flow Doppler could have helped us to identify the artery and avoid a possible intra-arterial injection of the LA, but this was omitted before the administration of ropivacaine. The rapid systemic absorption through the local capillaries could also explain this clinical presentation. However, the total dose administered (1 mL) was less than the maximum recommended dose in cats . The possibility that some of the LA was injected intra-arterially and some reached the subarachnoid space through penetration of the sheath could explain the quick onset of symptoms in combination with the increased duration of apnoea. In a similar case report in a human, an intra-arterial injection was suspected due to the instant occurrence of CNS toxicity. The same patient remained apnoeic for 40 min after seizures had resolved. The authors speculated that systemic absorption from the local capillaries or the spread of the LA in the subdural space along with the optic nerve could explain the sustained respiratory arrest . Following recovery from anaesthesia, the cat in the present case report had symptoms in the contralateral eye that could potentially be explained by brainstem exposure to the LA . Although nystagmus may be present during recovery from anaesthesia and opioids cause mydriasis in cats, the above symptoms were still present the following morning, although to a lesser degree. In contrast with humans, where convulsions or seizures are common, the cat in this report did not show any twitches, probably because general anaesthesia was masking this response. It is not clear why, upon administration of flumazenil, the cat started breathing spontaneously immediately, as midazolam causes minimal respiratory depression, and it did not compromise ventilation after administration in our case. However, when diazepam is administered with buprenorphine in rats, respiratory depression has been reported to be greater . Potentially, the impact of benzodiazepines on central ventilatory control after CNS dysfunction, as occurred in our case, is more pronounced compared with in a normal CNS. Additionally, the use of flumazenil has been reported to reduce the recovery time from anaesthesia in humans when no benzodiazepines have been administered . It is unknown as to whether it was the pharmacological action of flumazenil or the time that had elapsed since the retrobulbar block that caused the return to spontaneous breathing. Lipid emulsion therapy has been proven to be effective for treating local-anaesthetic-induced systemic toxicity , including a case of local-anaesthetic-induced seizures after a peribulbar block in humans . When brainstem anaesthesia was suspected in our case, the administration of a lipid emulsion could have potentially helped to resolve the cardiovascular and respiratory complications. 3. Conclusions We have presented a case report of a potential exposure of the CNS to ropivacaine after an US-guided RBB in a cat that showed sustained apnoea, cardiovascular instability, as well as nystagmus, mydriasis, and an absent PLR postoperatively in the contralateral eye. The early onset of symptoms, prior to needle withdrawal, indicates that an inadvertent intra-arterial injection of the LA was the possible cause, although we cannot exclude subarachnoid injection or a combination of the two. Although an US-guided technique was used and negative pressure was applied before the injection, this complication could not be avoided, and we highlight the importance of using colour-flow Doppler for better visualisation of the vasculature. Any medications that can be reversed could be beneficial for similar cases. Author Contributions Conceptualisation, A.P. and E.R.; writing--original draft preparation, A.P.; writing--review and editing, E.R.; supervision, E.R. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The presented case is not a prospective study and for that reason does not require ethical approval. The owner of the animal in the presented case gave consent to anonymously share the details of the case for publication purposes. Please see a blank consent form attached to the nonpublished materials section. Informed Consent Statement Informed consent was obtained from the owner of the animal involved in the case report. Data Availability Statement The presented case report is not a study, and no data were collected. Conflicts of Interest The authors declare no conflict of interest. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000030

Different cereal types, in combination with different protein sources, are fed to pigs after weaning, but their interactions and possible implications are not well researched. In this study, 84 male weaned piglets were used in a 21-day feeding trial to investigate the effects of feeding either medium-grain or long-grain extruded rice or wheat, in a factorial combination with protein sources of either vegetable or animal origin, on postweaning performance, shedding of b-haemolytic Escherichia coli, and the coefficient of total tract apparent digestibility (CTTAD). Pigs fed either rice type performed the same (p > 0.05) as wheat-fed pigs after weaning. The use of vegetable protein sources reduced growth rate (p < 0.001) and feed intake (p = 0.007) and deteriorated the feed conversion ratio (p = 0.028) in weeks two and three compared to pigs fed animal protein sources. The number of antibiotic treatments given for clinical diarrhoea was similar (p > 0.05). However, the faecal E. coli score showed a trend for the main effect of protein source, with pigs fed animal proteins showing a higher E. coli score than pigs fed vegetable proteins (0.63 vs. 0.43, p = 0.057). There was also a tendency for an interaction (p = 0.069) between cereal type and protein source (p = 0.069), with this difference being associated with a greater faecal score in pigs fed diets with long-grain rice plus animal proteins and wheat plus animal proteins. Significant interactions occurred for the CTTAD when assessed in week three. In general, pigs fed diets with medium-grain rice or long-grain rice with animal proteins had a higher (p < 0.001) CTTAD for dietary components than pigs fed all other diets, and vegetable proteins depressed (p < 0.001) CTTAD compared to animal proteins (main effect of protein: p < 0.001). In summary, pigs tolerated the extruded rice-based diets well and performed equivalently to pigs fed wheat as the sole cereal, and the use of vegetable proteins decreased the E. coli score. pigs weaning rice performance diarrhoea digestibility Rice Sub-Program of the Rural Industries Research and Development Corporation of Australia (now AgriFutures Australia)The Rice Sub-Program of the Rural Industries Research and Development Corporation of Australia (now AgriFutures Australia) is thanked for funding this research. pmc1. Introduction Postweaning diarrhoea (PWD), typically caused by the activity of enterotoxigenic and (or) enteropathogenic strains of Escherichia coli in the small intestine, remains a problem in commercial pig production in some parts of the world. This is exacerbated by bans or restrictions on the use of antimicrobial compounds such as medicinal zinc oxide and prophylactic antibiotics . In association with poorer performance after weaning, the postweaning growth check significantly impacts whole-of-life productivity. A plethora of nutritional strategies exists commercially to alleviate the negative impacts of weaning in the absence of these antimicrobial compounds . Among the numerous dietary options available to the industry, the choice of cereal and (or) protein sources in diets can have a major impact on PWD and performance. In this regard, rice, either as the sole cereal or in combination with others, can be used successfully in piglet diets as an alternative to other cereals such as maize, sorghum and wheat and can reduce the incidence of PWD and decrease postweaning mortality . Rice is characterised by its high starch content and varietal differences in the ratio of amylose:amylopectin, low non-starch polysaccharide (NSP) and oligosaccharide contents, and lower protein content in comparison with other cereals . Newly weaned pigs are typically fed a mixture of plant (vegetable) and animal protein sources to provide amino acids and energy for body growth and development. Animal protein sources are generally used more efficiently by the weaned pig than vegetable protein sources due mainly to the lack of anti-nutritive factors present and associated negative physiological effects in the gastrointestinal tract (GIT) . Past studies have typically shown differences between individual cereal sources and protein sources on postweaning performance and aspects of GIT structure and function and PWD; however, we are unaware of studies specifically comparing different types of extruded white rice with wheat in conjunction with either animal or vegetable protein sources. The hypotheses tested in this study were: (a) rice-based diets using animal protein sources will cause less excretion of b-haemolytic E. coli than diets based on vegetable proteins; (b) diets based on extruded rice will cause faster growth and less faecal excretion of b-haemolytic E. coli than a diet based on wheat; and (c) diets based on a medium-grain rice, with a lower amylose:amylopectin ratio, will cause better performance, irrespective of the protein source, than diets based on a long-grain rice having a higher amylose:amylopectin ratio. 2. Materials and Methods 2.1. Ethics Statement The Murdoch University Animal Ethics Committee and the Animal Ethics and Experimentation Committee of the WA Department of Agriculture approved this experiment (Number 05-02-22). 2.2. Animals, Procedures, and Housing Eighty-four entire male pigs (Large White x Landrace) aged ~24 days and weighing 6.7 +- 0.13 kg (mean +- SEM) were used in this experiment. On arrival at the Medina Research Centre, the pigs were ear-tagged, weighed, and stratified into pens of four pigs, each according to treatment and live weight. Pigs were offered their respective diets (Table 1) in groups of four for the first 7 days after weaning to accustom them to their new surroundings. For the final 2 weeks of the study, pigs were housed individually for the collection of faeces from the pen floor. Pens were of wire-mesh construction with slatted metal floors and measured 1.68 m2 in floor area (0.42 m2 per pig). Each pen was equipped with a nipple water drinker and a stainless-steel feed trough. The ambient temperature was maintained between 26 and 28 degC throughout the study using two reverse-cycle air conditioning units. The room containing the pens was cleaned daily. 2.3. Experimental Design, Diets, Feeding, and Sample Collection The 21-day experiment was designed as a 3 x 2 factorial arrangement of treatments with the respective factors being (a) three cereal types, i.e., a medium-grain, lower-amylose rice (cultivar Amaroo), a long-grain, higher-amylose rice (cultivar Doongara), and wheat, and (b) two protein sources, namely vegetable and animal protein sources (Table 1). Diets are subsequently referred to as: MGAP: medium-grain rice plus animal proteins; MGVP: medium-grain rice plus vegetable proteins; LGAP: long-grain rice plus animal proteins; LGVP: long-grain rice plus vegetable proteins; WAP: wheat plus animal proteins; WVP: wheat plus vegetable proteins. None of the diets contained any antimicrobial compounds. Isonitrogenous and isoenergetic diets were formulated to contain adequate levels of energy and nutrients for pigs of this genotype and age. Extruded rice was sourced as described previously and was passed through a hammer mill to reduce particle size before incorporation into meal-based diets. The diet was offered on an ad libitum basis to piglets in mash form (Table 1). Titanium dioxide (TiO2) was added as an inert marker for the estimation of the coefficient of total tract apparent digestibility (CTTAD). Faecal samples were collected from the wire-mesh floor of each pig at 0800, 1000, 1200, 1400, and 1600 h on days 18-21 of the experiment. Samples collected over the 3-day period were pooled, kept at -20 degC and later thawed, mixed, freeze-dried, and ground through a laboratory hammer mill (1 mm screen) prior to chemical analysis. 2.4. Microbiological Assessments Faecal swabs were taken to record initial E. coli presence upon arrival and then again on days 2, 5, 6, and 8 after weaning. Faecal swabs were cultured, and plates were assessed for b-haemolytic colonies displaying morphology characteristic of E. coli following overnight incubation, according to standard procedures . The presence of b-haemolytic E. coli was then scored from no growth (0) to heavy colonisation (5) . Piglets were monitored daily for clinical signs of diarrhoea . Affected pigs (as assessed by the stockperson, who was unaware of the treatment allocation of pigs) were treated for diarrhoea by intramuscular injection with Trisoprim-480 ((trimethoprim 80 mg/mL, sulfadiazine, 400 mg/mL), 1.5 mL/30 kg body weight; Troy Laboratories, Smithfield, NSW, Australia); treatment continued until the diarrhoea ceased. Records were kept of the duration of treatment required for each treated piglet. 2.5. Chemical Analyses The dry matter (DM), nitrogen (N), gross energy (GE), total starch, resistant starch (RS), amylose and amylopectin contents of extruded rice were determined as described previously . Crude protein (CP) content was calculated as N x 6.25. The DM, GE, and TiO2 content of diets and faecal samples were determined for the estimation of CTTAD . The GE content of the rice, diet, and faecal samples was determined using a Ballistic Bomb Calorimeter (SANYO Gallenkamp, Loughborough, U.K.). The TiO2 contents of diet and faecal samples were determined using the method described previously . 2.6. Statistical Analyses Treatment effects were analysed by two-way ANOVA for a factorial design, with the main effects being cereal type (medium-grain rice, long-grain rice and wheat) and protein type (vegetable and animal). Average daily gain (ADG) in the first week after weaning was evaluated using the pen as the unit of replication. The ADG, average daily feed intake (ADFI) and feed conversion ratio (FCR) in weeks two and three used the individual pig as the experimental unit. For the CTTAD of DM, starch, GE, and CP, the individual pig was considered the unit of replication. All effects were considered fixed effects in the model. Fisher's-protected least significant difference test was used (at a 5% significance level) for comparison between mean values of different variables. A p-value between 0.05 and 0.1 was considered a trend. All statistical analyses were conducted using the statistical package StatView 5.0 for Windows (AddSoft Pty. Ltd., Woodend, VIC, Australia). 3. Results 3.1. Antibiotic Treatments and Faecal Shedding of E. coli Both rice-based diets fed with vegetable proteins had fewer antibiotic administrations given for clinical diarrhoea than pigs fed diets WAP or WVP, but statistically, the number of treatments was similar (p > 0.05) across treatments. Shedding of b-haemolytic E. coli, ascertained via faecal swabs, showed a trend for the main effect of protein source, with pigs fed animal proteins having a higher E. coli score than pigs fed vegetable proteins (0.63 vs. 0.43, p = 0.057). There was also a tendency for interaction between cereal type and protein source (p = 0.069), with this difference being associated with the greater score recorded in pigs fed diets LGAP and WAP (Table 2). 3.2. Production Performance There were no statistically significant differences between treatment groups for ADG in the first week after weaning (Table 3). In weeks two and three, no main effects of cereal type on any indices were recorded (p > 0.05). However, pigs fed animal proteins rather than vegetable proteins were heavier (p = 0.01) at the end of the experiment (11.8 vs. 10.4 kg) because they grew faster (317 vs. 242 g/day, p < 0.001). This was a consequence of a higher ADFI (580 vs. 500 g/day, p = 007) and improved FCR (1.87 vs. 2.31, p = 0.028). No interactions (p > 0.05) occurred for any of the production indices (Table 4). 3.3. Coefficient of Total Tract Apparent Digestibility Significant main effects for cereal type (rice vs. wheat) were observed in the CTTAD for DM, energy, and CP. Nevertheless, significant interactions occurred between cereal type and protein source for DM, energy, and CP. The CTTAD for DM was higher in pigs fed diets MGAP and LGAP than in the wheat-based diet (WAP) (0.92 and 0.92 vs. 0.85, p < 0.001); however, DM digestibility was similar (p > 0.05) between all three diets when vegetable proteins were fed to pigs rather than animal proteins (0.83, 0.82 and 0.80 for diets MGVP, LGVP, and WVP, respectively). A similar interaction occurred between cereal type and protein source for the CTTAD of energy, with diets MGAP and LGAP having the highest coefficients compared to diet WAP (0.92 and 0.91 vs. 0.83, p < 0.001) (Table 5). In general, the CTTAD for starch was very high in all diets (range 0.989 to 0.999) and higher (main effect, p < 0.001) in both rice-based diets than in the wheat-based diets. The CTTAD for starch was higher in diet WVP than in diet WAP (0.993 vs. 0.989), which resulted in a significant interaction (p < 0.001). The CTTAD for CP was higher in pigs fed diet WVP compared to those fed diets MGVP and LGVP (0.76 vs. 0.67 and 0.66, respectively, p = 0.016) (Table 5). 4. Discussion The hypotheses proposed in this study were, in general, supported by the experimental findings. Feeding vegetable proteins (as a main effect) showed a tendency to reduce faecal shedding of b-haemolytic E. coli in the first 8 days after weaning compared to pigs fed animal protein sources, as evidenced by the greater faecal swab score recorded in pigs fed diets LGAP and WAP. This suggests that in these two cereal sources, the presence of vegetable proteins reduced intestinal colonisation of pathogenic E. coli. Within a few days of piglets being weaned, enterotoxigenic and (or) enteropathogenic strains of E. coli may proliferate within the intestinal tract and induce diarrhoea . Virtually all E. coli strains that cause PWD produce an alpha-haemolysin and show characteristic haemolysis on blood agar. These haemolytic strains also produce virulence factors that allow them to adhere to enterocytes (e.g., F4; F18; AIDA (Adhesin Involved in Diffuse Adherence)), and they generate one or more toxins (e.g., stable toxin (ST)a; STb; labile toxin (LT); Enteroaggregative E. coli heat-stable enterotoxin (EAST1)) that are responsible for inducing the diarrhoea . In the current study, we did not investigate the individual attributes of the recovered E. coli strains but relied instead on the presence of characteristically strong b-haemolysis as a marker of strains capable of causing PWD. Feeding extruded rice failed to translate into a reduced number of therapeutic antibiotic treatments given for clinical diarrhoea as this was statistically the same for all treatments, although it was evident that pigs fed both rice-based diets with vegetable proteins received fewer antibiotic administrations than pigs fed diets WAP or WVP. The incidence of PWD was generally low in this study, which contributed to the lack of statistically significant differences in these indices between diets. These results concur in part with those reported previously, where no correlation was found between faecal shedding of b-haemolytic E. coli and the number of antibiotic treatments required for PWD in the first 14 days after weaning in pigs fed either rice- or wheat-based diets , suggesting that while the presence and activity of enterotoxigenic E. coli (ETEC) are of central importance in the aetiology of PWD, dietary, and (or) physiological contributions also may have an important impact on disease expression in post-weaned piglets. The vegetable proteins used in the current study contained considerable levels of soluble non-starch polysaccharides (NSP) and oligosaccharides, which have been shown to exacerbate the shedding of E. coli and cause diarrhoea . Data from the present study showing an ameliorative effect of feeding vegetable proteins on faecal shedding of b-haemolytic E. coli suggesting that the types and quantities of dietary fibre (DF) fed in the postweaning period have a significant impact on the expression of diarrhoea. In this regard, data from the current study contrast with previous work using rice-based diets where the addition of different sources of DF increased the number of antibiotic injections required for the treatment of diarrhoea . In some previous studies, increased diarrhoea and faecal E. coli scores have been observed in pigs fed very highly digestible rice-based diets after weaning , but the use of a (mostly) insoluble NSP source such as oat hulls was found to ameliorate the condition . Oat hulls included in an extruded rice-based diet with animal proteins decreased total biogenic amine concentrations commensurate with lower plasma urea concentrations , suggesting that oat hulls were able to decrease diarrhoea where a misbalance of carbohydrate to protein entering the hindgut may occur. Alternatively, adding oat hulls to a rice-based diet might not influence fermentation behaviours in the large intestine due to its highly insoluble and lignified nature, but rather, it may have modified motility and transit time of digesta that, in turn, reduced the availability of substrate for bacterial growth . While DF was added to diets in the current study either in the forms of vegetable proteins or wheat rather than oat hulls, the observations are generally consistent with the current industry philosophy that the inclusion of fibre sources in postweaning diets aids in reducing the proliferation of pathogenic bacteria such as ETEC . Weaned piglets fed extruded medium-grain rice or long-grain rice performed similarly to pigs fed wheat in the first 3 weeks after weaning, indicating that extruded rice can replace wheat as the sole cereal in piglet diets after weaning. These data are consistent with previous studies and reinforce the excellent nutritional value of rice for young pigs, especially with mild cooking that can enhance diet digestibility and ileal morphology . Pigs fed rice-based diets also display lighter gastrointestinal organ weights and a greater carcase weight . Vegetable protein sources in the diet depressed growth rate and feed intake and caused a deterioration in FCR in weeks two and three of the study compared to animal protein sources. These data are consistent with numerous previous observations. Weanling pigs fed a mixture of animal proteins (whey-protein concentrate and fish meal) performed better in the 2 weeks after weaning than pigs fed a mixture of plant proteins, including soybean meal, fermented soy protein and rice protein concentrate , commensurate with higher digestibilities of energy, DM, and CP. Moreover, post-weaned pigs fed an increased content of animal protein in an extruded rice-based diet displayed improved performance . Animal proteins are more digestible than vegetable proteins , which are richer in anti-nutritive carbohydrate fractions, and hence more nutrients became available for body growth and development. Seemingly in contrast to some previous findings, Montagne et al. reported no differences in ADG or FCR when pigs were fed diets based on cooked (autoclaved) white rice with either animal or vegetable proteins. The reason(s) for this difference is (are) hard to explain but could be attributable to the fact that these authors infected pigs experimentally with F4:ETEC that could have disturbed the intestinal milieu associated with digestion and absorption, and (or) there was an effect of cooking form on digestibility and subsequent growth rate. Extruded rice contains very low levels of RS, whereas cooked (autoclaved) white rice that is then cooled contains approximately 20 times the RS content of extruded rice . The RS level of the extruded products was not considered in the derivation of the energy value of extruded rice used in the formulation of these diets; hence, there could have been a misbalance in energy contributions between the protein sources and the extruded rice that contributed to the inferior performance of the pigs fed vegetable proteins. In this regard, Montagne et al. found no difference in faecal shedding of b-haemolytic E. coli in pigs fed a wheat and vegetable-proteins-based diet compared to pigs fed medium-grain rice-based diets with either animal or vegetable proteins. This may also reflect the difference in the form (i.e., extrusion vs. autoclaving) of rice used in the different experiments. Be this as it may, the presence of significant interactions between cereal and protein sources for CTTAD in the current study indicates that the dietary component responded differently to both dietary factors. For example, CTTAD for energy was significantly higher in both extruded rice-based diets irrespective of whether animal or vegetable proteins were added compared to diets WAP and WVP, whereas for CP digestion, the significant difference was caused by an apparently higher digestibility in pigs fed diet WVP compared to pigs fed diets MGVP and LGVP. It is difficult to explain the higher CP digestibility in pigs fed diet WVP compared to pigs fed diets MGVP and LGVP, given the higher DM and energy digestibilities observed in the extruded rice-based diets when vegetable protein sources were added. This might be attributable to a higher formation of microbial protein, causing an overall depressed total tract digestibility. Interpretation of total tract apparent digestibility coefficients for CP is fraught regardless because the formation of protein by the microbiota provides no real indication of the ileal digestibility of CP and absorption of amino acids. Rice-based diets fed to piglets after weaning provide a ready form of energy in the form of glucose through starch digestion. In a previous study , the effects of different types of cooked white rice on starch digestion, digesta and fermentation characteristics, shedding of b-haemolytic E. coli, and performance after weaning were examined. Pigs received one of three rice-based diets: (i) medium-grain, (ii) long-grain, and (iii) waxy, all with animal protein sources, and a fourth diet contained mainly wheat, barley, and Australian sweet lupins. The apparent digestibility of starch measured in the ileum 14 d after weaning was highest in the medium-grain and waxy rices containing the lower amylose contents and lowest, but the same, with the other two cereal sources, similar to findings in the current study, albeit that digestibility was measured in faeces. Starch digestibility in faeces was highest in all rice diets, and digesta viscosity was highest in pigs fed the wheat-based diet in both the ileum and caecum . 5. Conclusions Weaned piglets fed the extruded rice-based diets with either animal protein sources or vegetable protein sources performed equivalently to pigs fed either of the wheat-based diets in the first 3 weeks after weaning, confirming that extruded rice is an excellent cereal for young pigs. The use of vegetable proteins compared to animal proteins decreased production performance in weeks two and three after weaning. Vegetable protein sources displayed a trend to reduce faecal E. coli shedding, as evidenced by lowered faecal scores, implicating a role for implicated a role for DF sources in PWD. The CTTAD of dietary components differed according to interactions between cereal and protein sources, although the CTTAD of DM, starch and energy was generally improved by the use of extruded rice compared to wheat and was increased by the use of animal rather than vegetable proteins. Acknowledgments Appreciation is extended to Bob Davis and Richard Seaward of the Medina Research Centre, Department of Agriculture and Food WA, and Fiona Cavaney of Murdoch University for technical assistance. Author Contributions Conceptualisation and methodology, J.R.P., J.C.K., B.P.M., and D.J.H.; Data curation, J.R.P. and J.C.K.; Formal analysis and investigation, J.R.P. and J.C.K.; Funding acquisition and visualisation, J.R.P. and D.J.H.; Project administration, J.R.P. and J.C.K.; Resources, J.R.P., J.C.K., B.P.M. and D.J.H.; Writing--original draft, J.R.P.; Writing--review and editing, J.R.P., J.C.K., B.P.M. and D.J.H. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The present study was approved by the Murdoch University Animal Ethics Committee and the Animal Ethics and Experimentation Committee of the WA Department of Agriculture (Number 05-02-22). All procedures were performed according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Informed Consent Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. animals-13-00863-t001_Table 1 Table 1 Composition of the experimental diets (g/kg as-fed). Medium-Grain Rice Long-Grain Rice Wheat Ingredient Animal Protein Vegetable Protein Animal Protein Vegetable Protein Animal Protein Vegetable Protein Rice 705.6 528.4 705.6 528.4 - - Wheat - - - - 780 532.8 Meat and bone meal 51.6 - 51.6 - 50 - Whey 100 - 100 - 50 - Bloodmeal 30 - 30 - 25 - Fishmeal 100.4 - 100.4 - 50 - Sweet lupins - 100 - 100 - 100 Canola meal - 150 - 150 - 150 Full-fat soybean meal - 185.2 - 185.2 - 151.6 Canola oil 5 - 5 - 28.3 30 L-lysine 2.78 6.4 2.78 6.4 6.04 6.84 DL-methionine 0.36 1.12 0.36 1.12 1.2 1.47 L-threonine 1.43 2.68 1.43 2.68 2.7 2.54 L-tryptophan 0.28 0.34 0.28 0.34 0.42 0.23 Choline chloride 0.4 0.4 0.4 0.4 0.4 0.4 Dicalcium phosphate - 18.7 - 18.7 0.7 17 Limestone - 4.4 - 4.4 - 5.2 Salt 1 1 1 1 1 1 Premix 1 0.7 0.7 0.7 0.7 0.7 0.7 TiO2 2 1 1 1 1 1 1 Calculated analysis: DE (MJ/kg) 15.3 15.4 15.3 15.4 15.0 15.3 CP, g/kg 200 200 200 200 197 215 SID lysine, % 1.30 1.31 1.30 1.31 1.28 1.30 Calcium % 1.2 0.8 1.2 0.8 0.91 0.8 Available P, % 0.6 0.45 0.6 0.45 0.49 0.45 1 Provided the following nutrients (per kg of air-dry diet): vitamins: A 1500 IU, D3 300 IU, E 37.5 mg, K 2.5 mg, B1. 1.5 mg, B2 6.25 mg, B6 3 mg, B12 37.5 mg, calcium pantothenate 25 mg, folic acid 0.5 mg, niacin 30 mg, biotin 75 mg; minerals: Co 0.5 mg (as cobalt sulfate), Cu 25 mg (as copper sulfate), iodine 1.25 mg (as potassium iodine), iron 150 mg (as ferrous sulfate), Mn 100 mg (as manganous oxide), Se 0.5 mg (as sodium selenite), Zn 0.25 mg (as zinc oxide) (Hogro Bronze Weaner and Grower, Rhone-Poulenc Animal Nutrition Pty Ltd., Queensland, Australia).2 Titanium dioxide (TiO2; Sigma Chemical Company, St. Louis, MO, USA). animals-13-00863-t002_Table 2 Table 2 Interaction means for the number of antibiotic treatments and the faecal swab score for piglets offered different diets after weaning. Dietary Treatment Number of Antibiotic Treatments Faecal Swab Score 2 Cereal Type Protein Source Medium-grain rice Animal 1.4 0.5 Vegetable 0.7 0.7 Long-grain rice Animal 1.0 0.6 Vegetable 0.6 0.3 Wheat Animal 1 0.8 Vegetable 1 0.3 Pooled mean 0.9 0.5 SEM 1 0.072 0.30 Probability, p = Cereal type 0.792 0.461 Protein source 0.230 0.057 Cereal x Protein 0.638 0.069 1 SEM: standard error of the mean. 2 Faecal swab score is the mean score per pig determined from swabs taken on days 2, 5, 6, and 8 after weaning. animals-13-00863-t003_Table 3 Table 3 Interaction means for average daily gain (ADG) of pigs kept in groups in the first week after weaning. Dietary Treatment Start Liveweight, kg Liveweight after 7 Days, kg ADG, g Cereal Type Protein Source Medium-grain rice Animal 6.71 7.32 87 Vegetable 6.66 7.04 54 Long-grain rice Animal 6.68 7.31 91 Vegetable 6.69 6.98 42 Wheat Animal 6.97 7.46 55 Vegetable 6.81 7.03 32 Pooled mean 6.73 7.15 60 SEM 1 0.131 0.177 110.2 Probability, p = Cereal type 0.900 0.991 0.625 Protein source 0.952 0.465 0.152 Cereal x Protein 0.996 0.984 0.903 1 SEM: standard error of the mean. animals-13-00863-t004_Table 4 Table 4 Interaction means for the performance of pigs fed different diets in weeks two and three of the experiment. Dietary Treatment Liveweight at Start of Week 2, kg Liveweight at End of Week 3, kg ADG, g ADFI, g Day-1 FCR (g Feed:g Gain) Cereal Type Protein Source Medium-grain rice Animal 7.31 11.66 311 586 1.92 Vegetable 6.98 10.39 243 527 2.69 Long-grain rice Animal 7.32 11.69 312 586 1.98 Vegetable 7.04 9.97 219 488 2.32 Wheat Animal 7.46 12.06 329 569 1.71 Vegetable 7.03 10.71 264 485 1.92 Pooled mean 7.15 11.07 278 540 2.13 SEM 1 0.170 0.284 10.4 15.1 0.122 Probability, p = Cereal type 0.981 0.723 0.330 0.712 0.191 Protein source 0.322 0.010 <0.001 0.007 0.028 Cereal x Protein 0.987 0.942 0.803 0.874 0.545 1 SEM: standard error of the mean. animals-13-00863-t005_Table 5 Table 5 The CTTAD of dry matter (DM), starch, energy and crude protein (CP) in pigs fed different diets after weaning. Dietary Treatment CTTAD of: Cereal Type Protein Source DM Starch Energy CP Medium-grain rice Animal 0.92 0.999 0.92 0.79 Vegetable 0.83 0.998 0.82 0.67 Long-grain rice Animal 0.92 0.999 0.91 0.78 Vegetable 0.82 0.997 0.81 0.66 Wheat Animal 0.85 0.993 0.83 0.78 Vegetable 0.80 0.989 0.80 0.76 Pooled mean 0.86 0.996 0.85 0.74 SEM 1 0.006 0.0012 0.006 0.009 Probability, p = Cereal type <0.001 <0.001 <0.001 0.022 Protein source <0.001 0.837 <0.001 <0.001 Cereal x protein <0.001 0.016 <0.001 0.016 1 SEM: standard error of the mean. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000031

In recent years, therapy dogs in medical and assisted living facilities have become popular in Japan, and the demand for such dogs has increased. However, some owners have their dogs take this test, which evaluates the dog's talent, without understanding what is required of the test. The system needs to teach owners in an understandable way whether their dog is suitable to become a therapy dog so that the owners can determine if their dog is ready to be tested. Therefore, we suggest that easy at-home testing is likely to encourage dog owners to apply for their dog to take the aptitude test. If more dogs take the test, more therapy dogs can be born. The purpose of this study was to identify the personality traits of therapy dogs that pass the aptitude test by using the Canine Behavior Assessment and Research Questionnaire (C-BARQ). The C-BARQ was administered to dogs that previously passed the aptitude test for therapy training at the Hokkaido Volunteer Dog Association, assessing their behavioural displays. A factor analysis was conducted for each questionnaire item, and a total of 98 items were analyzed. Data were collected from the results of 110 dogs encompassing 30 dog breeds, with the most common breeds including Labrador Retrievers, Golden Retrievers, and Toy Poodles. Factor analysis revealed that 14 extracted factors should be evaluated. Given these personality traits and the fact that breed and age did not influence aptitude, we believe that a variety of dogs have the potential to become therapy dogs. animal-assisted intervention C-BARQ dogs factor analysis This research received no external funding. pmc1. Introduction Dogs have long been familiar to humans and are animals that can provide psychological healing through their interactions with people . In recent years, this healing effect has been scientifically demonstrated in several animal species, and awareness of animal-assisted intervention (AAI) has increased . Interactions with dogs or other animal species are used in medical and assisted living facilities (i.e., hospitals and nursing homes) to improve people's quality of life (QOL) . A variety of animals, from horses to dolphins, have been used in animal therapy, but dogs have become mainstream in recent years . Specific effects of animal therapy have been reported, including decreased progression of depression , stabilization of blood pressure , and increased motivation to socialize . In expectation of these effects, many hospitals and facilities have introduced dog visits, and the demand for these services has been increasing. AAI can be divided into three categories according to its methods. The first is animal-assisted therapy (AAT). This is a type of subsequent therapy led by medical professionals. The second is animal-assisted education (AAE). This refers to activities that involve animals in educational fields. The third is animal-assisted activity (AAA), which involves activities that do not have a set therapeutic purpose and are mainly focused on interacting with dogs with the aim of improving the QOL of the subjects . Currently, AAA is the most common activity in Japan, and the dogs involved are generally called "therapy dogs" there. In Japan, there is no legal certification for a therapy dog, but dogs and handlers with a "therapy dog certification" are active in this field. To become certified, dogs must pass the Therapy Dog Aptitude Test. In this test, conducted by the Hokkaido Volunteer Dog Association, candidates are evaluated on the following content: gentleness of the dog's reactions to other dogs, a person touching the dog's body, loud sounds, and appearance of wheelchairs and obedience to the sit command and to walk at their owner's side. If the dog barks, engages in any dangerous behavior, urinates or defecates during the test, the test is terminated immediately . Opportunities for this test are limited to twice a year, and the aptitude test seems to be difficult for most dogs. Therefore, it is challenging to recruit a large number of candidate therapy dogs. To provide a wider selection of therapy dogs, a new selection system is required that encourages owners to have their dogs take an aptitude test. This study presents an instant test that can be conducted before the aptitude test to allow dog owners to judge at home whether their dogs have the potential to be therapy dogs. There are some tests that judge dogs' personality by questionnaires. The Canine Behaviour Assessment and Research Questionnaire (C-BARQ) is a well-known dog personality test that allows owners to evaluate their dogs. The C-BARQ is also applied for therapy dog assessment in other countries . We suggest that the C-BARQ could be a useful tool for identifying dogs with suitable characteristics to be therapy dogs. Based on the above background, the purpose of this study is to assess whether the C-BARQ could be used at home by owners to see whether their pet dog has the right characteristics to pass the aptitude test. In this study, we extracted the personality factors of certified therapy dogs through a questionnaire survey using the C-BARQ. 2. Materials and Methods 2.1. Dogs Dogs that passed the Hokkaido Volunteer Dog Association's therapy dog aptitude test and were currently or had previously been involved in therapy dog activities were surveyed with regard to their behaviour patterns under various circumstances. The dogs included were active dogs, retired dogs, and deceased dogs. For deceased dogs, owners were asked to recall the dog's behavior when answering the questionnaire. This aptitude test assesses only the dog's aptitude, not that of the owners. Dogs that pass the aptitude test must be retested every two years, because their temperament can change. Contact information of people with dogs that had passed the aptitude test was obtained with the help of the Hokkaido Volunteer Dog Association. The Hokkaido Volunteer Dog Association is a non-profit organization (NPO) established in 1996 that visits various places, including medical institutions and assisted living facilities (i.e., hospitals and nursing homes) . 2.2. Questionnaire The C-BARQ was used for the survey (version released in 2020). It was developed by the University of Pennsylvania researchers Serpell et al. in 2003 and has been used in many behavior analysis studies as a diagnostic index of canine personality . The questionnaire consists of 101 questions divided into 13 categories: stranger-directed aggression, owner-directed aggression, dog-directed aggression/fear, trainability, chasing, stranger-directed fear, nonsocial fear, dog-directed fear, separation-related behavior, touch sensitivity, excitability, attachment/attention-seeking, and energy level. Surveys were conducted twice, once in July 2020 via direct e-mail to 112 dog owners, and again in October 2021 via direct e-mail to 115 dog owners. The responding owners rated and answered the C-BARQ questions on a five-point scale from 0 to 4. Other basic information was gathered including the dog's name, date of birth, and breed. This study was conducted with the approval of the Ethical Regulations for Animal Experiments of Rakuno Gakuen University (VH19B9). 2.3. Factor Analysis Methods After compiling the collected data into a spreadsheet, a factor analysis was conducted on the questionnaire items. The statistical software R ver. 3.5.3. (The R Foundation, Vienna) was used for factor analysis. Samples with a response rate of at least 75% to the survey questions and questions with an overall response rate of at least 85% were used in the factor analysis . The samples obtained from these criteria were used to determine the number of factors based on the MAP (Minimum Average Partial) criteria. After estimating the factor loadings by the minimum residual method, oblique rotation of the axes was performed by the oblimin method. Items with factor loadings of 0.5 or higher and commonality of 0.3 or higher were selected, and the factors obtained were named. 3. Results In this study, therapy dog aptitude factors were extracted from dogs that passed the therapy dog aptitude test using the C-BARQ questionnaire. 3.1. Questionnaire Collection Rate A total of 114 questionnaires were collected as a result of the aggregated first- and second-round questionnaire data. Four questionnaires with insufficient data were excluded; thus, data from 110 dogs were used for factor analysis. The collection rate was 59.7%. Of these, 63 were dogs from multi-dog homes. 3.2. Dog Breed and Age Distribution The three most common breeds of therapy dogs in the Hokkaido Volunteer Dog Association were represented: the Labrador Retriever (LR), Golden Retriever (GR), and Toy Poodle (TP). Other breeds included mixed breed, Shih Tzu, Miniature Dachshund, Cavalier King Charles Spaniel, Yorkshire Terrier, Samoyed, Beagle, Miniature Poodle, Miniature Schnauzer, Shetland Sheepdog, Border Collie, Shiba Inu, Maltese, Papillon, Newfoundland, Standard Poodle, Boston Terrier, Australian Labradoodle, Pembroke Welsh Corgi, Pomeranian, Chihuahua, Bernese Mountain Dog, Australian Shepherd, Pug, Lowchen, Goldendoodle, and Brussel Griffon, for a total of 27 breeds in addition to the three main ones. Regarding age, the active dogs were between 2 and 15 years old, with a mean age of 8.53 years (+-2.82) . There were 9 retired dogs and 27 deceased dogs, which were not included in the age distribution. 3.3. Results of Factor Analysis In total, data for 110 animals and 98 questions were used for factor analysis. Factor analysis was performed on the 98 questions, and 51 items were selected (Table 1). These 51 items were grouped into 14 factors containing similar items, and named according to the included items. Two of the factors were obtained only from dogs in multi-dog households. The 14 extracted factors (Table 1) were as follows: trainability and obedience, separation-related anxiety behaviors, separation-related physiological reactions, aggression toward people, fear and anxiety toward strangers, fear and anxiety toward dogs, fear and anxiety in unfamiliar situations, territorial aggression, hyperactivity, aggression toward approaching unknown dogs, resource guarding-related aggression, abnormal behavior, excitability, attachment to family members, vigilant aggression toward family dogs, and resource guarding-related aggression toward family dogs. The results of the average scores (Table 2) indicated that therapy dogs tended to score highly on factors of trainability and obedience, low to moderate on attachment behavior to family members, and low on excitability. In contrast, therapy dogs tended to have low scores on the following factors: separation-related anxiety behaviors, separation-related physiological reactions, aggression toward people, fear and anxiety toward strangers, fear and anxiety toward dogs, fear and anxiety in unfamiliar situations, territorial aggression, hyperactivity, aggression toward approaching unknown dogs, resource guarding-related aggression, abnormal behavior, vigilant aggression toward family dogs, and resource guarding-related aggression toward family dogs. 4. Discussion 4.1. Breed, Age, and Housing Environment The fact that dogs of various breeds and a wide range of ages are used as therapy dogs suggests that dogs of many age groups may have aptitude for the role. Regarding breeds, the reason the LR, GR, and TP are the top three might be related to each dog breed's personality and popularity. LRs and GRs show low aggression toward people and dogs, and high trainability . The TP is the most popular dog breed in Japan and is considered "faithful and trainable" in terms of personality, which may have helped place it at the top of the list. Regarding the age distribution of the therapy dogs, the mean age was higher than that of general house dogs . The age distribution was also higher than that of general dogs, which may be due to the lack of new therapy dogs, especially the lack of young individuals. Additionally, 55.3% of the therapy dogs were kept in multi-dog households. Kubiny et al. reported that "more experience with prior dogs is associated with higher calmness", which suggests dogs in multi-dog households may have suitable temperaments for animal-assisted intervention. 4.2. Extracted Factors and Their Relevance to the Therapy Dog Temperament Test The factors were divided into two groups: one was included in the aptitude testing, and the other was not included in the test. 4.2.1. Factor Content Tested in the Aptitude Test For high "trainability and obedience", dogs must obey their owner's command to "sit" and walk the course with the owner properly. If the dog's training and obedience are at a low level, the dog may act selfishly due to a lack of control, and therapy activity cannot be carried out smoothly. Therapy animals are required to be obedient to their handler's commands while paying constant attention to them . For low levels of "aggression toward people" and "fear and anxiety toward strangers/dogs/unfamiliar situations", the dogs should not show aggression or fear/anxiety toward "any dogs, ordinary people or suspicious people", "approaching any dogs, ordinary people or suspicious people" or "a person who touches any part of their body" in an unfamiliar place (the testing room). Touching by residents is an essential part of AAI activity in care facilities, but sometimes the residents grasp the dog firmly. The dogs were evaluated for their reaction to simulated residents, especially those in wheelchairs or with canes. In addition, the locations of activities vary, and unfamiliar situations elicit fear and anxiety in dogs. If these factor scores are high, the dog is not suitable as a therapy dog. In the worst-case scenario, the dog will hurt people as a defensive behavior during the activity. Therapy dogs are required to have a temperament that allows them to "remain calm and gentle, and prefer to stay close by people", be "adaptable to unfamiliar situations such as novel scenes, sounds, or smells", "accept unfamiliar persons without fear", "ignore neutral dogs" and "never show aggressive behavior" . 4.2.2. Factors Not Included in the Aptitude Test With regard to low levels of "separation-related anxiety behaviors" and "separation-related physiological reactions", dogs with separation anxiety and separation-related physiological reactions may have stressful feelings in their ordinary life and may feel anxiety in unfamiliar situations. Gerrard et al. noted that separation anxiety is significantly related to excessive attachment to owners. Neither attachment to household members nor separation anxiety should be high in therapy dogs. In terms of low levels of "territorial aggression", dogs that show territorial aggression may show aggression toward other dogs or people who approach them. For low levels of "hyperactivity", if a dog cannot control itself and reacts excessively, it may not be able to pay proper attention to the owner's commands. If the dog has high hyperactivity, people may feel uncomfortable and may be injured. It is necessary for therapy dogs to regain control even after playing or excitement . Low levels of "resource guarding-related aggression" are also necessary. A dog that exhibits aggressive behavior to avoid having food or other necessary items taken away is highly unlikely to be suitable as a therapy dog, since the dog may have a strong tendency toward anxiety . Low levels of "aggression toward approaching unknown dogs" are also necessary; therapy dogs should not show aggression regardless of the proximity of other dogs. With regard to low levels of "abnormal behavior", compulsive behavior and instinctual drive disorder may disturb peaceful contact with people in a care home. Low levels of "vigilant aggression toward family dogs" and "resource guarding-related aggression toward family dogs" are necessary; among the therapy dogs in the present study, more than half (55.3%) were in multi-dog households. Mild "excitability" is necessary for therapy dogs. Dogs show excitement in response to playing, walking outside, food rewards, and hunting instinct stimuli, as well as owners returning home and car rides. However, dogs with high excitability behave intensely and are at risk of injuring people in care homes. It is necessary for therapy dogs to retain control even during excitable moments . With regard to mild to moderate "attachment behavior to family members", high-attachment dogs, such as those that sleep with their owners, tend to show separation anxiety and aggression , or tend to show attention-seeking behavior and separation anxiety . If the degree of attachment of a pet dog to its owner is low, "aggressive behavior" is often observed . This suggests that a mild to moderate level of attachment is necessary because attachment that is too high leads to behaviors such as barking, scratching and urination. These extracted factors are the aspects of temperament that owners should check to determine whether their dogs may be candidate to act as therapy dogs. Therefore, if the C-BARQ could be placed on the website of the Hokkaido Volunteer Dog Association, owners could independently evaluate whether their dogs have suitable temperaments to be therapy dogs. This might lead to an increase in the number of dogs that take the aptitude test. Furthermore, the results showed that low aggression is important for therapy dogs. However, some owners are not aware that a lack of aggression is a prerequisite for becoming a therapy dog . Therefore, the C-BARQ may help owners to understand the requirements of therapy dogs and to train their dogs to pass the therapy dog aptitude test. In addition, only some of the factors are included in the therapy dog aptitude test. Thus, the current test method does not fully evaluate therapy dog aptitude. Therefore, the C-BARQ may also be used as an initial screening instrument for the therapy dog aptitude test to better select dogs suitable for becoming therapy dogs. Furthermore, ordinary dog owners can complete the entire C-BARQ, which includes these questions. Uninterested owners can be directed to AAA. Additionally, the expression of temperament (i.e., behavioral displays) can change over time, despite the general perception that temperament is genetic and thus remains consistent over time, activities and environment. Therefore, therapy dogs must renew their certification every two years. These factors must be interpreted for future therapy dogs as well as active dogs. Since only dogs that passed the therapy dog aptitude test were included in this study, future studies should compare their C-BARQ scores with those of dogs that failed the therapy dog aptitude test to obtain more substantial results. 5. Conclusions As part of our efforts to promote therapy dogs, we used the C-BARQ to identify the characteristics of therapy dogs. We extracted factors that appear to indicate which dogs have the potential to become therapy dogs. Based on these factors, we believe that the C-BARQ will contribute to increasing the number of dog owners who take therapy dog aptitude tests by allowing owners to independently evaluate whether their dogs have suitable temperaments to become therapy dogs. The C-BARQ may also help owners to understand the requirements of therapy dogs and to train their dogs to pass the therapy dog aptitude test. Acknowledgments We are grateful to Takase, Chairman of the Board of Directors, Takahashi, Secretary General, and all members of the NPO Hokkaido Volunteer Dog Association for their cooperation in the questionnaire survey. Author Contributions The idea for the article was conceived by T.K. The experiments were designed by T.K. The survey was performed by M.S., M.I., Y.N. and T.K. The data were analyzed by M.S., M.I. and Y.N. The article was written by M.I. and M.S. and reviewed by T.K. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethical Committee of Rakuno Gakuen University, Hokkaido, Japan (VH19B9). Informed Consent Statement Informed consent was obtained from all owners of the animals who belong to the NPO Hokkaido Volunteer Dog Association involved in the study. Data Availability Statement The data presented in this study are not publicly available. Please contact the corresponding author with any enquiries. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Age distribution of 74 active dogs. animals-13-00834-t001_Table 1 Table 1 Named factors of therapy dog talent and their original questions with factor scores. Factors and Questionnaires Score Mean +- SD Factor Loadings Factor Contribution Factor Contribution Rate Cumulative Contribution Rate Cronbach's a 1. Trainability and obedience 2.7 (+-0.91) 2.052 0.256 -- 0.6 Obeys a stay command immediately. 3.4 (+-0.65) 0.68 When off the leash, returns immediately when called. 3.2 (+-0.81) 0.595 Slow to learn new tricks or tasks. 1.4 (+-0.81) -0.584 2. Separation-related anxiety behaviours 0.4 (+-0.11) 1.89 0.24 0.24 0.71 Restlessness/agitation/pacing. 0.5 (+-1.02) 0.71 Whining. 0.4 (+-0.86) 0.76 Barking. 0.3 (+-0.67) 0.6 3. Separation-related physiological reactions 0.1 (+-0.04) 1.76 0.22 0.46 0.75 Excessive salivation. 0.0 (+-0.21) 0.81 Chewing/scratching at doors, floor, windows, curtains, etc. 0.1 (+-0.40) 0.54 Loss of appetite 0.1 (+-0.47) 0.88 4. Aggression towards people 0.1 (+-0.03) 2.768 0.12 0.482 0.8 When stepped over by a member of the household. 0.0 (+-0.16) 0.804 When approached directly by an unfamiliar child while being walked or exercised on a leash. 0.1 (+-0.30) 0.638 When stared at directly by a member of the household. 0.0 (+-0.13) 0.623 When approached directly by an unfamiliar adult while being walked or exercised on a leash. 0.1 (+-0.28) 0.524 When an unfamiliar person tries to touch or pet the dog. 0.1 (+-0.26) 0.511 5. Fear and anxiety towards strangers, dogs, unfamiliar situations 0.6 (+-0.29) 5.74 0.3 -- 0.87 When approached directly by an unfamiliar male adult while away from the home. 0.2 (+-0.55) 0.73 When approached directly by an unfamiliar child while away from the home. 0.3 (+-0.57) 0.8 When an unfamiliar person tries to touch or pet the dog. 0.2 (+-0.46) 0.7 When approached directly by an unfamiliar dog of the same or larger size. 0.6 (+-0.77) 0.75 When approached directly by an unfamiliar dog of a smaller size. 0.4 (+-0.72) 0.67 When first exposed to unfamiliar situations (e.g., first car trip, first time in elevator, first visit to veterinarian, etc.). 0.9 (+-1.03) 0.76 In response to wind or wind-blown objects. 0.5 (+-0.94) 0.65 When having nails clipped by a household member. 0.9 (+-1.11) 0.51 When barked, growled, or lunged at by an unfamiliar dog. 1.0 (+-1.12) 0.62 6. Territorial aggression 0.3 (+-0.11) 5.28 0.23 0.23 0.91 When strangers walk past the home while the dog is in the yard. 0.3 (+-0.65) 0.923 When mailmen or other delivery workers approach the home. 0.4 (+-0.70) 0.905 Toward unfamiliar persons visiting your home. 0.3 (+-0.59) 0.868 When joggers, cyclists, roller skaters, or skateboarders pass the home while the dog is in the yard. 0.3 (+-0.57) 0.857 When an unfamiliar person approaches the owner or a member of the owner's family at home. 0.2 (+-0.49) 0.712 Toward cats, squirrels, and other animals entering its yard. 0.5 (+-0.76) 0.507 7. Hyperactivity 0.8 (+-0.19) 2.948 0.123 0.123 0.86 Active, energetic, always on the go. 0.9 (+-0.98) 0.805 Playful, puppyish, boisterous. 1.0 (+-1.08) 0.766 Hyperactive, restless, has trouble settling down. 0.5 (+-0.86) 0.707 8. Aggression towards approaching unknown dogs 0.4 (+-0.0) 3.036 0.132 0.362 0.99 When approached directly by an unfamiliar male dog while being walked or exercised on a leash. 0.4 (+-0.71) 0.953 When approached directly by an unfamiliar female dog while being walked or exercised on a leash. 0.4 (+-0.73) 0.951 9. Resource guarding-related aggression 0.1 (+-0.44) 2.274 0.099 0.581 -- When food is taken away by a member of the household. 0.1 (+-0.44) 0.618 10. Abnormal behavior 0.6 (+-0.02) 2.868 0.12 0.242 0.5 Licks people or objects excessively. 0.6 (+-0.84) 0.619 Rolls in animal droppings or other "smelly" substances. 0.5 (+-0.89) 0.554 11. Excitability 1.5 (+-0.09) 3.226 0.538 -- 0.87 When visitors arrive at its home. 1.6 (+-1.14) 0.838 Just before being taken for a walk. 1.5 (+-1.13) 0.793 Just before being taken on a car trip. 1.6 (+-1.18) 0.78 When you or other members of the household come home after a brief absence. 1.4 (+-1.14) 0.713 When playing with you or other members of your household. 1.4 (+-1.01) 0.637 When the doorbell rings. 1.6 (+-1.36) 0.61 12. Attachment behaviour to family members 1.8 (+-0.49) 2.629 0.438 -- 0.8 Tends to sit close to or in contact with a member of the household when that individual is sitting down. 2.4 (+-1.21) 0.83 Tends to nudge, nuzzle, or paw a member of the household for attention when that individual is sitting down. 1.7 (+-1.19) 0.733 Becomes agitated when a member of the household shows affection for another dog or animal. 1.0 (+-1.26) 0.676 Tends to follow a member of household from room to room about the house. 1.9 (+-1.26) 0.636 13. Vigilant aggression towards family dogs 0.2 (+-0.01) 1.689 1.69 0.42 0.89 When approached at a favorite resting/sleeping place by another (familiar) household dog. 0.2 (+-0.70) 1.015 Towards another (familiar) dog in your household. 0.3 (+-0.64) 0.773 14. Resource guarding-related aggression towards family dogs 0.4 (+-0.07) 1.340 1.34 0.76 0.78 When approached while playing with/chewing a favorite toy, bone, object, etc., by another (familiar) household dog. 0.5 (+-0.73) 0.914 When approached while eating by another (familiar) household dog. 0.3 (+-0.64) 0.67 Score Mean +- SD: The response scores (0-4) for each factor and question are averaged, and SD indicates standard deviation. Factor Loadings: Value indicating the extent to which each question is reflected in the factor. Factor Contribution: The square of the factor loading indicates the amount of variance in the observed variable that the factor can explain. Factor Contribution Rate: Factor contribution divided by number of items. Cumulative Contribution Rate: Cumulative total of factor contributions. Cronbach's a: reliability factor. animals-13-00834-t002_Table 2 Table 2 Trends in each factor of therapy dog temperament traits. Factor Trend Trainability and obedience High Attachment behaviour to family members Mild to moderate Excitability Mild Separation-related anxiety behaviours Low Separation-related physiological reactions Low Aggression towards people Low Fear and anxiety towards strangers, dogs, unfamiliar situations Low Territorial aggression Low Hyperactivity Low Aggression towards approaching unknown dogs Low Resource guarding-related aggression Low Abnormal behaviour Low Vigilant aggression towards family dogs Low Resource guarding-related aggression towards family dogs Low Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000032

(1) Background: Devriesea (D.) agamarum is a potential cause of dermatitis and cheilitis in lizards. The aim of this study was to establish a real-time PCR assay for the detection of D. agamarum. (2) Methods: Primers and probe were selected targeting the 16S rRNA gene, using sequences of 16S rRNA genes of D. agamarum as well as of other bacterial species derived from GenBank. The PCR assay was tested with 14 positive controls of different D. agamarum cultures as well as with 34 negative controls of various non-D. agamarum bacterial cultures. Additionally, samples of 38 lizards, mostly Uromastyx spp. and Pogona spp., submitted to a commercial veterinary laboratory were tested for the presence of D. agamarum using the established protocol. (3) Results: Concentrations of as low as 2 x 104 colonies per mL were detectable using dilutions of bacterial cell culture (corresponding to approximately 200 CFU per PCR). The assay resulted in an intraassay percent of coefficient of variation (CV) of 1.31% and an interassay CV of 1.80%. (4) Conclusions: The presented assay is able to detect D. agamarum in clinical samples, decreasing laboratory turn-around time in comparison to conventional culture-based detection methods. Devriesea agamarum polymerase chain reaction Uromastyx sp. Pogona vitticeps bearded dragon lizard reptile cheilitis dermatitis Laboklin GmbH & CO KGThe research received support in the form of salaries for authors [M.B., C.L., M.G., R.E.M.] from Laboklin GmbH & CO KG, a veterinary laboratory offering diagnostic services, including bacteriological and molecular biological examinations. The author E.M. is the head of the funding company. pmc1. Introduction Devriesea (D.) agamarum is a bacterial species known to cause dermatitis and cheilitis in lizards. Disease has most often been described in Uromastyx spp. . However, D. agamarum can also infect other lizards . It has been reported in captive as well as in free-ranging lizards . Clinical signs of disease generally include dermatitis or cheilitis, often described with a yellow crusty appearance . Disease outbreaks with extensive mortality have also been reported, especially if lizards developed septicaemia. . Bearded dragons have been described to asymptomatically carry D. agamarum in their oral cavities . Treatment of affected animals usually includes debridement of dermal lesions and systemic use of antibiotics--especially cephalosporines are considered effective --and may also require disinfection of the enclosure . Autovaccines have also been discussed as a treatment method . Therefore, a fast and reliable diagnostic approach is an important consideration, both in clinical disease with suspected D. agamarum infection and in entry controls. D. agamarum is relatively easily cultured at 37 degC but also grows at temperatures of 25-42 degC on Columbia agar with 5% sheep blood . Diagnosis can, however, be complicated in laboratories with limited experience with this pathogen. Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry (MALDI-TOF MS), one of the most frequently used standard techniques for the identification of bacteria in routine laboratory diagnostics, may not (yet) be able to identify D. agamarum when working with standardized databases . However, this issue is likely to be overcome as databases expand. Currently, laboratories can improve D. agamarum identification by implementing and expanding their own MALDI-TOF MS databases or using 16S rRNA gene sequencing to identify cultured but unidentified isolates. Another option would be a specific PCR assay for D. agamarum, which might prove especially valuable if other infectious agents, such as viral or fungal pathogens, are also suspected, allowing concurrent testing from the same sample. The aim of this study was, therefore, to develop a PCR assay for the detection of D. agamarum. 2. Materials and Methods 2.1. Bacterial Isolates Used to Establish the qPCR In total, 14 D. agamarum isolates were used in this study as positive controls. Three D. agamarum isolates (GenBank accession numbers: MT664091-93) were obtained from routine diagnostic submissions at Laboklin GmbH & Co. KG (Bad Kissingen Germany) in 2019 , while 11 were isolated between 2005 and 2009 at the Faculty of Veterinary Medicine, Ghent University (Table 1). Non-D. agamarum isolates (n = 34) were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Some were included in order to determine the ability of the assay to exclude a broad spectrum of different bacterial species. Others, like Brachybacterium sp. or Dermabacter sp., were included as their sequences were described to be highly similar to D. agamarum (Table 2). These 34 isolates served to determine the specificity of the PCR. 2.2. DNA Preparation Pure cultures of each strain were incubated in 750 mL lysis buffer (MagNA Pure DNA Tissue Lysis Buffer, Roche, Mannheim, Germany) and 75 mL proteinase K (proteinase K, lyophilisiert, >=30 U/mg, Carl Roth GmbH & Co KG, Karlsruhe, Germany) for one hour at 65 degC. From this, 200 mL were utilized for automated nucleic acid (NA) extraction using the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. The resulting NAs were eluted in a volume of 100 mL. The isolated NAs were kept at -18 degC until the PCR tests were performed. The DNA used for the dilution series was extracted manually using the QIAamp(r) DNA MicroKit (50) (Qiagen, Hilden, Germany), and the DNA concentration was measured with a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, Inc., Wilmington, NC, USA). 2.3. Design of Primers and Oligonucleotide Probe, PCR Protocol and Optimisation of Annealing Temperature The 16S rRNA gene was selected as the target region based on the availability of sequence data from a variety of isolates. Sequences of D. agamarum (NZ_LN849456.1, LN849456.1, NR_044368.1, EU009865.1, KF647330.1) were retrieved from GenBank, and multiple sequence alignment was performed with other sequences of different bacterial species (e.g., Agromyces species, Arthobacter species, Brachybacterium species, Dermabacter species, Pseudomonas species) using MUSCLE last accessed on 20 February 2023). The primers and probe were designed using primer3 last accessed on 20 February 2023). Reactions included 1.0 mL of each primer (10 mM), 1.0 mL of the probe (2 mM), 4.0 mL DNA Process Control Detection Kit qPCR Reaction Mix and 5.0 mL template DNA in a total volume of 20.0 mL. Amplification was performed with a LightCycler 96 (Roche, Mannheim, Germany) in a 96-well format. The following protocol was used: Preincubation at 95 degC for 30 s followed by 40 cycles of two-step-amplification (95 degC for 5 s and 60 degC for 30 s). PCR-grade water (Roche, Mannheim, Germany) served as a negative control. While all other bacterial samples were negative, Dermabacter hominis produced positive PCR results at an annealing temperature of 60 degC . Therefore, DNA of Dermabacter hominis (DSM 30958) from the DSMZ as well as DNA of D. agamarum (GenBank Accession number: MT664092.1/0919Ur) , was tested in duplicate with different annealing temperatures (protocol 1: 65.0 degC, protocol 2: 66.0 degC, protocol 3: 67.0 degC) using a LightCycler 96 (Roche, Mannheim, Germany). 2.4. Determination of Specificity at 66 degC and of Repeatability and Sensitivity of the Assay Various non-D. agamarum bacterial species (Table 2) were tested in duplicate for positive reactions at 66 degC. The following protocol was used: Preincubation at 95 degC for 30 s followed by 40 cycles of two-step-amplification (95 degC for 5 s and 66 degC for 30 s). PCR-grade water (Roche, Mannheim, Germany) served as a negative control. DNA of 14 D. agamarum isolates (Table 1) was also tested. To assess the intraassay repeatability of the PCR, standard deviations were calculated for 10-fold serial dilutions in triplicate on a single plate. For the interassay reproducibility, standard deviations were calculated for a 10-fold serial dilution series which was amplified three times daily for two days. The standard deviations of the CT values were used to calculate the percent of coefficient of variation (CV%). Detection limit for bacterial cell dilutions: In order to determine the sensitivity of the assay, a culture of D. agamarum (isolate MT664091.1/0219Bf) was used, starting with a dilution (D0) of 0.5 McFarland (1.5 x 108 per mL). This suspension (D0) was 10-fold serially diluted (D1-D10) in duplicate, and 1 mL of each dilution was inoculated onto Columbia agar with defibrinated sheep blood (Becton Dickinson GmbH, Heidelberg, Germany/Oxoid GmbH, Wesel, Germany), incubated at 36 degC and checked for growth after 30 h. Colony forming units (CFU) were counted if the result was expected to be between 0 and 300 CFU. These results were then used to determine the limit of detection of the assay. DNA was extracted from 200 mL of each dilution (D1-D10) as described above, and PCR was carried out in duplicate. The detection of Dermabacter hominis (DSM 30958) was quantified in the same way. Detection limit for DNA from pure culture: To evaluate the assay's sensitivity, PCR was carried out in triplicate using serial 10-fold dilutions of DNA prepared from colonies of D. agamarum (isolate MT664091.1/0219Bf). Spiked-in matrix: The assay was also evaluated using a spiked-in matrix (D. agamarum-negative-tested lizard skin with a known concentration of target DNA). For these assays, 10 mL of the above-described dilutions D0 to D6 were inoculated onto D. agamarum-negative-tested lizard skin. The skin was incubated in 500 mL lysis buffer, and 50 mL proteinase K and NA were extracted from 200 mL of this suspension as described above and eluted in a total volume of 100 mL NA. The PCR was carried out in duplicate as described above. 2.5. Testing of Samples Submitted to a Commercial Veterinary Laboratory Clinical samples from lizards for which appropriate material (skin, crusts, dry swab) was submitted to a commercial veterinary laboratory between March 2022 and December 2022 and for which the submitting veterinarian indicated an interest in D. agamarum diagnostics were tested using the established protocol to evaluate the PCR for use with clinical samples. Some of the samples were derived from animals showing clinical signs, others from asymptomatic animals tested in the context of a health check--often when D. agamarum had been isolated from animals in the group previously. If suitable material was available (skin or swab in a transport medium), bacteriology was performed (as previously described ). Identification of isolates was based on growth characteristics on agar plates (Columbia Agar with defibrinated sheep blood and Endo Agar, Becton Dickinson GmbH, Heidelberg, Germany), biochemical parameters and MALDI-TOF MS. In doubt, colonies were also re-checked via PCR. If the results of the bacteriological culture were available, the results of the PCR and culture would be compared. Samples were considered PCR positive if the cycle threshold (CT) was <35.0 and equivocal if the CT was >=35.0, but a signal was obtained. If swabs of different origins regarding the localisation (e.g., dermal and oral) of the same animal were received, all samples were tested separately. Amplicons from positive samples were sequenced (ABI PRISM 3130 XL Genetic Analyser, Applied Biosystems, Foster City, CA, USA) and sequences were analysed by BLAST last accessed on 20 February 2023). 3. Results 3.1. Development of the PCR The selected primer and probe sequences (Eurofins MWG Operon, Ebersberg, Germany) are shown in Table 3. The product size was expected to be 246 base pairs. Optimisation of annealing temperature was performed using three protocols with different annealing temperatures. In protocol 1 (65 degC), D. agamarum DNA was detected with CT values of 14.58 and 16.49 and Dermabacter hominis with CT values of 29.43 and 29.62. In protocol 2 (66 degC), D. agamarum DNA was detected with CT values of 16.22 and 16.14, and Dermabacter hominis showed values of 34.28 and 35.19. In protocol 3 (67 degC), D. agamarum was detected with CT values of 15.57 and 16.57, while Dermabacter hominis was not detected. Therefore, an annealing temperature of 66 degC was chosen for all further analyses as it was considered sufficient to discriminate between pure cultures of D. agamarum and Dermabacter hominis DNA without losing sensitivity for the detection of D. agamarum. 3.2. Specificity, Repeatability and Sensitivity of the Assay Specificity of the assay using the protocol with 66 degC as an annealing temperature was determined using 14 D. agamarum isolates (Table 1) and 34 non-D. agamarum bacterial isolates (Table 2) in duplicate. No signal was obtained from any bacterial DNA from isolates other than D. agamarum except for Dermabacter hominis. The CT value obtained using the DNA from pure cultures of Dermabacter hominis were high (34.55 and 34.48) in comparison to those reached using DNA from D. agamarum (12.08-18.14) but still below the threshold set for clinical samples. As these are the results for DNA extracted from pure culture, 66 degC was considered sufficient for further testing of samples without prior cultivation as samples without prior cultivation are expected to yield a lower pathogen level. The intraassay CV was calculated to be 1.31%, while the interassay CV was 1.80%. Detection limit for bacterial cell dilutions: A positive PCR signal was detected for D. agamarum dilutions D0-D4. An equivocal signal was detected for D5 and D6. In culture, D4 corresponded to 2 x 104 colonies per mL. Therefore, the assay sensitivity was 2 x 104 colonies per mL with dilutions of bacterial cell culture serving as a template. This corresponds to approximately 200 CFU per PCR. For the serially diluted culture of Dermabacter hominis, no positive PCR signals were observed. Two dilutions (D0 and D1) resulted in equivocal CT values, with D0 being set to 0.5 McFarland (1.5 x 108 per mL). Detection limit for DNA from pure culture: The DNA concentration was determined to be 42.5 ng/mL (A260/A280: 1.94). D. agamarum DNA was detectable in dilutions up to 1:105. Therefore, DNA concentrations of as low as 425 fg/mL were detectable in the PCR. Spiked-in matrix: Spiked-in matrixes produced clearly positive results up to skin spiked with 10 mL of D2 with D2 corresponding to 2 x 106 colonies per mL (approximately 360 CFU per PCR considering dilution during sample preparation). 3.3. Testing of Clinical Samples In order to test the use of the developed PCR for clinical samples, a total of 48 samples from 38 lizards were tested for the presence of D. agamarum (Table 4). The samples were derived from several species, mostly agamids (Pogona spp. and Uromastyx spp.), and were of different origins (zoological collection, animal rescue centre, private owner) from Germany and the Netherlands. Some of these animals were asymptomatic and were tested in the context of a health check. Others showed clinical signs such as skin lesions, hyperkeratosis or stomatitis (Table 4). Samples were mostly derived from the oral cavity or skin/crusts. Of the 38 animals tested, D. agamarum was detected by PCR in 16 animals (42.10%). A further five animals (13.16%) were considered to have equivocal results, and 17 animals (44.74%) were negative for D. agamarum. One animal (animal 3) that tested negative proved to be infected with a fungus of the family Onygenaceae. A bacteriological examination was performed for 33 of the 38 animals, but D. agamarum was not cultured. However, in most of these cases, various other bacterial species (Table 4) were cultured when bacteriology was performed. 4. Discussion D. agamarum is an important pathogen causing skin lesions and, in some cases, systemic disease in lizards. Depending on the species, some animals can be inapparent carriers, while others may develop severe diseases. Diagnosis of the causative agent is therefore important in order to facilitate treatment as well as to prevent the spread of disease. Since animals may suffer when untreated and the risks of spreading increase with time, a fast diagnostic approach is important. The detection of D. agamarum is commonly achieved via culture, followed in some cases by 16S rRNA gene sequencing . The PCR developed in this study provides a time-saving tool compared to culture and bacterial identification. Detection of D. agamarum and concurrent bacteriological examination was performed in 33 of the 38 animals, resulting in 13 of 33 clearly PCR-positive animals but no culture-positives. D. agamarum is expected to be abundantly present in symptomatic animals. Culturing of D. agamarum is not considered difficult and has been successfully performed in this laboratory before . However, a successful culture depends on the quality of the submitted samples. Appropriate samples include affected tissue below hyperkeratotic crusts or inside of the crusts as well as organs in septicaemic lizards and subcutaneous granulomas. In asymptomatic animals as well as in symptomatic animals, isolation from the oral cavity, gastrointestinal tract or healthy skin may be challenging. D. agamarum was cultured in the laboratory in which the study was performed during the study period, but these samples were excluded from the study as no suitable material for concurrent PCR testing (e.g., dry swab, skin) was available. In this study, six animals (1, 2, 21 and 22-24) were known to have been symptomatic and had positive PCR results. Bacteriological culture was performed in three of these animals (22-24). In animal 24, a positive PCR result was only obtained from the skin sample, which was not tested by bacteriological culture. Animal 6 might have been symptomatic (no information was received, but due to the reported previous treatment, it seemed likely). It was treated with antibiotics prior to sampling, which might have influenced the bacteriology results. Possible reasons for the failure of culturing D. agamarum out of positive clinical samples in this study include previous antibiotic treatment, incorrect sampling techniques, contamination with (oral) microbiota, increased transport time, inappropriate transport conditions or overgrowth by other bacteria. The latter is especially important as in the presented cases, no selective media for gram-positive bacteria were used, and various different bacterial species are expected to be present on the skin . PCR analysis is useful if the performance of bacterial culture is difficult, e.g., due to previous treatment with antibiotics, inadequate preanalytical conditions (such as increased or decreased temperature, increased transport time, inadequate transport medium), or in cases in which overgrowth by other bacteria make detection challenging or impossible. The detection of D. agamarum via PCR can also simplify concurrent PCR testing for other known pathogens, e.g., viral or fungal pathogens known to cause dermatitis , since the same extracted nucleic acids can be used. In general, PCR is advantageous when culturable samples are unavailable, for example, when older samples are tested or stored DNA is examined. However, bacterial DNA can persist in the environment , and D. agamarum has been shown to survive for several months in the environment, depending on the conditions . A PCR could therefore detect bacteria even in cases in which these were not responsible for clinical signs or in which no replication-competent bacteria were present. The PCR developed here was not 100% specific. Pure cultures of Dermabacter hominis did result in a weak positive signal. However, if diluted, only equivocal results were observed. Dermabacter hominis is genetically closely related to D. agamarum . Dermabacter hominis is associated with the human microbiome . It is occasionally described in human clinical samples such as abscesses or blood cultures but is usually found to be of minor clinical significance . So far, its clinical importance in reptiles is very unclear. Contamination during sampling or sample preparation should be considered a possible option leading to false equivocal results. However, clinical samples are expected to yield less bacterial DNA, making false equivocal results less likely. The 16S rRNA gene is known to be highly conserved between bacterial species, which makes it a useful target if the aim is to identify different bacterial species. It is a commonly used target for bacterial detection, and therefore a large amount of sequence data is available for a wide range of bacterial species. However, it may not be ideal for differentiating closely related bacteria. Currently, the availability of sequence data for D. agamarum other than the 16S rRNA gene is limited, but in the future, other targets may prove to be better options. In the meantime, especially equivocal CT values should be evaluated with caution in the face of clinical signs, sampling and sample preparation, and ideally, retesting is recommended. Possibly, skin samples might prove more useful than swabs as they yielded lower CT values in two of the three animals for which both sample types were available, but this might be highly dependent on the sampling method. The PCR protocol developed in this study proved helpful for the detection of D. agamarum in clinical samples. D. agamarum was detected in oral swabs from clinically healthy Pogona species and serrated casquehead iguana (Laemanctus serratus), while the results in which equivocal results were obtained were also from clinically healthy Pogona species as well as from clinically healthy black hardun (Laudakia stellio picea). Pogona species have previously been shown to be possible inapparent carriers of D. agamarum and a possible source of infection for more sensitive species . Therefore, this PCR protocol may not only be useful for clinical cases but also as a screening tool. However, the number of tested samples is still small, and testing of larger sample numbers is necessary in order to confirm the usefulness of this method for clinical practice. 5. Conclusions A real-time PCR was developed that is able to detect D. agamarum in clinical samples. The assay provides a fast method for the detection of this important pathogen of lizards but should be evaluated with further samples in the clinical context. Author Contributions Conceptualization, M.B., E.M. and R.E.M.; Data curation, M.B., C.L. and R.E.M.; Formal analysis, M.B.; Methodology, M.B., M.G. and R.E.M.; Resources, M.B., C.L., T.H., A.M. and F.P.; Supervision, R.E.M.; Validation, M.B., M.G. and R.E.M.; Visualization, M.B.; Writing--original draft, M.B.; Writing--review & editing, C.L., T.H., A.M., F.P., M.G. and R.E.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Data is contained within the article. Conflicts of Interest Laboklin GmbH & Co. KG is a veterinary laboratory offering diagnostic services, including bacteriological and molecular biological examinations. This does not alter our adherence to sharing data and materials. Figure 1 Two-step amplification carried out at 60 degC: All Devriesea agamarum isolates tested resulted in positive signals with CT values ranging from 12.61 to 18.54. Dermabacter hominis (blue curve) also gave a positive signal with a CT value of 26.29. animals-13-00881-t001_Table 1 Table 1 Devriesea agamarum isolates used to establish the PCR. No. Isolate Host Sample Material Laboratory 1 MT664091.1/0219 Bf Brachylophus fasciatus Swab (skin) Laboklin GmbH & Co. KG, Germany 2 MT664092.1/0919 Ur Uromastyx sp. Swab (skin) Laboklin GmbH & Co. KG, Germany 3 MT664093.1/0319 Ur Uromastyx sp. Swab (skin) Laboklin GmbH & Co. KG, Germany 4 IMP2 vacc Agama impalearis Swab (dermatitis), liver Faculty of Veterinary Medicine, Ghent University 5 30.7 Uromastyx dispar Swab (dermatitis, cheilitis) Faculty of Veterinary Medicine, Ghent University 6 34.1 Uromastyx acanthinura Swab (dermal abscess) Faculty of Veterinary Medicine, Ghent University 7 23 Pogona vitticeps Swab (oral cavity) Faculty of Veterinary Medicine, Ghent University 8 24 Pogona vitticeps Swab (oral cavity) Faculty of Veterinary Medicine, Ghent University 9 25 Pogona vitticeps Swab (oral cavity) Faculty of Veterinary Medicine, Ghent University 10 26 Crotaphytus collaris Swab (dermatitis) Faculty of Veterinary Medicine, Ghent University 11 28 Eublepharis macularius Swab (oral cavity) Faculty of Veterinary Medicine, Ghent University 12 30b Ctenonotus gingivus Swab (cloaca) Faculty of Veterinary Medicine, Ghent University 13 4d Iguana delicatissima Swab (dermal abscess) Faculty of Veterinary Medicine, Ghent University 14 L26 Pogona vitticeps Swab (oral cavity) Faculty of Veterinary Medicine, Ghent University animals-13-00881-t002_Table 2 Table 2 Bacterial species used as negative controls to determine the specificity of the PCR. No. Bacterial Species DSMZ Number Original Depositor (Acc. to DSMZ) 1 Acinetobacter baumannii DSM 30007 J.V. Cook 2 Bacillus atrophaeus DSM 675 E. McCoy 3 Cytobacillus firmus DSM 359 G. Bredemann 4 Brachybacterium faecium DSM 4810 H.E. Schefferle 5 Dermabacter hominis DSM 30958 C. Moissl-Eichinger 6 Enterococcus faecalis DSM 2570 Kaiser-Permanente 7 Enterococcus faecium DSM 20477 A. Grumbach 8 Enterococcus faecium DSM 2146 J.M. Skerman (Streptococcus faecalis) 9 Escherichia coli DSM 1103 F. Schoenknecht 10 Escherichia coli DSM 1576 G.C. Crooks 11 Flavobacterium psychrophilum DSM 21280 J.-F. Bernardet 12 Klebsiella oxytoca DSM 5175 R. Hugh 13 Klebsiella pneumoniae DSM 26371 H. Dalton 14 Klebsiella pneumoniae DSM 30104 CDC, Atlanta; 298-53 15 Listeria monocytogenes DSM 19094 H. Seeliger 16 Mycobacterium phlei DSM 750 IPH 17 Nocardia nova DSM 44481 N. F. Conant 18 Proteus hauseri DSM 30118 K.B. Lehmann 19 Proteus mirabilis DSM 4479 CDC PR 14 20 Pseudomonas aeruginosa DSM 1128 C.P. Hegarty 21 Pseudomonas aeruginosa DSM 1117 A. Madeiros 22 Salmonella enterica DSM 19587 CDC 23 Salmonella enterica DSM 17420 CDC 24 Staphylococcus aureus DSM 2569 E.H. Gerlach 25 Staphylococcus aureus DSM 1104 F. Schoenknecht 26 Staphylococcus aureus DSM 46320 W. Witte 27 Staphylococcus aureus DSM 799 AMC 28 Streptococcus dysgalactiae DSM 20662 T.M. Higgs 29 Staphylococcus epidermidis DSM 1798 FDA 30 Streptococcus equi ssp. equi DSM 20561 R.E.O. Williams 31 Staphylococcus felis DSM 7377 S. Igimi 32 Staphylococcus intermedius DSM 20373 V. Hajek 33 Streptococcus pyogenes DSM 11728 E. Mortimer 34 Yersinia enterocolitica DSM 4780 J.M. Coffey animals-13-00881-t003_Table 3 Table 3 Primers and probe used in the PCR for detection of Devriesea agamarum. Forward Primer Devag16_For GATGACTGCAGAGATGTGGTG Reverse Primer Devag16_Rev TTTGTACCGGCCATTGTAGCAT Oligonucleotide probe FAM BHQ1 CATGTTGCCAGCACTTCGG animals-13-00881-t004_Table 4 Table 4 Results of bacteriological culture and Devriesea agamarum PCR of clinical samples from 38 lizards. Animal No. Species Clinical Signs and History and Additional Information Country of Origin Time from Sampling to Sample Preparation: Sample Material Bacterial Culture PCR Result 1 Uromastyx sp. Suspicious skin lesion Same wildlife park as animal 2 NL unknown Skin N.D. Positive (CT 23.71) 2 Uromastyx sp. Suspected lesions/ dermatitis Same wildlife park as animal 1 NL unknown Swabs (skin) N.D. Positive (CT 30.53) 3 Pogona sp. Black discoloration of the scales after shedding, especially on the tail. NL 5d Skin + Glutamibacter creatinolyticus + Micrococcus sp. + aerobic spore-forming bacteria Negative Swab (skin) Negative 4 Uromastyx sp. Partner to animal 5 NL 2d Swab (oral mucosa) ++ Pantoea agglomerans ++ Pseudomonas aeruginosa ++ aerobic spore-forming bacteria Positive (CT 28.47) 5 Uromastyx sp. Partner to animal 4 NL 2d Swab (oral mucosa) ++ Exiguobacterium mexicanum ++ Kluyvera intermedia ++ Pseudomonas chlororaphis ++ aerobic spore-forming bacteria Negative 6 Uromastyx sp. Reported to have been treated with antibiotics NL 2d Swab (oral mucosa) ++ Acinetobacter variabilis +++ Arthrobacter globiformis + Bacillus cereus Positive (CT 28.44) 7 Pogona vitticeps No clinical signs DE 1d Swab (oral mucosa) ++ Proteus mirabilis Positive (CT 33.17) 8 Pogona vitticeps No clinical signs DE 1d Swab (oral mucosa) +++ Enterobacter cloacae + Pseudomonas aeruginosa Equivocal (CT 35.71) 9 Pogona vitticeps No clinical signs Partner to animal 10 Confiscated DE 1d Swab (oral mucosa) ++ Bordetella hinzii Negative 10 Pogona vitticeps No clinical signs Partner to animal 9 Confiscated DE 1d Swab (oral mucosa) + Bordetella hinzii + Peribacillus muralis ++ Enterococcus faecalis + Pantoea agglomerans Negative 11 Pogona vitticeps No clinical signs Abandoned DE 1d Swab (oral mucosa) ++ Morganella morganii ++ Klebsiella oxytoca Positive (CT 27.56) 12 Pogona vitticeps No clinical signs Abandoned DE 1d Swab (oral mucosa) ++ Klebsiella oxytoca + Proteus mirabilis Negative 13 Pogona henrylawsoni No clinical signs Abandoned juvenile same enclosure as animals 14 and 15 DE unknown Swab (oral mucosa) + Aeromonas hydrophila (+) aerobic spore-forming bacteria Negative 14 Pogona henrylawsoni No clinical signs Abandoned juvenile same enclosure as animals 13 and 15 DE unknown Swab (oral mucosa) ++ Proteus mirabilis (+) aerobic spore-forming bacteria Negative 15 Pogona henrylawsoni No clinical signs Abandoned juvenile same enclosure as animals 13 and 14 DE unknown Swab (oral mucosa) + Aeromonas hydrophila (+) alpha-hemolytic streptococci Negative 16 Pogona vitticeps No clinical signs DE unknown Swab (oral mucosa) + Aeromonas hydrophila + Pseudomonas aeruginosa (+) aerobic spore-forming bacteria Positive (CT 27.20) 17 Pogona vitticeps No clinical signs DE unknown Swab (oral mucosa) +++ Klebsiella oxytoca Equivocal (CT 35.26) 18 Pogona vitticeps No clinical signs DE unknown Swab (oral mucosa) ++ Klebsiella oxytoca Equivocal (CT 35.07) 19 Scincidae Unknown DE 2d Skin N.D. Negative 20 Iguanidae Hyperkeratosis (dorsal) DE 2d Skin N.D. Negative 21 Uromastyx sp. Skin lesion DE 2d Skin N.D. Positive (CT 21.34) 22 Uromastyx sp. Skin lesion Animals 22, 23 and 24 kept in the same enclosure DE unknown Swab (outer skin of the mouth) + Pseudomonas aeruginosa + Serratia marcescens Positive (CT 28.45) Swab (oral mucosa) + Pseudomonas aeruginosa + Serratia marcescens Positive (CT 34.89) Skin N.D. Positive (CT 31.56) Swab (skin) N.D. Positive (CT 30.88) 23 Uromastyx sp. Skin lesion Animals 22, 23 and 24 kept in the same enclosure DE unknown Swab (outer skin of the mouth) + Enterobacter cloacae Positive (CT 32.20) Swab (oral mucosa) + Enterobacter cloacae Positive (CT 32.06) Skin N.D. Positive (CT 31.97) Swab (Skin) N.D. Positive (CT 34.05) 24 Uromastyx sp. Skin lesion Animals 22, 23 and 24 kept in the same enclosure DE unknown Swab (outer skin of the mouth) + Pseudomonas synxantha Equivocal (CT 35.24) Swab (oral mucosa) + Pantoea agglomerans Negative Skin N.D. Positive (CT 33.84) Swab (Skin) N.D. Equivocal (CT 35.75) 25 Corucia zebrata Multiple animals with minimal to moderate stomatitis NL 2d Swab (skin) N.D. Negative Skin +++ Pseudomonas aeruginosa Negative 26 Chlamydosaurus kingii No clinical signs Kept with animal 27 DE 5d Swab (oral mucosa) + Proteus mirabilis ++ Serratia marcescens Negative 27 Chlamydosaurus kingii No clinical signs Kept with animal 26 DE 5d Swab (oral mucosa) + Klebsiella oxytoca + Stenotrophomonas maltophilia + aerobic spore-forming bacteria Negative 28 Laemanctus serratus No clinical signs Kept with animals 29 and 30 DE 5d Swab (oral mucosa) + Klebsiella oxytoca + Morganella morganii Positive (CT 31.20) 29 Laemanctus serratus No clinical signs Kept with animals 28 and 30 DE 5d Swab (oral mucosa) + Deinococcus proteolyticus + Morganella morganii + Serratia marcescens Positive (CT 34.35) 30 Laemanctus serratus No clinical signs Kept with animals 28 and 29 DE 5d Swab (oral mucosa) + Morganella morganii + Proteus mirabilis Positive (CT 29.10) 31 Laudakia stellio picea No clinical signs Kept with animal 32 DE 5d Swab (oral mucosa) (+) aerobic spore-forming bacteria Equivocal (CT 35.80) 32 Laudakia stellio picea No clinical signs Kept with animal 31 DE 5d Swab (oral mucosa) + Pseudomonas sp. + Serratia marcescens + aerobic spore-forming bacteria Equivocal (CT 36.87) 33 Pogona vitticeps No clinical signs Kept with animal 34 DE 5d Swab (oral mucosa) (+) Staphylococcus epidermidis Positive (CT 34.07) 34 Pogona vitticeps No clinical signs Kept with animal 33 DE 5d Swab (oral mucosa) + Staphylococcus aureus (+) aerobic spore-forming bacteria Positive (CT 32.61) 35 Pogona vitticeps Juvenile No clinical signs Kept with animals 36, 37 and 38 DE 5d Swab (oral mucosa) (+) aerobic spore-forming bacteria Negative 36 Pogona vitticeps Juvenile No clinical signs Kept with animals 35, 37 and 38 DE 5d Swab (oral mucosa) + Achromobacter xylosoxidans Negative 37 Pogona vitticeps Juvenile No clinical signs Kept with animals 35, 36 and 38 DE 5d Swab (oral mucosa) + Serratia marcescens (+) aerobic spore-forming bacteria Negative 38 Pogona vitticeps Juvenile No clinical signs Kept with animals 35, 36 and 37 DE 5d Swab (oral mucosa) + Proteus mirabilis Negative Legend: N.D. = not done, d = days, DE = Germany, NL = the Netherlands. 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PMC10000033

Different bedding materials have important effects on the behavioristics, production performance and welfare of buffalo. This study aimed to compare the effects of two bedding materials on lying behavior, production performance and animal welfare of dairy buffaloes. More than 40 multiparous lactating buffaloes were randomly divided into two groups, which were raised on fermented manure bedding (FMB) and chaff bedding (CB). The results showed that the application of FMB improved the lying behavior of buffaloes, the average daily lying time (ADLT) of buffaloes in FMB increased by 58 min compared to those in CB, with a significant difference (p < 0.05); the average daily standing time (ADST) decreased by 30 min, with a significant difference (p < 0.05); and the buffalo comfort index (BCI) increased, but the difference was not significant (p > 0.05). The average daily milk yield of buffaloes in FMB increased by 5.78% compared to buffaloes in CB. The application of FMB improved the hygiene of buffaloes. The locomotion score and hock lesion score were not significantly different between the two groups and all buffaloes did not show moderate and severe lameness. The price of FMB was calculated to be 46% of CB, which greatly reduced the cost of bedding material. In summary, FMB has significantly improved the lying behavior, production performance and welfare of buffaloes and significantly reduce the cost of bedding material. bedding material buffalo behaviororistics milk yeild animal welfare Modern Agro-industry Technology Research SystemCARS-36 This work was supported by the Modern Agro-industry Technology Research System: CARS-36. pmc1. Introduction The most used bedding materials in current barns are wood chips and sand . Other materials, including straw and peanut shells, are also often used . A study noted that the increasing demand for bedding materials has led to the increasing price of common bedding materials . Therefore, recycled manure bedding materials are increasingly preferred by farmers . The fermented manure bedding can be produced by ectopic fermentation with buffalo manure as raw material . At present, there are three main approaches to the cattle manure as bedding material: the solid-liquid separation direct utilization model , anaerobic fermentation bedding production model and aerobic fermentation bedding production model . Research shows that bedding plays a key role in improving bovine welfare and lying behavior . Beef kept on rubber mats have longer lying time, significantly better body hygiene and less joint damage compared to concrete floors . A study compared the effects of three bedding materials, concrete, soft rubber and sand, on the lying behavior of cows. The results showed that cows spent the longest time lying on the rubber bedding . Another study compared the effects of peanut shell and rice husk on the lying behavior of dairy cows, and the results showed that dairy cows had more lying time and lying frequency on rice husk (Li P et al., 2021) . Generally, the milk yield of cows is positively correlated with the lying time, and the longer the lying time, the higher the milk yield . Cows spent more time lying on the bed when the bedding materials were dry and soft . A study noted that, after transferring cows housed in concrete bedding to barns with deep fermented manure bedding, milk production per dairy cow increased by an average of 13.3% . A study compared the effects of recycled manure, new sand, recycle sand and foam-core mattress bedding material on the milk yield of dairy cows. The results showed that dairy cows raised on new sand and recycled manure bedding had a higher milk yield . Different bedding materials have an important impact on welfare indices, such as the hoof health and hygiene of cattle . Fernandez et al. concluded that compost-bedded packs significantly improved the welfare and comfort of cows. Housing bulls on deep fermented manure bedding significantly improved the hock lesion and injuries and body hygiene compared to the concrete floors . Moreover, deep fermented manure bedding was more effective in protecting cattle from joint injury and less risk of cattle limb and hoof disease . Research showed that the use of deep recycled manure bedding in free-stall barns reduced the prevalence of lameness and joint wear, and it can improve body hygiene in dairy cattle . Therefore, the aim of this study was to compare the effects of chaff bedding (CB) and fermented manure bedding (FMB) on buffalo lying behavior, milk production and animal welfare. By recording and observing dairy buffaloes for up to one year, we collected a series of indicators, such as lying behavior, milk production and animal welfare of buffaloes to evaluate the application prospect of fermented manure bedding. 2. Materials and Methods This study was conducted from December 2020 to December 2021 in Jinniu Animal Husbandry Co LTD, Jingmen city, Hubei province, China (Longitude: 112.07438; latitude: 30.72365). The protocol of this experiment was approved by the Scientific Ethics Committee of Huazhong Agricultural University (HZAUBU-2020-0003) and the animal trial was conducted in accordance with the National Institute of Health Guidelines for the Care and Use of Experiment Animals (Beijing, China). 2.1. Manufacture of Fermented Manure Bedding The process of using buffalo manure as a raw material and harmless ectopic fermentation treatment as bedding material is shown in Figure 1. Firstly, fresh buffalo manure and rice chaff were collected. The moisture content of the chaff was less than 5%, and the moisture content of the mixture was adjusted to approximately 60% by adding chaff into the buffalo manure and mixing it evenly with a forklift. Then, the microbial agents were evenly sprinkled at 125 g/m3 and mixed to build a strip chopped heap with a length of 8 m, a width of 6 m and a height of 1.5 m (The microbial agent mainly contained bacillus subtilis DK068, lactic acid bacteria, yeast MX0126, cellulase and lignin enzyme, with viable bacteria count > 1.0 x 1010 CFU/g). After that, the temperature of the pile will start to rise. After 7-10 days, the temperature will reach 55 degC, turn the pile with forklift every 4 days thereafter. The high temperature period (>55 degC) can be maintained for about 20 days, and the highest temperature can reach more than 75 degC. The fermentation period is about 40 days. The moisture content of FMB is reduced from 60% to 35-40%. The fermented manure bedding and chaff bedding were laid flat in the stall. FMB was laid in the experimental group and CB in the control group, and the thickness of the bedding was 50 cm. 2.2. Animal Management More than 40 multiparous lactating buffaloes were randomly divided into 2 groups according to age. All the buffaloes in this study were lactating Mediterranean hybrids, and all were in early or early-middle lactation stage. There were 20 lactating buffaloes in FMB group and 21 in CB group from January to June. There were 21 lactating buffaloes in FMB group and 23 in CB group from July to December. The barn is an east-west oriented open barn with a head-to-head double-column interior. The cowshed was a semi-enclosed barn with pens, and the water tank was located outside the barn. The two stalls on the south and north sides of the same barn were selected; each stall was about 35 m long and 10 m wide with an area of about 350 m2, and the exercise area of each buffalo was not less than 15 m2. The trial period was one year with a full mixed diet twice daily (07:00 and 15:00), ad libitum feeding and watering. Both stalls were supplemented with the appropriate bedding periodically every two months. 2.3. Measurement Index 2.3.1. Lying Behavioral Index Using a combination of cameras and human observation, high-definition cameras were installed at two opposite corners of each stall to continuously observe and count buffaloes throughout the day (0:00-24:00). These HD cameras were connected to a digital video recorder for recording (two cameras were installed at each diagonal of the cattle stall). HD cameras for daytime and nighttime monitoring of buffaloes are shown in Figure 2. Behavioral indicators of buffaloes were observed throughout 2021, recording 12 days per month. The 4th-7th, 14th-17th, and 24th-27th of each month were chosen to record the behavioral indicators of the buffaloes. The main observation and statistical items: lying time, standing time and buffalo comfort index. Lying: the belly of the body is in contact with the ground, and the body is supported by the ground rather than the hooves and legs. Standing: the body is supported by at least three legs on the ground. Buffalo Comfort Index: 1 h after each buffalo is milked, returned and fed, the number of buffalos lying in the stall/total number of buffaloes. 2.3.2. Milk Yield Buffaloes were milked twice a day, usually once in the morning at 3-4:00 a.m. and once in the afternoon at 15-16:00 p.m. The daily milk yield of each buffalo was collected and recorded. 2.3.3. Animal Welfare Index During the experiment, the two groups' body hygiene score, locomotion score and hock lesion score of the buffaloes were scored individually every month by 1 trained observer, 12 times in total. The buffalo body hygiene score (BHS) was assessed by the amount of dirt on the udder and lower hind legs based on a 5-point scale with 1 = clean and 5 = dirty . Buffalo body hygiene score < 3 is regarded as clean, and >=3 is regarded as dirty. The average body condition and hygiene score were calculated for each pen for analysis. Buffaloes were evaluated for lameness using a 5-point locomotion scoring method . Locomotion score (LS) was as follows: 1 = normal locomotion, 2 = imperfect locomotion, 3 = lame, 4 = moderately lame and 5 = severely lame. The severity of back leg hock lesion (HL) was measured using the 6-point scale scoring system as described previously . Hock lesions were classified as 1 = no lesion, 3 = hair loss (mild lesion) and 6 = swollen hock with hair loss (severe lesion). 2.4. Statistical Analysis Statistical analysis was performed by SPSS software (SPSS v. 29, SPSS Inc.; Chicago, IL, USA). This study analyzed the production performance, lying behavior indexes and animal welfare indexes of buffaloes and were conducted by two-way ANOVA in SPSS. p < 0.05 was used to indicate a significant difference. 3. Results 3.1. Lying Behavioral Index The factors affecting the lying time of buffaloes are shown in Table 1. The results showed that different months and bedding materials have an effect on the lying time of buffaloes (p < 0.001), and there is an interaction between month and bedding (p < 0.01). The results of the average daily lying time of buffaloes with different bedding are shown in Table 2; the buffalo ADLT was 606 min in the CB group and 664 min in the FMB group, and the FMB group increased the buffaloes' ADLT by 58 min compared with the CB group. The ADLT of buffaloes raised in the FMB group was higher than that in the CB group in July, but the difference was not statistically significant (p > 0.05). The difference was significant (p < 0.01) in the rest of the months. Buffaloes had the least ADLT in August-September, with only 542 min and 550 min in the CB group and 615 min and 616 min in the FMB group, respectively. The reason should be that at this time the ambient temperature is high, and buffalo are most severely affected by heat stress resulting in a decrease in ADLT. The buffalo had the longest ADLT in April-May when the ambient temperature was optimal, with 633 min and 646 min in the CB group and reaching 722 min and 713 min in the FMB group, respectively. The factors affecting the standing time of buffaloes are shown in Table 3. The results showed that different months and bedding materials have an effect on the lying time of buffaloes (p < 0.001), and there is an interaction between month and bedding (p < 0.05). The results of the average daily standing time of buffaloes with different bedding are shown in Table 4. The ADST was 354 min in the CB group and 324 min in the FMB group, and the FMB group decreased by 30 min compared with the CB group. The ADST of buffaloes raised in the FMB group was lower than that in the CB group in February and June, but the difference was not statistically significant (p > 0.05). The difference was significant (p < 0.05) in the rest of the months. The results showed that buffaloes in the CB group had the longest ADST in August-September, reaching 378 min and 371 min, respectively, while buffaloes in the FMB group had the most ADST in July-September, reaching 341, 340 and 342 min, respectively. The results showed that buffaloes had the least ADST in April-May, with only 346 min and 327 min in the CB group and 307 min and 308 min in the FMB group, respectively. The results of the BCI of buffaloes with different bedding are shown in Table 5. The BCI (a.m.) of buffaloes was 86.56% in the CB group and 89.45% in the FMB group. The BCI (a.m.) in July was 82.53% in the CB group and 86.91% in the FMB group, with significant differences (p < 0.05); the differences were not significant (p > 0.05) in other months. The BCI (p.m.) of buffaloes was 85.82% in the CB group and 88.33% in the FMB group. The BCI (p.m.) in October was 85.64% in the CB group and 90.40% in the FMB group, with significant differences (p < 0.05); the differences were not significant (p > 0.05) in other months. 3.2. Milk Yield The results of the average daily milk yield of buffaloes with different bedding materials are shown in Table 6. The buffalo ADMY was 4.39 kg in the FMB group and 4.15 kg in the CB group, and the difference was significantly. The buffalo ADMY increased by 5.78% in the FMB group compared to the CB group. The FMB group was higher than the CB group in June, but the difference was not significant (p > 0.05), and the difference was significant (p < 0.05) in other months. The results showed that the ADMY of lactating buffalo was lowest in July-September, which were 3.8 kg, 3.9 kg and 3.75 kg in the CB group, respectively; the FMB group were 4.05 kg, 4.13 kg and 4.05 kg, respectively. It is speculated that the high ambient temperature in this period, which causes the buffalo to be seriously affected by heat stress, and the reduced lying time resulted in lower milk yield. 3.3. Animal Welfare Index The results of the body hygiene score of buffaloes with different bedding are shown in Table 7, and the body hygiene of the buffalo is dirty when the BHS >= 3. The BHS of buffaloes in the FMB group was 1.81 and 1.89 in the CB group, with no significant difference (p > 0.05). However, the proportion of dirty buffaloes in the CB group was 29.93%, which was significantly higher than 24.80% in the FMB group (p < 0.01). The results of the locomotion score of buffaloes with different bedding are shown in Table 8. The LS of buffaloes in the FMB group was 1.41 and 1.45 in the CB group, with no significant difference (p > 0.05). All buffaloes in both groups did not show moderate and severe lameness during the experiment. The results of the hock lesion score of buffaloes with different bedding are shown in Table 9. During the experiment, the HLS of buffaloes in the FMB group was 0.87 and 0.90 in the CB group, with no significant difference (p > 0.05). All buffaloes in both groups did not show moderate and severe damage to the hock joints. 3.4. Costing The cost of the chaff bedding material was calculated to be 52 USD/ton; the cost of fermented manure bedding materials was 6.67 USD/ton. The area of the two stalls is similar: approximately 350 m2 and the thickness of bedding is 50 cm. The cost of chaff bedding spread over the whole stall is approximately USD 3633. The cost of the bedding materials of fermented manure bedding spread over the whole stall is approximately USD 1631, which can save USD 2002 on bedding cost. 4. Discussion 4.1. Lying Behavior Cattle spend a lot of time lying on bedding every day; therefore, the comfort level of the bedding material in the stall has a direct impact on the quality of their rest. A study showed that the ADLT for cows on straw bedding beds was 749 min, significantly higher than 678 min on sand bedding beds . Angus bulls raised on rubber bedding had significantly more lying time than those on concrete floors . The cows raised on deep sand bedding had significantly more lying time and lying frequency than those on rubber bedding . The cows raised on dry manure bedding had a 2.2 h higher lying time and 2 times higher lying frequency than the cows on rubber bedding . Fernandez et al. and Marcondes' et al. studies showed that compost bedded pack barns have a positive impact on cows' performance, animal welfare and comfort. The results of this experiment showed that the ADLT of buffaloes raised in the FMB group was 664 min, and was 606 min in the CB group. The ADLT of buffaloes in the FMB group increased by 58 min compared to the CB group. The difference in ADLT was not significant (p > 0.05) in July, but the difference was significant (p < 0.05) in the rest of the months. The ADST of buffaloes was 324 min in the FMB group and 354 min in the CB group, and the ADST of buffaloes in the FMB group was reduced by 30 min compared with that in the CB group. The difference in ADST in February and June was not significant (p > 0.05), but the rest of the months were significantly different (p < 0.05). Studies have shown that ADLT is shortest in cattle in summer, significantly lower than in spring and autumn . The ADLT of cows during the strong heat stress period in June-August was significantly lower than during the mild heat stress period . Smith et al. found that, under heat stress conditions, ADLT and lying frequency were higher in cows with evaporative pads than in cows without evaporative pads. Studies have shown that the more severe the heat shock, the lower the average daily lying time and frequency of cows. Cow core body temperature rises when lying down and decreases when standing . This is consistent with the results of this study, where buffaloes had the least ADLT and the longest ADST in August-September. In the CB group, ADLT was only 542 min and 550 min, and ADST was 378 min and 371 min. In the FMB group, ADLT was 615 min and 616 min, and ADST was 340 min and 342 min. The reason should be that at this time the ambient temperature is high and buffaloes are most severely affected by heat stress, resulting in a decrease in ADLT and an increase in ADST. In April and May, buffaloes had the most ADLT and the least ADST. The CB groups' ADLT was 633 min and 646 min, and ADST was 346 min and 327 min. In the FMB group, ADLT reached 722 min and 713 min, and ADST was 307 min and 308 min. The Cow Comfort Index (CCI) was proposed by Nelson in 1996 and is now widely used as a stall comfort assessment. In general, the maximum value of the comfort index for cows occurs in the morning and 1 h after returning from afternoon milking ; a CCI > 85% indicates high cow comfort, and this index can also be applied to buffaloes (Buffalo Comfort Index). In this study, both groups of buffalo BCI (a.m.) were greater than 85%, indicating high comfort level of buffalo. However, the BCI (p.m.) of the CB group was less than 85% in July-September, and the BCI (p.m.) of the FMB group was greater than 85%, indicating that fermented manure bedding can improve the comfort of buffalo. In summary, the application of FMB improved the lying behavior of buffaloes compared to CB bedding. 4.2. Milk Yield A study showed that the milk yield of cows is positively correlated with the lying time, and the longer the lying time, the higher the milk yield . Dry and soft bedding in the barn provided an excellent and comfortable environment for cattle to rest and grow . Graunke et al. found that growing holstein cows raised on soft rubber mats increased daily weight gain by 9.09% compared to cows raised on concrete floors. The average daily milk yield of cows raised on recycled manure bedding was extremely significantly higher than that on hardened floors . Milk yield per cow in the barn increased by an average of 13.3% after transferring cows housed in concrete floor barns to barns with deep recycled manure bedding . The results of this study showed that the average milk yield of buffaloes in the CB group was 4.15 kg/d and the average milk yield of buffaloes in the FMB group was 4.39 kg/d. The average daily milk yield of buffaloes in the FMB group increased 5.78% compared to buffaloes in the CB group. As the ADLT of buffaloes in the FMB group was 60 min higher than that of the CB group, this was the reason that the milk yield of buffaloes in the FMB group was higher than that of the CB group. 4.3. Welfare Indexes Cattle body hygiene is one of the important animal welfare indexes and it is influenced by several factors, the type of bedding being one of the key factors . Previous studies have shown that cows raised on the deep sand bedding had better hygiene than those in the regular straw bedding ; both used recycled cow manure as bedding material, and cattle in the deep bedding group had better body hygiene than the shallow bedding group, but the difference was not significant . Guarin et al. compared the effect of four bedding materials (new sand, recycled sand, deep-bedded manure solids and shallow-bedded manure solids over foam core mattresses) on udder cleanliness of cows. The udder cleanliness of cattle raised on deep-bedded manure solids bedding reached 95%. In this study, the body hygiene score of buffaloes in the FMB group was lower than that in the CB group, the difference was not significant (p > 0.05). However, the percentage of dirty buffaloes was 29.93% in the CB group and 24.80% in the FMB group, the difference was significant (p < 0.01). The reason might be that the bedding in the FMB group was made from buffalo manure and fermented with microbial agents. Fermented manure bedding contains a large number of beneficial microorganisms. After buffalo treading and plowing to mix fresh manure and urine, it facilitates the microorganisms in the bedding to decompose the organic matter in manure and urine and accelerate the evaporation of water in the bedding. The locomotion score and hock lesion score can identify the early lameness of cattle, determine the hoof health of cattle and make reference for early management adjustment . It has been shown that cows raised on rubber mats have lower levels of joint damage compared to concrete floors . Similarly, less hock injury and joint wear and healthier limbs and hooves have been reported in pastures with deep sand or deep recycled manure bedding . Barrientos et al. surveyed 79 farms, and cows with bedding depths of at least 10 cm had healthier hoof limbs. By testing serum biomarkers of joint damage in cows, the degree of lameness and joint damage was more severe in cows raised on concrete floors than in deep sand bedding beds and deep fermented manure bedding . In this study, the bedding thickness was 50 cm for both the FMB and CB groups, and the results showed that the locomotion score of buffaloes was 1.41 in the FMB group and 1.45 in the CB group, with no significant difference between the two groups (p > 0.05). Moreover, all buffaloes did not show moderate or severe lameness during the experiment. No moderate and severe joint injuries were observed in all buffaloes. It indicates that the deep bedding played a cushioning and supporting role for the limb and hoof of cattle, which can effectively protect the limb and hoof of cattle. 5. Conclusions In this study, we compared the effects of two bedding materials, FMB and CB, on lying behavior, performance and animal welfare of dairy buffaloes. The results showed that FMB significantly improved the lying behavior of dairy buffaloes compared with CB and significantly increased the milk production of dairy buffaloes. The application of FMB improved the hygiene welfare of dairy buffaloes. The differences in locomotion scores and hock lesion scores between the two groups were not significant, and all dairy buffaloes showed no moderate and severe joint damage. In addition, the price of FMB was calculated to be 4.74 USD/m2 and the price of CB was 10.38 USD/m2. The price of FMB was 46% of the price of CB, which greatly reduced the cost of bedding material. The results of this study provide a reference for the rational utilization of buffalo manure resources, reducing bedding costs and achieving the coordinated development of cattle breeding and ecological environment. Acknowledgments The authors would like to thank the Buffalo Farm of Jinniu Animal Husbandry Co. LTD, China for their experimental animals and all farmers for their cooperation and their contribution is acknowledged. Author Contributions L.Y. and K.N.; methodology: L.Y., K.N. and Z.A.; investigation, K.N., C.C. and Z.Y.; writing--original draft preparation and writing--review and editing: K.N. and Z.A.; supervision and project and funding acquisition: L.Y. and J.X. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Buffaloes involved in the experimental protocols have been approved by the Huazhong Agricultural University Animal Care and Use Committee (HZAUBU-2020-0003). Informed Consent Statement All authors approved the final manuscript. Data Availability Statement The datasets used and or analyzed during the current study are available from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The production process of harmless fermentation bedding material of buffalo manure. (A) Collect fresh cow dung, adjust the moisture content and add microbial agent. (B) Turning the pile regularly. (C) Fermented manure bedding material. (D) Spread the FMB over the experimental group barn. (E) Spread the CB over the control group barn. Figure 2 Daytime barn conditions (A), night barn conditions (B). animals-13-00842-t001_Table 1 Table 1 The factors influencing lying time of buffaloes and their interaction. Dependent Variable SS df MS F p-Value Month 194,168.13 11 17,651.65 28.76 p < 0.001 Bedding 190,519.56 1 190,519.56 310.43 p < 0.001 Month * Bedding 16,864.05 11 1533.10 2.50 p < 0.01 Error 117,834.22 192 613.72 Total 519,385.96 215 Abbreviations: SS, sum of squares; df, degree of freedom; MS, mean square; *, interaction effect. animals-13-00842-t002_Table 2 Table 2 Average daily lying time of buffaloes with different bedding and month. Month Environment Temp (degC) Average Daily Lying Time (min) p-Value CB FMB January 3.6-11.6 603 +- 4.20 646 +- 7.35 p < 0.01 February 5.0-13.1 614 +- 4.39 658 +- 9.70 p < 0.01 March 7.9-16.7 624 +- 5.13 693 +- 12.17 p < 0.01 April 12.4-18.5 633 +- 5.23 722 +- 9.79 p < 0.01 May 17.5-24.1 646 +- 6.75 713 +- 10.79 p < 0.01 June 22.4-32.0 614 +- 6.09 658 +- 12.29 p < 0.01 July 25.0-32.4 610 +- 6.03 632 +- 9.79 p = 0.068 August 23.3-29.9 542 +- 6.29 615 +- 8.34 p < 0.01 September 21.5-32.3 550 +- 8.22 616 +- 10.32 p < 0.01 October 14.2-21.2 599 +- 8.32 674 +- 10.70 p < 0.01 November 3.6-11.6 613 +- 5.42 678 +- 6.75 p < 0.01 December 3.2-11.2 619 +- 5.05 673 +- 10.20 p < 0.01 Abbreviations: CB, chaff bedding; FMB, fermented manure bedding. animals-13-00842-t003_Table 3 Table 3 The factors influencing standing time of buffaloes and their interaction. Dependent Variable SS df MS F p-Value Month 31,282.72 11 2843.88 10.48 p < 0.001 Bedding 49,111.34 1 49,111.34 181.01 p < 0.001 Month * Bedding 5795.61 11 526.87 1.94 p < 0.05 Error 52,094.22 192 271.32 Total 138,283.89 215 Abbreviations: SS, sum of squares; df, degree of freedom; MS, mean square; *, interaction effect. animals-13-00842-t004_Table 4 Table 4 Average daily standing time of buffaloes with different bedding and month. Month Environment Temp (degC) Average Daily Standing Time (min) p-Value CB FMB January 3.6-11.6 355 +- 6.43 331 +- 6.41 p < 0.05 February 5.0-13.1 347 +- 4.98 327 +- 9.83 p = 0.097 March 7.9-16.7 350 +- 3.99 312 +- 3.14 p < 0.01 April 12.4-18.5 346 +- 3.62 307 +- 3.54 p < 0.01 May 17.5-24.1 327 +- 3.50 308 +- 4.37 p < 0.01 June 22.4-32.0 350 +- 4.91 335 +- 8.30 p = 0.13 July 25.0-32.4 361 +- 6.31 341 +- 5.79 p < 0.05 August 23.3-29.9 378 +- 2.21 340 +- 4.69 p < 0.01 September 21.5-32.3 371 +- 5.39 342 +- 7.88 p < 0.01 October 14.2-21.2 362 +- 7.29 317 +- 4.08 p < 0.01 November 3.6-11.6 355 +- 3.83 310 +- 3.85 p < 0.01 December 3.2-11.2 343 +- 6.40 312 +- 3.89 p < 0.01 Abbreviations: CB, chaff bedding; FMB, fermented manure bedding. animals-13-00842-t005_Table 5 Table 5 BCI of buffaloes with different bedding. Month Environment Temp (degC) BCI a.m. (%) p-Value BCI p.m. (%) p-Value CB FMB CB FMB January 3.6-11.6 87.78% +- 1.25% 90.07% +- 1.70% p = 0.30 85.50% +- 1.79% 86.28% +- 1.38% p = 0.73 February 5.0-13.1 87.40% +- 1.48% 91.21% +- 2.28% p = 0.18 86.28% +- 1.38% 88.58% +- 2.03% p = 0.36 March 7.9-16.7 86.91% +- 1.70% 92.06% +- 1.95% p = 0.064 86.80% +- 1.64% 89.80% +- 1.65% p = 0.22 April 12.4-18.5 86.84% +- 1.30% 89.07% +- 1.92% p = 0.35 87.26% +- 1.71% 89.72% +- 1.24% p = 0.26 May 17.5-24.1 87.14% +- 2.12% 87.29% +- 1.98% p = 0.95 86.75% +- 1.68% 89.21% +- 1.32% p = 0.26 June 22.4-32.0 86.56% +- 1.84% 90.16% +- 2.01% p = 0.21 85.15% +- 0.87% 88.31% +- 1.56% p = 0.09 July 25.0-32.4 82.53% +- 1.06% 86.91% +- 1.75% p < 0.05 84.94% +- 1.76% 86.17% +- 1.23% p = 0.57 August 23.3-29.9 85.16% +- 1.62% 87.78% +- 1.48% p = 0.25 84.43% +- 1.25% 85.37% +- 1.34% p = 0.62 September 21.5-32.3 86.52% +- 1.17% 89.52% +- 2.01% p = 0.22 84.61% +- 1.62% 87.34% +- 1.83% p = 0.28 October 14.2-21.2 86.93% +- 1.30% 89.88% +- 2.01% p = 0.24 85.64% +- 1.01% 90.40% +- 1.48% p < 0.05 November 3.6-11.6 86.83% +- 1.43% 89.74% +- 1.13% p = 0.13 86.06% +- 1.84% 89.20% +- 1.24% p = 0.17 December 3.2-11.2 88.11% +- 1.51% 89.74% +- 1.13% p = 0.41 86.49% +- 1.52% 89.64% +- 1.51% p = 0.16 Abbreviations: CB, chaff bedding; FMB, fermented manure bedding. animals-13-00842-t006_Table 6 Table 6 Average daily milk yield of buffaloes with different bedding materials. Month Environment Temp (degC) Average Daily Milk Yield (kg) p-Value CB FMB January 3.6-11.6 4.29 +- 0.082 4.57 +- 0.062 p < 0.01 February 5.0-13.1 4.24 +- 0.060 4.51 +- 0.061 p < 0.01 March 7.9-16.7 4.39 +- 0.047 4.56 +- 0.074 p < 0.05 April 12.4-18.5 4.27 +- 0.044 4.51 +- 0.053 p < 0.01 May 17.5-24.1 4.22 +- 0.063 4.52 +- 0.054 p < 0.01 June 22.4-32.0 4.13 +- 0.075 4.24 +- 0.071 p = 0.28 July 25.0-32.4 3.80 +- 0.048 4.05 +- 0.053 p < 0.01 August 23.3-29.9 3.90 +- 0.071 4.13 +- 0.056 p < 0.05 September 21.5-32.3 3.75 +- 0.065 4.05 +- 0.048 p < 0.01 October 14.2-21.2 4.26 +- 0.041 4.43 +- 0.072 p < 0.05 November 3.6-11.6 4.27 +- 0.056 4.52 +- 0.043 p < 0.01 December 3.2-11.2 4.25 +- 0.059 4.54 +- 0.061 p < 0.01 Abbreviations: CB, chaff bedding; FMB, fermented manure bedding. animals-13-00842-t007_Table 7 Table 7 Body hygiene score of buffaloes with different bedding materials. Month Environment Temp (degC) Body Hygiene Score p-Value Proportion of Dirty Buffalo SEM p-Value CB FMB CB FMB January 3.6-11.6 1.75 +- 0.19 1.86 +- 0.20 p = 0.70 25.00% 33.33% 1.08% p < 0.01 February 5.0-13.1 1.85 +- 0.20 1.81 +- 0.19 p = 0.16 30.00% 28.57% March 7.9-16.7 1.80 +- 0.19 1.95 +- 0.19 p = 0.57 25.00% 33.33% April 12.4-18.5 1.75 +- 0.18 1.86 +- 0.19 p = 0.69 20.00% 28.57% May 17.5-24.1 1.70 +- 0.19 1.90 +- 0.19 p = 0.46 25.00% 33.33% June 22.4-32.0 1.85 +- 0.18 1.81 +- 0.18 p = 0.87 25.00% 23.81% July 25.0-32.4 1.86 +- 0.17 1.96 +- 0.17 p = 0.69 23.81% 30.43% August 23.3-29.9 1.86 +- 0.19 2.00 +- 0.18 p = 0.58 28.57% 34.78% September 21.5-32.3 1.86 +- 0.19 1.87 +- 0.18 p = 0.96 28.57% 30.43% October 14.2-21.2 1.76 +- 0.18 1.87 +- 0.17 p = 0.67 23.81% 26.09% November 3.6-11.6 1.81 +- 0.16 1.91 +- 0.17 p = 0.66 19.05% 26.09% December 3.2-11.2 1.86 +- 0.17 1.91 +- 0.18 p = 0.82 23.81% 30.43% Abbreviations: SEM, overall standard error on the mean; CB, chaff bedding; FMB, fermented manure bedding. animals-13-00842-t008_Table 8 Table 8 Locomotion score of buffaloes with different bedding materials. Month Environment Temp (degC) Locomotion Score p-Value CB FMB January 3.6-11.6 1.40 +- 0.11 1.48 +- 0.11 p = 0.63 February 5.0-13.1 1.45 +- 0.11 1.43 +- 0.11 p = 0.89 March 7.9-16.7 1.35 +- 0.10 1.43 +- 0.11 p = 0.62 April 12.4-18.5 1.45 +- 0.11 1.48 +- 0.11 p = 0.87 May 17.5-24.1 1.35 +- 0.11 1.43 +- 0.11 p = 0.62 June 22.4-32.0 1.40 +- 0.11 1.48 +- 0.11 p = 0.63 July 25.0-32.4 1.38 +- 0.11 1.43 +- 0.10 p = 0.72 August 23.3-29.9 1.48 +- 0.11 1.52 +- 0.11 p = 0.77 September 21.5-32.3 1.43 +- 0.11 1.48 +- 0.11 p = 0.75 October 14.2-21.2 1.48 +- 0.11 1.43 +- 0.11 p = 0.79 November 3.6-11.6 1.43 +- 0.11 1.39 +- 0.11 p = 0.81 December 3.2-11.2 1.38 +- 0.11 1.43 +- 0.11 p = 0.72 Abbreviations: CB, chaff bedding; FMB, fermented manure bedding. animals-13-00842-t009_Table 9 Table 9 Hock lesion score of buffaloes with different bedding materials. Month Environment Temp (degC) Hock Lesion Score p-Value CB FMB January 3.6-11.6 0.85 +- 0.18 0.86 +- 0.17 p = 0.97 February 5.0-13.1 0.85 +- 0.19 0.90 +- 0.18 p = 0.84 March 7.9-16.7 0.95 +- 0.18 0.90 +- 0.18 p = 0.86 April 12.4-18.5 0.80 +- 0.17 0.86 +- 0.18 p = 0.82 May 17.5-24.1 0.90 +- 0.19 0.86 +- 0.17 p = 0.87 June 22.4-32.0 0.80 +- 0.19 0.90 +- 0.18 p = 0.69 July 25.0-32.4 0.86 +- 0.18 0.91 +- 0.18 p = 0.83 August 23.3-29.9 0.90 +- 0.18 0.87 +- 0.18 p = 0.89 September 21.5-32.3 0.81 +- 0.19 0.96 +- 0.17 p = 0.57 October 14.2-21.2 0.95 +- 0.17 0.91 +- 0.17 p = 0.88 November 3.6-11.6 0.86 +- 0.18 0.87 +- 0.18 p = 0.96 December 3.2-11.2 0.90 +- 0.17 0.96 +- 0.17 p = 0.83 Abbreviations: CB, chaff bedding; FMB, fermented manure bedding. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000034

Hepatitis E is a viral infectious disease in pigs, wild boars, cows, deer, rabbits, camels, and humans as hosts caused by Paslahepevirus. Recently, it has been detected in a wide variety of animals including domestic small ruminants. Mongolia is a land of nomadic people living with livestock such as sheep, goats, and cattle. Due to how Mongolian lifestyles have changed, pork has become popular and swine diseases have emerged. Among them, Hepatitis E disease has become a zoonotic infectious disease that needs to be addressed. The HEV problem in pigs is that infected pigs excrete the virus without showing clinical symptoms and it spreads into the environment. We attempted to detect HEV RNA in sheep which had been raised in Mongolia for a long time, and those animals living together with pigs in the same region currently. We also conducted a longitudinal analysis of HEV infection in pigs in the same area and found that they were infected with HEV of the same genotype and cluster. In this study, we examined 400 feces and 120 livers (pigs and sheep) by RT-PCR in Tov Province, Mongolia. HEV detection in fecal samples was 2% (4/200) in sheep and 15% (30/200) in pigs. The results of ORF2 sequence analysis of the HEV RT-PCR-positive pigs and sheep confirmed genotype 4 in both animals. The results suggest that HEV infection is widespread in both pigs and sheep and that urgent measures to prevent infection are needed. This case study points to the changing nature of infectious diseases associated with livestock farming. It will be necessary to reconsider livestock husbandry and public health issues based on these cases. hepatitis E pig sheep prevalence Mongolia phylogenetic analysis JSPS Core-to-Core Program, B. Asia-Africa Science PlatformsInstitute of Veterinary and Medicine, MongoliaRakuno Gakuen University, JapanThis research was partly supported by the JSPS Core-to-Core Program, B. Asia-Africa Science Platforms (2016-2018), and jointly sponsored by the Institute of Veterinary and Medicine, Mongolia, and Rakuno Gakuen University, Japan. pmc1. Introduction The hepatitis E virus (family Hepeviridae; subfamily Orthohepevirinae; genus Paslahepevirus) is one of the main viral causes of acute human hepatitis worldwide . Out of eight different genotypes, HEV-3 and HEV-4 are confirmed as zoonotic. Suidae are generally recognized as the main reservoirs of these genotypes. HEV-4 has been identified in China in goats , cows , cow's milk , and sheep . The disease is endemic in Asia, Africa, and Latin America . The presence of anti-HEV antibodies in sheep and goats has been reported worldwide . High similarity between human and ruminant HEV sequences has also been confirmed . This raises concerns about the zoonotic transmission of HEV from these animal species . In connection with this, it has been suggested that the consumption of contaminated milk, meat, and/or dairy products from sheep could be a source of HEV infection in humans . However, information about the relationship between other livestock and sheep is not well understood. Mongolia is a country of animal husbandry with 30 million sheep . It also has the third-largest population of sheep in the world . Sheep meat is considered a staple food for Mongolians. Due to recent dietary changes, pig farming is being promoted in Mongolia. Most of the pig farms are located in Tov Province. Currently, ham, bacon, liver paste, and sausages are common foods for Mongolians that are prepared from pork at pig farms in Tov Province. This situation must be considered an important public health issue for food safety. There have been several studies on the molecular characterization of HEV in both pigs and human patients in Mongolia . In a report on Mongolians, HEV prevalence was 12%, which is higher than in Russia (1.2%) and Japan (3%), despite lower pork consumption in Mongolia compared to the other countries. Although there have been reports of HEV infection in sheep from China, there is no information regarding HEV transmission from sheep in Mongolia. The purpose of this study was to determine the risk of HEV infection in native sheep associated with the introduction of pigs into livestock in Mongolia. 2. Materials and Methods 2.1. Samplings This was a cross-sectional study conducted in Tov Province and Ulaanbaatar city between 2020 and 2022. Considering a 95% confidence interval (95% CI) and the desired precision of +-5%, the sample size was calculated as 384. We sampled 400 feces (200 sheep feces and 200 pig feces), and 120 livers (60 sheep livers and 60 pig livers). Sheep fecal and liver samples were equally collected in the Bayanchandmani, Batsumber, Undurshireet, and Zaamar soums (districts) in Tov Province. The liver and fecal samples were transported to the laboratory on ice. The Bayanchandmani and Batsumber soums are close to Emeelt slaughterhouse, which is the main slaughterhouse in the central region of Mongolia. The study area for the sheep was set near Ulaanbaatar city, where pig farming is most prevalent in Mongolia. Sheep and free-roaming livestock were raised in the same region as the pig farms. The fecal samples were collected from 200 pigs and the liver samples were collected from 60 pigs slaughtered in Ulaanbaatar city. The ORF2 region of HEV RNA was a target sequence for viral detection . In addition, the farmers were asked questions that included general knowledge about HEVs and possible contact between livestock to analyze risk factors. 2.2. RNA Extraction and Detection of HEV RNA by RT-PCR Fecal samples from sheep and pigs were suspended 10% (w.p.v.) in sterile PBS. The fecal and liver samples were homogenized with zirconium beads in a tissue lyser (QIAGEN, Hilden, Germany). Next, the homogenized samples were centrifuged at 8000 rpm for 5 min, and the supernatants were collected in sterile tubes. RNA from pig livers, experimentally infected with HEV, was used as a positive control, and non-HEV-infected healthy pigs were used as a negative control . Viral RNA was extracted from fecal supernatants using a QIAamp Viral RNA extraction kit (QIAGEN, Hilden, Germany) according to the kit's protocol, and 50 mg from each liver tissue was lysed with 1 mL Trizol (Invitrogen, Carlsbad, CA, USA). RNA was precipitated with 0.5 mL isopropanol and washed with 1 mL of 75% ethanol. The RNA was solubilized in 20 mL RNase-free water . A one-step RT-PCR kit (QIAGEN, Hilden, Germany) was used to amplify the ORF2 region of the HEV (primer sequences used: ORF2-F1; (5772-5794) 5'-AATTATGCYCAGTAYCGRGTTG-3', ORF2-R1; (6416-6439) 5'-CCCTTRTCYTGCTGMGCATTCTC-3'). The reaction conditions were reverse transcription at 45 degC for 60 min and 95 degC for 15 min as a PCR cycle, denaturation at 94 degC for 30 s, annealing at 55 degC for 30 s, extension at 72 degC for 75 s for 35 cycles, and final extension at 72 degC for 7 min . Nested PCR: the RT-PCR products were then diluted and amplified again using Takara Ex-Taq (Takara Bio. Inc., Shiga, Japan) to check the PCR amplification of the F1-R1 region (primer sequences used : ORF2-F2 (5953-5975) 5'-GTWATGCTYTGCATWCATGGCT-3', ORF2-R2 (6341-6363) 5'-AGCCGACGAAATCAATTCTGTC-3') under the following conditions: 95 degC for 2 min (1 cycle); 94 degC for 30 s, 55 degC for 30 s, 72 degC for 30 s (35 cycles); and 72 degC for 5 min (1 cycle). The nested PCR products were electrophoresed on a 1.5% agarose gel, and amplified RNA bands were detected using a transilluminator (Toyobo-FAS-III, Toyobo Co., Ltd., Osaka, Japan) followed by ethidium bromide staining. The expected amplified HEV RNA band (348 bp) was sequenced after purification . 2.3. DNA Sequencing The PCR products from each DNA sample were purified using a FastGene Gel/PCR Extraction Kit (NIPPON Genetics Co., Ltd., Tokyo, Japan) and sequenced using an ABI Prism Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosciences, Foster City, CA, USA). The sequences were analyzed using a 3500 Genetic Analyzer (Life Technologies, Carlsbad, CA, USA) . 2.4. Phylogenetic Analysis The ORF2 gene sequences were then compared with those in the NCBI GenBank database using the multiple alignment function of the online ClustalW tool. Phylogenetic trees were constructed using the neighbor-joining method in MEGA (version X). A total of 28 sequences were submitted to GenBank through an online submission system and accession numbers were obtained and compared with previously reported sequences . The same data were utilized to generate an ML phylogenetic tree, which was initially used to conduct the Bayesian maximum clade credibility (MCC) host a discrete traits tree by using the software BEAST v1.8.4 accessed on 12 June 2022). The strict clock and the best fit GTR + G + I nucleotide substitution model with a constant population size coalescent tree prior were used. The MCMC was run at 50,000,000 generations and sampled at every 5000 generations. The effective sample size (ESSs) of the analysis was checked by using the software Tracer v1.6 accessed on 12 June 2022). The MCC host discrete traits output tree was generated by using TreeAnnotator v1.10.4 accessed on 12 June 2022) afterburn 10% of the first trees. The host phylogenetic tree was reconstructed by using the software FigTree v.1.4.3 accessed on 12 June 2022) . 2.5. Anti-HEV Antibody Detection from Sheep and Pigs Serum samples from sheep (n = 42) and pigs (n = 45) collected in the study area were investigated for antibodies to HEV. In-house ELISA was used in this study as previously described . Anti-HEV antibodies were detected by enzyme-linked immunosorbent assay (ELISA) using VLPs derived from HEV genotype 3 as antigens. For the detection of antigen-bound IgG, an anti-pig IgG antibody-HRP conjugate and an anti-sheep IgG antibody-HRP conjugate (Bethyl Laboratories Inc., Montgomery, TX, USA) were applied as a secondary antibody according to the method described in a previous report . The serum samples were diluted 1:100 with PBS containing 0.05% Tween 20, and 10% of Block Ace (DS Pharma Promo Co., Ltd., Osaka, Japan) and incubated for 1 h at room temperature. After the secondary antibody reactions, 50 mL of TMB (3,3',5,5'-tetramethylbenzidine) (Kirkegaard & Perry Laboratories Inc., Baltimore, MD, USA) was added, and after 10-min incubation at room temperature, 50 mL of 2 M sulfuric acid was added to stop the reactions. The optical density (OD) value at 450 nm was measured by a microplate spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Positive control sera were obtained from pigs experimentally infected with HEV, and negative sera were obtained from non-infected, healthy pigs. The cut-off value was set at 0.295 by calculating the average value + 2 standard deviation of the negative samples (n = 5). The sensitivity and specificity of this ELISA test in 20 experimentally infected and 24 negative pigs were 90.0% (95% CI: 68.30-98.77) and 91.67% (95% CI: 73.0-98.97), respectively. 2.6. Statistical Analysis The age of the pigs was categorized as piglet less than 6 months; older than 6 months; over 1 year old. All epidemiological data were triple-entered and cleaned in Microsoft Office Excel 2010 (Microsoft Co., Redmond, WA, USA). We performed univariable and multivariable analysis using IBM SPSS Statistics 26 (IBM, Chicago, IL, USA) and Fisher's exact test calculation . Categorical variables were compared using Fisher's exact test and kh2 test. A statistically significant difference was considered when the p-values were <0.05. The adjusted odds ratio (OR) and the 95% confidence interval (CI) for HEV risk factors were analyzed using univariate and multivariable logistic regression. 3. Results 3.1. Prevalence of HEV RNA in Sheep in Tov Province, Mongolia All of the fecal and liver samples were examined for the HEV ORF2 region using nested RT-PCR. The HEV ORF2 region was detected in sheep liver and fecal samples (Table 1). A total of 3/60 (5%) positive sheep livers were found near farms, which is in Bayanchandmani soum, Tov Province. Three RT-PCR-positive sheep liver samples were sequenced, and these sequences belonged to genotype 4 (accession numbers LC752752, LC752753, and LC702420). The sequence was highly homologous (98%) to those detected from pig samples (PF121, PF101, and PF175, which is located in the Songino Khairkhan district of Ulaanbaatar city; accession numbers LC413554, LC413553, and LC413557) (Table 1). The HEV prevalence in the feces samples was 2% (4/200) in sheep. 3.2. Prevalence of HEV RNA among Pigs in Tov Province, Mongolia Pig feces (PF) and pig livers (PL) were collected from a farm (sample numbers: MGL-PF 1-200, MGL-PL 1-60). A total of 200 pig feces samples and 60 pig liver samples were examined for the HEV ORF2 region using nested RT-PCR, and the HEV ORF2 regions were detected in fecal samples (30/200, 15%) and liver samples (4/60, 6.6%) using nested RT-PCR (Table 2). 3.3. Genetic Analysis of the HEV from Pigs and Sheep The nucleotide sequence analysis of the HEV ORF2 region detected in pigs identified genotypes 3 and 4. The pig-derived HEVs belonging to genotype 3 were divided into two clusters. The viruses belonging to genotype 4 were divided into two clusters, one of which showed high homology to the sheep sequence (Supplementary Tables). The accession numbers of the HEV-3 type were LC413551, LC413555, LC413556, LC413558, and LC413573. The accession numbers of the HEV-4 type were LC413550, LC413552, LC413553, LC413554, LC413557, and LC413574. The HEV detection rate for those older than 6 months was 12.3%, with 75% of the positive samples belonging to genotype 3 and 25% belonging to genotype 4. Pigs over 1 year of age were slaughtered and RT-PCR for HEV in the liver was performed. The results showed that 6.6% (4/60) of the samples were positive and that the positive samples belonged to genotypes 3 and 4, with two animals in each (accession numbers LC413570, LC413569, LC413550, and LC413574) (Table 2, Supplementary Tables). Phylogenetic analysis showed that 19 out of the 25 sequences belonged to genotype 3 . The sequence analysis of the ORF2 region of three of the HEV-detected sheep belonged to genotype 4 (accession numbers: LC702420, LC752752, and LC752753). The sequence was identical to the genotypes (LC413553, LC413554, and LC413557) detected in pigs from the same region. Their amino sequences were also 99% identical to a previously reported sequence (ABO93596). Homology comparison of the sequences of HEV genotype 4 identified in Mongolian sheep and China (KU904269) showed a homology of 86%. Therefore, the HEV genotype 4 identified in Mongolian sheep was different from the HEV genotype 4 in China. The Bayesian MCC hosts a discrete traits tree for 302 bp HEV ORF2 nucleotide sequences (nt position 6022-6324) of a total of 40 Mongolian strains, including 28 sequences from our study and reference strains (six swine HEV-ORF2 sequences, genotype 3 , four human HEV-ORF2 sequences , and two Bactrian camel HEV-ORF2 (BcHEV1, BcHEV2) sequences ) obtained from the GenBank database . The phylogenetic host tree indicated transmission between HEV hosts, the host at the node indicated the ancestor of the sub-group, and the number at the node indicated the posterior probability. Genetic analysis of HEV determined in Mongolian people suggested that it was derived from genotype 4 of HEV in pigs and sheep in that country. In addition, six swine HEV-ORF2 sequences, three sheep HEV-ORF2 from our study, and four human HEV-ORF2 sequences from a previous study belong to genotype 4 of HEV. The other 25 pig HEV-ORF2 sequences belong to genotype 3, including 19 sequences from our study and 6 sequences from F. Lorenzo's study . 3.4. Anti-HEV Antibody Detection from Sheep and Pigs Serum samples from sheep (n = 42) and pigs (n = 45) collected in the study area were investigated for anti-HEV IgG. HEV-3 genotype VLP protein was used as the ELISA antigen. The HEV seropositive animals were 11.9% (5/42) of the sheep and 35.5% (16/45) of the pigs. 3.5. Statistical Results Univariable analysis was performed (Supplementary Table S3). Categorical variables were compared using Fisher's exact test and kh2 test. Age and foreign introduction of pigs are statistically significant (p < 0.05), while gender and feeding source is not statistically significant. A risk assessment was performed for each variable (Table 3). The odds ratio between the age ranges was calculated when the age of 1 year old is the reference, and the highest was a less than 6 months old piglet, OR = 3.42. The odds ratio between the feeding source was calculated when mill offal is the reference; the highest OR equals 2.6 in the small ruminant offal. The odds ratio between whether a foreign introduction is introduced or not is the reference, the highest OR equals 3.06 in the foreign introduction. These factors led to the higher risk for HEV cases in Mongolia. These three risk factors were statistically significant (p < 0.02-0.003, 95% CI). Gender was not statistically significant. 4. Discussion HEV has been detected in pigs and other animals all over the world including in Asia . The HEV genotypes predominantly detected in Asia are genotypes 3 and 4, which are known to cause zoonoses. In this study, we used molecular methods to demonstrate the prevalence of HEV in Mongolian pigs and sheep. HEV genotype 3 has been detected in Mongolia previously ; however, the sequences of the viruses obtained in this study were different from those previously reported. Moreover, our results have shown the efficient detection of HEV RNA in feces. Virus shedding is an important factor in virus transmission among pigs on farms . Thus, these results imply that sanitary control on pig farms is an important issue for healthy livestock management in the country. Since meat is the staple diet of the Mongolian people, there is concern that meat contaminated with HEV could cause adverse situations, especially for pregnant women and other hepatitis virus carriers. The prevalence of HEV in the swine liver requires caution for handlers at slaughterhouses and in meat processing factories. The phylogenetic tree analysis showed that genotypes 3 and 4 were detected in pigs. The HEV gene sequence detected in pigs in Mongolia is highly homologous to previously reported cases, which may be due to the establishment of the virus based on the importation of live pigs to Mongolia. The history of live piglet imports from China suggests that Mongolian HEVs originate from two different sources. HEV genotypes 3 and 4 are similar to Asian isolates, including those from Japan and China . One HEV-4 genotype cluster was also identified in sheep-derived HEVs, suggesting that HEVs can be transmitted between pigs and sheep. From the results of the phylogenetic analysis, 19 sequences belonged to cluster HEV-3 and six sequences belonged to cluster HEV-4. The HEV strain may be transmitted between farms via human and pig movement. Pigs are usually fed on the by-products of slaughterhouse-derived sheep's internal organs. The pigs had no restrictions on their movement. Phylogenetic analysis of the ORF2 region revealed that the HEV genotypes in the infected pigs in the vicinity of Ulaanbaatar city are homologous to those in sheep. This could be because sheep co-graze near pig farms in the same area. In comparison with HEVs from sheep detected in Tov Province, phylogenetic analysis showed genetic similarity with PF121, PF101, and PF175 from pigs. The geographical distance between Bayanchandmani soum (the sheep sample area) and Songino Khairkhan district intersects with livestock movement and feed distribution. Therefore, it is quite possible that HEV of livestock origin between the same regions could be transmitted via various routes. Mongolian sheep and goats usually co-graze in pasturelands together with other livestock, including cattle. We assume that sheep are not HEV reservoirs but are spillover infected animals. Sheep are most likely spillover infected animals due to HEV infection from pigs. The maximum clade credibility tree analysis in this study suggests that sheep HEV is of porcine origin. Chinese and Italian researchers have detected HEV in fecal and milk samples from goats and sheep . HEV isolates have been detected in the southern and eastern parts of China, including Yunnan Province, Tai'an region, and Shandong Province . With regard to HEV detection efficiency and the age of the tested pigs, the detection rate was low in subjects aged approximately 1 year old but high in younger populations (less than 6 months old) . Fecal shedding of the virus is frequently detected in young pigs under 6 months of age and this age is a risk factor for environmental shedding of the virus. On the other hand, since the virus is detected in the liver even at the age of over 6 months, there is a risk of HEV infection in the livers of pigs shipped to the market. The main concern is that the virus shed from infected pigs is transmitted to other pigs. Each year, 10-20% of pigs are replaced by foreign introductions, mainly from China. Fattened pigs are slaughtered for meat, and piglets are fattened on farms. The statistical analysis indicated that the foreign introduction of pigs was a strong factor and that another possible cause of infection could be the feeding source. Except for piglets, all of the pigs were fed with mill offal as a fattening food. According to a cross-sectional study, there was a statistically significant difference in terms of the use of small ruminant offal and mill offal. Free-range pigs can contaminate the water or hay resources of sheep that graze near pig farms. Gender was not found to be associated with the risk estimation analysis. Improvements are needed to reduce the opportunities for contact with other livestock in the future. In our study, HEV-antibody-positive sheep were detected (positivity at about 11.9% (5/42) in sheep and 35.5% (16/45) in pigs), and previous reports of HEV infection epidemiology around the world also suggest that ruminant livestock is at risk of HEV infection . If there is the contamination of HEV from the internal organs of livestock, the possible transmission risk of the virus in Mongolia will be high. Mongolian people's main source of food is meat. HEV-contaminated food can lead to bad situations, especially in pregnant women and other hepatitis virus carriers. HEV has been reported to have been detected in sheep from neighboring China. Anti-HEV antibodies were detected in 35.2% of sera and 57.7% of slaughter meat samples from Xinjiang, which borders the Mongolian west border. All of the isolates belonged to genotype 4 . Moreover, various types of livestock are mixed-reared in China, and it has been pointed out that HEVs are transmitted between livestock . To date, sheep infected with HEV-4 have been reported from China as follows: KU904269 sheep HEV. In our study, the MCC analysis suggested that the pig and sheep HEV detected in this study could be transmitted to humans, presenting a public health issue. A detailed investigation is required for virus transmission among livestock, and investigation and management of HEV-infected animals are also important for public health. The risk verification of the use of pig feed from Mongolian livestock would be a further research point from the perspective of zoonotic diseases. 5. Conclusions The present results suggest that the spread of HEV in Mongolian livestock is a transmission risk owing to the management of introductions among pigs. Furthermore, HEV of the same genotype was shared among pigs and sheep reared in the same area. As sheep offal is fed to pigs, livestock husbandry management to interrupt HEV transmission and prevent its spread appears necessary. The virus was also confirmed in the livers of slaughtered pigs and sheep, suggesting the need for a call to attention with regard to food hygiene. These data show that measures are needed to improve prevention and control strategies for food safety. Acknowledgments The present study was selected as part of a grant for young researchers and supported by "Project for Strengthening the Practical Capacity of Public and Private Veterinarians", MJ-VET, JICA. The authors are thankful to Norikazu Isoda, for his assistance in the statistical analysis of this study. Supplementary Materials The following supporting information can be downloaded at: Table S1: Sample genotype and HEV homology % (NA) detected in this study; Table S2: Sample genotype and HEV homology % (AA) detected in this study; Table S3: Univariate analysis of HEV in farm pig in Ulaanbaatar city; Table S4: HEV genotypes used in this phylogenetic tree; Table S5: Reference Information of Research Pigs; Table S6: Reference Information of Research Sheep. Click here for additional data file. Author Contributions Sample collection, E.B. and A.S.; molecular biological analysis, E.B. and B.B.; preliminary processing, E.B. and A.S.; investigation, E.B., B.B. and Y.K.-M.; writing--original draft, E.B. and B.B; data curation, E.B. and B.B.; software, E.B., B.B. and Y.K.-M.; methodology B.B., Y.K.-M. and K.H.; formal analysis B.B. and Y.K.-M.; visualization, Y.K.-M.; validation, Y.K.-M. and K.H.; conceptualization, K.H.; supervision, K.H.; writing--reviewing and editing, K.H.; project administration, K.H.; funding acquisition, K.H. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All animal sampling was performed according to the ethical guidelines of the Institute of Veterinary Medicine of Mongolia (sampling ethical approval number: MEBAUS-18/02/10). Informed Consent Statement Not applicable. Data Availability Statement The datasets presented in this study are publicly available. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Sampling locations in Mongolia. The upper left figure shows the whole Mongolian territory with two neighboring countries and the locations of Tov Province and the capital city, Ulaanbaatar. The lower left figure shows the whole territory of Ulaanbaatar city and the location of the pig farm in Songino Khairkhan district in the red rectangle. The figure on the right shows the whole territory of Tov Province and the locations of four soums are shown in pink (Zaamar, Undurshireet, Bayanchandmani, and Batsumber soums) where we collected the feces and liver samples of the sheep. In addition, the figure shows that the Bayanchandmani and Batsumber soums are near to the pig farm located in Songino Khairkhan district. Emeelt--the main slaughterhouse in Mongolia--is also in Songino Khairkhan district. The red rectangles show the areas where the pig feces samples were collected. Figure 2 Phylogenetic analysis of HEV-ORF2 regions detected in animals in Mongolia with Smith's prototype sequences. Phylogenetic tree (neighbor-joining method) of the open reading frame (ORF) 2 gene region of hepatitis E virus (HEV) genotypes 3 and 4. The numbers and letters before the accession number in the table indicate the HEV subtype, and the genotype (HEV-3, HEV-4) is shown after the sample number identified in this study. Blue dots represent pig feces samples, orange dots represent pig liver samples, and red dots represent sheep liver samples (ShL-0-2). A total of 28 samples were sequenced for HEV ORF2. MEGA X was used for multiple alignments and phylogenetic tree construction. Based on the phylogenetic tree results, 9/28 sequences belonged to HEV-4, whereas 19/28 sequences belonged to HEV-3. The ORF2 gene region of the JQ001749.1_Bat HEV/GE/2009 (GE--Germany) complete genome was used as an outgroup. The bar represents 0.05 nucleotide substitutions per site. Figure 3 The Bayesian maximum clade credibility (MCC) host discrete traits tree for 302 bp HEV ORF2 nucleotide sequences (nt position 6022-6324) of 40 Mongolian strains, including 28 sequences from our study and reference strains (six swine HEV-ORF2 genotype 3, reprinted with permission from reference , four human HEV-ORF2 sequences , and two Bactrian camels HEV-ORF2 (BcHEV1, BcHEV2) sequences ) obtained from the GenBank database. The phylogenetic host tree indicated transmission between HEV hosts, the host at the node indicated the ancestor of the sub-group, and the number at the node indicated the posterior probability. The bar at the bottom of the figure denotes evolutionary distance. animals-13-00891-t001_Table 1 Table 1 Detection of HEV RNA of sheep in Tov Province. Samples Numbers Positive Nos. Rate (%) 95% CI Liver 60 3 5.0 4.75-5.25% Feces 200 4 2.0 1.25-2.75% animals-13-00891-t002_Table 2 Table 2 Detection of HEV RNA on pig farms. Pig Age Male/Female Positive in Feces Positive in the Liver 95% CI Less than 6 months 10/30 10/40 (25%) NT 20-30% Older than 6 months 32/98 16/130 (12,3%) NT 7.3-17.3% Around 1 year 8/22 4/30 (13.3%) NT 8.3-18.3% Over 1 year 15/45 NT 4/60 (6.6%) 1.6-11.6% Total 65/195 30/200 (15%) 4/60 (6.6%) NT: not tested. animals-13-00891-t003_Table 3 Table 3 Risk factor analysis of HEV in farm pigs in Ulaanbaatar city. Variables Category Total Positive Negative Positive (%) OR p-Value Sex Female 195 31 164 15.9 1.86 0.183 Male 65 6 59 9.2 Ref Age Less than 6 months 40 10 30 25 3.42 0.014 More than 6 months 130 16 114 12.3 1.44 0.424 1 year old 90 8 82 8.9 Ref Feeding source Sheep offal 40 10 30 25 2.6 0.02 Mill offal 220 25 195 11.4 Ref Foreign introduction Yes 51 13 38 25.5 3.06 0.003 No 209 21 188 10 Ref Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000035

The correct interpretation of an animal's emotional state is crucial for successful human-animal interaction. When studying dog and cat emotional expressions, a key source of information is the pet owner, given the extensive interactions they have had with their pets. In this online survey we asked 438 owners whether their dogs and/or cats could express 22 different primary and secondary emotions, and to indicate the behavioral cues they relied upon to identify those expressed emotions. Overall, more emotions were reported in dogs compared to cats, both from owners that owned just one species and those that owned both. Although owners reported a comparable set of sources of behavioral cues (e.g., body posture, facial expression, and head posture) for dogs and cats in expressing the same emotion, distinct combinations tended to be associated with specific emotions in both cats and dogs. Furthermore, the number of emotions reported by dog owners was positively correlated with their personal experience with dogs but negatively correlated with their professional experience. The number of emotions reported in cats was higher in cat-only households compared to those that also owned dogs. These results provide a fertile ground for further empirical investigation of the emotional expressions of dogs and cats, aimed at validating specific emotions in these species. emotion expression dog cat owner perception welfare This research received no external funding. pmc1. Introduction Dogs and cats may provide a wide range of health, emotional, behavioral, cognitive, educational, and social benefits to humans, and support economic growth . To maximize these animals' social and economic value, we need to understand and interpret their emotions appropriately so that high-quality human-animal interaction and animal welfare can be achieved. A misinterpretation can lead to suffering in these animals (e.g., behavioral problems), owner distress and mental health issues, reduced societal benefits, and possible physical harm to humans . For instance, dog bites often occur because humans cannot correctly identify the early signs of discomfort or distress in dogs . Many dog owners also believe their dogs can feel guilt , yet studies have shown that so-called 'guilty' behavior exhibited by dogs after a 'prohibited' behavior may instead be linked to owners' scolding behavior . If the dog cannot understand why their owner is scolding them, it could cause them to feel unnecessary distress and negatively impact its welfare. Considering the importance of understanding emotion in human-animal interactions, we know surprisingly little about what extent dogs and cats express a typical range of emotions and what behavioral signs are indicative to humans for potentially detecting these emotions. This lack of information may be due to inherent challenges in studying emotion in nonhuman animals, as their behavior and communicative signals vary greatly from our own and we cannot rely on language to help bridge the gap. Furthermore, human emotion perception and processes seem to be preferentially adapted to conspecifics, but we may adopt an identical or similar process to perceive and interpret both conspecific and heterospecific emotional cues . For instance, humans displayed qualitatively similar gaze distributions by applying the same local facial features when viewing human and dog faces or judging the approachability of these faces , and by gazing predominantly at the faces when assessing whole-body human and dog emotional expressions . Considering that humans and dogs facially express the same emotion differently and many non-facial cues (e.g., tail movements, ear and body postures) are informative indicators of dog affective states but are less evident or absent in humans, the same gaze strategy, which is adapted for the effective perception of human emotions, may not function well in perceiving dog emotions, subsequently leading to poor human performance in recognizing dog emotions . However, regardless of their ability or performance in identifying animal emotions, pet owners are arguably an important potential data source for studying companion animal emotions because of their intensive interaction with their pets, their experience of emotional triggers and contexts associated with specific pet emotions, and their knowledge or sensitivity to subtle pet emotional behaviors. Although this data source is naturally subjective and susceptible to anthropomorphic bias (i.e., attributing human emotion characteristics to animals), owner perception of pet emotions may provide consistent trends and collective evidence that could provide a fertile source for guiding further empirical research to determine whether these owner observations are accurate. To date, only a few studies have examined owners' beliefs about their pet's emotions, with large variances between individual owners' reports and between studies. Generally, dog and cat owners believed their pets could express a wide range of emotions, which can be grouped into primary/basic and secondary/complex emotions . Primary emotions (e.g., anger, disgust, fear, joy, sadness, and surprise) are more widely supported in animals as they are viewed as evolutionarily adaptive and biologically predisposed . In contrast, the occurrence of secondary emotions, which can be a mixture of primary emotions and may embrace both positive and negative effects concurrently alongside cognitive elaborations (e.g., compassion, disappointment, embarrassment, empathy, envy, and pride) , are considered more controversial. This is usually because many of these secondary emotions involve meta-cognitive (e.g., cognitive appraisal of own and/or other's mental states, such as jealousy) and/or self-conscious evaluative processes (e.g., evaluation of own behavior against learned and internalized rules or standards, such as guilt or shame) that are often believed to be beyond the representational capacities of human infants and nonhuman animals . Nevertheless, roughly half of the pet owners reported some secondary emotions in their pets . Furthermore, dog and cat owners may report differently about their pet's emotions. In a recent survey, participants attributed primary emotions to both dogs and cats roughly equally (88% dogs vs. 87% cats); however, secondary emotions were only reported by 44% of cat owners, compared to 55% of dog owners . Furthermore, cats were more frequently attributed the emotions of anger and disgust, while all other emotions surveyed were reported more frequently in dogs (also see ). However, these studies mainly used a 'between-subject' design in which participants were asked to report the pet they have lived with the longest, even if they owned both dogs and cats. It is unclear whether these findings could be extended to those people with experience and knowledge of the emotional behavior of both dogs and cats. Regarding the large variances in the reported number and type of emotions across dog and cat owners, this might be caused by both individual differences in owners and breed differences in dogs/cats. According to recent studies, the number of owner-reported pet emotions or the accuracy in recognizing pet emotional expression images could be affected by an owner's gender and age , cultural background , mental health condition (e.g., anxiety and depression) , personal or professional experience with the species , and attachment with the species . Additionally, wide variations in morphology across dog and cat breeds, such as coat color, coat length, ear shape, and tail shape , could affect the visibility of animal facial and/or bodily expressions and subsequently owners' abilities in reading these animal emotions. However, no owner perception studies to date have collated all of these owner and pet breed factors to assess their potential impact on the identification of animal emotions. To address these potential confounding factors in previous research, in the current study we aimed to directly compare the number of emotions reported by dog and cat owners, both in homes with only one species and homes with both species, using an extended emotion list including primary and secondary emotions commonly reported in both human and animal emotion research e.g., . To our knowledge, it is the first study to collect data on multiple species from the same owners. If significant differences are found, a detailed analysis could reveal which individual emotions are driving the variation. Based on previous research , we hypothesized that more emotions (especially secondary emotions) would be reported in dogs than in cats, and this trend would be qualitatively similar for owners that keep both species. We also requested owners to indicate the common animal arousal, signaling, and behavioral tendency signs used by them to detect specific animal emotions . Given that human primary and secondary emotional expressions are associated with distinctive patterns of dynamic multistage and multimodal behavior (including facial action, head/body gesture, movements, vocalization, etc.) , it is plausible that different dog/cat emotional expressions could also be differentiated based on the information contained within these behavioral changes. Our final aim was to collect data containing a large group of owners (e.g., varying in age, gender, and pet experience) with pets varying in age, sex, and morphology so that we could determine those factors which may influence the number of emotions reported within a species. 2. Materials and Methods 2.1. Participants Dog and cat owners over 16 years old were mainly recruited via social media (Facebook, Instagram, and LinkedIn). A recruitment poster was uploaded to the authors' personal social media pages as well as relevant Facebook group pages (found via searches for 'dog owners', 'cat owners', 'dog', and 'cat'). In addition to social media, some participants were recruited via dog training and behavior businesses. The survey was open for 6 weeks and there was no incentive for participation. To minimize anthropomorphic attribution, participants had to have lived with their pets for at least two years so that they could answer the survey questions by using their direct and intimate experience with their pets rather than their general ideas about animal emotions . Participants that had both a dog and a cat were asked to complete the survey for both species. Participants that had multiple dogs/cats could choose to complete the survey multiple times if their pets were different breeds. Prior to the study, the research purpose and survey tasks had been explained to the participants, and informed consent was obtained from each of them. All procedures complied with the British Psychological Society Code of Ethics and Conduct. The study was approved by the Ethical Committee of the University of Lincoln (2022-1069). 2.2. Questionnaires The online survey was created in Qualtrics and included three sections: (1) owner demographic information, (2) pet demographic information, and (3) an emotion questionnaire. Owner demographic questions were a mixture of free-type and multiple choice and included questions on age, gender, cultural background, years of pet ownership, professional experience with dogs/cats (e.g., trainer, behaviorist, groomer, kennel/cattery worker, dog walker, dog/cat sitter, rescue work, fosterer, education), and mental health information (e.g., autism, anxiety, and depression). Pet demographic questions were also a mixture of free-type and multiple choice (with picture guides for dog ear and muzzle shape), and included age, sex, breed, and morphological characteristics (for dogs, they included fur color, muzzle size (short, medium, and long), ear shape (cropped, pricked/bat, drop/folded, V-shaped, rose-shaped, and semi-pricked/button), and tail shape (docked/bobbed, tightly curled over back, and other); for cats, they included fur color, fur length (hairless, short, and long), fur pattern (tabby, solid, bi-color, tortoiseshell/calico, and colorpoint), ear shape (pricked, curled, and folded), and tail shape (no tail, bobbed, curly, other)). The emotion questionnaire included 6 primary emotions (anger, disgust, fear, joy, sadness, and surprise) and 16 secondary emotions (amusement, anxiety, boredom, confusion, curiosity/interest, disappointment, embarrassment, empathy, frustration, grief, guilt/shame, jealousy, love/affection, pain, positive anticipation, and pride). Owners were asked whether their pet could express a certain emotion (Yes/No). If the owners answered yes to a particular emotion, they were then asked to choose the behavioral cues they used to identify this emotion. They chose from a list of behavioral cues that included proximity to the owner, facial expression, head posture, ear posture, body posture, tail height, tail wagging, vocalization, eye contact, speed of movement, physical contact and action, and "others" (adapted from ), and could select as many as they deemed applicable. 2.3. Data Analysis All data analyses were conducted in R 4.1.2. Linear models were used to analyze whether the number of owner-reported animal emotions (i.e., the total number of reported emotions, number of primary emotions, and number of secondary emotions) varied between dogs and cats, both in dog/cat-only homes and in homes with both species. For each emotion, binary generalized linear models were then used to compare the percentage of 'yes' and 'no' responses reported for dogs and for cats (dogs in dog-only homes vs. cats in cat-only homes, dogs vs. cats in homes that owned both species). Furthermore, multifactor linear models (one for owner factors and one for pet factors) were used to assess whether individual differences in owners and breed differences in dogs/cats could significantly alter the total number of emotions reported for dogs or cats. For owners that had professional experience with dogs or cats, their professions were categorized to facilitate post hoc comparisons (all other data were analyzed in their original form). Specifically, trainers and behaviorists were grouped together, all veterinary professional staff were grouped together, and the remaining respondents were divided into 'hands-on' (e.g., kennel/cattery workers, rescue workers, groomers, and pet sitters) and 'hands-off' groups (e.g., education and research). Binary generalized linear models were used to analyze the proportion of 'yes' and 'no' responses for each emotion in cat/dog-only vs. cat and dog homes to understand which emotions were driving the results of the owner multifactor linear model. 3. Results In total, 453 completed survey responses were collected. Fifteen were excluded from further analysis because the respondents had lived with their pets for less than 2 years, leaving 438 responses to be analyzed. Of these, 277 were from dog-only owners, 93 were from cat-only owners, and 68 were from owners of both species. No respondents completed the survey for multiple dogs or cats of different breeds. 3.1. Do Owners Believe Dogs Have More Emotions than Cats? Dog-only owners reported a significantly higher number of emotions than cat-only owners (F(1, 368) = 12.65, p < 0.001; Figure 1). On average, 65% of dog owners and 58% of cat owners believed their pets could express a given emotion. Regardless of species, the five most frequently reported animal emotions were curiosity (98%), happiness (96%), love (96%), fear (89%), and anxiety (86%); the five least reported emotions were guilt (17%), grief (20%), embarrassment (24%), pride (28%) and disgust (32%). There also existed significant variations in individual emotions reported between dog and cat owners. Specifically, for primary emotions, happiness and sadness were reported significantly more frequently in dogs, whereas anger was reported more frequently in cats (all p-values < 0.05). For secondary emotions, anxiety, boredom, confusion, envy/jealousy, frustration, guilt/shame, pain, and positive anticipation were reported more frequently in dogs (all p-values < 0.05). All other emotions were reported with comparable frequencies between dog and cat owners (all p-values > 0.05). Respondents that owned both cats and dogs also reported significantly more emotions in their dogs (F(1, 134) = 83.54, p < 0.001; Figure 2). Unlike the dog-only versus cat-only comparison, these owners consistently reported more dog expressions for both primary (F(1, 134) = 20.21, p < 0.001) and secondary emotions (F(1, 134) = 99.62, p < 0.001). Referencing the 6 primary emotions, owners reported fear, happiness, sadness, and surprise more frequently in their dogs than in their cats (all p-values < 0.05). Almost all secondary emotions were reported significantly more frequently in dogs (all p-values < 0.05), except for curiosity, love, and pride (all p-values > 0.05). 3.2. Do Owners Rely on the Same Cue Sources to Identify Dog and Cat Emotions? Table 1 compares common cue sources used by over 50% of respondents to identify a given emotion expressed by dogs and cats. Across all emotions, body posture, facial expression, and head posture were the three most frequent sources of cues used by both dog and cat owners (reported by 66% vs. 59% vs. 52% of dog owners, and 63% vs. 47% vs. 43% of cat owners). Although there existed quantitative differences in the reported frequency for a given source of information, dog and cat owners used some common sources to detect 16 out of 22 animal emotions and relied on the same set of sources in identifying 5 emotions (disgust, empathy, pride, confusion, and frustration; Table 1). However, when differentiating (at least some) different emotions in either dogs or cats, a distinctive combination of sources of behavioral cues tended to be informative for owners (e.g., facial expression and body posture for dog embarrassment vs. proximity to the owner and physical contact for dog empathy), and this tendency was more evident for dog owners than cat owners. Furthermore, there were no clear differences in common sources of behavioral cues (e.g., up to three most frequently reported cues) used to detect different animal emotions between dog-only owners and dog + cat owners or between cat-only owners and cat + dog owners. 3.3. Which Owner or Pet Factors Affect the Number of Reported Animal Emotions? The analyzed owner factors included owner's age (1% of owners were 16-20 years old, 30% were 20-29, 21% were 30-39, 17% were 40-49, 18% were 50-59, 11% were 60-69, and 3% were over 70), gender (87% female, 12% male, and 1% nonbinary), cultural background (90% Caucasian, 3% Asian, 0% African, 4% others, and 3% preferred not to say), mental health profile (24% with autism, anxiety, depression, etc., 72% none, and 4% preferred not to say), years of pet ownership, years of professional experience with dogs/cats, type of dog-/cat-related profession, and pet species (Table 2). For dogs, the number of reported emotions (varying between 3 and 22) was only affected by the years of experience owners had, both in a personal (varying between 2 and 70 years; F(1, 269) = 6.87, p = 0.009) and professional (varying between 0 and 70 years, F(1, 269) = 7.35, p = 0.007) capacity. Specifically, the number of dog emotions reported tended to increase with increasing years of personal experience but decreased with increasing professional experience . These patterns appear quite weak visually and are not significant with a simpler Pearson correlation analysis (personal experience: r = 0.05, p = 0.17; professional experience: r = -0.06, p = 0.13). For cats, the number of reported emotions was only affected by pet species owned (i.e., whether owners also owned dogs; F(1, 84) = 28.87, p < 0.001; Figure 4). Cat-only owners reported more emotions in their cats compared to owners of both cats and dogs (F(1, 159) = 54.73, p < 0.001). This pattern occurred for both primary (F(1, 159) = 31.53, p < 0.001) and secondary emotion categories (F(1, 159) = 53.39, p < 0.001). Specifically, compared to owners of both species, cat-only owners more frequently reported the primary emotions of anger, fear, sadness, and surprise, and secondary emotions of anxiety, curiosity, embarrassment, empathy, envy/jealousy, guilt, amusement, boredom, confusion, disappointment, and frustration (all p-values < 0.05). Our analyzed pet factors included pet age, sex, breed, ear shape, tail shape, dog muzzle shape, dog coat color, cat coat length, and cat coat pattern (Table 3 and Table 4). The multifactor linear model analysis revealed that the number of reported dog or cat emotions was not affected by any of these factors (all p-values > 0.05). 4. Discussion The three objectives of this exploratory study were to (1) directly compare dog and cat owners' beliefs about the number of emotions their pets could express, (2) evaluate what behavioral changes were associated with each emotional expression by owners, and (3) analyze the influence of various owner and pet-related factors on the owners' belief. Regarding the 1st objective, our analysis revealed that pet owners only reported more anger in cats than in dogs and a more frequent expression of two other primary and eight secondary emotions in dogs . The latter trend was exaggerated in owners of both dogs and cats in which dogs were believed to express 4 primary and 13 secondary emotions more frequently than cats . Such variation in the perceived pet emotions between dogs and cats may partly be due to differences in the emotional bond between owners of the two species, as owners of both dogs and cats have reported a greater level of emotional closeness with their dogs than their cats . Cat owners were also less likely to consider their cats as 'family members' than dog owners, indicating a lower level of owner-cat attachment than owner-dog attachment . However, caution in making such generalizations is warranted as recent work has indicated a wide disparity in the nature of cat-owner relationships, with some owners forming very strong, emotionally dependent relationships . Our novel observation of the exaggerated belief of dog emotion capabilities (especially) from owners of both dogs and cats may further suggest the crucial role of human-pet attachment in perceiving pet emotions. Future work could consider the effect of the nature of the pet-owner relationship on the number of perceived emotions of a pet. Nonetheless, domestication history may also be important. Dogs were historically selected to work for humans, in roles typically requiring frequent heterospecific communication and understanding . Cats, on the other hand, had a more commensal relationship with humans, typically hunting rodents independently . As semi-solitary animals, their need for heterospecific communication and the ability to express emotion in a way that others can easily interpret may not have been as evolutionarily important . Cats also tend to look at their owners less frequently . Owners may thus perceive cats' behaviors as generally less sociable and communicative or simply independent , all of which could reduce the level of perceived emotion in this species by owners. The fact that cats are semi-solitary animals may also explain why anger was the only emotion reported significantly more frequently in cats than in dogs by cat-only owners. For instance, aggressive behavior may be more common in cat-only households with multiple cats competing for key resources and territorial boundaries . Cat-only owners may also tend to spend more time interacting with their cats compared to owners of both species, which could increase the prevalence of petting-related aggressive incidents . These possibilities could be addressed in future research by more in-depth and structured interviews with cat and dog owners, yielding a more detailed understanding of processes underlying those believed variations in emotional capability between the two species. Regarding the second objective, our analysis revealed that both dog and cat owners tended to rely on a comparable set of sources of behavioral cues, especially body posture, facial expression, and head posture, to detect their pet's emotions, indicating the important role of nonfacial cues in identifying animal emotions. As the facial expression is generally the dominant channel of emotional expression in human-human emotional perception , humans predominantly rely on facial cues to recognize other humans' emotions and tend to adopt a similar strategy to detect animal emotion by mainly gazing at the faces when assessing whole-body animal emotional expressions . This strategy, adapted for the effective perception of human emotions, may lead to poor human performance in recognizing dog emotions . This is because some nonfacial cues (e.g., ear and body posture) are highly informative indicators of dog emotional states . Indeed, the experienced pet owners (>=2 years of pet ownership) in this study consistently reported body posture and head posture as two of the most frequently used behavioral cues to decipher their pet's emotions, indicating the adoption of a learned and animal-specific strategy in perceiving pet emotions. It would be informative to objectively measure, in future research, the effectiveness of these owners' strategies in recognizing different animal emotions, especially from unfamiliar dogs/cats. The pet (especially dog) owners further believed that many different categories of animal emotions were associated with distinctive combinations of behavioral cues, such as changes in facial expression and body posture for dog embarrassment, and changes in proximity to the owner and physical contact for dog empathy (Table 1). These beliefs are consistent with previous empirical observations of dogs displaying distinctive facial movements, coded through an objective, anatomically based dog facial action coding system in response to different categories of emotional triggers . Hence, it is plausible that, like humans , dogs can display a distinctive range of primary and secondary emotions that are recognizable through emotion-specific dynamic changes in facial and bodily expressions. This hypothesis could also be empirically examined in future research. Regarding the third objective, our analysis revealed that the number of reported dog emotions had a tendency to increase with owners' increasing personal experience with dogs, but decrease with increasing professional experience with dogs. As reading a dog's body language and multimodal emotional behavior is likely to involve a social learning process, longer dog ownership may allow the owners to pick up on subtle changes in their dog's behavior and to be familiar with (or sensitive to) different emotional triggers and contexts. This might enable them to report more (especially complex secondary) dog emotions. It could also be argued that this longer dog ownership would result in a stronger emotional bond between owner and dog with a concomitant increased risk of anthropomorphism and humanization bias toward their dogs , which may lead to an increased willingness to attribute human-like emotions to their dogs and an over-estimation of dogs' emotional capabilities. On the other hand, longer professional experience with dogs may allow individual humans to be familiar with possible individual and/or breed variances in dog emotional behaviors triggered by the same emotional context, hence reducing the possibility of attributing different dog behaviors to different emotions and anthropomorphism bias toward their own dogs. However, most of our participants had a relatively long personal experience but relatively short professional experience with dogs. This skewed sample distribution might have biased our findings. These hypotheses could be systematically examined in future research. It should also be noted that these relationships between the number of reported dog emotions and owners' personal/professional experiences were relatively weak, as they were not revealed by correlation analysis. Further research could re-examine these relationships by recruiting a larger cohort of dog owners. Furthermore, the number of reported cat emotions was reduced significantly if cat owners also had a dog at home (the results for dogs were similar regardless of any cats in the house). One interpretation is that cats may behave differently (e.g., being repressed and less expressive) if they live with dogs compared to if they live alone or with other cats. Owners may also report fewer cat emotions if they spend less time with the cat because the dog is always close by. However, it is worth mentioning that most dogs and cats can share a house peacefully, and tend to choose to spend time in the same room every day . In addition to possible changes in cat behaviors, owners' behaviors or attitudes may also lead to this variation in reported cat emotions. Perhaps owners of both species are overlooking cats' emotional capabilities and/or are inferring dogs' emotional states more frequently than cats because they believe that they have a greater level of emotional closeness with their dogs and are more likely to treat dogs (but not cats) as 'members of the family' . These hypotheses could also be systematically examined in future research. It should be noted that the generalization of these reported research findings might be constrained by the limits of our sample population. For instance, the participating pet owners were dominated by female (87%) and Caucasian (90%) participants, as is common in such work. Given that the owners' gender and cultural background may affect their ability to perceive dog emotions , future research could recruit a larger cohort of dog and cat owners, or one that is more focused on a more balanced gender and culture distribution, to systematically examine the impact of owner factors on the perception of animal emotions. Nevertheless, the variation in the number of emotions reported between species has several implications, both for animal welfare and human health and safety. One potential implication is that the emotional states of cats, particularly those living with dogs, are being overlooked or interpreted incorrectly. This could mean that the welfare needs of cats are not being met in terms of reducing the incidences of negative emotions (sadness, frustration, boredom, etc.) and increasing the opportunities to elicit positive emotions (happiness, amusement, etc.). If the findings are driven by owners having a relatively less positive belief in cats compared to dogs, this could also affect how they treat their cats (e.g., the misattribution of cat fear as anger may lead to the use of positive punishment, negatively impacting the cat's welfare). Furthermore, being unable to interpret subtle cat behavioral cues may put an owner at risk. For example, petting-related aggression often occurs because owners miss those subtle cues indicating that the cat wants the owner to stop touching them . Conversely, it is also possible that the emotional capabilities of dogs are being exaggerated and/or misinterpreted because of increased anthropomorphism and humanization trends in dog owners. Believing dogs are just another member of the family can lead to welfare issues. If owners do not appreciate how different dogs are, particularly concerning their communicative signals, they may interpret dog signals in a human-like way more frequently. For example, the reported sources of behavioral cues for detecting guilt/shame in dogs were 'head posture', 'body posture', and 'eye contact', with the most likely scenario being a dog that is holding its head low, contracting its body and avoiding eye contact. In fact, these behaviors are typically associated with a fearful, deferent, or submissive dog, and so scolding the dog when he exhibits these behaviors may exacerbate the dog's fear/anxiety and have a negative impact on its welfare. Humanization can also increase the risk to humans, as it increases the likelihood of people ignoring signs of discomfort or putting their dog in situations they cannot cope with, believing that their dog would never hurt them. For example, most dog bites involving children occur with familiar dogs when unsupervised in the home environment, and the behavior of the child toward the dog is the most common trigger . 5. Conclusions This online study compared owners' perceptions of their dogs' and cats' emotional expressions. Overall, more emotions were reported in dogs compared to cats, both from owners that owned just one species and those that owned both. While dogs and cats used a comparable set of behavioral signs to express the same emotion, distinct combinations tended to be associated with specific emotions in both cats and dogs. Although owner beliefs and anthropomorphisms are problematic in many situations, they are helpful as a starting point for an objective definition of animal emotions. With current technology, it is difficult to confirm animal emotional states; however, validating or refuting these owner beliefs could be the next step in this direction. This should be a high-priority research area as it is evident that regardless of whether owners are underestimating the emotional abilities of cats or overestimating that of dogs (or perhaps both), there are potentially important consequences for both humans and the animals they hope to care for. Author Contributions Conceptualization, O.P., D.S.M. and K.G.; methodology, O.P. and K.G.; formal analysis, O.P.; investigation, O.P.; writing--original draft preparation, O.P.; writing--review and editing, D.S.M. and K.G.; visualization, O.P. and K.G.; supervision, K.G. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethical Committee of the University of Lincoln (protocol code: 2022-1069 and date of approval: 17 May 2022). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement Data are available from the authors. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Percentage of participants reporting individual emotions as a function of species in dog-only (n = 277) and cat-only (n = 93) homes. * p < 0.05, ** p < 0.01, *** p < 0.001. Figure 2 Percentage of participants reporting individual emotions as a function of species in homes with both dogs and cats (n = 68). * p < 0.05, ** p < 0.01, *** p < 0.001. Figure 3 The number of emotions reported in dogs by owners with varying levels of personal (A) and professional experience with dogs (B). Trendlines show the direction of significance. Figure 4 Percentage of participants reporting individual emotions in their cats, comparing cat-only homes (n = 93) with those that also own dogs (n = 68). * p < 0.05, ** p < 0.01, *** p < 0.001. animals-13-00820-t001_Table 1 Table 1 Sources of behavioral cues were reported by over 50% of respondents for each emotion and species. Those that are common to both species are highlighted in bold. Emotion Dogs Cats Anger Facial expression, body posture, tail height, vocalisations Facial expression, ear posture, body posture, wagging tail, vocalisations Disgust Facial expression Facial expression Fear Proximity to owner, facial expression, head posture, ear posture, body posture, tail height Ear posture, body posture, tail height, speed of movement Happiness/Joy Facial expression, head posture, ear posture, body posture, tail height, wagging tail, vocalisations, speed of movement Facial expression, head posture, body posture, tail height, vocalisations Sadness Facial expression, head posture, ear posture, body posture, tail height Body posture Surprise Facial expression, head posture, ear posture, body posture Facial expression, body posture, speed of movement Anxiety Proximity to owner, facial expression, ear posture, body posture, tail height Body posture Curiosity/Interest Facial expression, head posture, ear posture, body posture, tail height Facial expression, head posture, ear posture, body posture Embarrassment Facial expression, body posture Body posture Empathy Proximity to owner, physical contact Proximity to owner, physical contact Envy/jealousy Proximity to owner None Grief Facial expression, body posture, physical contact None Guilt/shame Head posture, body posture, eye contact None Love/affection Proximity to owner, facial expression, body posture, eye contact, physical contact Proximity to owner, head posture, body posture, vocalisations, physical contact Pride Facial expression, head posture, body posture Facial expression, head posture, body posture Amusement Facial expression, wagging tail, vocalisations Body posture Boredom None None Confusion Facial expression, head posture Facial expression, head posture Disappointment Facial expression, body posture None Frustration Vocalisations Vocalisations Pain Facial expression, body posture, vocalisations, speed of movement Body posture, speed of movement Positive anticipation Facial expression, head posture, ear posture, body posture, tail height, wagging tail, vocalisations Facial expression, head posture, body posture, vocalisations animals-13-00820-t002_Table 2 Table 2 Pet ownership demographic details. Category % of Dog Owners % of Cat Owners Personal experience with dogs or cats (Years) 2-10 years 43% 40% 11-20 years 23% 22% 21-30 years 15% 19% >30 years 18% 19% Professional experience with dogs or cats Yes 40% 24% No 60% 76% Profession Veterinary staff 23% 44% Trainer/Behaviourist 42% 8% Other (hands-on) 23% 31% Other 12% 18% Professional experience (years) <5 years 39% 46% 5-10 years 34% 21% >10 years 27% 33% animals-13-00820-t003_Table 3 Table 3 Dog demographic details. Dog Demographic Category % of Dogs Age Junior (2-7 years old) 45% Senior (>7 years old) 55% Sex Female 48% Male 52% Coat colour Black 15% Brown/Tan 10% Cream/White 8% Golden/Red 12% Grey/Blue 2% Fur with 2 colours 37% Fur with 3+ colours 16% Ear shape Cropped 1% Drop/Folded 25% Pricked/Bat 17% Rose shaped 7% Semi-pricked/Button 20% V-shaped 30% Muzzle length Long 7% Medium 85% Short 8% Tail shape Docked/bobbed 9% Other 82% Tight curl 8% animals-13-00820-t004_Table 4 Table 4 Cat demographic details. Dog Demographic Category % of Cats Age Junior (2-7 years old) 37% Senior (>7 years old) 63% Sex Female 57% Male 43% Coat length Long hair 19% Short hair 81% Coat pattern Bi-color 30% Color point 2% Solid 20% Tabby 31% Tortoiseshell/Calico 17% Ear shape Pricked ears 98% Folded ears 2% Tail shape Bobbed 7% Curled tail 10% All others 83% Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000036

Manganese is a trace element with essential physiological functions that should be supplied to animals and humans through diet. Goose meat is prevalent in many regions of the world. Therefore, the aim of the study was a systematic review (PRISMA statement, 1980-2022) of the content of Mn in raw and cooked goose meat and their relation to the recommended intake at the level of adequate intake (AI) and the nutrient reference values-requirements (NRV-R). The literature analysis shows that the content of Mn in goose meat depends on the breed, type of muscles, the presence of skin, and the cooking method used. AI level recommendations for Mn intake range from 0.003 to 5.50 mg/day, depending on the country, age, and gender. Consumption by adults (regardless of sex) of 100 g of domestic or wild goose meat covers the daily AI per Mn in various percentages, depending on the type of muscles (more Mn in leg muscles), presence of skin (more Mn in skinless muscles), and thermal treatment (pan fried with oil, grilled, and cooked meat contains more). Placing information on the Mn content in goose meat and the percentage of NRV-R on the packaging may be valuable information for the consumer in making food choices to diversify the diet. There are few studies on the content of Mn in goose meat. Therefore, it is reasonable to conduct research in this area. goose meat manganese thermal treatment adequate intake reference values-requirements PRISMA This research received no external funding. pmc1. Introduction Manganese (Mn) is one of the essential minerals in animal and human nutrition. It is classified as a micronutrient or trace element. Manganese (Mn) is an essential dietary mineral for mammals and is a component of metalloenzymes such as arginase, glutamine synthetase, and pyruvate carboxylase . Because it activates manganese superoxide dismutase (MnSOD), manganese is necessary for normal antioxidant defenses. In addition, it is an activator for many hydrolases, kinases, decarboxylases, and transferases. Manganese is involved in amino acid-, lipid- and carbohydrate metabolism, and proteoglycan synthesis in bone formation. Its role in regulating and transforming thyroid hormones is indicated . It is necessary for utilizing biotin, vitamin B1, and vitamin C. Metabolic association between manganese and choline affects fat metabolism in the liver . Manganese competes directly with cobalt (Co) and iron (Fe) for binding sites in the digestive tract. Therefore, an excess of Co or Fe may result in lower absorption of Mn and its potential deficiency. The amount of manganese absorbed is inversely related to the concentration of manganese in the diet. The human body content of manganese is estimated to be 10-20 mg. The concentration is relatively high in bone and organs rich in mitochondria, such as the liver, pancreas, and kidney, and concentrations are low in muscle and plasma . This regulation seems to be part of the adaptive changes to the amount of dietary manganese intake, which allow the maintenance of manganese homeostasis over a wide range of intakes. Humans obtain manganese from the air, water, and food. Plant sources have much higher manganese concentrations than animal sources. Whole grains (wheat germ, oats, and bran), rice, and nuts (hazelnuts, almonds, and pecans) contain the highest amounts of manganese. Chocolate, tea, mussels, clams, legumes, fruit, leafy vegetables (spinach), seeds (flax, sesame, pumpkin, sunflower, and pine nuts), and spices (chili powder, cloves, and saffron) are also rich in manganese . Manganese is mainly absorbed as Mn(II), and absorption is reported to be below 10% of ingested manganese. Manganese uptake in the intestine is mediated by high-affinity metal transporters, such as divalent metal transporter-1 (DMT1, also called DCT1), which is also involved in transporting other metals . Absorbed manganese is transported to the liver and distributed to other tissues bound to transferrin, a2-macroglobulin, and albumin. The main route of elimination of manganese from the body is via bile to the small intestine, whereas very little is excreted in the urine. The half-life in men varies from 13 to 37 days, and the half-life in women is longer, but sizeable inter-individual variation exists . Low dietary manganese intake results in increased manganese absorption relative to intake . Vegetarians often have diets richer in manganese than those who select omnivorous diets because they consume more fiber and phytates. Dermal changes and hypercholesterolemia are possible signs of manganese deficiency, as well as diffuse bone demineralization and poor growth in children . Geese are waterfowl consumed in some regions of the world, especially Asia, some countries of Europe, and the USA . The quality of meat obtained from them is a complex combination of characteristics such as appearance, color, texture, functionality (e.g., cooking loss), taste, and nutritional value, including mineral content . Goose meat can be a valuable source of minerals in the human diet . However, their amount of meat depends on many factors: poultry species, age, sex of birds, rearing system and region, time of fattening, content in the feed, drinking water, premixes, and even medicines. Deficiency of Mn in young birds causes perosis (swelling and deformation of the tibia-metatarsal joints, "slipping" of the Achilles tendon, and lameness). Manganese in plant materials is available in 50-60%, but its bioavailability in poultry feed mixtures is reduced in excess calcium and phosphorus. Because its content and bioavailability in plant feed fluctuate significantly, it is often added to premixes in the form of inorganic trace minerals (ITM), for example, oxides, carbonates, chlorides, and sulphates. Manganese has a low potential for toxicity due to its poor intestinal absorption and efficient biliary elimination. Still, it can interact with several other dietary nutrients, such as Zn and Fe, by competing with Fe for intestinal absorption sites or reducing Fe and Zn tissue concentrations . Historically, manganese sulfate has been the most common source of manganese in animal supplements. Manganese sulphate monohydrate is generally used as the standard reference for assessing Mn bioavailability. Still, the actual absorption of the Mn in this compound is 2% to 8%, depending on the species and the diet to which Mn is added. Manganese can be added to feed in the form of organic trace minerals (OTM), for example, metal proteinate and metal amino acid chelate, which are more expensive than ITM. Relative bioavailability values for manganese in poultry can range from 29% for manganese dioxide MnO (ITM) to 174% for manganese methionine Mn-Met (OTM) compared to the sulphate standard . However, an excessive supply of this ingredient in the feed reduces the absorption of iron, magnesium, and phosphorus. The minimum nutritional requirements of geese is 60 mg/kg in a grower and 40 mg/kg in a breeder. The manganese content in goose feed, its chemical form, and bioavailability may be reflected in its range in goose meat. In the literature, there are few published research results on the content of Mn in goose muscles in terms of its consumption and, more often, in biomonitoring as a bioindicator of environmental pollution . In addition, in the literature, the content of minerals in poultry meat is often given without dividing it into breast and leg meat, which as culinary portions, are most often consumed by consumers. However, muscles differ in their histological structure and the nature of metabolic changes, which may affect the content of minerals, including manganese . Similarly, considering the sex of poultry, males, and females differ in the growth rate, which affects the amount of feed and manganese intake, and thus its use in the body, as well as excretion. Despite these facts, research conducted in the pectoral muscles of the Egyptian goose by Geldenhuys et al. shows that the manganese content in males and females did not differ significantly, amounting to an average of 0.06 mg/100 g dry basis. In addition, as part of preventing cardiovascular disease or weight reduction, consumers are encouraged to remove the skin and subcutaneous fat from carcass elements before cooking. The skin is a source of fat, cholesterol, sulfur amino acids, collagen, elastin, fat-soluble vitamins, and minerals . For the consumer, it may be essential to know the manganese content in the elements of a goose carcass with or without skin. This systematic review aims to (1) analyze the original animal studies from 1980-2022 on the manganese content of raw and culinary processed goose, (2) present chosen recommendations for adequate intakes (AI) for manganese (mg/day) depending on age and sex; (3) compare manganese in goose meat content with adequate intake and nutrient reference values-requirements for humans. 2. Methods The review was prepared following PRISMA guidelines to ensure the transparency of the research conducted . An electronic-based search was performed in the scientific libraries Scopus, Web of Science, and ScienceDirect . Searches comprised a combination of MeSH terms and keywords, applying quotes and field tags with BOOLEAN operators. For all databases, four primally exclusion steps were determined: 1. keywords ("goose AND meat") AND (manganese or mineral OR Mn), 2. years (1980-2022), 3. language (English), 4. publication type (article). The AGRO database was used as an additional source . AGRO bibliographic database has been created by the members of the main library of the University of Life Sciences in Poznan since 1993. The database includes bibliographic descriptions of articles of 1010 Polish titles published in Polish and English. The screening was made in the areas: title, abstract, keywords (Scopus), and topic (Web of Science, Science Direct). The search and selection process was performed by two reviewers working independently and in parallel. The results from each database were exported to files with the appropriate extension of CSV or excel, and summaries were created for each database containing publication information, including abstracts. The files are included in Supplementary Materials. Initial searches returned 61 results, and 46 peer-reviewed papers were selected for consideration based on relevance after removing duplicates. Two independent researchers reviewed the title, abstracts, and papers that did not meet the criteria for inclusion in the review were excluded through discussion. Two independently working researchers analyzed the results obtained to avoid errors. Any inconsistencies were resolved through discussion. Exclusion criteria were as follows: reviews, research notes, book chapters, and no data on the content of manganese in goose meat in the article's abstract and contents. Additional searches were performed on the FoodData Central U.S. website Department of Agriculture (USDA) . A total of 11 original studies, 5 reports from the USDA database, and 2 reports from Tables of composition and nutritional value of food were selected for review (Table 1). Figure 1 presents a diagram of the literature search and selection criteria. 3. Manganese Content in Raw Goose Meat The manganese content (mg/kg of tissue) in raw goose muscles is shown in Table 2. In Poland, a series of studies on the content of, for example, manganese, as an element necessary in animal organisms but also as a bioindicator of environmental pollution was conducted by Falandysz et al. . In the studies carried out in the years 1978-1983, in the raw meat of geese for slaughter from northern Poland, 0.17 mg Mn/kg was found ; in 1984, 0.38 mg Mn/kg was found ; and in 1985, 0.26 mg Mn/kg was found . In 1986, the average content of Mn in the raw muscles of slaughter geese from northern Poland was comparable to that found in earlier years (1983-1985) (1.9 mg/kg) , and in 1987 the mean value obtained was 0.27 mg Mn/kg . In subsequent studies, in the meat of geese randomly selected from slaughterhouses during 1988-1991, Falandysz et al. determined 0.25 mg Mn/kg. The content of manganese in the muscles of Polish geese, stated by the above-cited authors, was characteristic of this animal species and the type of tissue. Kunachowicz et al. give 0.2 mg Mn/kg of tissue in the muscles of Polish geese. Chen et al. , examining goose meat purchased in markets and supermarkets in Taipei (Taiwan), found the average Mn content at 0.268 mg/kg of tissue. Considering the type of muscles, Oz and Celik determined the manganese content in Turkish goose's breast and leg muscles, which was 0.2 and 5.0 mg/kg tissue, respectively. Considering the presence of skin, data from the U.S. Department of Agriculture) show that raw goose with skin has less of this mineral than meat without skin (0.20 vs. 0.24 mg/kg tissue, respectively). Similarly, Goluch et al. analyzed the Mn content in raw breast muscles with and without skin from White Koluda(r). The mean Mn content in breast muscles was 1.6 mg/kg dry mass (DM) and did not differ significantly between with skin and without skin (1.5 vs. 1.7 mg/kg DM). Game geese are also a source of energy and nutrients for the population in many parts of the world. For example, in South Africa hunted (during spring and autumn hunting) Egyptian geese (Alopochen aegyptiacus) are eaten. In the Eastern James Bay Cree of Quebec in Canada, goose (Branta canadensis) is traditionally eaten by local rural communities. A study by Geldenhuys et al. in the pectoral muscles of the Egyptian goose showed that the Mn content did not differ significantly in terms of sex and season (winter vs. summer) and was, on average, 0.6 mg/kg dry basis. Canada's goose raw breast muscles without skin contained 0.50 mg Mn/kg of tissue . 4. Manganese Content in Goose Meat after Thermal Treatment Because people rarely eat raw meat, the mineral content after it has been subjected to various thermal treatments is important. Cooking meat is essential to achieve a tasty and safe product. Different ways of processing meat strengthen its taste and delicacy and improve its hygienic quality by inactivating pathogenic microorganisms. During the heat treatment, cooking losses due to mass transfer depend on not only the cooking conditions, such as cooking method, cooking surface, cooking temperature, and time but also the meat properties, such as water content, fat content, protein content, pH value of the raw meat, and the meat portion size . The internal temperature endpoint, during boiling, significantly affects mineral content . Losses of minerals during the thermal treatment of meat depend on the form in which they occur. Mineral components, which can be found as soluble dissociated salts (part of sodium, small amounts of phosphorus, calcium, and potassium), go to the leakage. Components, such as iron, that combine with proteins remain in the meat . The thermal treatment can lead to the loss of a part of the mineral matter, thereby reducing the product's nutritional value. The most significant mineral matter reduction is generated when the meat is thermally treated in the aquatic environment . The manganese content in goose meat subjected to thermal processing is presented in Table 3. USDA data analysis shows the identical Mn content in raw and cooked skinless carcasses (0.24 mg/kg tissue), whereas in cooked carcasses with skin higher than in raw carcasses with skin (0.24 vs. 0.23 mg/kg tissue). The research conducted by Kunachowicz et al. showed a higher content of Mn in cooked and roasted geese carcasses with skin than in raw carcasses (0.30 vs. 0.20 mg/kg tissue). Oz and Celik researched the breast and leg muscles of Turkish geese slaughtered at 24 weeks. Their estimates of manganese content were made both in raw meat (Table 2) as well as that submitted to boiling (<100 degC), grilling (180 degC), pan-frying without fat or oil (180 degC), pan-frying with oil (180 degC), deep-fat frying (180 degC), oven roasting (200 degC), and automatic microwave cooking. These various thermal treatments were applied for 5 to 35 min, depending on the cooking method and type of muscle. The content of Mn in the breast and leg muscles of geese was changed under the influence of various types of thermal processing; however, these changes were not statistically significant. Goluch et al. studied the effect of various methods of heat treatment (water bath cooking WBC, oven convection roasting OCR, grilling G, pan frying PF) on manganese content in White Koluda(r) goose breast muscles with and without skin. They found significantly (p <= 0.05) the highest Mn content in skinless WBC muscles compared to PF muscles (2.7 vs. 1.1 mg/kg DM). However, in muscles with skin, significantly (p <= 0.01), the highest content of Mn was found in grilled muscles (3.8 mg/kg DM), compared to raw muscles and other methods of heat treatment (raw 1.5, WBC 1.4, ORC 1.1, PF 1.3 mg/kg DM). In terms of Mn, the interaction between the type of breast muscle (with or without skin) and the heat processing method was statistically significant (p <= 0.001). However, the authors did not note significant differences in this ingredient retention, regardless of the muscle type (with or without skin) and the applied heat treatment. Considering the wild goose, Geldenhuys et al. found 1 mg Mn/kg of tissue in the breast muscles of Egyptian geese cooked in a preheated oven (160 degC). According to Sorbal et al. , boiling and frying lower the mineral content of meat, whereas baking, grilling, and microwaving increases it. Grilling and boiling pork and beef decrease the contents of Na, K, P, Ca, and Mg while increasing the contents of Fe and Zn . Deep-frying, pan-frying, oven-cooking, and microwaving decrease the mineral content of cooked beefsteak, and microwaving causes the highest loss . Purchas et al. compared the mineral content in uncooked and cooked lean beef and reported a decrease in the contents of Na and K and an increase in Ca, Cu, Fe, Mn, and Zn in cooked meat in comparison with raw meat. These results indicate that divalent minerals are better retained during cooking than Na and K. The lower loss of divalent minerals during cooking is due to their greater association with protein. For manganese, Goluch et al. observed no significant differences in the retention of this component, regardless of the type of goose muscle (skinless or with skin) and the applied heat treatment. 5. Recommendation for Manganese Intake and Its Consumption in Different Countries Balance studies have suggested that an intake of 0.74 mg/d should be sufficient to replace daily manganese losses . Intakes over 1 mg/d generally result in a positive manganese balance . According to the data (Table 4) of the American Institute of Medicine (IOM, now National Academy of Medicine), the recommended intake of manganese for adults, at the level of adequate intake (AI), is 1.8 mg/day for women and 2.3 mg/day for men . In 1993, the EU Scientific Committee for Food suggested 1-10 mg/d to be an acceptable intake of manganese . The World Health Organization, the Food and Agriculture Organization of the United Nations (WHO/FAO), and the Nordic countries (Nordic Council of Ministers--NCM) have not established recommended intakes of manganese . The European Food Safety Authority (EFSA) concluded that data is insufficient for deriving average requirements (ARs) or population reference intakes (PRIs) for manganese. EFSA proposed an adequate intake (AI) at 3 mg/day for adults based on observed mean intake from a mixed diet in the EU as stated in dietary reference values (DRVs) for manganese. An AI of 3 mg/day is proposed for adults, including pregnant and lactating women. For infants aged 7 to 11 months, an AI of 0.02-0.5 mg/day is suggested, reflecting the wide range of manganese intakes that appear to be adequate for this age group. The main contributor to dietary manganese intake is cereals (57%), followed by fruit, vegetables, nuts, and coffee/tea . The societies for nutrition in Germany, Austria, and Switzerland recommend a manganese intake of 2.0-5.0 mg per day for adults and children above the age of 10 years . Higher AI values have been determined in Australia and New Zealand: 5 mg/day for women and 5.5 mg/day for men . Manganese standards in Poland have been adopted according to the IOM at the AI level . The Scientific Committee on Food (SCF) could not set any observed adverse effect level (NOAEL) because no relevant dose-response animal studies were found. Consequently, SCF did not assign a tolerable upper intake level (UL) for manganese. animals-13-00840-t004_Table 4 Table 4 Adequate Intakes (AI) for manganese (mg/day) concerning the chosen recommendations. Age, Both Sexes IOM (2001) NIZP-PZH (2020) EFSA (2013) DACH (2021) NHMRC, AGDHA NZMH (2006) Infants 0-6 months 0.003 4-12 months 0.6-1.0 0.003 7-12 months 0.6 7-11 months 0.02-0.5 0.600 Children and adolescents 1-3 years 1.2 1-3 years 0.5 1-3 years 1.0-1.5 2.0 4-8 years 1.5 4-6 years 1 4-6 years 1.5-2.0 2.5 9-13 years 1.9 1.6 7-10 years 1.5 7-9 years 2.0-3.0 3.0 2.5 14-18 years 2.2 1.6 11-14 years 2.0 >=10 years 2.0-5.0 3.5 3.0 15-17 years 3.0 Adults >18 years 2.3 1.8 >=18 years 3.0 5.5 5.0 Pregnant all ages 2.0 3.0 5.0 Lactation all ages 2.6 3.0 5.0 AI--adequate intake; IOM--Institute of Medicine (now National Academy of Medicine); NIPH-NIH--National Institute of Public Health-National Institute of Hygiene; EFSA--European Food Safety Authority; NHMRC--National Health and Medical Research Council; AGDHA--Australian Government Department of Health and Ageing; NZMH--New Zealand Ministry of Health; DACH--Nutrition Societies in Germany and Austria and Switzerland. In some countries, the intake of manganese is not often estimated. In the EU, adults' estimated mean manganese intakes range from 2 to 6 mg/day, with most values around 3 mg/day. Estimated mean manganese intake ranges from 1.5 to 3.5 mg/day in children and 2 to 6 mg/day in adolescents . The Polish population's estimated intake of manganese was 4.7 mg/day. The average content of manganese in women's diets was 4.10 mg/day, whereas in men's diets, it was 5.45 mg/day . In contrast, the Spanish population's estimated daily manganese intake was 2.372 mg/day. The population of the island of El-Hierro presented the highest intake (2.717 mg/day), and the one in Fuerteventura (1.986 mg/day) showed the lowest intake . Results from the Swedish Market Basket study, 2015, indicate an average daily manganese intake of 4.2 mg per person and day and was well above estimated adequate intakes (AI) of 1.8 mg per day for women and 2.3 mg per day according to the Institute of Medicine U.S. . The average daily per capita intake of manganese from meat was 0.07 mg . Calculations based on data from Denmark, 2013 and 2015, evaluate the mean dietary intake of manganese to 3.9 mg/day for adults and up to 6.9 mg/day in the higher intake groups. The manganese intake of Finnish children 3-18 years of age was 3-7 mg/d calculated from food consumption and composition . In a study conducted among the communities of Northern Italy, the average intake of manganese was 2.34 (1.46-3.52) mg/day . A pilot study of 20,000 people in Germany shows that the average intake of manganese in the general German population aged 14-80 is about 2.8 mg per day (for a 70 kg person) and is in the 2-5 mg per person per day as recommended by the German, Austrian and Swiss nutrition societies . 6. Coverage of the Adequate Daily Intake of Manganese (AI) and Nutrient Reference Values-Requirements by Goose Meat in Adults For the consumer purchasing food products, the information on the label, which relates to energy and nutritional value, is essential. This information should also include the reference value of the daily intake (NRV). These recommendations are based on the best available scientific knowledge of the daily amount of energy or nutrient needed for good health . The nutrient reference values (NRVs) for manganese, 2 mg/day for adults, is listed in Annex XIII to Regulation (EU) No 1169/2011 of The European Parliament and of The Council of 25 October 2011 on the provision of food information to consumer . According to this regulation, the nutritional value of products, including mineral content, is given per 100 g or 100 mL. The Codex Committee on Nutrition and Foods for Special Dietary Uses has determined that NRV-R for manganese is 3 mg . Table 5 presents calculations of the daily coverage of AI on Mn recommended by selected countries for adults, taking into account its content in raw and thermally treated meat. Consumption by adults (regardless of sex) of 100 g of raw Turkish goose leg muscles in the highest percentage (9.09-27.8%) will cover the daily AI per Mn, taking into account the presented recommendations of 1.8-5.5 mg/day. In the case of consumption of 100 g of raw breast muscle without skin, the highest percentage of AI implementation (3.09-9.44%) was calculated for meat from Polish White Koluda(r). However, considering wild goose meat, more AI will cover 100 g of raw muscle from Egyptian Geese (1.2-3.33%). In many countries, various thermal treatment of goose meat is used, which is associated with changes in the content of minerals and their retention . According to our calculations, the largest percentage of the daily AI for adults will cover 100 g of Turkish goose leg meat fried in a pan with oil (32.5-81.5%) and Polish White Koluda(r) breast muscles grilled with skin (7.6-21.1%). Also, the consumption of 100 g of cooked wild Egyptian goose breast meat will cover a significant percentage (10.9-33.3%) of the daily AI. The higher Mn content in fried and pan-roasted meat is due to the specific nature of these treatments, which do not require water, allowing for greater water retention. It has already been shown that during frying, the meat surface temperature quickly reaches 115-120 degC or above 120 degC, and the protein forms a solid layer on the surface of the meat, thanks to which the loss of soluble substances such as inorganic salts is reduced . Taking into account the marking of products with the NRV-R value (Table 5), raw goose meat without skin covers the daily demand of the consumer (regardless of gender) in 1.2-25% and with skin in 1.0-7.5% (in a higher percentage of leg meat). Manganese requirements for an adult are met to the greatest extent by pan-fried with oil (NRV-R = 81.5%) and grilled (NRV-R = 25%) Turkish goose leg muscles, as well as cooked muscles of domestic Turkish goose and wild Egyptian goose (NVR-R 25 to 30%). When analyzing the presence of skin, Polish White Koluda(r) goose grilled breast meat with skin covers 19% of the consumer's NRV-R. A deficiency of manganese in the diet can cause changes in the osteoarticular system. Research has now established that it is the most efficient divalent cation in activating the glycosyltransferase enzymes, essential in the chondroitin sulfate formation, the major polysaccharide of the cartilage synthesis of cartilage and bone collagen, as well as in bone mineralization. Women with osteoporosis have been shown to have lower serum Mn levels than women with normal bone mineral density . A link between low dietary Mn and impaired insulin secretion and glucose metabolism has also been shown . Mn is an essential component of MnSOD for reducing mitochondrial oxidative stress. Oxidative stress is a common risk factor for the development of metabolic diseases. Low manganese intake is correlated with metabolic syndrome (MS) and its components: abdominal obesity, hypertriacylglycerolaemia, and high blood pressure . Manganese is essential for many vital processes, including nerve and brain development and cognitive function. In humans, manganese deficiency has also been linked with reduced levels of the neurotransmitter dopamine. However, excess Mn has neurotoxic effects . Manganese is considered an essential and critical nutrient, and its deficiency or overabundance may cause depressive disorders. People with depression were observed to have lower urinary manganese concentrations or MnSOD activity compared to the control group . Several studies have shown that manganese concentrations in patients with schizophrenia were lower than in control groups , but there are no consistent findings. That is why it seems important to balance the dietary content of this trace element. 7. Conclusions The primary source of manganese in the human diet is tea and plant products. Diversifying the diet with goose meat is also worthwhile because, unlike plant food, it does not contain substances that diminish bioavailability, such as fiber or phytates. As a natural source of this element, goose meat does not increase exposure to overconsumption, as can happen when consuming dietary supplements. As little as 100 g of goose meat can cover the daily AI for Mn for a wide range of adults, depending on the heat treatment used. Consumption of goose meat containing manganese may be justified in people suffering from mental disorders (depression, anxiety disorders), lipid (hypocholesterolemia), and/or carbohydrate (reduced glucose tolerance) disorders, in whom reduced concentration of this element has been confirmed in the blood. Placing information on the content of Mn in goose meat and the percentage of NRV-R on the packaging may be valuable information for the consumer in terms of making food choices to diversify the diet. Since there are few studies on the content of Mn in goose meat, it is reasonable to research the content of the impact of various thermal processing techniques on its concentration and retention. Acknowledgments We would like to thank Joanna Kasprzyk-Machata and Barbara Grzelczak, custodians of the Library of the University of Economics and Business in Wroclaw, for her help in searching the databases. Supplementary Materials Bibliographic query in Repository for Open Data Author Contributions Conceptualization, Z.G.; methodology, Z.G. and G.H.; investigation, Z.G. and G.H.; writing--original draft preparation, Z.G. and G.H.; writing--review and editing Z.G. and G.H. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the writing of the manuscript; or in the decision to publish. Figure 1 Identification, screening, and exclusion criteria of research included in the review. animals-13-00840-t001_Table 1 Table 1 Description of studies included in the systematic review. First Author Year Source References Falandysz et al. 1986 Agro Falandysz et al. 1987 Agro Falandysz et al. 1989 Agro Falandysz et al. 1989 Scopus Falandysz J. 1991 Scopus, WoS Falandysz et al. 1994 Scopus, WoS Geldenhuys et al. 2013 Scopus, WoS Geldenhuys et al. 2015 Scopus, WoS, Science Direct Chen et al. 2013 Scopus, WoS Oz and Celik 2015 Scopus Goluch et al. 2021 Scopus, WoS, Science Direct Kunachowicz et al. 2020 Tables of composition and nutritional value of food. USDA Food Data Central 2022 Websites animals-13-00840-t002_Table 2 Table 2 Manganese content of goose meat raw (mg/kg tissue). Meat Goose n Age Carcass Breast Leg/Thigh Reference Meat raw Anser Anser 29 n.d. 0.17 +- 0.04 (0.12-0.24) n.d. n.d. Meat raw Anser Anser 18 n.d. 0.38 (0.14-2.41) n.d. n.d. Meat raw Anser Anser 18 n.d. 0.25 (0.17-0.31) n.d. n.d. Meat raw Anser Anser 39 n.d. 0.26 (0.14-0.48) n.d. n.d. Meat raw Anser Anser 32 4-7 months 0.27 (0.15-0.42) n.d. n.d. Meat raw Anser Anser 16 4-7 months 0.25 +- 0.06 (0.15-0.30) n.d. n.d. Meat raw Anser Anser n.d. n.d. 0.20 n.d. n.d. Meat raw Anser Anser 10 n.d. 0.268 +- 0.073 (0.151-0.367) n.d. n.d. Meat only, raw Anser Anser n.d. n.d. 0.24 n.d. n.d. Meat and skin, raw Anser Anser n.d. n.d. 0.20 n.d. n.d. Meat raw Turkish goose 16 16 weeks n.d. 0.2 5.0 Meat raw: White Koluda(r) 24 17 weeks n.d n.d Without skin 1.7 With skin 1.5 Meat raw Egyptian goose Alopochen aegyptiacus 36 n.d. n.d. 0.6 +- 0.01 0.6 +- 0.08 season winter/summer n.d. Meat raw, skinless Canada goose Branta canadensis 6 n.d. 0.50 n.d. n.d. n.d.--no data. animals-13-00840-t003_Table 3 Table 3 Manganese content of goose meat after thermal treatment (mg/kg tissue). Meat Goose n Age Carcass Breast Leg/Thigh Reference Meat only, cooked, roasted Anser Anser n.d. n.d. 0.24 n.d. n.d. Meat and skin, cooked, roasted Anser Anser n.d. n.d. 0.23 n.d. n.d. Meat and skin, cooked, roasted Anser Anser n.d. n.d. 0.30 n.d. n.d. Meat: Turkish goose 16 16 weeks n.d. Boiled 0.7 +- 0.2 8.0 +- 8.0 Grilled 0.4 +- 0.1 5.0 +- 3.0 Pan fried without fat or oil 4.5 +- 6.0 3.4 +- 3.1 Pan with oil 0.5 +- 0.5 16.3 +- 18.1 Deep-fat fried 0.6 +- 0.7 2.1 +- 0.3 Oven cooked 0.3 +- 0.3 2.4 +- 0.9 Microwave 0.1 +- 0.1 1.15 +- 2.0 Meat: White Koluda(r) 48 17 weeks n.d. n.d. Water bath cooking Without skin 2.7 With skin 1.4 Grilled Without skin 2.0 With skin 3.8 Oven convection Roasting Without skin 1.3 With skin 1.1 Pan fried Without skin 1.1 With skin 1.3 Meat breast, cooked Egyptian goose Alopochen aegyptiacus 6 n.d. n.d. 1.0 n.d. n.d.--no data. animals-13-00840-t005_Table 5 Table 5 Percentage of the coverage of the AI for manganese of adults by the consumption of 100 g of goose meat, with regard chosen the recommendation, and nutrient reference values-requirements. Meat Goose Manganese Content [mg/100 g] Reference IOM (2001) NIPH-NIH (2020) EFSA (2013) NHMRC, AGDHA NZMH (2006) DACH (2021) NRV-R 1.8 mg 2.3 mg 3.0 mg 5.0 mg 5.5 mg 2.0-5.0 mg 2.0 mg Raw Meat raw, only Anser Anser 0.024 carcass 1.33 1.04 0.8 0.48 0.44 1.2-0.48 1.2 Meat and skin, raw Anser Anser 0.020 carcass 1.11 0.86 0.67 0.4 0.36 1.0-0.4 1.0 Meat raw Turkish 0.020 breast 0.50 leg 1.11 27.8 0.86 21.7 0.86 16.7 0.4 10.0 0.36 9.09 1.0-0.4 10.0 1.0 25.0 Meat raw without skin White Koluda(r) 0.17 breast 9.44 7.39 5.67 3.4 3.09 8.5-3.4 8.5 Meat raw with skin White Koluda(r) 0.15 breast 8.33 6.52 5.0 3.0 2.73 7.5-3.0 7.5 Meat raw, skinless Canada goose 0.05 carcass 2.78 2.17 1.67 1.0 0.91 2.5-1.0 2.5 Meat raw Egyptian goose 0.06 3.33 2.60 2.0 1.2 10.9 3.0-1.2 3.0 Thermal treatment Meat only, cooked, roasted Answer Anser 0.024 carcass 1.33 1.04 0.8 0.48 0.44 1.2-0.48 1.2 Meat and skin, cooked, roasted Anser Anser 0.023 carcass 1.28 1.0 0.77 0.46 0.42 1.15-0.46 1.15 Meat: Turkish goose Breast/leg Boiled 0.07/0.08 3.89/4.44 3.04/3.48 2.33/2.67 1.4/1.6 1.27/1.27 3.5/4.4-1.4/1.6 3.5/4.0 Grilled 0.04/0.50 2.22/27.8 1.74/2.17 1.33/16.7 0.8/10.0 0.73/9.09 2.0/25.0-0.8/10.0 2.0/25.0 Pan fried without fat or oil 0.45/0.34 25.0/18.9 19.6/14.8 15.0/11.3 9.0/6.8 8.18/6.18 22.5/17.0-9.0/6.8 22.5/17.0 Pan with oil 0.05/1.63 2.78/90.6 2.17/70.9 1.67/54.3 1.0/32.5 0.91/29.6 2.5/81.5-1.0/32.5 2.5/81.5 Deep-fat fried 0.06/0.21 3.33/11.7 2.61/9.13 2.0/7.0 1.2/4.2 1.09/3.82 3.2/10.5-1.2/4.2 3.0/10.5 Oven cooked 0.03/0.24 1.67/13.3 1.30/10.4 1.0/8.0 0.6/4.8 0.55/4.36 1.5/12.0-0.6/4.8 1.5/12.0 Microwave 0.01/0.15 0.56/3.33 0.43/6.52 0.33/5.0 0.2/3.0 0.18/2.73 0.5/7.5-0.2/3.0 0.5/7.5 Meat: White Koluda(r) breast Water bath cooking Without skin 0.27 15.0 11.7 9.0 5.4 4.91 13.5-5.4 13.5 With skin 0.14 7.8 6.09 4.67 2.8 2.55 7.0-2.8 7.0 Grilled Without skin 0.20 11.1 8.70 6.67 4.0 4.0 10.0-4.0 10.0 With skin 0.38 21.1 16.5 12.7 7.6 6.91 19.0-7.6 19.0 Oven convection Roasting Without skin 0.13 7.22 5.65 4.33 2.6 2.36 6.5-2.6 6.5 With skin 0.11 6.11 4.78 3.67 2.2 2.0 5.5-2.2 5.5 Pan fried Without skin 0.11 6.11 4.78 3.67 2.2 2.0 5.5-2.2 5.5 With skin 0.13 7.22 5.65 4.33 2.6 2.36 6.5-2.6 6.5 Meat breast, cooked Egyptian goose breast 0.60 33.3 26.1 20.0 12.0 10.9 30.0-12.0 30.0 AI--adequate intake; IOM--Institute of Medicine (now: National Academy of Medicine); NIPH-NIH--National Institute of Public Health-National Institute of Hygiene; EFSA--European Food Safety Authority; NHMRC--National Health and Medical Research Council; AGDHA--Australian Government Department of Health and Ageing; NZMH--New Zealand Ministry of Health; DACH--Nutrition Societies in Germany and Austria and Switzerland; NRV-R--nutrient reference values-requirements. 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PMC10000037

The red crab, Pleuroncodes planipes, is a decapod crustacean abundant off the Pacific coast of the Baja California Peninsula. This species is caught and used in preparing animal feed, such as flour, particularly for aquaculture. Levels of calcium (Ca), cadmium (Cd), copper (Cu), iron (Fe), lead (Pb), magnesium (Mg), manganese (Mn), nickel (Ni), phosphorus (P), and zinc (Zn) were measured in red crabs collected from three geographic zones during three cruises in different seasons. Significant differences were found in the levels of Ca, Cd, Cu, Fe, Mg, Ni, P, and Zn between the two El Nino years (cruises C1 and C3, based on a threshold of +-0.5 degC for the Oceanic Nino Index). The highest concentrations of most elements were observed in the south of the Baja California Peninsula, a highly productive area influenced by upwelling events. Our findings suggest that while environmental temperature plays a central role in the benthic or pelagic distribution of red crabs, their content and variability of trace and macro elements appear to be associated with the presence of oceanic conditions, such as upwelling and potential changes in the composition of their diet associated with the depth in which these crustaceans are collected. crustacean marine economic resource health risk marine pollution El Nino Southern Oscillation Secretaria de Agricultura, Ganaderia, Desarrollo Rural, Pesca y Alimentacion (SAGARPA)Consejo Nacional de Ciencia y Tecnologia2003-002-019 This study was supported by Secretaria de Agricultura, Ganaderia, Desarrollo Rural, Pesca y Alimentacion (SAGARPA) and Consejo Nacional de Ciencia y Tecnologia (CONACYT, grant 2003-002-019). pmc1. Introduction Zinc (Zn), copper (Cu), manganese (Mn), iron (Fe), calcium (Ca), magnesium (Mg), and phosphorus (P) are essential elements required to perform different metabolic processes. Consequently, their levels and the biochemical mechanisms that regulate these levels in the organism vary across species . When in excess, chemical elements can have adverse health effects, and these trace elements have been associated with severe effects in animals, including humans . The chemical composition and bioavailability of trace elements in the aquatic environment and the organisms that thrive in it are influenced by the local geochemistry (volcanism, mineral deposits) and anthropogenic activities (agriculture, mining) that can introduce high amounts of metallic and non-metallic elements . One of the largest phosphorite deposits worldwide is found on the coast of the Baja California Peninsula. Common impurities in phosphate-rich rocks are cadmium (Cd), lead (Pb), Cu, and Zn . In addition, in the Pacific Ocean off the Baja California Peninsula, there are sites where upwelling events contribute to enriching elements such as Cd and P in the water column . The red crab Pleuroncodes planipes Stimpson, 1860 (Decapoda, Anomura, Galatheoidea) is abundant in the Pacific coast upwelling system of the Baja California Peninsula . During the first year of life, P. planipes is part of the micronekton . In the larval and juvenile stages, it is found in pelagic and benthic environments, while the adult stage (upon 32 mm length) becomes strictly benthic, found at depths between 200 m and 500 m . Depending on the conditions in the ocean, the feeding habits of this crab vary due to its mass bathymetric migration. The red crab is a key food item for many predators, including cetaceans, pinnipeds, birds, turtles, and fishes , being an important component in the trophic web in the Pacific Ocean. The estimated annual abundance of the red crab along the Pacific coast of the Baja California Peninsula was 460,217 tons in 1995 , increasing to 611,525 tons by 2014 . In addition to its ecological importance, this species is a major economic resource. Once red crabs are collected, they are processed (whole, without further preparation) to obtain flour used as a feed ingredient for cultured shrimp. This flour has been evaluated with encouraging results . Dried whole red crab contains about 43% protein, 8% lipids, and 7.1% astaxanthin . Given the need to find protein sources for humans, red crab flour could be included as a food ingredient for human nutrition . The red crab feeds mainly on organic matter (approximately between 60% and 70%) and the composition of its diet can vary over time and between sites . The oceanic conditions vary between years, inducing major faunal shifts , mainly changes in zooplankton abundance , that affect the marine trophic ecology and lead some species to use alternative food sources. Aurioles-Gamboa et al. found that in winter, red crabs are distributed across the entire continental shelf. However, in summer, they are found only on the outer shelf (100-200 m depth), and their stomach contents are markedly lower than those recorded during winter at the same depths; these authors concluded that this pattern is correlated with the lower intensity of the coastal upwelling system and the weakening of the California Current. Also, the red crab nutritional status (fatty acid, lipid and astaxanthin contents) varies across feeding sites . Therefore, if the nutrient content of red crabs varies over time and seasonally, the content of chemical elements may also vary. Bioaccumulation of trace metals in marine organisms can eventually lead to adverse health effects and can be a potential health risk to humans if contaminated seafood is consumed in the human diet . Red crabs are used mainly for animal feed, as they are a protein source, and they could also be used for human consumption. Seafood is a good source of animal protein for humans because it usually contains all of the essential amino acids and also has a low fat content . An increasing trend to include seafood in the human diet has been reported; for example, the average annual European Union consumption per person is 21.97 kg . Trace-metal concentrations in commercial marine organisms should be evaluated for seafood safety and public health reasons, as well as for sustainable management of the coastal environment . Therefore, the objective of this study was to assess temporal and spatial variations in the content of trace and macro elements in red crabs. 2. Materials and Methods 2.1. Study Area The study area (latitude 23deg19' N-longitude 110deg45' W and latitude 28deg51' N-longitude 114deg42' W) is located in the Pacific Ocean, off the west coast of the Baja California peninsula. In this area , three research cruises were conducted onboard the research vessel "BIP XII". The first cruise (C1) took place between 21 October and 10 November 2004 . Autumn of 2004 was considered to be under the influence of an El Nino event, lasting from June 2004 to February 2005 based on a threshold of +-0.5 degC in the Oceanic Nino Index (ONI; accessed on 8 march 2015). The second cruise (C2) occurred from 15 March to 29 March 2005 . Spring of 2005 was declared a neutral year (see the website above). The third cruise (C3) was conducted between 21 November and 4 December 2006 . Autumn of 2006 was considered to be under the influence of an El Nino event. The oceanographic dynamics of the west coast of the Baja California peninsula are influenced primarily by the California Current, the California Subcurrent, and the North Equatorial Current. The prevailing winds are strong in coastal zones, triggering cold upwelling events that foster plankton blooms and abundant sea life, including species that are important for fisheries . 2.2. Sampling Sampling was performed following a stratified random design along evenly spaced transects (of variable length, depending on the topography) running perpendicular to the coast . At each station, bottom trawls were performed as described by . The region in which red crabs were collected was split into three zones: south, center, and north . The transects sampled at each site were used as replicates of a zone for statistical analyses (south: Todos Santos-Bahia Magdalena; center: Gulf of Ulloa; north: Punta Abreojos-Bahia Sebastian Vizcaino). The specimens of P. planipes collected were stored at -20 degC until analyses. 2.3. Analysis of Trace and Macro Elements When red crab is used as feed for aquaculture, specimens are processed whole, i.e., the viscera, muscle, and shell are not separated to produce the flour used to feed aquatic organisms. For this reason, a total number of 279 whole adult specimens were selected and analyzed, with body size ranging between 32 mm and 40 mm; samples were dried for 72 h at 70 degC. For the analysis of trace and macro elements, specimens were subjected to acid digestion in nitric acid (HNO3, concentrated) and hydrogen peroxide (H2O2, 30%) in Teflon vials using a microwave oven (Mars 5x, CEM, Matthews, NC, USA). The concentrations of Ca, Cd, Cu, Fe, Mg, Mn, Ni, Pb, and Zn were quantified using an atomic absorption spectrophotometer (GBS Scientific AVANTA, Dandenong, Australia) with air-acetylene flame . Phosphorus (P) content was measured with the molybdovanadate method , which was validated using blanks tested in parallel . High-purity reagents were used throughout the testing process. Standardized reference material (TORT-2, DORM-2 and DORM-4 from the National Research Council of Canada; Ottawa, Canada) samples spiked with known concentrations of trace elements and macro elements, and blanks were also analyzed as a quality control . The analyses of metal content yielded recovery values ranging from 93% to 116% for the entire process. The highest recovery value corresponded to magnesium, which probably included the contribution of air pollution. The runs by element analyzed were for triplicate. The limits of detection and of quantification (mg g-1), respectively, were as follows: Ca: 0.08 and 0.10; Cd: 0.01 and 0.02; Cu: 0.01 and 0.02; Fe: 0.65 and 1.35; Mg: 0.05 and 0.08; Mn: 0.04 and 0.07; Ni: 0.03 and 0.05; Pb: 0.02, and 0.07; Zn: 0.02 and 0.06. All the concentrations are expressed in dry weight. 2.4. Statistical Analyses The datasets were tested for normality and homoscedasticity using the Shapiro-Wilk and Levene tests. To explore significant differences in the content of trace elements (Cd, Cu, Fe, Mn, Ni, Zn) and macro elements (Ca, Mg, and P), concentrations were grouped by cruise (C1, C2, and C3) and zone (south, center, north). To this end, trace element concentrations were log-transformed. For elements recorded at concentrations below the limit of detection, the value corresponding to one-half of the respective limit of detection was used for statistical analyses . Since the number of samples differ between cruises and zones, the analyses were carried out by testing the Type-1 hypothesis (Type-1 decomposition), which is particularly useful for complex unbalanced designs . Statistical significance was defined at p < 0.05. Data for all variables met the normality and homoscedasticity assumptions, except for P (usually, slight deviations from the normality and homoscedasticity assumptions do not bias the F-test). Lindman showed that the F statistic is quite robust against deviations of the homoscedasticity assumption. However, P failed to comply with the assumption of no correlation between means and standard deviations. A two-way analysis of variance (ANOVA) was performed, using cruise and zone as fixed main factors and including one two-way interaction term, followed by a comparison of cruises C1 and C3 (El Nino) combined vs. C2 (non-El Nino). Bonferroni's post hoc test was used to determine significant differences between group means . Discriminant analysis followed by a Factor analysis was carried out to identify how the different trace and macro elements grouped together in each cruise. The variable "zone" as ordinal variable was transformed to numerical value (South = 1; Central = 2; North = 3) and included in the factorial analysis according to Robitzsch . All statistical analyses were run using the software STATISTICA version 13.5.0.17 . 2.5. Health Risk The results obtained in this study were compared with guidelines and results of previous studies to assess the potential health risk of human consumption of red crabs. With this purpose, element concentrations were converted from dry weight (dw) to fresh weight (fw) as follows:Element fw = Element dw x (100 - % moisture)/100(1) using the percentage of moisture in red crabs (range 73.8-79.4%). The estimated daily intake (EDI, mg trace element kg-1 BW day-1) was calculated as follows :EDI = (Cm x CR)/BW(2) where Cm = mean concentration of the chemical element in red crabs, expressed as a fresh weight (mg g-1); CR = mean daily per-capita consumption rate of crabs (41 g day-1 per person); BW = mean body weight of an adult person (70 kg) . From a nutritional standpoint, we used the recommended daily intake (RDI) for Fe (8 mg day-1), Zn (11 mg day-1), Cu (0.9 mg day-1), Mn (2.3 mg day-1), Ca (1000-1200 mg day-1), Mg (400-420 mg day-1), P (700 mg day-1), and a tolerable intake of Ni (1.0 mg day-1) to value the contribution of the estimated RDI for these elements. No established RDI has been set for cadmium and lead because these metals are considered toxic to humans. To measure the possible toxicological risk of the intake of the elements mentioned, from the consumption of red crabs, each EDI was compared with its respective reference oral dose (RfD): Cd: 1.0 mg kg-1 body weight day-1; Cu: 40.0 mg kg-1 bodyweight day-1; Fe: 700 mg kg-1 bodyweight day-1; Mn: 0.140 mg kg-1 bodyweight day-1; Ni: 20.0 mg kg-1 bodyweight day-1; and zinc: 300 mg kg-1 bodyweight day-1 . The tolerable upper intakes (DRIs) for Ca were been set as 2500 g day-1; for Mg: 350 g day-1; and for P: 4.0 g day-1 . 3. Results No detectable levels of Pb (<0.07 mg g-1) were found in red crabs from any location or cruise. Thus, this element was not included in the statistical analyses. The results of the multivariate test of trace (Cd, Cu, Fe, Mn, Ni, Zn) and macro element (Ca, Mg, P) concentrations showed significant differences in the different groups (cruise and zone), including their interaction (p < 0.05) (Table 1). Post hoc comparisons of means with the Bonferroni test (Table 2) showed significant differences in Cd, Zn, Cu, Fe, and Mg content between C1 vs. C2. In addition, significant differences were observed in Zn, Cu, Ni, Fe, Ca, and P between C2 vs. C3, as well as in Cd, Zn, Cu, Ni, Fe, Ca, Mg, and P levels between the two El Nino years (C1 and C3). Although both C1 and C3 showed El Nino conditions (positive anomalies of up to 0.9 degC and 1.1 degC, in October-November 2004 and November 2006, respectively; NOAA data), the highest concentrations of Cd and Zn, significantly different (p < 0.05) compared to C3, were recorded in red crabs collected during C1, and Ni and Mg content was significantly higher in C3 (Table 2). On the other hand, the highest significant (p < 0.05) concentrations of Cu, Fe, Ca, Mg, and P were recorded in C2 (normal environmental conditions with positive temperature anomalies of 0.3 degC in March 2005; NOAA data), although Mg content in C2 was not significantly different from the concentration recorded in C3, and neither were Ca and P concentrations in C2 versus C1 (Table 2). Spatially (south, center and north), the lowest Cd and Fe content was found in the north (Punta Abreojos--Bahia Sebastian Vizcaino). The lowest concentrations of Zn, Ca, and Mg were found in the center, and the highest concentrations of Cu, Mn, Fe in the south (Todos Santos--Bahia Magdalena). In addition, the highest Cd levels (up to 23.10 mg g-1) were found in the south. Ni and P content did not show significant differences between the three zones. The central zone (Golfo de Ulloa) generally showed the lowest concentrations of all the trace and macro elements analyzed in red crabs during the three cruises (C1, C2, and C3), especially towards the south (Table 3). 3.1. Discriminant Analysis All trace and macro element concentrations included in the discriminant function analysis were highly significant in discriminating between the cruises (Wilks' Lambda = 0.149; F = 47.30; p < 0.000; Table 4). The first root distinguished C1 and C3 and explained 91% of the cumulative variance in elemental concentrations; the variables with the greatest contribution were Mn, Ni, Fe, Ca, Mg, and P content (Table 5). In addition, the second root differentiated cruises C1 and C2 and explained 9% of the cumulative variance in elemental concentrations; the contributing variables were Cd, Zn, and Cu levels, with a relatively high correlation between the concentrations of Cd and Cu (0.44) (Table 5). The highest levels of these elements were recorded in crabs from the south zone. However, only the highest concentrations of Cd and Cu coincided with the fact that they were found in organisms from C1 and C2, respectively. The canonical scores, the discriminant function mainly differentiated between C1 and C2; the distribution of the values for C1 are below the central line (0) with a mean of -1.73, while C2 values show a mean of 0.40 . These results discriminate clearly and significantly between C3 and C1-C2, and between C1 and C2 with the first and second discriminant functions, respectively. 3.2. Factor Analysis In the red crab, the loadings of the trace and macro elements that were assessed varied across cruises (Table S1 in Supplementary Material). In the first cruise (C1), the first three factors accounted for 84.4% of the variance, while in cruises 2 and 3 (C2 and C3), the first three factors explained 65.3% and 64.5% of the variance, respectively (Table S1). However, the groups and loadings of some elements in the factors that were derived from the factor analysis were constant: Cd, Cu, and Mn were the elements with the greatest loadings to Factor 1 in C1 and C2. In C3, these three elements were associated with Factor 2, where Ca and Mg are the elements with the greatest loadings, followed by Mn. In C1 , an inverse relationship was recorded between the group formed by Mg, Ca, and Fe (red circle) vs. the zone and Zn (blue circle); that means the highest concentrations of Mg, Ca, and Fe but the lower of Zn were in the south during C1. The other elements did not show a consistent association with the other elements, nor were their greatest loadings found in the same factor during the cruises. For its part, Fe contributed to Factor 2 along with Ca, Mg, and Zn in C1, and to Factor 3 along with P and Zn in C3, but showed no significant loading related to any factor in C2. In C3, Fe and slightly P showed an inverse contribution with the zone; zone 1 showed the highest Fe and P concentrations, while the lowest Fe and P concentrations were grouped together in zone 3. In addition, in C3, Zn slightly showed a direct contribution to the zone. 3.3. Health Risk Assessment In relation to the % RDI of trace elements associated with the consumption of 41 g of red crab, the contribution of these elements to the human diet is low (<1%), Cu being the element with the highest contribution (~0.7%), followed by Fe (~0.2%), Zn (~0.09%), Mn (~0.03%), and Ni (~0.03%) (Table 6). In addition, the contributions of these five elements plus Cd and Pb (the concentrations of the latter below detection limits) from the consumption of red crab are several orders of magnitude below the RfD established for each specific element (Table 6). As for the macro elements, Ca concentrations in red crab contribute approximately 1% to the recommended RDI for a 70 kg person, followed by magnesium with 0.3% and phosphorus with 0.2% (Table 6). 4. Discussion Except for Cd, the concentrations of the elements measured in red crabs collected in all cruises, particularly Pb (below 0.07 mg g-1), were within the normal range observed in organisms living in non-polluted areas and, therefore, the levels of these trace elements were not of dietary concern . Although the Cd concentrations recorded in this study may seem high compared with the content recorded in other marine organisms, they are within the range considered normal for crabs , in which values as high as 13 mg g-1 and even up to 50 mg g-1 wet weight have been reported. Red crabs are found mainly in areas influenced by upwelling events because these provide a rich source of nutrients and optimum water conditions (below 16 degC) for this species . During upwelling events, particulate material enriched with Ca, Cu, and especially Cd is mobilized from the bottom to the surface of the water column . Particulate matter, which accounts for about 60% to 70% of the diet of red crabs, contains diatoms . This is relevant because up to 17 genera of diatoms present in plankton have been reported in the stomach contents of red crabs . The high Cd content found in red crabs can be associated with the consumption of diatoms, which are a major source of Cd . Although Cd is bioaccumulated by P. planipes, this species has the capacity to make it non-bioavailable through detoxification mechanisms that include Cd binding to high-affinity low-molecular-weight proteins known as metallothioneins . Along with Cd, the highest Cu and Mn concentrations in red crab were found in the south, an area characterized by submarine phosphorite deposits . In phosphate rocks, Cd, Cu, Ni, and Mn are common impurities . Cu is a particularly important element for crustaceans for being a component of hemocyanin, the protein responsible for transporting oxygen in the metabolism of these organisms. The factor analysis showed that Cd and Cu grouped together in the different factors. Cu metabolism also involves metallothioneins, as their synthesis can be stimulated by Cu homeostasis . The importance of Cu in crustaceans likely explains the fact that it is the trace element supplied in highest levels to the human diet. Cd is another element present at high concentrations in red crabs. Cadmium has similar metabolism pathways to copper in these organisms, which poses a potential risk for human health given its toxicity. However, the amount of trace elements contained in 41 g of red crab is lower than 1%, which poses a low risk from its consumption by humans. After copper, Fe is the second most important trace element for its content in red crabs. In the present study, the results of the factor analysis show that in C3, Fe and Zn content in these organisms may be associated with the area where they were collected. Additionally, the highest Fe concentrations were also found in red crabs collected in the south zone. Fe is an indicator of both terrigenous materials and the resuspension of sediments from the continental shelf , which can also be associated with upwelling events. De Anda-Montanez et al. , in a spatio-temporal analysis of red crabs collected in the same cruises, reported that during the spring of 2005 (C2) under environmental conditions of a non-El Nino year, red crabs were highly abundant and widely distributed from 51 m to 300 m depth. The red crabs collected during C2 (in spring) recorded the highest Cu, Fe, Ca, Mg, and P levels relative to the other cruises. This finding is likely due to the fact that the most intense upwelling period occurs in spring and early summer (March-June), although upwelling events occur less frequently throughout the year in the area studied in this work . C1 and C3 were carried out in autumn under El Nino conditions. In these cruises, red crabs were collected at 200 m depth in C1, in contrast with C3, when the abundance and distribution of P. planipes increased at 400 m to 500 m depth . El Nino events are associated with a shift in salinity and thermal stratification along the water column . In general, a linear and significant relationship was observed, that is, higher temperatures were associated with lower red crab catches . P. planipes alternates between pelagic and benthic environments according to seawater temperature , migrating to deeper layers during El Nino years . Therefore, environmental variability plays a central role in the distribution of P. planipes and the variability of its content of trace and macro elements. Although El Nino conditions prevailed during C1 and C3, the concentrations of Cd, Zn, Ca, and P were lower in crabs collected during C3, when these organisms were found at deeper layers than in C1 . Zooplankton and inorganic matter are the main components in the diet of P. planipes collected at depths of 150 m to 200 m . In the study area, zooplankton biomass and chemical composition are influenced mainly by the California Current that converges in upwelling areas . Elements with potentially enriched concentrations in these areas, such as Fe and Mg, interfere with the absorption of P due to the formation of insoluble phosphate salts . In addition, other food components, such as lipids, can affect Ca and P uptake . Robinson et al. reported that 90% of the total abundance of red crabs in their study was found in areas where chlorophyll-a concentrations increased as a result of upwelling events, which are a major source of trace metals, and where submarine phosphorite deposits are present . 5. Conclusions Most of the significant differences observed in the levels of trace and macro elements in red crabs were associated with environmental changes that probably influence the diet of this crustacean species, such as zooplankton abundance. The organisms collected during C3 (El Nino year), when red crabs were found at deeper levels, recorded the lowest Ca, P, and Zn content. The highest Cd, Cu, Mn, Ni, Fe, and P levels were recorded in the south, probably due to the presence of submarine phosphorite deposits and upwelling events, which boost primary productivity. Although the content of trace and macro elements changes in organisms, some associations between elements were observed in the three cruises, such as those between Ca and Mg, and between Cd and Cu. In contrast, Fe, Ni, Mn, P, and Zn showed no consistent associations with other elements in the three cruises. Previous studies show that flour from red crabs is a protein source. Our findings suggest that although its consumption poses no risks to human health, it is not an important source of minerals of nutritional value since 41 g of red crab consumed per day represent less than 1% of the recommended daily dose of trace elements of nutritional value. For the consumption of red crab to pose a human health risk associated with Cd toxicity, a much higher amount of this crustacean would have to be consumed. As for the macro elements, Ca concentrations in red crab contribute approximately 1% to the recommended RDI for a 70 kg person, followed by magnesium with 0.3% and phosphorus with 0.2%. Continued monitoring of the contents of trace and macro elements in red crabs is recommended to guarantee the safe consumption of this crustacean by humans. Acknowledgments The authors appreciate the assistance of Angelica Barrera Garcia in the construction of a database of test results, of Gerardo Hernandez Garcia in the design of the map and graphical abstract, and of Griselda Pena Armenta and Emerson Zuniga for their technical assistance. The authors are especially grateful to the crew of BIP XII for their collaboration during the cruises, particularly with captain Gabriel Rivera Velazquez. Isela Vazquez, Arturo Tecuapleta, Marisol Perez, Alejandro Ramos, Ismael Mascarenas, and Manuel Calderon provided valuable assistance in the sampling work onboard. This study was supported by Secretaria de Agricultura, Ganaderia, Desarrollo Rural, Pesca y Alimentacion (SAGARPA) and Consejo Nacional de Ciencia y Tecnologia (CONACYT, grant 2003-002-019). Maria Elena Sanchez-Salazar edited the English manuscript. Supplementary Materials The following supporting information can be downloaded at: Table S1: Loadings of trace and macro elements in red crab by cruise on varimax rotate using factor analysis. The variables with the greatest loadings to each factor are market with an asterisk. Click here for additional data file. Author Contributions Conceptualization: J.A.D.A.-M., T.Z.-S., E.F.B. and L.C.M.-R.; formal analysis, B.A.-V. and L.C.M.-R.; funding acquisition, J.A.D.A.-M.; research, J.A.D.A.-M., T.Z.-S., E.F.B. and L.C.M.-R.; methodology, J.A.D.A.-M., B.A.-V. and L.C.M.-R.; project management, J.A.D.A.-M.; resources, J.A.D.A.-M.; software, J.A.D.A.-M. and R.G.-R.; oversight, J.A.D.A.-M., E.F.B. and L.C.M.-R.; validation, E.F.B.; writing--original draft, J.A.D.A.-M. and L.C.M.-R.; writing--review and editing, J.A.D.A.-M., T.Z.-S., E.F.B., B.A.-V., R.G.-R. and L.C.M.-R. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Sampling was performed under the collection development fishing permit issued by the Mexican government (DGOPA-PF-01, Folio No. 5502; National Fisheries Institute CRIP-LP-06/880). This permit was issued based on the research protocol described in the project entitled "Estimation of population parameters and biomass evaluation of galateid crustaceans (Pleuroncodes planipes and Munida spp.) on the western coast of the Peninsula of Baja California (SAGARPA: 11371.101106.-5502; CONACYT 2003-002-019; CIBNOR 119C)". This study was performed following the demands, requirements, and protocols required by the various institutions involved (SAGARPA, CRIP; CONACYT, CIBNOR). No experimentation tests using red crabs (bioassays) were carried out during this study. Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request addressed to Dr. Juan Antonio de Anda-Montanez ([email protected] (J.A.D-M)). Conflicts of Interest The authors declare no conflict of interest. The financial sources (SAGARPA, CRIP and CONACYT) did not participate in the development of this research. Figure 1 Geographic location of the sampling stations of the research cruises along the west coast of the Baja California Peninsula, Mexico. (A) Cruise 1, October-November 2004; (B) Cruise 2, March 2005; (B) Cruise 3, November 2006. The numbers over the peninsula indicate the transect number. Figure 2 Scatterplot of canonical scores for two discriminant functions (roots) between cruises (C1-C3). Figure 3 Loadings of trace and macro elements in red crab by a cruise on varimax rotate using factor analysis. The variables with significant loadings to each factor were grouped. In C1 (A), the elements in the blue circle have an inverse relationship with the elements in the red circle. No inverse relationship were recorded in C2 (B) or C3 (C). animals-13-00822-t001_Table 1 Table 1 Multivariate significance tests of the concentrations of trace and macro elements in red crabs, Pleuroncodes planipes, using cruise and zone as fixed main factors, including one 2-way interaction term (combined effect: Cruise x Zone). Effect Test Value F Effect df Error df p Intercept Wilks 0.000085 341071.4 9 262.0 0.00 Cruise Wilks 0.114642 56.9 18 524.0 0.00 Zone Wilks 0.470140 13.3 18 524.0 0.00 Cruise x Zone Wilks 0.154107 17.7 36 983.6 0.00 animals-13-00822-t002_Table 2 Table 2 Concentrations of trace and macro elements in red crabs, Pleuroncodes planipes, collected during three cruises (C1, C2, C3) off the west coast of the Baja California Peninsula, Mexico. Data are shown as mean plus/minus standard error (SE), with the concentration interval in parenthesis. Concentrations are expressed in dry weight and N is sample size per cruise. Rows with different superscript letters denote statistically significant differences (p < 0.05). Cruise C1 (N = 29) Mean +- SE C2 (N = 119) Mean +- SE C3 (N = 131) Mean +- SE Cadmium (mg g-1) 12.07 +- 0.76 a 9.27 +- 0.24 b 8.97 +- 0.28 b (8.01-23.1) (0.24-18.1) (0.12-20.9) Zinc (mg g-1) 71.34 +- 3.04 a 63.00 +- 0.76 b 56.88 +- 1.03 c (52.4-111) (44.7-84.7) (39.9-131) Copper (mg g-1) 40.46 +- 4.44 a 49.35 +- 1.75 b 41.21 +- 1.03 c (15.1-92.3) (27.5-88.5) (18.0-82.1) Manganese (mg g-1) 4.47 +- 0.58 a 6.64 +- 0.57 a 4.92 +- 0.33 a (<0.04-10.2) (<0.04-27.7) (<0.04-17.4) Nickel (mg g-1) 1.23 +- 0.26 a 1.56 +- 0.16 a 3.19 +- 0.23 b (<0.03-4.43) (<0.03-6.52) (<0.03-13.79) Lead (mg g-1) <0.07 a <0.07 a <0.07 a Iron (mg g-1) 127 +- 11 a 162 +- 5 b 111 +- 8 c (54.1-247) (45.3-389) (21.8-532) Calcium (mg g-1) 8.41 +- 0.22 a 9.05 +- 0.007 a 7.27 +- 0.12 b (5.84-10.7) (4.34-12.0) (4.59-12.7) Magnesium (mg g-1) 0.97 +- 0.03 a 1.09 +- 0.02 b 1.09 +- 0.01 b (0.68-1.25) (0.53-1.46) (0.84-1.46) Phosphorus (mg g-1) 0.97 +- 0.04 a 1.13 +- 0.02 a 0.67 +- 0.02 b (0.06-1.77) (0.75-2.59) (0.4-1.10) animals-13-00822-t003_Table 3 Table 3 Concentrations of trace and macro elements in red crabs, Pleuroncodes planipes, collected and grouped by zone (south, center, and north) during three cruises (C1, C2, C3) off the west coast of the Baja California Peninsula, Mexico. Data are shown as mean plus/minus standard error (SE), with the concentration interval in parenthesis. Concentration is expressed in dry weight. N is sample size per zone. Rows with different superscript letters denote statistically significant differences (p < 0.05). Zone South (N = 142) Mean +- SE Center (N = 81) Mean +- SE North (N = 56) Mean +- SE Cadmium (mg g-1) 10.10 +- 0.32 a 9.31 +- 0.28 a 7.84 +- 0.19 b (0.12-23.1) (4.95-20.9) (4.36-11.0) Zinc (mg g-1) 61.58 +- 0.99 a 57.32 +- 0.77 b 64.82 +- 2.12 a (39.9-131) (40.1-74.8) (43.6-111) Copper (mg g-1) 48.78 +- 1.26 a 39.35 +- 1.33 b 41.61 +- 1.75 b (18.0-92.3) (20.0-82.2) (15.1-71.0) Manganese (mg g-1) 7.59 +- 0.46 a 4.20 +- 0.45 b 2.60 +- 0.19 b (0.06-27.7) (<0.04-15.3) (<0.04-5.63) Nickel (mg g-1) 2.70 +- 0.23 a 1.91 +- 0.21 a 1.81 +- 0.23 a (<0.03-13.8) (<0.03-7.3) (<0.03-6.7) Lead (mg g-1) <0.07 a <0.07 a <0.07 a Iron (mg g-1) 155 +- 7 a 100 +- 6 b 134 +- 10 c (38.4-532) (21.8-247) (32.4-389) Calcium (mg g-1) 8.22 +- 0.13 a 7.32 +- 0.13 b 9.17 +- 0.24 a (4.34-12.7) (4.59-10.5) (5.45-12.0) Magnesium (mg g-1) 1.09 +- 0.01 a 1.04 +- 0.01 b 1.12 +- 0.01 a (0.53-1.46) (0.68-1.30) (0.61-1.40) Phosphorus (mg g-1) 0.95 +- 0.03 a 0.82 +- 0.02 a 0.91 +- 0.02 a (0.5-2.58) (0.06-1.8) (0.5-1.6) animals-13-00822-t004_Table 4 Table 4 Chi-square tests with successive roots removed. Roots Removed Eigenvalue Canonical R Wilks' Lambda X 2 d.f. p-Value 0 3.847 0.891 0.149 517.37 18 0.000 1 0.382 0.526 0.723 88.06 8 0.000 animals-13-00822-t005_Table 5 Table 5 Raw coefficients for canonical variables. An asterisk denotes the variables with the greatest contribution to each root. Variable Root 1 Root 2 Cadmium 0.005 -0.878 * Zinc -0.271 -0.484 * Copper -0.187 0.527 * Manganese 0.318 * 0.289 Nickel 0.469 * -0.204 Iron -0.422 * 0.181 Calcium -1.401 * -0.289 Magnesium 1.081 * 0.570 Phosphorus -0.744 * 0.215 Eigenvalue 3.847 0.382 Variance explained 0.910 1.000 animals-13-00822-t006_Table 6 Table 6 Contribution of trace and macro elements from the consumption of Pleuroncodes planipes, (mg day-1) relative to the recommended daily intake (RDI) (mg day-1, ) and reference dose RfD (mg kg-1 bodyweight day-1, ). Element EDI Calculated RDI Theoretical RDI % RDI RfD % RfD Cadmium 1.33 x 10-03 na 0.07 1.90 x 10+00 Copper 6.31 x 10-03 2.05 x 10-04 0.9 0.70 2.8 2.25 x 10-01 Iron 1.90 x 10-02 7.32 x 10-04 8 0.24 49 3.88 x 10-02 Manganese 7.86 x 10-04 5.42 x 10-05 2.3 0.03 9.8 8.02 x 10-03 Nickel 2.96 x 10-04 2.34 x 10-05 1.0 0.03 1.4 2.11 x 10-02 Zinc 9.59 x 10-03 1.70 x 10-04 11 0.09 21 4.57 x 10-02 Calcium 1.23 x 10+01 3.10 x 10-01 1200 1.03 na Magnesium 1.57 x 10+00 2.98 x 10-02 420 0.37 na Phosphorus 1.40 x 10+00 4.29 x 10-02 700 0.20 na na: not available. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000038

Increased knowledge of the developmental processes during gestation could provide valuable information on potential alterations in embryonic/fetal development. We examined the development of ovine conceptus between the 20th and 70th day of gestation with three convergent analyses: (1) uterus ultrasound examination and measurement (eco) of crown-rump length (CRL) and biparietal diameter (BPD) of the conceptus; (2) direct measurement (vivo) of CRL and BPD of the conceptus outside the uterus (3) osteo-cartilage dynamics during development by differential staining. No significant differences were observed between eco and vivo measurements for CRL and BPD in all examined concepti. CRL and BPD, instead, showed a significant positive linear correlation with gestational age. The study of osteogenesis dynamics has demonstrated a completely cartilaginous ovine fetus at up to 35 days of gestation. The ossification begins in the skull (40th day) and is almost complete between the 65th and the 70th of pregnancy. Our study highlighted that CRL and BPD are accurate parameters for gestational age estimation in the first part of sheep pregnancy and provides an overview of osteochondral temporal dynamics. Furthermore, tibia ossification is a valid parameter to estimate fetal age by ultrasound. prenatal growth osteogenesis dynamics ultrasound sheep This research received no external funding. pmc1. Introduction Since the beginning of studies on reproduction in animal and human models, the knowledge of the mechanisms and the description of the main events related to gestation have represented an intense field of study and research. Gestation in mammalians is a complex phenomenon and any physiological changes that occur during fetal development can affect the subsequent health and well-being into adulthood. Prenatal development is influenced by several factors, such as genetic background, size and age of the mother, size of the offspring, determination of the sex and function of the placenta. On the other hand, many pathological conditions or changes in homeostasis such as metabolic disorders, infectious events, stress and temperature could negatively affect the growth rate of embryonic or fetal development . The first part of pregnancy is considered particularly sensitive because it concerns related physiological processes, such as the development of the corpus luteum (as a temporary endocrine reservoir), the implantation of the conceptus in the uterus, its growth, and the development of an efficient functional placenta . Increased knowledge of the developmental processes during gestation could provide valuable information on potential alterations in embryonic/fetal development. Sheep have been largely used as an experimental model to study prenatal growth during gestation . Indeed, ovine conceptus development closely resembles the human one in terms of weight and number of births . Furthermore, sheep have a good tolerance to uterus manipulation and its pregnancy is considered a useful model for the study of skeletal development, because the bone tissue is very similar to human in terms of density and strength . Bones represent the framework of support and protection of the body and are considered the passive organs of movement . In this way, the fetal sheep can also be used as a practical model to study the skeletal system development during the uterine period . Ultrasonography is an advanced technique that provides greater quality images and is useful to identify and to study embryonic and fetal anatomical structures. The ultrasonographic assessment of skeletal growth represents an important marker to evaluate the correct development and health of the conceptus and to determine the gestational age . Despite being a well-validated technique, the reliability of ultrasonography can be affected by the quality of the ultrasonic equipment, the technique for obtaining the images, the frequency of examinations, the evaluation period, and the experience of the operator . Furthermore, sheep represents a useful and low-cost model to study dynamic fetal growth due to the possibility of finding fetuses of different gestational age from regularly slaughtered pregnant animals. In these samples, a precise ultrasound fetal growth can be performed, combined with a direct measurement of the concepti and evaluation of skeletal development using specific staining. For this purpose, we have examined the development of the ovine conceptus between the 20th and 70th day of gestation in regularly slaughtered ewes. Conceptus growth was measured by ultrasound of the pregnant uterus and then by direct measurement of the conceptus after removal from the uterus. The development of the skeletal system and the dynamics of ossification were studied by clarification and differential cartilage-bone staining of the conceptus. 2. Materials and Methods All chemicals in this study were purchased from Sigma Chemical CO (St. Louis, MO, USA) unless stated otherwise. 2.1. Conceptus Collection and Measurements A total of 70 concepti of different gestational ages were obtained at local slaughterhouses from pregnant female Sarda sheep. These pregnant females were regularly slaughtered in accordance with European Regulations (Regulation of European Parliament and of the Council 853/2004; 627/2019; 1/2005; 1255/97) and the pregnant uteri were recovered after opening the abdominal cavity. The uteri were subjected to ultrasound using a Philips HD 11 and a 5-8 MHz micro convex probe. The probe was positioned on the ventral side of the pregnant uterus and the conceptus was carefully measured. According to , the morphometric parameters crown-rump length (CRL) and bi-parietal diameter (BPD) were measured in each conceptus and used to date the gestational age . After ultrasound examination, each conceptus was recovered by an incision along the uterine body and both horns. The measurement of CRL was recorded directly by a metal ruler and the conceptus was processed for differential staining as described below. 2.2. Conceptus Staining for the Study of Osteogenesis Dynamics The development of the skeletal system and the dynamics of ossification were assessed by a selective staining for bone and cartilage, according to , with some modifications. In brief, specimens were prepared by removing skin and organs, especially liver, intestine, kidneys and were then dehydrated and fixed in 95% ethanol for at least 7 days. It was very important to use ethanol as a fixative as well as other fixatives such as formalin to avoid non-specific staining of embryonic structures. For further removal of fatty tissue and for tissue permeabilization, specimens were exposed to 100% acetone overnight at room temperature. Then, they were transferred to Alcian blue stain (Ab), specific for cartilage. In detail, the specimens were immersed in a dye solution containing 10 mg of Alcian blue dissolved in 4 mL of H2O MilliQ, 80 mL of absolute ethanol and 20 mL of (glacial) acetic acid, for three days. It is also important to specify that embryos with an age of less than 30 days cannot be processed due to the richness of glycosaminoglycans of the young connective tissue that interferes with the staining of the cartilage matrix. After that, specimens were exposed to decreasing concentrations of ethanol (ethanol 70% for 3 h, ethanol 70% for 3 h, ethanol 40% for 3 h, ethanol 15% for 3 h) for rehydration and then transferred to 1% potassium hydroxide (KOH) for 2 days for clarification. Then the specimens were immersed in dye solution specific for bone tissue containing 1 mg of Alizarin red stain (Ar) in 100 mL of 1% KOH solution, for at least 3 days and this solution had to be changed daily. For concepti aged 45 days or more it was necessary to double the Ar concentration. After that, the specimens were exposed to 1% KOH for 12 h to overnight for hydrolysis of soft tissues, leading to transparency and allowing visualization of stained skeletal elements. Finally, the samples were transferred to 100% glycerol for long-term storage. After staining, the specimens were observed using a stereomicroscope with 60X magnification and photographed. 2.3. Statistical Analysis The normality of data distribution was analyzed using a Kolmogorov-Smirnov test. A General Linear Model method (GLM) with Tukey pairwise comparisons were used to analyze the morphometric measurements (CRL and BPD) of each conceptus where: Y = m + measurements (by ultrasound vs. in vivo) + time (days of pregnancy) + measurements x time. The measurements and time were considered as fixed factors. All results are expressed as mean +- S.E.M. Statistical correlation between each morphometric measurements and gestational age were investigated by using the Pearson's correlation coefficient. A p-value <= 0.05 was considered the minimum level of significance. Data were analyzed using Statistical Software Minitab(r) 18.1 (2017 Minitab). 3. Results 3.1. Concepti Measurements As shown in Figure 1, the increase in CRL was constant and statistically significant from day 22 to day 45 of pregnancy in all measured concepti (p < 0.001). No significant differences were observed between the measurements of the concepti performed by ultrasonography (eco) and by direct measurement (vivo), so the values of CRL obtained by the two different measurement methods are consistent . Similarly, BPD growth was constant and statistically significant from day 30 to day 70 of pregnancy in all measured concepti (p < 0.001). The two morphometric measurements were significantly (p < 0.05) correlated with the gestational age, showing a positive linear correlation with the increasing gestational age, and more specifically CRL eco R2 = 0.9729, CRL in vivo R2 = 0.9736 and BPD R2 = 0.9838. 3.2. Study of Osteogenesis Dynamics We examined N = 20 concepti aged between 25 and 70 days of gestation, stained with the method described above. As shown in Figure 3A, the ovine fetus remained completely cartilaginous up to 35 days of gestation. The ossification began in the skull, starting from the mandible, towards the 40th day of pregnancy , continued to the maxilla around the 45th day, then to the rest of the splanchnocranium between the 50th and the 60th day of gestation, and finally to the neurocranium between the 60th and the 70th days . The process of ossification of the limbs also began around the 40th day of gestation, with the appearance of ossification nuclei in the humerus and radius diaphysis. At 45 days, the process became more evident, the nuclei elongate and were also evident in the ulna and distal portion of the scapula. At 50 days, the diaphysis of the humerus, radius and ulna are ossified . At 60 days, the ossification of the middle third of the metacarpal was observed , while the remaining segments do not vary from the previous one. At 70 days of gestation, ossification nuclei appear in the phalanges. The process of ossification of the hind limbs is observed starting from the 45th day of gestation, when small areas of ossification are visualized in the middle third of femur and tibia. From the 50th day of gestation, the tibia underwent considerable and rapid development, becoming the most developed bone segment of the entire body . The elongation of this segment takes on a characteristic trapezoidal shape and remains linear between the 60th and the 70th day of gestation. The ossification of the spine occurs after 50 days and begins from the thoracic, lumbar, and sacral portions. The cervical portion ossified at 60 days while the coccygeal one after 65 days. The ossification of the ribs is evident starting from approximately the 45th day of gestation in the dorsal portions . It is more marked on the 50th day, always in the dorsal portion , while between 65 and 70 days it extended to the ventral portion. The costochondral junctions and the sternal ribs remained cartilaginous . The ossification of the sternum occurred later compared to the ribs. The centers of ossification appeared around the 65th day and at the 70th day of gestation the structure is yet not completely ossified. Table 1 provides a summary of the dynamics of ossification during conceptus growth between day 30 and 70 of gestation in sheep. 4. Discussion The present study described the development of the ovine conceptus by ultrasound analysis and the dynamics of ossification during the first 70 days of gestation. Three convergent analyses were performed in different stages of gestation: (1) ultrasound examination of the uterus and measurement of crown-rump length (CRL) and biparietal diameter (BPD) of the conceptus; (2) direct measurement of CRL and BPD of the conceptus outside the uterus and (3) osteo-cartilage dynamics during development by differential staining. Ultrasonography is widely used in many domestic species to measure fetal development and to determine the gestational age . In our study, sheep concepti ranging between 22 and 70 days of presumptive gestation were examined by ultrasound. In this time window, the most representative morphometric parameter was the CRL, due to the linearity of the shape of the embryo. Indeed, the CRL is considered one of the selection parameters to calculate the gestational age, especially in the first stages of pregnancy . Our data are in accordance with the foregoing statements; we have highlighted a high correlation between CRL, and gestational age and no significant differences were detected between ultrasonography (eco) and direct (vivo) measurement . This result is very interesting as it demonstrates the reliability of ultrasound analysis for the determination of gestational stages in the ovine species at the beginning of pregnancy. Recently, several studies investigated the usefulness of advanced ultrasound approaches in the estimation of fetal age. Three-dimensional (3D) ultrasound nevertheless represents a valuable tool for measuring fetal volumes, providing precise assessment of fetal structures. Although widely used in human clinical obstetrics, it has had limited applications in veterinary reproduction. In the mare and the cow, 3D ultrasound has been tested for the acquisition of fetal measurements, sexing and predicting weight at birth . However, the need to standardize acquisition procedures and the costs for the equipment may limit the application in clinical practice. Another promising approach could be represented by quantitative ultrasound. Aimed at investigating the interactions between ultrasound waves and tissue microstructure, quantitative ultrasound has been employed in humans to assess fetal lung maturity and bone mineralization dynamics . In the ovine, quantitative ultrasound has been employed to investigate morphophysiological changes in maternal-fetal tissues such as placentomes, lungs, liver and kidneys during pregnancy paving the way to future applications in farm animals. As already described in the ovine species, during clinical routines the CRL measurement by ultrasound is less reliable after the 40th day of pregnancy, as a consequence of rapid fetal growth, the positions of the concepti are more variable and their movements significantly increase, making the measurement less accurate. In this regard, the measurement of the largest transverse diameter between both parietal bones, evaluated as the biparietal diameter (BPD), shows greater reliability. Previous studies by ultrasound showed that the BPD is the most representative parameter of the gestational age during the second third of pregnancy in sheep . The strong correlation of BPD with the gestational age shown in our study confirms this parameter as a reliable indicator of fetal age, as previously observed in other domestic ungulates such as pig , sheep and cattle . At this stage of development, in addition to the use of the BPD, the search and observation of specific anatomical structures could be advisable. In this respect, the development of the skeleton is of great importance. In the last years, various studies tried to investigate the dynamics of the development of the conceptus in mammals and in sheep, several studies focused on the skeletal development, since this species is considered an excellent model for human(s) . In the past, most of the studies used radiology to describe skeletal development in ruminants, pigs, cats and humans . The spread of the use of ultrasound has allowed a change in the investigation approaches. Ultrasonography is a well-validated technique and largely used to detect mineralization and to measure fetal bones and ossification centers . However, even if significant progress has been made, the precise detection of skeletal ossification loci and dynamics needs to be confirmed by other estimation methods. Our study represents an innovative attempt to describe fetal skeleton development during the first part of gestation in sheep using ultrasonography associated with a differential staining technique that allows the observation of the first ossification processes. The first step was to adapt the staining and clarification protocols to the ovine fetuses, especially the fixation times and methods. Indeed, the conceptus must be fixed exclusively in ethanol and not in formalin, to avoid non-specific staining of embryonic structures. Furthermore, embryos with an age of less than 30 days cannot be processed due to the richness of glycosaminoglycans of the young connective tissue that interfere with the staining of the cartilage matrix. As a result, we obtained an overview of osteochondral temporal dynamics in the early stage of development from 30 days up to 70 days. In effect, while a previous study described single skeletal components , no research has previously reported data on the complete skeleton in sheep with this technique. The first evidence of ossification (bone mineralization) in our concepti was observed in the skull towards the 40th day of gestation (26.6% of the total gestational period (GP)) starting from the mandible, earlier compared to that described in sheep by Harrys in 1937, but similar to observations in domestic pig (37 days of gestation, 26% of GP; , cow (25% of GP; ) humans (26% of GP; ) and some wild mammals such as lowland paca (28% of GP; ), collared and white-lipped peccary and African elephant . The development of the forelimbs and hind limbs was evidenced by the early appearance of ossification (40 and 45 days of gestation, 26,6% and 30% of GP, respectively) like other precocial animals (cow , African elephant , collared and white-lipped peccary ), contrary to what happens in altricial animals as rabbit , rats , domestic carnivores , and primates such as marmosets , where the first mineralization of the appendicular skeleton was identified at 66%, 75%, 72.6% and 70% of GP, respectively. In mammals, except for humans, the gestation period seems to be correlated with the size of the offspring and its competence (altricial vs. precocial; ); furthermore, there seems to be a correspondence between the length of gestation and the timing of ossification . Sheep, as a mammal with a long gestation, is precocial and begins ossification earlier than other mammalian species with a long gestation and altricial offspring. In accordance, our data on osteo-chondral dynamics have shown fetuses with the ossification process extended to the whole skeleton already at 70 days of pregnancy, an early phase of gestation equal to 46.6% of GP. The timing of ossification and the growth rate of skeletal components are very important to assess the normal prenatal growth, as indicated in different species (humans , pigs , dogs , mice ). The correct bone development provides an adequate structural support to the whole organism and its locomotive function ; therefore, adequate skeletal development during gestation is essential for the good performance of the offspring after birth, which has implications for the survival rate of lambs . The study of the dynamics of ossification of the limbs provides a great help in estimating the age. Especially in the early period of pregnancy, when in the ovine species the bone growth is very fast, this knowledge may be useful for gestational management and early diagnosis of skeletal diseases. 5. Conclusions Our study using staining and clarification techniques combined with systematic ultrasound has confirmed that biparietal diameter is the most accurate parameter for estimation of gestational age in sheep. Furthermore, our study suggests that the ossification of the tibia, due to its growth characteristics and its particular shape, could be considered a valid parameter to estimate fetal age by ultrasound. To our knowledge, this study is the first observation on the overall skeletal development of the ovine species. Supplementary Materials The following supporting information can be downloaded at: Table S1: Table 1. Values of Crown Rump Length (CRL) expressed as mean +- SEM, measured by ultrasonography (eco) and directly by a ruler (vivo) in ovine conceptus from day 30 to day 70 of pregnancy; Table S2: Values of Biparietal Diameter (BPD) in cm expressed as mean +- SEM, measured by ultrasonography (eco) in ovine conceptus from day 30 to day 70 of pregnancy. Click here for additional data file. Author Contributions Conceptualization, S.L., S.S. and E.C.; methodology, E.C., L.F. and S.M.N.; validation, S.L., S.S., D.B. and S.D.G.; formal analysis, S.S., S.L., D.B. and S.D.G.; investigation, E.C., S.M.N. and L.F.; resources, S.L. and S.S.; writing--original draft preparation, S.S.; writing--review and editing, S.S., S.L., D.B. and S.D.G. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study was not conducted on experimental animals. The material used (ovine embryos and fetuses) was collected post-mortem at local slaughterhouses in Sardinia, Italy, from regularly slaughtered pregnant animals, which does not require ethics approval. All aspects relating to animal welfare are the responsibility of the staff employed in slaughterhouses, according to the EU guidelines (Regulation of European Parliament and of the Council 853/2004; 627/2019; 1/2005; 1255/97). Informed Consent Statement Not applicable. Data Availability Statement The data produced during the current study are available from the corresponding author on reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 (A): crown rump length (CRL) in ovine conceptus from day 22 to day 70 of pregnancy measured by ultrasonography (eco) or directly (vivo) with a ruler. No significant difference was shown in the measurements obtained by the two different systems (eco vs. vivo). (B): Ultrasound measurement of CRL of a pregnant uterus at about 30 days of gestation (on the left) and the direct measurement of the same embryo (on the right). Figure 2 (A): Biparietal diameter (BPD) growth in ovine concepti from day 30 to day 70 of pregnancy measured by ultrasonography (blue continuous line) and linear correlation between BPD and gestational age (black dashed line; R2 = 0.98; p < 0.05). (B): Ultrasound measurement of BPD in a conceptus at 38 days of gestation. Figure 3 (A): ovine fetus at about 35 days of gestation (on the left). On the right, the same fetus stained with Alizarin red stain + Alcian blue stain. The skeleton is totally cartilaginous; indeed, it is colored blue. (B): ovine fetus at about 40 days of gestation (on the left). On the right, the same fetus stained with Alizarin red stain + Alcian blue stain. It is possible to observe the initial ossification of the mandible, in red (black arrow). Figure 4 (A): ossification of the mandible and maxilla and extension of the ossification to the splanchnocranium (50 days), black arrows. (B): intra-membranous ossification of the neurocranium (approximately 60 days), black arrowhead. Figure 5 Progression of the ossification of the forelimbs and hindlimbs at 35 (A), 50 (B) and 65 (C) days of gestation. (A): The ovine fetus remained completely cartilaginous up to 35 days of gestation. (B): Ossification of radius, ulna and tibia is observed starting from the 50th day of gestation (black arrows). (C): progressive ossification of the metacarpal and metatarsal is noticeable at 65 days (black arrows). Figure 6 Ossification of the ribs at 45 (A), 50 (B) and 65 (C) days of gestation. The arrows indicate the cranio-caudal and the dorsal ventral axes of the conceptus in (A-C). animals-13-00773-t001_Table 1 Table 1 Dynamics of ossification during fetal growth between day 30 and 70 of gestation in sheep. CRL (cm) BPD (cm) Day of Pregnancy (% of Gestational Period) Dynamic of Ossification 2.01 +- 0.045- 2.54 +- 0.022 0.49 +- 0.002- 0.66 +- 0.007 30th-35th (20-23) Skeleton cartilaginous 3.34 +- 0.088- 4.05 +- 0.058 1.03 +- 0.01- 1.12 +- 0.026 40th-45th (26.6-30) Beginning of jaw ossification and humerus and radius diaphysis 7.06 +- 0.037- 8.15 +- 1.32 1.38 +- 0.026- 1.63 +- 0.055 50th-55th (33.3-36.6) Beginning of ossification of rachis, ossification of ribs, diaphysis of femur, and tibia. Beginning of intra-membranous ossification of the skull. 8.65 +- 0.058- 9.2 +- 0.153 2.41 +- 0.024- 2.52 +- 0.03 65th-70th (43.3-46.6) Almost all skeleton is ossified, except ends of the limbs, cervical vertebra and chondrocostal junctions. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000039

The effect of breed on milk components--fat, protein, lactose, and water--has been observed to be significant. As fat is one of the major price-determining factors for milk, exploring the variations in fat QTLs across breeds would shed light on the variable fat content in their milk. Here, on whole-genome sequencing, 25 differentially expressed hub or bottleneck fat QTLs were explored for variations across indigenous breeds. Out of these, 20 genes were identified as having nonsynonymous substitutions. A fixed SNP pattern in high-milk-yielding breeds in comparison to low-milk-yielding breeds was identified in the genes GHR, TLR4, LPIN1, CACNA1C, ZBTB16, ITGA1, ANK1, and NTG5E and, vice versa, in the genes MFGE8, FGF2, TLR4, LPIN1, NUP98, PTK2, ZTB16, DDIT3, and NT5E. The identified SNPs were ratified by pyrosequencing to prove that key differences exist in fat QTLs between the high- and low-milk-yielding breeds. milk fat whole-genome sequencing SNPs genomic variation variant calling indigenous breeds Genomics for conservation of indigenous cattle breeds and for enhancing milk yieldBT/PR26466/AAQ/1/704/2017 This research was funded from the project "Genomics for conservation of indigenous cattle breeds and for enhancing milk yield (BT/PR26466/AAQ/1/704/2017)". pmc1. Introduction India has become the largest milk producer in the world . Several schemes involving crossbreeding have been implemented to enhance milk production in the country. As a result, the number of crossbred cattle increased and contributed to around 28 percent of total milk production in India (ca. 188 million tons), surpassing the contribution of indigenous cattle . However, indigenous cattle breeds are well known for their heat tolerance and disease resistance , and the crossbreds have been found to be susceptible to tropical diseases and harsh climatic conditions and require constant good management practices. To strike a balance between increasing demand for milk and the change in the environment due to global warming, exploring the genomic merit of indigenous cattle/breeds becomes even more important. Milk being a polygenic trait with medium heritability, the majority of animal breeding research has centered on quantitative trait loci (QTLs) with moderate to large effects on milk production traits. The DGAT1 on chromosome 14 , the growth hormone receptor (GHR) on chromosome 20 , and the ABCG2 or SPP1 (Osteopontin) on chromosome 6 are well-known QTL genes that have been fully characterized with a strong putative or well-confirmed causal mutation. The two QTLs DGAT1 (K232A) and ABCG2 (Y581S) in Bos taurus have been suggested to be associated with increased fat yield and fat and protein percent in milk with a decrease in milk yield . The GHR mutation F279Y has been observed to have a significant effect on milk composition (fat and protein percentage) and milk yield . The locus c.8514C > T in the intronic region of SPP1 has also been found to have a significant effect on milk production and milk composition . However, the DGAT1 and ABCG2 genes have been found to be fixed among Indian breeds Sahiwal, Rathi, Deoni, Tharparkar, Red Kandhari, and Punganur . Currently, the animal QTL database (QTLdb) contains 1,93,216 QTLs for different bovine traits, out of which 83,458 QTLs have been reported for milk traits . Milk is the primary source of nutrition for infants, as well as adults. Besides its nutritional value, it has a major role in imparting growth and immunity through intrinsic milk components such as growth factors, chemokines, anti-inflammatory molecules, antioxidants, prebiotics, and probiotics . Milk has four major components, fat (3.6%), protein (3.2%), lactose (4.7%), and water (87%), along with other various kinds of minerals, enzymes, vitamins, and dissolved gases. Various research studies have shown that several factors, such as lactation stage, genetics, environmental factors, and diet management, influence milk quality. The variability of milk composition among popular dairy breeds Brown Swiss, Holstein Friesian, Jersey, Simmental, Grey Alpine, and Rendena under the same dairy management practices has been explored, and Holstein Friesian had higher milk yield with lower fat content (27.45 kg/d, 4.04%) , whereas Jersey had lower milk yield with relatively higher fat content (17.27 kg/d, 5.65%) . A low fat percentage has also been reported for Ayrshire, Brown Swiss, Guernsey, Holstein Friesian, and Jersey breeds in the United States . Furthermore, the effect of breed has been found to significantly influence the water (p <= 0.0001), protein (p <= 0.05), total solids (p <= 0.05), fat (p <= 0.05), milk urea nitrogen (p <= 0.001), and ash (p <= 0.0001) content of milk . India, with a huge diversity of 50 cattle breeds, forms an ideal ground to study genetic variation at the genomic level vis-a-vis milk traits . The cost of the milk world-over varies with the percentage of fat present in the milk. Exploring the variations in fat QTLs across breeds would shed light on the variable fat content in their milk. No such studies have been reported in the past for indigenous cattle breeds to evaluate the variation across indigenous breeds within the fat QTLs. Therefore, the objective of the present study was to explore genomic variation(s) within the fat QTLs that were identified to be differentially expressed in lactation across indigenous breeds, which were divided into high- (Sahiwal and Gir) and low-milk-yield (Gaolao, Deoni, Pulikulam, Hallikar, Dangi, and Amritmahal) groups. 2. Materials and Methods 2.1. Data Retrieval from the Public Repository QTL genes associated with milk fat traits (milk fat content and percentage) and metabolism were extracted from the Animal QTLdb database , and the duplicates were removed. QTL genes (286 and 256 (total of 542)) were identified for milk fat yield and milk fat percentage, respectively, with 417 unique genes for both traits (Supplementary File 1). Functional annotation of the 125 QTL genes commonly associated with milk fat yield and milk fat percentage was performed in g:Profiler and ShinyGO to identify the enriched biological processes. Protein interaction network analysis of the 417 genes was performed using the Search Tool for the Retrieval of Interacting Genes Search Tool for the Retrieval of Interacting Genes 11.0 (STRING 11.0) database at a confidence score value of 0.5 against model species Bos taurus . The interaction network was imported into the Cytoscape 3.8.0 software (Institute for System Biology, CA, USA) for visualization. The hub and bottleneck genes were identified in the interaction network using the Cytohubba plugin of Cytoscape considering the degree of association between the genes and by taking the bottleneck approach, which takes into account the top 20% of the degree of distribution of the proteins in the network . A total of 74 QTL genes were identified to be hub/ bottleneck genes. The hub genes were the genes that had the highest degree of association, and the bottleneck genes were the key connectors having a high betweenness (measure the centrality of the nodes) among different clusters in protein interactions . 2.2. Bioinformatics Analysis of Milk Transcriptome The publicly available milk transcriptome bioproject ID (PRJNA419906) was used to analyze the expression of QTL genes associated with milk fat traits. This bioproject was considered in this study as it has data generated from Jersey (a breed with high fat content ranging from 4.10-4.86%) and Kashmiri (a breed with low fat content ranging from 3.20-3.94%) . The data were generated from mammary epithelial cells (MECs) collected on Day 15 (D15), D90, and D250 from six lactating cows (three Jersey and three Kashmiri cattle). These days represent early, mid-, and late lactation, respectively . The data were downloaded from the Sequence Read Archive (SRA) of the NCBI database, and the fastq-dump program of SRAtoolkit was used to extract the fastq reads. Quality assessment and control of RNA-seq data were performed through Fast QC Version 0.11.5 , MultiQC Version 1.8 , and trimmomatic Version 0.39 . All the high-quality reads were mapped to the Bos indicus genome (GCF_003369695.1) using STAR Version 2.5.4b with the default parameters . Gene expression was estimated using RSEM , and differential gene expression was performed through the DESeq2-R package . The differentially expressed genes among the 74 fat QTL hub and bottleneck genes were identified. 2.3. Breed Selection, Sampling, Genomic DNA Extraction, and Whole-Genome Sequencing Indigenous cattle breeds for the proposed study were grouped into high- and low-milk-yield-breed groups. The high-milk-yield group (avg milk yield per day 8 kg) included Sahiwal (n = 4) and Gir (n = 4), and the low-milk-yield group (avg milk yield per day 2.5 kg) included 6 animals representing the breeds Gaolao, Deoni, Hallikar, Dangi, Pulikulam, and Amritmahal . Animals of different breeds were considered in the study to have a true representation of both the high- and low-milk-yield groups. Genomic DNA (gDNA) was extracted from the blood samples of the animals from these breeds using the nucleospin blood L-kit (Macherey-Nagel), and the integrity of the genomic DNA was checked on agarose. After estimating the concentration of gDNA (Nanodrop2000, ThermoFischer Scientific), DNA libraries were prepared as per the manufacturer's protocol (Illumina sequencing platform) for paired-end sequencing (2150 bp). 2.4. Bioinformatic Analysis (Variant Calling, SNP Annotation, and Functional Enrichment) Sequencing data generated on an Illumina platform were pre-processed for quality assessment and improvement (base quality, nucleotide distribution, GC content, adaptor sequence, duplication, length distribution, etc.) by FastP . All high-quality reads were mapped to the Bos indicus reference genome (Brahman-GCF_003369695.1) using BWA aligner . Variant calling was performed from the aligned data using freebayes and GATK . For the GATK- and freebayes-generated vcf files, only SNPs were selected, leaving aside all indels and insertions. After freebayes variant calling, the low-quality variants were filtered by vcftools Version 1.10 for Q > 20. In the GATK pipeline, the paired-end Illumina Hi-Seq raw reads for each individual were first converted into an unaligned bam; the illumina adapters were marked; the sam was converted back to FASTQ; the reads were mapped to the reference Brahman genome (GCF 003369695.1); the unaligned and mapped bams were combined. Finally, the duplicate marked clean bam was used to generated GVCF for each animal using GATK haplotypecaller. The GVCFs generated for all the animals were combined to call the variants using the genotypeGVCF module. The parameters for GATK VariantRecalibration to generate the VQSLOD score were QD < 2.0, MQRankSum < -8.5, ReadPosRankSum < -8.0, FS > 60.0, MQ < 40.0, SOR > 3.0, and DP 30x (depth or coverage). The final set of SNPs after recalibration by GATK included the selection of SNPs that passed. We further used the GATK-filtered set and the freebayes-filtered set to identify the common SNPs across these variant callers. From this vcf file, the SNPs in the differentially expressed hub and bottleneck genes (i.e., genes that are hub/bottleneck and are differentially expressed as identified in Section 2.2) were extracted using an in-house perl script. The non-synonymous SNPs (nsSNPs) in the coding regions of these were identified through the SnpEff tool . 2.5. SNP Validation through Real-Time Sequence-Based Pyrosequencing Three nsSNPs that were found to be distinctly different between the high- and low-milk-yield groups were selected for validation and were genotyped in PyroMark Q48 (Qiagen) as per the manufacturer's protocol. These nsSNPs were found in the differentially expressed hub and bottleneck genes GHR, LPIN1, and TLR4. GHR was one of the genes having the maximum number of interactions in the network. LPIN1 had the maximum SNP count of 10, whereas TLR4 was one of the top 10 highly upregulated genes. PCR and sequencing primers were designed using PyroMark Assay Design Software 2.0 (Qiagen). The PCR amplification was performed in a 20 mL reaction, with the thermal cycling conditions, which included an initial denaturation of 95 degC for 3 min followed by 40 cycles of 95 degC for 30 s, 65 degC for 30 s, 72 degC for 1 min, and a final extension of 72 degC for 10 min. Sequence analysis was performed by PyroMark Q48 Autoprep software Version 2.4.2 in SNP analysis assay mode for 14 animals. The schematic representation of the study is given in Supplementary File 1: Figure S1. 3. Results 3.1. Meta-Analysis of Cattle-Milk-Fat-Component-Associated QTLs and Related Genes Animal QTLdb was used to extract fat-trait-associated QTL genes. In the QTLdb, after the removal of duplicate genes (Supplementary File 1), 286 and 256 genes were found associated with milk fat yield and percentage, respectively, whereas 125 common genes were found between milk fat yield and percentage . A total of 417 unique genes were found to be associated with milk fat and other milk traits. These genes were also found to be annotated for other milk traits such as milk yield, protein yield, and percentage. Among all genes, 24 genes were found to be associated with both fat yield and fat percentage traits only (Supplementary File 1). The common genes (125) were found enriched in biosynthetic-, catabolic-, regulatory-, transportation-, and cellular-response-associated metabolic processes. Among the metabolic genes associated with milk fat traits, the genes associated with milk fatty acid metabolism were FASN, GPAT4, DGKG, ELOVL6, and LIPIN1 (Supplementary File 5: Table S1). 3.2. Milk Transcriptome Data Processing and Gene Expression Analysis of Fat QTL Genes The publicly available RNA-seq bioproject (PRJNA419906) has library sizes ranging from 7764992200-13682843400 bp and 6842583600-12088807400 bp, for Jersey and Kashmiri, respectively . Further, gene expression counts per million (CPM), principal component analysis (PCA), and multidimensional scaling (MDS) of the samples were assessed. The PCA and MDS plots of sequenced RNA-seq libraries showed a high level of similarity within breeds and relatively low variation between the lactation stages of breeds. A total of 70 genes were found to be upregulated and 52 genes downregulated in the Jersey breed in comparison with the Kashmiri breed. Differentially expressed transcripts (DETs) were also explored for fat QTL genes (Supplementary File 2). The volcano plots of differentially expressed genes (DEGs) and DETs depicting the distribution of upregulated and downregulated genes are shown in Figure 2. In both the Jersey and Kashmiri breeds, Beta-lactoglobulin (LOC113901792), Casein beta (CSN2), and Casein alpha s1 (CSN1S1) were identified to be among the highly expressed top 20 genes (Supplementary File 5: Table S2). The DETs are listed in Supplementary File 2. In the Jersey breed, CXCL-8, TLR4, and OLR1 were among the highly upregulated genes. CXCL-8/IL8, produced by macrophages, epithelial cells, and airway smooth muscle cells, is a neutrophil chemotactic factor that induces chemotaxis in target cells and other granulocytes to initiate movement toward infection sites, whereas OLR1, a receptor on macrophages, epithelial cells, and airway smooth muscle cells, is involved in rapid oxidization of low-density lipoprotein (LDL), which is more readily recognized by the TLR4 receptor. The list of top 10 upregulated and downregulated DEGs is given in Supplementary File 5: Table S3. 3.3. Protein Interaction Network Analysis of Milk Fat QTL Genes A protein interaction network analysis was performed among 417 milk fat QTL genes, and the interaction network was generated with 403 nodes and 671 edges. Based on the degree of association, 50 hub and 50 bottleneck genes were selected from the network (Supplementary File 2). A total of 74 QTL genes were found to be either hub or bottleneck genes . Out of these, 25 genes (which accounted for 18 hubs/17 bottleneck genes) were differentially expressed in Jersey and Kashmiri . Out of these genes, ten genes possessed both hub and bottleneck gene characteristics. The SRC and DGAT1 genes were among the top differentially expressed hub and bottleneck genes (Table 1). SRC had the highest degree of association (30) with a log2 fold change of 1.480587, followed by DGAT1 with a degree of 25 and a log2FC of 0.921104. 3.4. Variant Analysis of Hub and Bottleneck Genes among Indigenous Breeds Illumina short read (Paired end) data of 14 samples from both groups of high- and-low-milk-yield breeds had 12.77 billion reads. After preprocessing, clean data included 11.02 billion reads, which is ca. 1516 Gb data. Each dataset had a minimum sequencing depth of >=30x with an average GC content of 45.26%. The processed datasets contained on average 97.91% Q20 bases and 93.97% Q30 bases. The high-quality trimmed data aligned to the Brahman reference genome with an overall alignment rate of >95%. Initial variant calling on the aligned data provided 63,357,363 variants, which were filtered for high quality. After quality filtering on Q20, a total of 33,976,892 SNPs were identified across the genomes (Supplementary File 3). Upon GATK analysis, 39,625,917 variants were found to pass the variant calibration. A total of 25,956,231 SNPs were found to be common among the variant callers. From these, SNPs in the 25 differentially expressed hub and bottleneck milk fat QTL genes were extracted, out of which 20 genes were found to have non-synonymous substitutions in the coding regions (Table 2). The variants identified in these 20 genes were further explored for two kinds of genomic variant patterns, i.e., fixed SNP pattern in the cattle of the high-milk-yield group vs. variable SNP pattern in cattle of the low-milk-yield group, or vice versa. The fixed SNP pattern in high-milk-yield breeds in comparison to low-milk-yield breeds was observed in the genes GHR, TLR4, LPIN1, CACNA1C, ZBTB16, ITGA1, ANK1, and NTG5E (Table 3), and the opposite was observed in the genes MFGE8, FGF2, TLR4, LPIN1, NUP98, PTK2, ZTB16, DDIT3, and NT5E (Table 4). SNPs C/G, C/A, and G/A were confirmed in GHR, TLR4, and LPIN1 in the Amritmahal, Pulikulam, and Dangi breeds (low-milk-yield) as against SNPs C/C, C/C, and G/G in the Gir and Sahiwal breed (high-milk-yield), respectively (Supplementary File 4). In the TLR4 gene, variant g.107083326A>C was found in the low-milk-yield group, but the same variant was fixed in the high-milk-yield group. Similarly, in the LPIN1 gene variant, g.85211528C>G was found in the low-milk-yield group, but this variant was fixed in the high-milk-yield group. In the NUP98 gene and LPIN1 variants, g.32707374G>A and g.85205642T>G, respectively, were found in the high-milk-yield group, but were found to be fixed (g.32707374G>G; g.85205642T>T) in the low-milk-yield group. Another LPIN1 variant, g.85205642T>G, was observed in the high-milk-yield group, but was fixed in the low-milk-yield group. LPIN1 and ITGA1 had the maximum SNP count of 10, and the genes PTK2, IGF1R, DDIT3, CXCL8, and LPL had the least SNP count (Table 2). 4. Discussion The Bos indicus genome is an interesting model to study the genomic potential of different indigenous cattle breeds such as Sahiwal, Gir, Amritmahal, Dangi, Gaolao, Deoni, Pulikulam, and Hallikar, which are highly adapted to different tropical conditions with varying milking potential. The availability of bovine QTL resources such as the Animal QTL database and the collection of QTLs for different traits have provided the opportunity to investigate genomic variation among indigenous breeds for milk-associated traits. Milk quality such as fat yield and percentage are highly variable traits among breeds. Jersey is one among the high-milk-producing breeds worldwide, whereas Kashmiri is one of the poorly performing breeds in the Kashmir region of India. Therefore, we aimed at differences in the expression of fat QTL genes between the two contrasting breeds, Jersey and Kashmiri. In this study, significantly expressed hub and bottleneck fat QTL genes were further analyzed to identify the genomic variants from the whole-genome sequence data between high- (Sahiwal and Gir) and low- (Amritmahal, Dangi, Gaolao, Deoni, Pulikulam, and Hallikar) milk-yield indigenous breeds. This understanding of low- and high-milk-yield breeds for milk fat quality may help in enhancing the quality of milk in the long run. To explore the fat QTL genes, MEC RNA-seq data were processed and analyzed. The high level of similarity within breeds and relatively low variation between lactation confirmed the selection of RNA-seq data to explore differences between breeds rather than to explore difference in lactation stages of breeds. Among the highly expressed genes identified, it was observed that the Jersey breed has allocated more resources for the immune system, whereas the Kashmiri breed for regulation of ribosomal proteins. Among the top 10 upregulated genes, CXC motif chemokine ligand 8 was the most-upregulated gene. It is reported to be involved in various biological pathways such as increased insulin resistance, uncoupling of the GH/IGF1 axis, and an increase in mammary cell proliferation to improve metabolic health and milk yield . Diacylglycerol kinase gamma (DGKG), another upregulated gene, is a member of the type I diacylglycerol kinases and is highly upregulated (log2FC = 4.03) in Jersey. It plays a role in lipid metabolism by modulating the balance between diacylglycerol and phosphatidic acid. Phosphatidic acid is a lipid second messenger to activate protein kinase C isoforms, ras guanyl nucleotide-releasing proteins, and some transient receptor potential channels . Most of the top-upregulated genes in Jersey have been found to have a role in adipogenesis (ETS2) , adipocyte differentiation (OLR1, PARM1) , glucose transport (SLC6A9) (log2FC = 4.49) , glucose uptake (SLC45A4) , thyroid hormone synthesis (TG) , and aldosterone secretion (KCNK9) . The upregulation of these genes in Jersey indicates their involvement in lipid biosynthesis in the mammary gland during lactation. Furthermore, UDP-glucose 6-dehydrogenase (UGDH), which is involved in the biosynthesis of glycosaminoglycans, hyaluronan, chondroitin sulfate, and heparan sulfate, has been found (log2FC = 0.75) to be upregulated in the Jersey breed. UGDH's expression pattern in liver cells has been associated with an indispensable role in the metabolism of carbohydrates, fats, and proteins in dairy cattle . Moreover, UGDH has been found close to two reported QTLs for fat yield, fat percentage, and protein yield . During lactation, various morphological changes happen in the mammary tissue to support cellular differentiation, tissue elasticity, and reduced fat storage capacity in the animal. Upregulated GRH13 is a transcription factor that mediates the proliferation of epithelial cells . Similarly, MTMR3/3-PAP, a catalytically inactive member of the myotubularin gene family that coprecipitates the activity of lipid phosphatidylinositol 3-phosphate-3-phosphatase, is upregulated in Jersey. Matrilin 2 (MATN2) and prolyl 4 hydroxylase (P4HA3), which are important to maintaining the newly synthesized collagen's stability, are also upregulated . P4HA3 catalyzes the formation of 4-hydroxyproline (Hyp), which ensures the proper folding of procollagens during post-translational modification. The upregulation of MATN2 and P4HA3 probably may help in increasing the elasticity of the udder gland in Jersey during lactation. Further, the downregulation of MFGE8 and ELOVL16 may be responsible for the high fat content in milk and the shift to C18 from C16 fatty acids, respectively, in Jersey. MFGE8 regulates the absorption of free fatty acids and increases intracellular triglycerides' hydrolase activity, thereby restricting the storage of fat , whereas ELOVL fatty acid elongase (ELOVL16) elongates C16 saturated and monounsaturated fatty acids to C18 fatty acids . Further, IDH1, which catalyzes the conversion of isocitrate to a-ketoglutarate and generates the primary source of NADPH for de novo fatty acid synthesis , has been to be found downregulated in Jersey (log2FC = -1.632). The dysregulation of the genes MFGE8, EVOLVL16, and IDH1 might be responsible for the variation in the fat yield and composition in Jersey and Kashmiri cows. In addition, the decreased expression of these genes may be linked to the increased expression of genes involved in metabolism, glucose transport, and other transport activities, leading to higher milk production performance in Jersey. Among the ten QTLdb milk and milk trait genes that were differentially expressed and had hub and bottleneck gene characteristics, four genes were found enriched in metabolic pathways (Supplementary File 5: Table S1). SRC was identified as the top hub gene interacting with several genes in the protein-protein interaction network. SRC, a non-receptor tyrosine kinase, performs a wide variety of cellular functions in terms of metabolism and is primarily involved in impaired glucose uptake . Genes Diacylglycerol O-acyltransferase 1 (DGAT1) and ecto-5'-nucleotidase (NT5E) possess hub and bottleneck gene features. DGAT1 encodes a protein that catalyzes the conversion of diacylglycerol and fatty acyl CoA to triacylglycerol. DGAT1 is one of the highly studied genes for milk yield and fat quality . The NT5E gene encodes a plasma membrane protein that catalyzes the conversion of extracellular nucleotides to membrane-permeable nucleosides. SRC and DGAT1 were found to be highly upregulated in Jersey with log2FC = 1.48 and log2FC = 0.92, respectively. In the network, DGAT1 was found to interact with Glycerol-3-phosphate acyltransferase 4 (GPAT4) (also known as AGPAT6). This gene has been found to be involved in triglyceride biosynthesis and is comprised of acyltransferase motifs, which are essential for binding to substrates and catalyzing acyltransferase reactions. The AGPAT6 gene, which is highly expressed in mammary gland epithelium during lactation , was also observed to be upregulated. NT5E was found to interact with BDNF, NTRK2, and ZNRF4. NT5E is involved in various biological process such as adenosine biosynthetic process, AMP catabolic process, leukocyte cell-cell adhesion, and negative regulation of inflammatory response . DGKG, which was upregulated, was found to be a bottleneck gene in the network and had interactions with PRKCG and RNT. Phosphatidate phosphatase (LPIN1), an enzyme involved in lipid metabolism , was upregulated and found as a hub gene in the network having interactions with PPARA and PPARGC1A. LPIN1 is involved in various biological processes related to lipid metabolism such as the triglyceride biosynthetic process, the fatty acid catabolic process, and the regulation of transcription by RNA polymerase II . The gene PTK2 is well known for its association with milk production traits , and CACNA1A has a role in hormone regulation of lactation . Similarly, ZBTB16 is involved in bovine adipogenesis . NUP98 is associated with protein percentage . TLR4 is a mastitis-associated marker . FGF2 expression is reported to be associated with milk production traits . In this study, breed (high-milk-yield and low-milk-yield groups) differences in nsSNPs within these hub and bottleneck genes (GHR, TLR4, LPIN1, CACNA1C, MFGE8, PTK2, ZBTB16, FGF2, and NUP98) were identified. These fat QTL nsSNPs may have a role in the existing fat and milk yield differences between the breed groups. However, further studies to evaluate the impact of these SNPs on fat yield/percentage or milk yield need to be carried out. 5. Conclusions In this study, initially, DEGs in Jersey epithelial cells were identified, and these were further explored in the QTL database as being the major hub and bottleneck genes. The transcriptome in Jersey indicated higher expression of genes involved in metabolism, glucose transport, and other transport activities, leading to higher milk production performance. The 20 differentially expressed hub and bottleneck fat QTL genes were explored for non-synonymous genomic variants in the whole-genome sequence data, which were generated from fourteen animals. The fixed SNP pattern in high-milk-yield breeds in comparison to low-milk-yield breeds was observed in the genes GHR, TLR4, LPIN1, CACNA1C, ZBTB16, ITGA1, ANK1, and NTG5E, and the opposite was observed in the genes MFGE8, FGF2, TLR4, LPIN1, NUP98, PTK2, ZTB16, DDIT3, and NT5E. The role of these SNPs needs to be further explored. Acknowledgments We are grateful to the Director of NIAB for providing support to carry out the study. The bioinformatics facility NIAB is acknowledged for the bioinformatics data analysis. S.S.M. is thankful for the support of the JCBOSE fellowship (Grant No. SERB-JCB/2017/000027). Supplementary Materials The following Supporting Information can be downloaded at: File 1: Meta-data of milk QTL genes, biological process, and pathways; File 2: Detailed description of differentially expressed QTL, hub, and bottleneck genes; File 3: Features of sequenced data before and after filtering; File 4: Detailed description of the performed variant analysis; File 5: Tables consisting of metabolic gene details, highly expressed genes, a list of top ten upregulated and downregulated differentially expressed gene between the Jersey and Kashmiri breeds, along with the QTL id and trait name; schematic representation of the study and figure of quality evaluation of transcriptome samples. Click here for additional data file. Author Contributions S.S.M., R.K.G., S.A. and S.K.K.: conceived of the idea as a team; N.A.T. and V.K.: contributed to the first draft of the manuscript; N.A.T.: performed the Q48 experiments; S.A.: generated the VCF file from the sequenced genotype for the variant analysis; R.K.G.: finalized the final draft; S.K.K. and M.K.: analyzed the transcriptome data, generated the plots, and helped in the first draft of the manuscript. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement All the used RNA-seq data are available at NCBI SRA under the project accession number PRJNA419906. Genomics for conservation of indigenous cattle breeds and for enhancing milk yield BT/PR26466/AAQ/1/704/2017. Conflicts of Interest The authors have declared that they have no competing interest. Figure 1 Venn diagram showing the hub and bottleneck genes: (A) QTL genes of milk fat percentage (256) and milk fat yield (286), (B) genes of top 50 hub and bottleneck genes, (C) genes of 17 hub and 18 bottleneck differentially expressed genes. MFP = milk fat percentage, MFY = milk fat yield. Figure 2 Differentially expressed milk-trait-associated QTL genes and transcripts in the milk transcriptome of Jersey and Kashmiri breeds (A) DEGs and (B) DETs. Volcano plot showing the significance versus the fold change. Blue dots (p < 0.05 and log2FC > 2): all Bos indicus genes; green dots (p < 0.05): QTL genes; orange dots (p < 0.05): bottleneck genes; yellow dots (p < 0.05): hub genes; red dots (p < 0.05): genes possessing hub and bottleneck features. Figure 3 Confirmation of mutations shown in pyrograms with PyroMark SNP analysis assays: (A) GHR Gy392Ala (C>G) in Pulikulam (low-milk-yield breed) and (C/C) in Gir (high-milk-yield breed); (B) LPIN1 a Arg772Lys (G>A) in Amritmahal (low-milk-yield breed) and (G/G) in Sahiwal (high-milk-yield breed); (C) TLR4 a Asn347Gly (A>C) in Dangi (low-milk-yield breed) and (C/C) in Gir (high-milk-yield breed); the pyroseq sequencing primer is in reverse. animals-13-00884-t001_Table 1 Table 1 Differentially expressed hub and bottleneck QTL genes of the protein interaction network of fat QTL genes. Gene ID log2FC p-Value Trait Name Description Hub genes ENSBTAG00000008938 (SRC) 1.480587 0.002561 MFY; MPP; MPY; TDMY SRC proto-oncogene, non-receptor tyrosine kinase ENSBTAG00000026356 (DGAT1) 0.921104 0.032625 MFP; MFY; MPP. MPY; MKCP; MY Diacylglycerol O-acyltransferase 1 ENSBTAG00000007867 (STAT1) 1.938208 0.0000648 MFP; MPP; MPY; MY Signal transducer and activator of transcription 1 ENSBTAG00000005691 (FGF2) -1.55871 0.042228 MFY; MY Fibroblast growth factor 2 ENSBTAG00000019716 (CXCL8) 7.097327 0.000000000000077 MFY; MPP; MPY;TDMY C-X-C motif chemokine ligand 8 ENSBTAG00000006240 (TLR4) 3.918383 0.0000000123 MFP; MPP; MY Toll like receptor 4 ENSBTAG00000001335 (GHR) -1.97171 0.02566 MFP; MFY; MPP; MPY; MY Growth hormone receptor ENSBTAG00000012855 (LPL) -1.61377 0.008224 MFP; MFY; MPP Lipoprotein lipase ENSBTAG00000009578 (PTK2) -0.97617 0.043672 MFP; MFY; MPP; MPY; MY Protein tyrosine kinase 2 ENSBTAG00000000546 (ERBB2) -1.89535 0.048867 MFP; MFY; MPP; MPY; MY Erb-b2 receptor tyrosine kinase 2 ENSBTAG00000021527 (IGF1R) 1.798566 0.003947 MFP; MFY; MPP; MPY; MY Insulin like growth factor 1 receptor ENSBTAG00000007476 (BTRC) 2.698919 0.0000384 MFY Beta-transducin repeat containing E3 ubiquitin protein ligase ENSBTAG00000014357 (SDC2) -1.96312 0.029226 MFP; MPP Syndecan 2 ENSBTAG00000048655 (NT5E) -2.97069 0.000136 MFP; MFY; MPY 5'-nucleotidase ecto ENSBTAG00000007689 (LPIN1) -2.38799 0.001822 MFP; MFY; MPP; MPY; TDMY Lipin 1 ENSBTAG00000003300 (MFGE8) -1.90426 0.015698 MFP; MPP Milk fat globule EGF and factor V/VIII domain containing ENSBTAG00000020536 (HERC6) 1.37883 0.00206 MFP; MFY; MPP; MPY HECT and RLD domain containing E3 ubiquitin protein ligase family member 6 Bottleneck Genes ENSBTAG00000026356 (DGAT1) 0.921104 0.032625 MFP; MFY; MPP; MPY; MKCP; MY Diacylglycerol O-acyltransferase 1 ENSBTAG00000008938 (SRC) 1.480587 0.002561 MFY; MPP; MPY; TDMY SRC proto-oncogene, non-receptor tyrosine kinase ENSBTAG00000008432 (NUP98) 1.307627 0.033165 MFP Nucleoporin 98 and 96 precursor ENSBTAG00000027629 (ANK1) -2.3949 0.000752 MFY; MPY Ankyrin 1 ENSBTAG00000011266 (ZBTB16) -1.84946 0.011504 MFY Zinc finger and BTB domain containing 16 ENSBTAG00000016525 (ITGA1) -2.10583 0.004815 MFY; MPP; MPY; MY Integrin subunit alpha 1 ENSBTAG00000019716 (CXCL8) 7.097327 0.000000000000077 MFY; MPP; MPY; TDMY C-X-C motif chemokine ligand 8 ENSBTAG00000007867 (STAT1) 1.938208 0.0000648 MFP; MPP; MPY; MY Signal transducer and activator of transcription 1 ENSBTAG00000000546 (ERBB2) -1.89535 0.048867 MFP; MFY; MPP; MPY; MY Erb-b2 receptor tyrosine kinase 2 ENSBTAG00000007476 (BTRC) 2.698919 0.0000384 MFY Beta-transducin repeat containing E3 ubiquitin protein ligase ENSBTAG00000010106 (CCND3) -1.78227 0.000135 MFP; MPP; MPY; MY Cyclin D3 ENSBTAG00000001335 (GHR) -1.97171 0.02566 MFP; MFY; MPP; MPY; MY Growth hormone receptor ENSBTAG00000006240 (TLR4) 3.918383 0.0000000123 MFP; MPP; MY Toll like receptor 4 ENSBTAG00000010660 (CACNA1C) -2.82445 0.0000723 MFY Calcium voltage-gated channel subunit alpha1 C ENSBTAG00000012855 (LPL) -1.61377 0.008224 MFP; MFY; MPP Lipoprotein lipase ENSBTAG00000031544 (DDIT3) 2.367342 0.0000047 MFP; MFY; MPP; MPY; MY DNA damage inducible transcript 3 ENSBTAG00000005091 (DGKG) 4.032464 0.000000442 MFP; MPP; MY Diacylglycerol kinase gamma ENSBTAG00000048655 (NT5E) -2.97069 0.000136 MFP; MFY; MPY 5'-nucleotidase ecto MFP, milk fat percentage; MFY, milk fat yield; MPP, milk protein percentage; MPY, milk protein yield; MKCP, milk kappa casein protein; MY, milk yield. animals-13-00884-t002_Table 2 Table 2 Summary of variant analysis of differentially expressed hub and bottleneck genes. Gene ID Chr Start End SNP Count BiSNP ENSBTAG00000007689 (LPIN1) NC_040086.1 85168990 85299051 10 9 ENSBTAG00000009578 (PTK2) NC_040089.1 2793205 2986179 1 1 ENSBTAG00000026356 (DGAT1) NC_040089.1 547336 558673 2 2 ENSBTAG00000014357 (SDC2) NC_040089.1 67885139 68006993 2 2 ENSBTAG00000008432 (NUP98) NC_040090.1 32662235 32749000 2 2 ENSBTAG00000011266 (ZBTB16) NC_040090.1 59519169 59723887 3 3 ENSBTAG00000005691 (FGF2) NC_040092.1 37900967 37980582 2 2 ENSBTAG00000005091 (DGKG) NC_040076.1 80595457 80821976 3 3 ENSBTAG00000016525 (ITGA1) NC_040095.1 25934013 26116012 10 9 ENSBTAG00000001335 (GHR) NC_040095.1 31683009 31993386 6 6 ENSBTAG00000003300 (MFGE8) NC_040096.1 20516116 20540642 4 4 ENSBTAG00000021527 (IGF1R) NC_040096.1 7856996 8161856 1 1 ENSBTAG00000027629 (ANK1) NC_040102.1 36035255 36276595 5 4 ENSBTAG00000010660 (CACNA1C) NC_040080.1 11268673 11659827 3 3 ENSBTAG00000031544 (DDIT3) NC_040080.1 64346323 64350540 1 1 ENSBTAG00000020536 (HERC6) NC_040081.1 36492384 36549974 9 9 ENSBTAG00000019716 (CXCL8) NC_040081.1 88364933 88368713 1 1 ENSBTAG00000006240 (TLR4) NC_040083.1 107075099 107086126 6 5 ENSBTAG00000012855 (LPL) NC_040083.1 66717576 66744131 1 1 ENSBTAG00000048655 (NT5E) NC_040084.1 64029657 64102659 6 6 BiSNP, biallelic SNP. animals-13-00884-t003_Table 3 Table 3 Genes with a fixed SNP pattern in high-milk-yield breeds (Sahiwal and Gir) and the respective variable SNP pattern in low-milk-yield breeds (Gaolao, Deoni, Pulikulam, Hallikar, Dangi, and Amritmahal). Gene GHR GHR TLR4 TLR4 LPIN1 LPIN1 LPIN1 CACNA1C ZBTB16 ITGA1 ANK1 ANK1 NT5E NT5E Genomic location 31685773 31685984 107083326 107083914 85187074 85209309 85211528 11271411 59717709 25983121 36054194 36076037 64035090 64065719 Genomic Variant Ref C A A C G C C C C C G G C G Alt G G C A A T G T T T A A T A Protein Variant Pos(Ref/Alt) 392 (G/A) 462 (N/D) 151 (A/T) 347 (N/G) 772 (R/K) 631 (A/E) 542 (R/P) 204 (E/K) 393 (V/M) 588 (V/M) 111 (P/S) 127 (R/H) 475 (C/F) 151 (A/V) Sahiwal 1 C/C A/A A/A C/C G/G C/C C/C C/C C/C C/C G/G G/G C/C G/G Sahiwal 2 C/C A/A A/A C/C G/G C/C C/C C/C C/C C/C G/G G/G C/C G/G Sahiwal 3 C/C A/A A/A C/C G/G C/C C/C C/C C/C C/C G/G G/G C/C G/G Sahiwal 4 C/C A/A A/A C/C G/G C/C C/C C/C C/C C/C G/G G/G C/C G/G Gir 1 C/C A/A A/A C/C G/G C/C C/C C/C C/C C/C G/G G/G C/C G/G Gir 2 C/C A/A A/A C/C G/G C/C C/C C/C C/C C/C G/G G/G C/C G/G Gir 3 C/C A/A A/A C/C G/G C/C C/C C/C C/C C/C G/G G/G C/C G/G Gir 4 C/C A/A A/A C/C G/G C/C C/C C/C C/C C/C G/G G/G C/C G/G Amritmahal C/C A/A A/A C/C G/A C/C C/C C/C C/C C/C G/G G/G C/C G/G Dangi C/G A/G A/C C/A G/G C/C C/G C/C C/T C/C G/G G/G C/C G/G Gaolao C/G A/A A/C C/C G/G C/T C/C C/C C/C C/C G/G G/G T/T G/G Deoni C/C A/A A/C C/C G/G C/C C/G C/T C/T C/C G/G G/G C/C G/A Pulikulam C/G A/A A/A C/C G/A C/C C/T C/C C/C C/T G/A G/G C/C G/G Hallikar C/C A/A A/A C/C G/A C/C C/G C/C C/C C/C G/G G/A C/C G/G animals-13-00884-t004_Table 4 Table 4 Genes with fixed SNP pattern in low-milk-yield breeds (Sahiwal and Gir) and respective variable SNP pattern in high-milk-yield breeds (Gaolao, Deoni, Publicum, Hallikar, Dangi and Amritmahal). Gene MFGE8 FGF2 TLR4 LPIN1 NUP98 PTK2 ZBTB16 ZBTB16 DDIT3 NT5E Genomic location 20518217 37920746 107080326 85205642 32707374 2973942 59717979 59718206 64346576 64102580 Genomic Variant Ref A G G T G A T G C G Alt T A A G A C C A T T Protein Variant Pos(Ref/Alt) 328 (S/R) 19 (G/R) 67 (R/K) 766 (S/P) 548 (H/Y) 904 (D/A) 598 (G/R) 627 (A/V) 87 (S/L) 8 (T/N) Sahiwal 1 A/A G/G G/A T/T G/G A/A T/T G/G C/T G/G Sahiwal 2 A/A G/G G/G T/G G/G A/A T/T G/G C/C G/T Sahiwal 3 A/A G/A G/G T/T G/A A/A T/T G/A C/C G/G Sahiwal 4 A/A - G/G T/T G/A A/A T/T G/G C/C G/G Gir 1 A/T - G/G T/T G/G A/A T/T G/G C/C G/G Gir 2 A/A - G/G T/T G/G A/A T/T G/G C/C G/G Gir 3 A/A G/G G/G T/T G/G A/C T/C G/G C/C G/G Gir 4 A/A G/G G/G T/T G/G A/A T/T G/G C/C G/G Amritmahal A/A G/G G/G T/T G/G A/A T/T G/G C/C G/G Dangi A/A G/G G/G T/T G/G A/A T/T G/G C/C G/G Gaolao A/A G/G G/G T/T G/G A/A T/T G/G C/C G/G Deoni A/A G/G G/G T/T G/G A/A T/T G/G C/C G/G Pulikulam A/A G/G G/G T/T G/G A/A T/T G/G C/C G/G Hallikar A/A G/G G/G T/T G/G A/A T/T A/A C/C G/G Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000040

Gut microbiota play an important role in impacting the host's metabolism, immunity, speciation, and many other functions. How sex and environment affect the structure and function of fecal microbiota in red deer (Cervus elaphus) is still unclear, particularly with regard to the intake of different diets. In this study, non-invasive molecular sexing techniques were used to determine the sex of fecal samples from both wild and captive red deer during the overwintering period. Fecal microbiota composition and diversity analyses were performed using amplicons from the V4-V5 region of the 16S rRNA gene sequenced on the Illumina HiSeq platform. Based on Picrust2 prediction software, potential function distribution information was evaluated by comparing the Kyoto Encyclopedia of Genes and Genome (KEGG). The results showed that the fecal microbiota of the wild deer (WF, n = 10; WM, n = 12) was significantly enriched in Firmicutes and decreased in Bacteroidetes, while the captive deer (CF, n = 8; CM, n = 3) had a significantly higher number of Bacteroidetes. The dominant species of fecal microbiota in the wild and captive red deer were similar at the genus level. The alpha diversity index shows significant difference in fecal microbiota diversity between the males and females in wild deer (p < 0.05). Beta diversity shows significant inter-group differences between wild and captive deer (p < 0.05) but no significant differences between female and male in wild or captive deer. The metabolism was the most important pathway at the first level of KEGG pathway analysis. In the secondary pathway of metabolism, glycan biosynthesis and metabolism, energy metabolism, and the metabolism of other amino acids were significantly different. In summary, these compositional and functional variations in the fecal microbiota of red deer may be helpful for guiding conservation management and policy decision-making, providing important information for future applications of population management and conservation. cervidae KEGG fecal microbiota 16s rRNA Rolling support Project for the provincial University scientific research platform team of the Guizhou Provincial Department of Education 031 Subsidy Project of Guizhou Science and Technology Department 5778-05 This research was funded by the Rolling support Project for the provincial University scientific research platform team of the Guizhou Provincial Department of Education ( 031) and Subsidy Project of Guizhou Science and Technology Department (Platform Talents 5778-05). pmc1. Introduction Red deer (Cervus elaphus), which belong to the family Cervidae, order Artiodactyla, distributed in Asia, Europe, North America, and North Africa . The red deer is a typical forest-inhabiting mammal in northeast China and has an important ecological status in the forest ecosystem . Owing to habitat fragmentation, the populations of red deer in the wild are currently in sharp decline . Using captive populations as reintroduction resources is an effective strategy to restore the populations of wild red deer . The complex gut microbiota systems in the mammalian gut are composed of large fractions of microbes . The gut microbiota are a complex product of the long-term evolution of hosts and microbes . Recent studies have shown that not only are gut microbiota a part of the host, but they also have a significant impact on the health of the host, such as promoting immunity, digestion, metabolism, and intestinal endocrine hormones, among others . Simultaneously, the complex and flexible gut microbiota can be affected by multiple environmental and host genotypes . Many studies have shown that diet is an important factor that affects the structure and function of the fecal microbiota . For example, changes in diet alter the function and diversity of fecal microbiota as well as the relative abundance of some microorganisms . Moreover, diet-induced loss of microbial function and diversity will increase the risk of diversity loss and extinction through generational amplification . It was necessary to investigate the gut microbiome by comparing differences between wild and captive red deer. However, to date, there has been a lack of studies comparing the gut microbiota between wild and captive red deer . Because of sex differences in behavior and physiology, sex as an important intrinsic factor leads to differences in gut microbiota among individuals within species . Although the results are inconsistent, animal species with significant sexual dimorphism and human studies have shown sex-related differences in gut microbiota. In mice (Mus musculus), poultry, and forest musk deer (Moschus berezovskii), the composition of the gut or fecal microbiota shows sex differences . At present, few studies have analyzed the sexual dimorphism of fecal microbiota in red deer. In order to save endangered populations, artificial breeding of wild populations is carried out. The food types and nutrient intake ratios obtained in captivity and wild environments are very different, especially for endangered cervidae . Therefore, monitoring the digestive system of captive animals and identifying standardized levels of nutritional requirements and fiber composition is critical for captive wild animals to determine whether they have acclimated to artificially provided food and new environments--a part of wildlife conservation's main problem . Using captive populations as reintroduction resources is an effective strategy to restore the populations of wild red deer. The composition of gut microbiota in wild populations can be a good indicator of the breeding direction of the captive population . Therefore, understanding the impact of dietary differences between wild and captive red deer on the fecal microbiota can help to assess and ensure the long-term viability of this species . At present, the research methods for fecal microbiota have also shifted from traditional methods to 16S rRNA gene sequencing technology, from simple microbial composition, community structure, and core microbiota research to microbial function research, which has become a hot frontier in ungulate research today . The main goal of this study was to characterize the composition of the fecal microbiota of red deer of different sex and feeding plus environment. We used high-throughput 16S rRNA sequencing technology to comprehensively analyze. Thus, we hypothesized that: (1) the fecal microbiota composition and function are different between wild and captive deer; and (2) under the wild or captive environment, the microbiota diversity and evenness are different between females and males. 2. Materials and Methods 2.1. Study Site, Subjects, and Sample Collection This study was conducted at the Gaogestai National Nature Reserve in Chifeng, Inner Mongolia (119deg02'30'', 119deg39'08'' E; 44deg41'03'', 45deg08'44'' N). The total area is 106,284 hm2. It is a typical transition zone forest-steppe ecosystem in the southern foothills of Greater Khingan Mountains, including forests, shrubs, grasslands, wetlands, and other diverse ecosystems. In February 2019, 75 line transects were randomly laid in the Gogestai protection area. Positive and reverse footprint chain tracking was carried out after the foodprints of red deer were found through line transect investigation. Disposable PE gloves were worn to collect red deer feces. While tracking the footprint chain, set 2 m x 2 m plant quadrate every 200 m to 250 m along the footprint chain, and collect all kinds of plant branches eaten by deer in the quadrate as far as possible . A total of 162 fecal samples were collected and stored at -20 degC within 2 h. The feces of red deer from different areas of the Reserve were identified as coming from different individuals, and 43 feces were identified individually in the laboratory. In February 2019, the HanShan Forest Farm in Chifeng City, Inner Mongolia, China (adjacent to the Gaogestai Nature Reserve) had a total of 11 healthy adult red deer of similar age and size. Ear tags were used to differentiate each individual red deer. Through continuous observation, feces were collected immediately after excretion by different red deer individuals and stored at -20 degC. We measured crude protein, energy, neutral detergent fiber (NDF), and total non-structural carbohydrates in red deer diets. 2.2. Individual Recognition and Sex Identification We used a qiaamp DNA Fecal Mini-kit (QIAGEN, Hilden, Germany) to extract host deoxyribonucleic acid (DNA) from the fecal samples of red deer as previously described . Microsatellite PCR technology was used with nine pairs of microsatellite primers (BM848, BMC1009, BM757, T108, T507, T530, DarAE129, BM1706, and ILST0S058) with good polymorphism that were selected based on the research results of previous studies. These nine pairs of primers can amplify fecal DNA stably and efficiently. A fluorescence marker (TAMRA, HEX, or FAM) was added to the 5' end of upstream primers at each site (Supplementary Table S1). Primer information, PCR amplification, and genotype identification procedures are described in the literature . Multi-tube PCR amplification was used for genotyping , and 3-4 positive amplifications were performed for each locus to determine the final genotype . The excel microsatellite toolkit was used to search for matching genotypes from the data. Samples are judged to be from the same individual if all loci have the same genotype or if only one allele differs at a locus. The microsatellite data were analyzed by Cervus 3.0 software, and the genotyping was completed . Male and female individuals were identified by detecting the existence of genes after the individual identification of red deer was completed. Sry gene primers (F:5'-3' TGAACGCTTTCATTGTGTGGTC; R:5'-3' GCCAGTAGTCTCTGTGCCTCCT) were designed, and the amplification system was determined. To minimize the occurrence of false positives or false negatives that could affect results, the Sry gene was repeated three times to expand and increase during the experiment, and samples with target bands that appeared on the second and third occasions were determined to be male . 2.3. Fecal Microbiota DNA Extraction, Amplification, and Sequencing The total microbial DNA of fecal samples was extracted using an E.Z.N.A(r) Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA). The DNA integrity of the extracted samples was determined by 1% agarose gel electrophoresis. Targeting a 420 bp fragment encompassing the V4-V5 region of the bacterial 16S ribosomal RNA gene was amplified by PCR using primers 515F (5'-GTG CCA GCM GCC GCG GTA A-3') and 907R (5'-CCG TCA ATT CMT TTR AGT TT-3'). NEB 154 Q5 DNA high-fidelity polymerase (NEB, Ipswich, MA, USA) was used in PCR amplifications (Supplementary Table S1). A 1:1 mixture containing the same volume of 1XTAE buffer and the PCR products were loaded on a 2% agarose gel for electrophoretic detection. PCR products were mixed in equidensity ratios. Then, the mixture of PCR products was purified using the Quant-iTPicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). Sequencing libraries were generated using the TruSeq Nano DNA LT Library Prep kit (Illumina, San Diego, CA, USA) following the manufacturer's recommendations, and index codes were added. The library's quality was assessed on the Agilent 5400 (Agilent Technologies Co. Ltd., Santa Clara, CA, USA). At last, the library was sequenced on an Illumina NovaSeq 6000 platform, and 250 bp paired-end reads were generated. Microbiome bioinformatics were performed with QIIME2 2019.4 with slight modification according to the official tutorials (accessed on 30 September 2022)). Briefly, raw data FASTQ files were imported into the format that could be operated by the QIIME2 system using the qiime tools import program. The DADA2 process is to obtain amplified variant sequences through de-duplication. In the process, clustering is not carried out based on similarity, but only de-duplication is carried out. Demultiplexed sequences from each sample were quality filtered and trimmed, de-noised, merged, and then the chimeric sequences were identified and removed using the QIIME2 DADA2 plugin to obtain the feature table of amplicon sequence variants (ASV) . The QIIME2 feature-classifier plugin was then used to align ASV sequences to a pre-trained GREENGENES 13_8 99% database (trimmed to the V4V5 around a 420bp region bound by the 515F/907R primer pair) to generate the taxonomy table . In order to unify the sequence effort, samples were rarefied at a depth of 25,318 sequences per sample before alpha and beta diversity analysis. Rarefaction allows one to randomly select a similar number of sequences from each sample to reach a unified depth. 2.4. Bioinformatics and Statistical Analyses Sequence data analyses were mainly performed using QIIME2 and R software (v3.2.0). ASV-level alpha diversity indices, such as the Chao1 richness estimator and Pielou's evenness, were calculated using the ASV table in QIIME2 , and visualized as box plots (R software, package "ggplot2"). Beta diversity analysis was performed to investigate the structural variation of microbial communities across samples using weighted or unweighted UniFrac distance metrics and visualized via principal coordinate analysis (PCoA) (R software, package "ape"). The significance of differentiation of microbiota structure among groups was assessed by PERMANOVA (permutational multivariate analysis of variance) . Random forest analysis (R software, package "randomForest") was applied to sort the importance of microbiota with differences in abundance between groups and screen the most critical phyla and genera that lead to microbial structural differences between groups using QIIME2 with default settings . Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (Picrust2) is software that predicts the functional abundance from the sequencing data of marker genes (typically 16S rRNA). An ASV's abundance table is used for standardization, and the corresponding relationship of each ASV is compared with the Kyoto Encyclopedia of Genes and Genomes (KEGG) library to obtain the functional information and functional abundance spectrum. 3. Results 3.1. Identification of Individuals and Sex A total of 22 red deer individuals were identified from 43 fecal samples, including 12 males and 10 females (Supplementary Table S2). The female captive deer were CF1, CF2, CF3, CF4, CF5, CF6, CF7, and CF8. The male captive deer were CM1, CM2, and CM3. We divided all the red deer (22 wild and 11 captive) into four groups: wild females (WF) (n = 10), wild males (WM) (n = 12), captive females (CF) (n = 8), and captive males (CM) (n = 3). The information about identification, location, sex, and diet is summarized in Supplementary Table S2. 3.2. Diet Composition and Nutritional Composition of Wild and Captive Red Deer Winter Diets The wild red deer were fed on 16 species of plants in the winter. The edible plants belonged to 16 species of 16 genera and 9 families. Since the frequency of occurrence of other edible plants in red deer, such as Mongolian oak (Quercus mongolica) and Chinese maple (Acer sinensis), was less than 7%, the nutrient content of these plants was not measured. In addition, we hypothesized that they had little influence on the nutritional strategy of red deer. Therefore, the primary nutrient contents of 14 types of edible plants were determined. The food and nutritional composition of wild red deer are shown in Supplementary Table S3. When the captive red deer were fed, each type of food was fed separately at different times. The nutritional content of the primary food of captive red deer from the farm (adjacent to the Gaogestai Nature Reserve) in winter is shown in Supplementary Table S4. Only one kind of diet were provided to captive deer at each feeding time with all captive deer feeding together. Captive red deer feed on leaves and high protein given by artificial feeding. Compared with captive red deer, wild deer have a wider feeding range and no dietary limitations. Substantial differences exist between these two feeding methods. 3.3. Sequencing Analysis and Clustering A total of 1,561,654 high-quality sequences were obtained from the fresh winter feces of 22 wild deer and 11 captive deer. Rarefaction curves based on the Chao1 diversity index reached asymptotes at 22,500. The results showed that with the increase in amount of sequencing, the curve tended to be flat and no longer changed, indicating that the amount of sequencing in this study basically reflected the diversity of red deer fecal microbiota in this study . A total of 15,228 ASVs were obtained using a 100% similarity clustering method. The WF, WM, CF, and CM groups included 3056 ASVs, 3924 ASVs, 6661 ASVs, and 1587 ASVs, respectively. 3.4. Microbial Composition and Diversity by Environment and Sex We found significant differences in fecal microbial composition between wild and captive red deer based on environment. The fecal microbial communities of four groups (WF, WM, CF, and CM) were dominated by the phyla Firmicutes and Bacteroidetes . The phylum Firmicutes was the most abundant in WF (81.12 +- 2.87%), followed by WM (79.03 +- 2.19%), CF (58.24 +- 3.17%), and CM (59.66 +- 0.47%). Secondly, Bacteroidetes was abundant in WF (15.19 +- 2.09), WM (16.89 +- 2.08%), CF (33.02 +- 5.48), and CM (31.55 +- 1.61%). At the genus level, the genera from the four groups with abundance > 1% were Oscillospira, a candidate genus 5-7N15 from the family Bacteroidaceae, Ruminococcus, Roseburia, Clostridium, and Prevotella . The chao1 diversity indices demonstrate a significant difference between the WF and WM groups (p < 0.01). There was no statistically significant difference between the CF and CM groups (p > 0.05). Pieluo's diversity index showed that no significant differences occurred between WF and WM groups (p > 0.05) or CF and CM groups (p > 0.05) . Wild and captive red deer also differed in beta-diversity. An PCoA plot based on the Unweighted Unifrac and Weighted Unifrac distance matrix revealed clear separation of the fecal microbiota between wild and captive red deer . The results of a PCoA analysis showed that the fecal microbial structures of the CF and CM groups were more similar than those of the WF and WM communities . A random forest analysis showed that Firmicutes and Bacteroidetes were the primary microorganisms that had differences between the wild and captive populations by (an importance > 0.1) . This analysis indicated that there were significant differences in the abundances of Firmicute and Bacteroidetes between the four groups (an importance > 0.1), which were the primary phyla that caused differences in the microbial communities between groups . Ruminococcus, Treponema, Akkermansia, a candidate genus 5-7N15 belonging to family Bacteroidaceae, and a candidate genus rc4-4 belonging to family Peptococcaceae were the main genera that caused differences in microbial communities between sex and environment . 3.5. Functional Modules of Fecal Microbial Communities Metabolism was found to be the most common function prediction performed on fecal microbial communities and included the most important pathways for microbial clustering (76.67%). The second pathway of metabolism included amino acid metabolism (17.26%), carbohydrate metabolism (17.85%), metabolism of cofactors and vitamins (16.57%), and metabolism of terpenoids and polyketides (12.66%) . A PCoA analysis showed that the WF and WM groups had more similar microbial function clusters . It was found that there were significant differences in the three metabolic pathways of glycan biosynthesis and metabolism (GBM), energy metabolism (EM), and metabolism of other amino acids (MAA) (p < 0.05) . 4. Discussion This is the first study to apply high-throughput sequencing to describe the fecal bacterial microbiota of wild and captive red deer by sex. Analysis of the differences in fecal microbiota is a key step in releasing captive red deer to help expand the wild population. In general, the fecal bacterial microbiota of red deer was similar to that of other cervidae, such as elk (Cervus canadensis), white tailed deer (Odocoileus virginianus) , and white-lipped deer (Cervus albirostris) , at least at the bacterial phylum level, with high proportions of the phyla Firmicutes and Bacteroidetes. In the digestive tract of herbivores, the role of Firmicutes is mainly to decompose cellulose and convert it into volatile fatty acids, thereby promoting food digestion and host growth and development. The enrichment of Firmicutes plays an important role in promoting the ability of red deer to obtain abundant nutrients from food and, at the same time, affects the metabolic function of the fecal microbiota. Bacteroidetes can improve the metabolism of organisms, promote the development of the gastrointestinal immune system, participate in the body's bile acid, protein, and fat metabolisms, and also have a certain regulatory effect on carbohydrate metabolism. It can also produce special glycans and polysaccharides, which have a strong inhibitory effect on inflammation . Differences in microbiota may be explained by changes in diet. Data from previous local and overseas studies have shown that diet is the main factor affecting the gut microbiota in mammals . It is likely that wild deer have a more varied diet, more than captive deer. These phyla, Firmicutes and Bacteroidetes, are involved in important processes such as food digestion, nutrient regulation and absorption, energy metabolism, and host intestinal defense against foreign pathogens . Alpha diversity alterations may be attributed to differential diet or hormonal influences on the gut microbiota. Fecal microbiota richness in wild populations is higher than that in captive animals, such as the Tibetan wild ass (Equus kiang), bharal (Pseudois nayaur), Tibetan sheep (Ovis arise), and yak (Bos mutus) . Nevertheless, other studies also found that captivity might increase the alpha diversity of fecal microbiota in most Cervidae compared with other animals, for example, sika deer (Genus Cervus), Pere David's (Elaphurus davidianus), and white-tailed deer (Odocoileus virginianus) . It may be that some environmental stresses in the wild or the special structure of the stomach and intestines in these deer lead to decreased alpha diversity of fecal microbiota in wild deer . This phenomenon needs further research to determine its cause. Our results showed that the richness of the fecal microbial community in wild red deer differed by sex . In wild deer, the microbiota diversity was higher for females than males. Microbial community alterations by sex could be attributed to hormonal . The sampling time was during the gestation period of red deer. Levels of female growth hormone during pregnancy may affect the fecal microbiota. Reproductive hormones have also been associated with sex and gut microbial changes in wild animals . Increased evidence indicates that sex steroid hormone levels are associated with the human gut microbiota . Futher, Edwards et al. reported that estrogen and progesterone had an impact on gut function . The captive deer also had the smallest sample size (n = 3 males and 8 females), which limited our ability to detect these differences. In this study, the functional pathway composition of wild red deer is more similar , which is completely opposite to the microbial structure . The change in microbial structure does not necessarily lead to the change in function, which may be due to the same function in different microbial communities . In recent years, studies have shown that gut microbiota are involved in various metabolic processes such as amino acids, carbohydrates, and energy, confirming their primary role in assisting host digestion and absorption . It has also been found to be involved in environmental information processing, suggesting that the gut microbiota plays an important role in facilitating acclimation to changing environments . The metabolism of gut microbiota is closely related to the feeding habits of the host. In the long-term evolution process, the gut microbiota will respond to changes in diet types or specific diets by adjusting the content of certain digestive enzymes . Studies have shown that the decrease of fecal microbial diversity can lead to a reduction in the functional microbiota, in the efficiency of the microbiota, and in the resistance to pathogen invasion . The decrease in fecal microbial diversity in captive populations resulted in a decrease in functional microbiota . Ruminococcaceae and Lachnospiraceae are two of the most common bacterial families within the Firmicutes phylum . It has been hypothesized that they have an important role as active plant degraders . According to our results, the level of Ruminococcaceae in the captive groups is significantly lower than that in the wild group, which could suggest that the fiber-reduced diet in captivity is modifying the ability of the fecal microbiota to degrade recalcitrant substrates such as cellulose, hemicellulose, and lignocellulose, among others, that are commonly found on the main resources of the wild red deer diet. The captive deer's consequent reduction of diet resources might trigger the decline of important metabolic pathways associated with nutrient use . 16S rRNA analysis constitutes a valuable and cost-efficient approach for surveillance and monitoring wild populations as well as captive individuals. Picrust2 prediction accuracy is dependent on the availability of closely related annotated bacterial genomes in the database and the phylogenetic distance from the reference genome. However, the prediction results are still uncertain, which does not mean that the correlation between the predicted genes and the real metagenome of the microbiota is 100% . At present, due to the difficulty of cultivation, the mechanism by which some functional bacteria exert their effects remain unclear. Therefore, in the follow-up work, it is necessary to repeatedly cultivate the conditions of some intestinal anaerobic bacteria, the most extensive of which are Firmicutes and some Bacteroidetes. The microbiota was cultured in vitro by simulating the gut environment, and its functions were speculated and further verified in combination with multiple groups of studies (metagenomics, meta transcriptome, and proteome, etc.). At the same time, the unknown functional microbiota and its genome sequence information can be explored and studied. These works will help to understand the metabolic activities of the complex microbiota and further explore the host physiological processes involved in gut microbiota. 5. Conclusions In conclusion, our study provided information on the structure and function of the fecal microbiome of red deer through the 16S rRNA gene of fecal samples. Comparing analyses identified significant variations of fecal microbiota composition and functions between captive and wild populations and also indicated that environment and sex have a great influence on these variations. These findings were of great significance for the reintroduction of captive red deer, given that the differences in fecal microbiota composition and functions between captive and wild red deer would greatly impact the ability of captive red deer to adapt to the wild environment. For further study, incorporating novel methods (e.g., transcriptome) to study the functional annotation of gene content and the functional traits of the host would be essential for better understanding the physiology and immunology of red deer. Acknowledgments We thank Qian of the Gaogestai Nature Reserve in Inner Mongolia and other staff for their support of the field work. We thank other students for their help and suggestions during the sample processing and collection process. We thank the Application development laboratory of characteristic microorganisms resources of Biological Sciences, Guizhou Education University. We thank the Key Laboratory of Forest Fire Ecology and Management of Guizhou Province. We thank the Key Laboratory of Development and Utilization of Biological Resources in Colleges and Universities of Guizhou Province, Guizhou Education University, Guiyang 550018 China. We thank all the reviewers who participated in the review for providing English editing services during the preparation of this manuscript. Supplementary Materials The following supporting information can be downloaded at: Figure S1: The rarefaction curve based on the Chao1 diversity index; Table S1: The amplification system of PCR based on 16S rRNA gene. Table S2: Individual information of 33 red deer. Table S3: The ratio and nutritional content of plants eaten by wild red deer of the Gaogestai Reserve in winter. Table S4: Nutritional content of the main food of captive red deer per 100 grams. Table S5: The results of PERMANOVA analysis based on weighted UniFrac and Unweighted UniFrac. Click here for additional data file. Author Contributions Methodology, Y.Y.; Software, L.Z.; Resources, M.Z.; Data curation, J.G.; Writing--original draft, Y.S. All authors assisted in the overall execution of the review, and in doing so, all authors agree to be accountable for the content of the work. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Data Availability Statement The datasets generated for this study can be found at NCBI/BioProject/PRJNA830551 (accessed on 1 March 2023)). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Relative abundance of the top 10 bacterial phyla and top 30 genera present in the fecal microbiota of wild and captive red deer (A) at the phylum level and (B) at the genus level (WF: n = 10, WM: n = 12, CF: n = 8, CM: n = 3). Figure 2 Boxplots of alpha-diversity indices showing differences between sex and environment (WF: wild female; WM: wild male; CF: captivity female; CM: captivity male). * represents significant (p < 0.05), ** represents very significant (p < 0.01), *** and represents highly significant (p < 0.001) (WF: n = 10, WM: n = 12, CF: n = 8, CM: n = 3). Figure 3 Faecal microbial structure of red deer. (A) Principal Coordinate Analysis based on weighted unifrac showing microbial samples clustered by sex and environment into different groups (in parenthesis, the explained variance of each axis; WF: wild female; WM: wild male; CF: captivity female; CM: captivity male). (B) Principal Coordinate Analysis based on unweighted unifrac showing microbial samples clustered by sex and environment into different groups (in parenthesis, the explained variance of each axis; WF: wild female; WM: wild male; CF: captivity female; CM: captivity male). (C) A random forest heatmap indicating intergroup differences at the phylum level. Importance means screening the most critical phylum or genus that leads to microbial structural differences between groups (WF, WM, CF, CM). (D) A random forest heatmap indicating intergroup differences at the genus level (WF: n = 10, WM: n = 12, CF: n = 8, CM: n = 3). Figure 4 (A) Metabolic composition of secondary function pathways. (B) A PCoA plot based on weighted UniFrac distance metrics associated with a functional dataset. The colors represent sample sets of microbial functions (WF: n = 10, WM: n = 12, CF: n = 8, CM: n = 3). Figure 5 Microbial functions related to energy metabolism (EM), glycan biosynthesis and metabolism (GBM), and metabolism of other amino acids (MAA) grouped by sex and environment (WF: wild female; WM: wild male; CF: captivity female; CM: captivity male). * represents significant (p < 0.05), ** represents very significant (p < 0.01), and *** represents highly significant (p < 0.001) (WF: n = 10, WM: n = 12, CF: n = 8, CM: n = 3). animals-13-00929-t001_Table 1 Table 1 Relative abundance > 1% at the genus level for four groups of red deer fecal microbiota. Tax_Name WF WM CF CM Oscillospira 4.71 +- 0.79 4.55 +- 0.73 4.55 +- 0.41 2.89 +- 0.31 5-7N15 3.81 +- 1.4 4.51 +- 1.53 4.51 +- 1.71 4.01 +- 1.29 Ruminococcus 3.05 +- 0.5 3.1 +- 0.56 3.1 +- 0.37 2.03 +- 0.6 Roseburia 2.27 +- 1.11 2.17 +- 1.08 2.17 +- 0.63 0.37 +- 0.35 Clostridium 1.55 +- 0.59 1.67 +- 1.01 1.67 +- 0.67 0.88 +- 0.68 Prevotella 1.32 +- 0.11 1.25 +- 0.12 1.25 +- 0.08 1.31 +- 0.31 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000041

For sows introduced into new groups, the aggressive behavior associated with establishing a social hierarchy represents a period of severe stress. The aim of this study was to investigate the effect of providing sows with an improved pen environment (straw in racks and ropes) on aggressive behavior after mixing and to understand the role played by sow back fat thickness and parity order. At 29 d post-service, sows were mixed into IMPROVED or CONTROL pens with individual feeding stalls (6 groups/treatment, 20 sows/group). Aggressive behavior was recorded for 2 h at mixing (T0) and 24 h (T1) and 3 weeks post-mixing (T21). Overall, the sows in the CONTROL pens performed more fighting behavior compared to the IMPROVED sows (p < 0.001). This difference was significant only at T21 (p < 0.001). Additionally, the sows in the CONTROL pens generally initiated more aggressive behaviors than the sows in the IMPROVED pens (p = 0.02). The sows with a low back fat thickness initiated more aggressive behaviors, but parity had no significant effect on any of the aggressive behaviors. These results indicate a beneficial effect of improvements to the pen environment on the aggression performed by group-housed sows between the time of mixing and three weeks later. The effect was reduced on the day of mixing, which is in accordance with the necessity for sows to employ aggression to establish the dominance hierarchy. gestating sows environmental enrichment group-housing welfare aggressive behavior Teagasc Walsh Scholarship ProgramUniversity of Padova (Italy)The work was conducted as part of a Teagasc-funded grant-in-aid project "Effects of chronic stress on sow welfare and performance, and on the resilience of their offspring (SowWeanWel)" (ref 0370). Martyna Lagoda was funded by the Teagasc Walsh Scholarship Program. Fondazione Cariparo supported Maria Costanza Galli by a doctoral fellowship at the University of Padova (Italy). pmc1. Introduction In current indoor gestation housing systems, sows experience several highly relevant stressors with welfare consequences. The major stressor for pregnant sows is being mixed into new groups . The associated aggression required for sows to establish the social hierarchy often has detrimental implications for sow welfare and production. In light of this and of the increased use of group housing systems for sows in response to the forthcoming European ban on stalls, as called for by the European Citizens' Initiative "End the cage age" , finding methods that reduce this stress would be necessary nowadays. Most agonistic behaviors resulting from mixing unacquainted groups of pigs occur within the first hours after mixing, and the dominance order tends to be stable within 48 h . Agonistic behavior includes offensive and defensive contact and non-contact events, such as fighting, biting, pushing, and pursuing, as well as communicative elements such as threats expressed through body movements and vocalizations . There are various measures that are effective in mitigating this aggression, such as the use of a specialized mixing pen, increasing the space allowance, or the provision of enrichment materials . Environmental enrichment effectively reduced post-mixing aggression in weaner and finisher pigs . However, the impact of environmental enrichment on sow aggression is less investigated , especially around mixing, and the limited literature works that are available report contradictory findings. Previous studies found that the provision of straw in individual racks for sows housed in small static groups with individual feeding cubicles or of point-source materials in floor-fed pens of 12 sows had no effect on aggression at mixing. Meanwhile, Durrell et al. reported that sows housed in small static groups with individual feeding stalls with spent mushroom compost in suspended racks showed a lower frequency of overall agonistic behavior on the day of mixing than sows in barren pens. In contrast, Stewart et al. found higher levels of post-mixing aggressive behavior when sows in a large dynamic group had access to straw provided in a single rack. Indeed, Commission Recommendation (EU) 2016/336 recommends that all pigs in a group should be able to access materials that are edible or feed-like, chewable, investigable, and manipulable. Straw is a valued material, as it has all the necessary characteristics to be effective and should satisfy the need to express foraging motivation . However, if it is only provided in limited amounts/circumstances to a large group of sows, it could be perceived as a limited resource and increase aggression between sows . In the present study, straw was provided at three different locations in the loose area of a pen where 20 sows each had access to their own feeding stall. In addition, we suspended a length of natural fiber rope in each stall such that all sows in the group had individual access to a pliable and easily destructible material. Animal factors such as body weight/size and age (e.g., sow parity) influence a pig's position in the dominance hierarchy. Some studies found a positive correlation between social rank and weight and parity , while others found no correlation or a negative correlation . However, only a few studies investigated the impact of these parameters on aggressive behavior, and these report contradictory results. Considering that the individual level of aggressiveness is not necessarily correlated with the pig's dominance rank , there is a need to better determine the role of animal factors in sow aggressive behavior. We hypothesized that sows housed in pens and provided with straw in racks and manila ropes, in addition to two blocks of wood and two chains, would engage with these materials, which would function as distractions to reduce aggressive behavior during hierarchy establishment. To test the above hypothesis, aggressive interactions were recorded immediately after mixing and 24 h and 3 weeks post-mixing. Moreover, we also investigated the role played by back fat thickness and parity order on the level of aggressive behavior and potential interactions with environmental complexity. 2. Materials and Methods This study was conducted with the approval of the Teagasc Animal Ethics Committee (Approval no. TAEC 2020-266). It did not require licensing under the European Communities Regulations (2002), as no invasive measures were used. 2.1. Animals and Treatments The study was carried out on a commercial 2000-sow farrow-to-finish farm in Co. Cork, Ireland, as part of a larger study described by Lagoda et al. (under review). The study was performed in six replicates using batches of sows weaned between July 2021 and November 2021. In total, 240 Large White X Landrace sows were used in the study. The sows were artificially inseminated in stalls in the service house within 24 h of displaying signs of oestrus post-weaning and remained in the stalls without environmental enrichment until day 28 post-insemination. On day 25 post-insemination, 40 sows within the replicate were selected for the experiment in such a way that both treatments were balanced according to the parity order (parity 1-5, mean +- standard deviation; 2.4 +- 1.03) and back fat thickness. The experiment started on the day that the sows were moved to gestation pens and mixed (day 28.9 +- 0.37 post-insemination) into one of the two following experimental treatments: CONTROL: A total of 20 sows per replicate were moved to a fully slatted gestation pen with two rows of ten individual free-access feeding stalls, in which two blocks of wood on chains and two simple chains were provided as enrichment in the middle of the loose slatted area. IMPROVED: A total of 20 sows per replicate were moved to similar pens but in which the floors of the feeding stalls were covered with rubber mats (EasyFix Rubber Products, Ballinasloe, Galway, Ireland). In the middle of the loose area, there were two blocks of wood on chains and two simple chains, and a portion of the floor was covered with rubber mats, in the middle of which a rooting tower holding straw was mounted. A straw rack was also mounted on the gates of the pen at either end of the loose area and suspended above a steel collection plate on the floor. Manila ropes (1 m manila rope; Marine Suppliers & Co., Ltd., Howth, Dublin, Ireland) were suspended at a height of one meter within each feeding stall. The racks were filled with straw each day throughout the trial, while manila ropes were replaced as often as necessary to provide continuous access. In each pen, the space allowance per sow was 2.62 m2/sow, and 20 individual free-access feeding/lying stalls (0.55 m width x 2.3 m length) were available. The sows were free to move around the remainder of the pen (7.2 m width x 7.3 m length; loose roaming area between two rows of feeding stalls: 2.7 m width x 7.3 m length), and they had free access in and out of the feeding stalls through a rear closing gate which could also be locked into position (open or closed) using a valve lever. The sows were fed a standard, restricted gestation diet twice daily (Table 1), while drinking water was available ad libitum with a ratio of 3 drinkers/20 sows per pen. All sows were maintained in their treatment group until approximately 110 d of gestation, when they were moved into the farrowing crates. 2.2. Aggressive Behaviors Aggressive behaviors were monitored by two trained observers, who alternated between the two treatments in each replication, at three times: immediately after mixing (T0), 24 h post-mixing (T1) and 3 weeks post-mixing (T21). All sows were identified by individual marks on their backs. Before the observations commenced, the animals were allowed 10 min to get used to the presence of the observer, who was positioned in the corridor outside the pen. During the observation days, all occurrences of various aggressive behaviors and the identities of the sows involved in the encounters were recorded for 2 h continuously from 8:00 to 10:00. The list of aggressive behaviors recorded was adapted from Stewart et al. (2008) and is summarized in Table 2. For each behavior, except for fighting, the sows receiving the aggressive behavior were also identified. The aggressive behaviors of biting, head knock, and chase were merged for the analysis and defined as "initiated" behaviors, while bitten, head knocked, and chased were considered as "received" behaviors. 2.3. Back Fat Thickness The back fat thickness (BFT) was recorded at 25 days post-service. BFT was measured at the last rib, 6 to 7 cm off the midline on the left and right side (P2 position), using an ultrasonic scanner (Lean-meter, Renco, Minneapolis, MN, USA). 2.4. Statistical Analysis All the behaviors recorded (fight, initiated and received aggressive behavior, threat, and avoidance) were calculated as the number of events per hour. For the purposes of the analysis, the sows were categorized on the basis of the BFT of the sows within each pen in "LOW", "MEDIUM", and "HIGH" sows. "MEDIUM" sows were the animals that belonged to the interquartile range of their pen (8.5-14.5 mm), while "LOW" and "HIGH" sows were the animals that belonged to the lower (5.5-10 mm) and upper (12.5-21 mm) quartiles of their pen, respectively. Regarding parity order, the sows were divided into young (parity 1-2) and old (parity 3-5). The statistical analyses were carried out using the software package SAS (SAS Institute, Inc., Cary, NC, USA). Normality tests of data distribution and residuals were performed for every variable evaluated with the PROC UNIVARIATE using the Shapiro-Wilk test. None of the variables recorded in the study were normally distributed; therefore, the GENMOD procedure with Poissson distribution was used for the data processing. The model considered the pen as the experimental unit, while the replicate, day of measure, treatment (control vs. improved), back fat thickness (low vs. medium vs. high), and parity order (young vs. old) and their interaction were considered as fixed effects. The pen by day was considered as a repeated effect. For all statistical tests, the significance level was established at p < 0.05. 3. Results Overall, there was an effect of treatment on the average number of fights, with the sows in the control pens performing more fighting behavior compared to the improved sows (p < 0.001). Within time, this difference was significant only at T21 . Moreover, there was an overall effect of time, with more fights on T0 compared to T1 and T21 (p < 0.001). However, in the control pens, a similar number of fights were recorded on T1 and T21, whereas the number of fights decreased significantly between T1 and T21 in the improved pens . There was an effect of treatment on the average number of initiated aggressive behaviors, being higher in the control pens compared to the improved pens . There was no treatment effect on threats and no interactive effects for either variable (p > 0.05). Moreover, there was an effect of time on both initiated aggressive and threat behaviors. There were more initiated aggressive behaviors on T0 than on any other day . There were more threat behaviors on d0 and d21 . There was no interaction between treatment and back fat thickness regarding the number of any of the different aggressive behaviors (p > 0.05). However, regardless of the environmental complexity, there was an effect of back fat thickness on the expression of initiated aggressive behaviors (Table 3), which were performed more by low-BFT sows (p < 0.0001). No interaction was found between treatment and parity order. Regardless of the environmental complexity, the parity order did not have a significant effect on the display of any of the aggressive behaviors (p > 0.05). 4. Discussion The present data support our hypothesis that improvements to the environment of a group housing system with free-access stalls reduces aggressive behavior including fighting, likely mediated by the distraction provided to the sows by good environmental enrichment. Given the need for sows to employ aggression to establish the dominance hierarchy, the reduced effect on aggression on the day of mixing is perhaps unsurprising. Regarding fighting behavior, the comparison between treatments revealed significant differences overall and specifically on d21, confirming the hypothesis that the sows in the improved pens would fight less frequently than the sows in the control pens. However, on d0 and on d1, despite the sows performing numerically fewer fights, the difference was not significant. This is likely because the dominance hierarchy was still in formation and is in line with previous research with sows . These studies and the current study confirm that, irrespective of the housing system, fighting at mixing is mostly unavoidable, and it is an essential component of the establishment of the dominance hierarchy . Indeed, given the evolutionary importance of the dominance hierarchy to group stability , it is not surprising that it takes precedence over other less valuable activities, such as, in this case, interacting with the enrichment materials or even eating the straw. This is also in spite of the sows used in the study not having prior experience with such materials, which would likely have heightened their interest in them . Our findings suggest that it is not possible to influence sows fighting at mixing by providing these materials as a distraction . However, it is important to note that, 21 days after mixing, there were significantly more fights in the control pens than there were in the improved pens, and the reduction in the frequency of fights between day 1 and day 21 was faster in the latter pens. This indicates that environmental enrichment has an important role later, when the social hierarchy was established. This is in contrast to Durrell et al. and Greenwood et al. , who found no differences between barren and enriched pens in terms of the number of fights observed between day 2 and day 14/20 after mixing. The type and the number of materials provided may be responsible. In the study of Greenwood et al. , no foraging substrate, such as straw, was provided, and the number of ropes was lower than the number of sows in the pen, probably decreasing the attractiveness of this enrichment material. Additionally, Durrell et al. only used one form of enrichment, whereas both straw and natural fiber ropes were used in the current study. As already shown by previous studies , it is clear that, regardless of the material provided, fights are frequent on the day of mixing and decrease considerably thereafter. This result is consistent with other findings, namely, the finding that, in domestic sows, the dominance order tends to stabilize within 24-48 h of mixing . Within time, no significant differences were found between treatments in terms of initiated aggressive behaviors. However, the overall number of these behaviors was significantly lower for sows in improved pens. In contrast, Greenwood et al. found that the material provided (ropes, plastic disk swings, and rubber mats) had no effect on bites and head knocks performed by sows. Durrell et al. found a significant difference on day 1 after mixing in the frequency of agonistic behavior, particularly the behavior "head-thrusting", with the sows in the barren pens showing a higher frequency than the sows in the enriched pens. Although fighting and aggressive behaviors were not distinguished separately by Stewart et al. , they showed that the provision of straw to sows in large dynamic groups increased the average proportion of aggressive behavior, while access to straw had no effect on the occurrence of aggressive behavior in the post-mixing period in small static groups. Regardless of the experimental treatments, initiated aggressive and threat behaviors showed a different trend over time. Initiated aggressive behavior decreased soon after the day of mixing, while threat behaviors showed the same frequency on d1 and on d21. These findings suggest that once the social hierarchy is established, sows replace aggressive behavior with threat displays. Regardless of the environmental complexity, there was an effect of back fat thickness on the expression of aggressive behavior only for initiated aggressive behaviors, which were performed more by low-BFT sows. This finding is in contrast with the few studies that investigated the role of body condition in determining the outcomes of aggression in pigs. Indeed, Andersen et al. and D'Eath et al. reported that heavier pigs initiated more aggressive acts and were more involved in fighting. Conversely Mount et al. found no correlation between the number of aggressive interactions initiated and received and body weight. So far, the scientific literature has mainly focused on the correlation between weight and the position of animals within the dominance hierarchy based on the success in winning agonistic interactions . The limited number of studies that have investigated the correlation between body condition and frequency of aggressive behavior makes it difficult to find an explanation as to why low-BFT sows initiated more aggressive behavior. However, a possible explanation could lie in the fact that fatter sows may engage in less aggressive behavior because their size alone scares the other sows away. Regardless of the environmental complexity, the parity order did not have a significant effect on the display of any of the aggressive behaviors. This is in line with the findings of Mount et al. . By contrast, Strawford et al. found that old sows were involved in a greater number of aggressive encounters than young and intermediate sows. In light of the small differences found in the present study in terms of the frequency of aggressive behavior depending on back fat thickness and parity order, it seems that, in pens with free-access, full-length individual feeding/lying stalls, these intrinsic factors have a marginal impact on aggressive behavior. 5. Conclusions This study confirms the benefit to sow welfare, through reduced aggression, of improving the housing environment. The benefit was likely largely driven by the sows' interest in the substrates provided. The findings indicate that aggression at mixing is unavoidable, as it is essential for the establishment of the dominance hierarchy and group stability, but enrichment materials could at least have an effect on reducing its frequency. The current study indicates that the body condition (measured as back fat thickness) and parity do not have a great impact on sow social behavior, even though the thinnest sows were those that most frequently performed initiated aggressive behaviors. Acknowledgments The authors would like to thank the farm owner and his staff for facilitating the work. The authors would also like to thank L. Markland, M. Harrison, and L. Serai for their help with the data collection. Author Contributions Conceptualization, M.C.G., L.A.B., F.G. and M.E.L.; methodology, M.C.G., L.A.B., F.G. and M.E.L.; validation, B.C.; formal analysis, B.C.; investigation, M.C.G. and M.E.L.; resources, L.A.B.; writing--original draft preparation, M.C.G.; writing--review and editing, F.G., L.A.B. and M.E.L.; visualization, M.C.G.; supervision, L.A.B.; project administration, L.A.B.; funding acquisition, L.A.B. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by the Teagasc Ethics Committee (Approval no: TAEC 2020-266). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Number of fights per hour in the CONTROL and IMPROVED pens on the day of mixing (T0), the day after mixing (T1), and three weeks later (T21). a, b, c: Different letters indicate significant differences (p < 0.05) between the treatment and time. Figure 2 Number of initiated aggressive behaviors and threats per hour in the CONTROL and IMPROVED pens. a, b: Different letters indicates significant differences (p < 0.05) between treatments. Figure 3 Average number of initiated aggressive behaviors and threats per hour according to the time after mixing: the day of mixing (T0), the day after mixing (T1), and three weeks later (T21). a, b: Different letters indicate significant differences (p < 0.05) over time. animals-13-00825-t001_Table 1 Table 1 Ingredients and composition of the gestation diet. INGREDIENT % Barley 41.1 Maize 7 Wheat 23 Suguar beet pulp 4 Hi pro soya 15 Soya hulls 4 Soya oil 2.5 Min 2.4 COMPOSITION C Protein 15.6 C Oil 4.9 C Fiber 5.3 C Ash 5.7 De 13.3 Lysine 0.9 M + C 0.53 Threonine 0.61 Calcium 0.95 Av Phos 0.36 Salt 0.58 animals-13-00825-t002_Table 2 Table 2 Description of aggressive behaviors recorded in the study (adapted from Stewart et al., 2008 ). Behavior Description Fighting Mutual pushing parallel or perpendicular; ramming or pushing of the opponent with the head; with or without biting in rapid succession. Lifting the opponent by pushing the snout under its body Initiated Biting Biting any part of another sow, but not as part of a head knock Head knock Ramming or pushing another sow with the head (with or without biting) Chase Moving rapidly in pursuit of another sow Received Bitten Being bitten by another sow, but not as part of a head knock Head knocked Being rammed or pushed by another sow with the head (with or without biting) Chased Moving rapidly/running away from another sow Threat Interaction expressed through body movements without physical contact, with a sow actively withdrawing (avoid) Avoid Active withdrawal of a sow being threated without physical contact animals-13-00825-t003_Table 3 Table 3 Number of aggressive behaviors per hour according to back fat thickness (low, medium, and high). Behavior Back Fat Thickness p Low Medium High Fight 0.003 +- 0.001 0.002 +- 0.000 0.002 +- 0.000 NS Initiated 0.13 a +- 0.03 0.06 b +- 0.01 0.02 b +- 0.01 <0.001 Received 0.06 +- 0.02 0.06 +- 0.01 0.05 +- 0.02 NS Threat 0.10 +- 0.02 0.05 +- 0.02 0.09 +- 0.02 NS Avoid 0.05 +- 0.02 0.09 +- 0.01 0.06 +- 0.02 NS a, b: Different letters within a row indicate significant differences between means. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000042

According to the current guidelines, watchful waiting (WW) is a feasible option for patients with good or intermediate prognosis renal-cell carcinoma (RCC). However, some patients rapidly progress during WW, requiring the initiation of treatment. Here, we explore whether we can identify those patients using circulating cell-free DNA (cfDNA) methylation. We first defined a panel of RCC-specific circulating methylation markers by intersecting differentially methylated regions from a publicly available dataset with known RCC methylation markers from the literature. The resulting RCC-specific methylation marker panel of 22 markers was subsequently evaluated for an association with rapid progression by methylated DNA sequencing (MeD-seq) in serum from 10 HBDs and 34 RCC patients with a good or intermediate prognosis starting WW in the IMPACT-RCC study. Patients with an elevated RCC-specific methylation score compared to HBDs had a shorter progression-free survival (PFS, p = 0.018), but not a shorter WW-time (p = 0.15). Cox proportional hazards regression showed that only the International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) criteria were significantly associated with WW time (HR 2.01, p = 0.01), whereas only our RCC-specific methylation score (HR 4.45, p = 0.02) was significantly associated with PFS. The results of this study suggest that cfDNA methylation is predictive of PFS but not WW. metastatic renal cell carcinoma watchful waiting DNA methylation cfDNA Dutch Cancer SocietyRUG 2012-5400 NKB-EMCR-2016-108154 This work was supported by the Dutch Cancer Society (Alpe d'HuZes Grant RUG 2012-5400). M.K. Bos was funded by the Dutch Cancer Society (no. NKB-EMCR-2016-108154). pmc1. Introduction Systemic treatment options for patients with metastatic renal clear cell carcinoma (mccRCC) have changed within the last two decades, with the arrival of angiogenesis inhibitors and immunotherapy . Those treatments can be given either sequentially as monotherapy or as combination therapy, depending on the patients' risk status, as assessed by the International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) criteria . However, the immediate initiation of systemic treatment at the diagnosis of metastatic disease is not always indicated. A selected subset of mccRCC patients have an indolent disease course that can last for years. As such, this subgroup of patients can benefit from active surveillance, also referred to as watchful waiting (WW). A period of WW postpones the potentially unnecessary treatment toxicity and is considered an initial treatment strategy in newly diagnosed patients with mRCC, according to the current clinical practice guidelines . In daily practice, it is challenging to identify patients with expected indolent disease and potential candidates for the WW strategy. It is clear that patients with a poor prognosis, according to the IMDC score, require immediate systemic treatment. However, the remaining patients with a good or intermediate prognosis based on the IMDC score form a heterogeneous population . This includes patients with more aggressive disease who progress within a few months after diagnosis, and patients with an indolent course of disease over several years. This indicates that the IMDC criteria alone do not suffice in selecting patients for long-term WW . While only limited prospective studies have studied WW in mccRCC patients, one prospective study has suggested that disease extent (number of involved organs) could be a valuable prognostic factor to discriminate between indolent and more aggressive disease . Recently, the data of the prospective Imaging Patients for Cancer drug Selection (IMPACT)-RCC study confirmed the predictive value of <2 IMDC risk factors and <=2 involved organ sites (or "W&W criteria") to select the patients who are eligible for a period of WW . In addition to these criteria, this study showed the potential of tumor [18F]FDG-uptake to predict the WW time beyond these "W&W criteria". In addition to imaging, cell-free DNA (cfDNA) is a biomarker that could be used to study tumor characteristics in a minimally invasive way. CfDNA is composed of DNA fragments originating from dying cells in body fluids, such as blood and urine . With respect to blood, cfDNA can be obtained from either serum or plasma, with a preference for the latter when available, given the higher background of leukocyte DNA from serum . Given the low fractions at which tumor-derived cfDNA is present in the circulation, especially in patients with RCC , highly sensitive techniques are required for its detection . Currently, different techniques are available to characterize cfDNA, focusing on either genetic or epigenetic alterations. Profiling of cfDNA methylation has not been studied extensively. In RCC, however, it was previously demonstrated to be a feasible technique to distinguish patients from healthy donors, also at an earlier stage . Here, we define an RCC-specific panel of cfDNA methylation markers using the cfDNA methylation profiles described by Nuzzo et al. in conjunction with the already described RCC methylation markers from the literature . Subsequently, this panel is evaluated in the serum of patients with mRCC who started WW in the context of a prospective study , with the aim of exploring whether cfDNA methylation profiling can be used to identify patients at high risk of rapid disease progression. 2. Materials and Methods 2.1. Patient Population All of the patients were recruited within the IMPACT-RCC study (NCT02228954), which was a prospective multicenter cohort study that included patients with histologically or cytologically proven RCC with a clear cell component, as well as recently (<6 months) diagnosed metastases and a good or intermediate prognosis according to the IMDC criteria. At inclusion patients had not received any prior systemic therapy. Patients with untreated central nervous system metastases, patients with symptomatic intra-cerebral metastases and pregnant or breastfeeding women were excluded. As for patients eligible for this study, there was no direct indication to start treatment, the IMDC criterion 'time from diagnosis to treatment <1 year' was adapted into 'time from primary diagnosis to diagnosis of metastatic disease <1 year'. WW was terminated if clinical and/or radiological disease progression was established, in combination with a clinical need to start systemic treatment. However, similarly to the current clinical practice, the treating physician could also decide to continue WW beyond radiological progression if it was found to be clinically appropriate. 2.2. Sample Collection and DNA Isolation Blood was collected in a 10 mL clot activator tube (CAT, BD Vacunatainer(r), Franklin Lakes, NJ, USA). Within four hours after collection, the blood was centrifuged for 15 min at 2000x g, and the serum was stored at -80 degrees until further processing. cfDNA was isolated from the total amount of serum, ranging between 1100 and 4600 mL, using the Maxwell(r) (MX) RSC ccfDNA Plasma Kit (Promega, Madison, WI, USA). In addition, the serum from 10 age-matched (5 female, 5 male) healthy blood donors (HBDs) was collected and processed following the same protocol. Although the distribution of males and females was not equal between the HBDs and the patient samples, this difference was not statistically different (Fishers exact p = 0.1105). 2.3. MeD-seq Analysis For the methylation analysis, 10 ng of cfDNA from the patients was analyzed using the MeD-seq technique, as described before . This technology relies on a methylation-dependent restriction enzyme, LpnPI, that binds to either methylated or hydroxy-methylated cytosines in the following contexts--GmCGC, CmCG, and mCGG--and subsequently cuts the DNA 16 basepairs upstream to generate 32 base pair fragments for sequencing. LpnPI is blocked by a fragment size smaller than 32 base pairs. This unique property prevents the over-digestion of methylation-dense DNA, observed with many other methylation-dependent restriction enzymes and, therefore, allows the accurate analysis of CpG methylation at a single nucleotide resolution. Libraries were multiplexed and sequenced on an Illumina HiSeq 2500 for 50 base pair single reads according to the manufacturer's instructions (Illumina, San Diego, CA, USA). The samples were first sequenced until ~2 M reads and continued to a total of ~20 M reads, only when the fraction of reads that passed the LpnPI filter (explained below) was at least 20%. 2.4. Data Processing Nuzzo et al. previously described cell-free methylated DNA immunoprecipitation (cfMEDIP) analysis in RCC patients . The authors kindly provided us with the raw sequencing data of 32 stage IV RCC patients and 32 HBDs, which we summarized into 2 kilobase (kb) regions surrounding all known transcription start sites (TSS) annotated in ENSEMBL. MeD-seq data processing was carried out as previously described . In short, the dual indexed samples were demultiplexed using the bcl2fastq software (version 2.20, Illumina, San Diego, CA, USA). Subsequent data processing was carried out using specifically created scripts in Python (version 3.9.11), which include a trimming step to remove the Illumina adapters and a filtering step based on LpnPI restriction site occurrence between 13 and 17 bp from the 5'- or 3' end of the read, after which only reads originating from the LpnPI digestion remain . The reads passing the filter were mapped to the genome (hg38) using Bowtie 2 (version 2.1.) . Using all of the unambiguously mapped reads, count scores were assigned to each individual LpnPI site in the genome. Subsequently, the count scores for individual CpG sites were summarized into the same 2 kb regions to facilitate comparative analyses. Only regions containing data in at least 75% of all samples were included in the analysis, resulting in a total of 36,474 regions on chromosome 1-22 for the Nuzzo data, which were all also detected in at least 75% of the IMPACT cohort. The data were normalized to the total number of reads passing the LpnPI filter per sample, after which square root transformation was applied to reduce the skewness in the data distribution. The sequencing data of the Nuzzo dataset is available upon request from the authors, whereas the MeD-seq data from the IMPACT cohort is available in the NCBI Sequence Read Archive (SRA; accessed on 1 January 2023) under Accession code PRJNA895206. 2.5. RCC-Specific Methylation Score To identify differentially methylated regions (DMRs) between patients and HBDs in the dataset from Nuzzo et al., we used LIMMA (version 3.40.6) . A false discovery rate (FDR) was calculated to correct for multiple testing. The resulting DMRs were intersected with a composed list of 53 potential RCC methylation markers from the literature , resulting in a panel of 22 validated RCC-specific cfDNA methylation markers . To generate an overall methylation score per patient based on these 22 markers, Z-scores were calculated per region for every patient relative to our normal control panel of 10 HBDs. These Z-scores per region were squared and summed into an RCC-specific methylation score, as described before and as explained in more details in our File S1 supplementary methods. In addition, a detailed description of this method, including the equations, have been added as File S1 supplementary methods. 2.6. Statistical Analysis The primary aim was to assess the predictive value of a cfDNA methylation score to predict the time to disease progression warranting systemic treatment, also referred to as the WW time, in patients with good or intermediate prognosis RCC. The WW time was defined as the time from the baseline CT to the initiation of treatment. The time to the RECIST-defined progression of disease, also referred to as progression-free survival (PFS), was defined as the time from baseline CT to radiological progression according to RECIST 1.1 . The median follow-up of patients who were still on watchful waiting was 36.2 months (range: 15.8-48 months). Descriptive statistics were calculated for the variables of interest. A Mann-Whitney U test was performed for the univariate analyses of the continuous variables and a chi-square test was used for the categorical variables. To analyze the association between the methylation score and WW time or PFS, univariate cox proportional hazards analyses were performed using the survival package in R (v3.6.3). To visualize the effect of the significant variables on the WW time and PFS, Kaplan Meier plots were made using the survminer package in R (v3.6.3). 3. Results 3.1. Building an RCC-Specific cfDNA Methylation Score When comparing the cfDNA methylation profiles from 31 patients to 31 controls in the Nuzzo dataset , we identified 16,713 DMRs with an FDR < 0.1. From these DMRs, we selected the known tissue RCC markers to come to a validated RCC-specific methylation marker panel that was also validated in cfDNA, rather than tissue alone. For this purpose, we composed a list of 53 potential RCC methylation markers from the literature and checked which of these markers showed significantly differential methylation in the cfDNA dataset obtained from Nuzzo et al. This resulted in an RCC specific, cfDNA specific methylation panel, including 22 RCC markers , which was subsequently summarized into one RCC-specific methylation score. 3.2. The RCC-Specific Methylation Score Associates with Clinical Outcome To evaluate the potential clinical value of this RCC-specific cfDNA methylation score, we turned to the IMPACT-RCC study, which included 42 mRCC patients eligible for watchful waiting (WW). Baseline serum was available in 34 of 42 (83%) patients and the clinical characteristics of the patients from whom baseline serum was available did not differ from the total cohort of patients included in the IMPACT-RCC study . The majority of the patients had a pure clear cell carcinoma (79%) and an intermediate prognosis (71%) based on the presence of one risk factor (n = 11) or two risk factors (n = 13). The median obtained cfDNA concentration was 4.8 ng/mL serum (range: 0.6-117 ng/mL). The MeD-seq analysis was successful in 33 out of 34 patients and all 10 HBDs. The resulting data for the selected 22 markers is shown in Figure 1 and was used to calculate the RCC-specific methylation score in these 33 patients, as well as in the 10 HBDs. Using the upper limit of the 95% confidence interval observed in our set of HBDs as a cut off for methylation positivity, five patients showed a positive RCC-specific methylation score . From the patients who experienced rapid progression, defined as progression within two months after inclusion (n = 4), one had an altered RCC-specific methylation score. However, as shown in Figure 3, we observed that the patients with a score above the cut off had a significantly shorter PFS time (longrank test p = 0.0088). For the WW time, the same trend was observed, but the difference in WW time was not significant. To investigate the potential value of our RCC-specific methylation score in addition to the known prognostic clinical parameters in RCC patients, we performed Cox regression analyses with either the WW time or PFS as the outcome measure. For the WW time, we observed that, in the univariate analyses, only the IMDC score was associated with outcome, whereas age, the number of affected organ sites, the sum of CT-measured diameter of lesions, and our methylation score were not (Table 2a). For PFS, only the methylation score showed a significant association with the outcome in the univariate analyses (Table 2b). As, for both outcomes, only one variable was significantly associated, no multivariable analyses were performed. 4. Discussion The aim of this study was to investigate whether cfDNA could be used as noninvasive biomarker to identify patients with mccRCC with a high risk of early disease progression during a WW period. Therefore, we first established an RCC-specific panel consisting of 22 methylation markers and subsequently evaluated the methylation score for this panel in 33 mRCC patients eligible for WW, based on a low or intermediate prognosis according to the IMDC score. We used a technique that applies a restriction enzyme to generate >=32 bp methylated fragments for sequencing (MeD-seq). This method was previously shown to enable cfDNA methylation profiling with small amounts of fragmented cfDNA at relatively low costs . Although MeD-seq provides a promising method for cfDNA methylation profiling and was found to generate reliable profiles in metastatic colorectal cancer patients, it should be noted the enzyme recognizes ~50% of human CpGs and does not discriminate between methylated cytosines and hydroxy-methylated cytosines. In addition, only methylated DNA fragments are sequenced. Particularly with cfDNA, which is highly fragmentated, the absence of a region could therefore indicate either that the fragment was not present in the sample or that it was not methylated. We found that an altered methylation score, relative to the HBDs, was significantly associated with a shorter PFS, but not associated with the WW time. The results from this relatively small cohort of patients suggests that the blood-based methylation scores might add to the IMDC score for the identification of patients with low- and intermediate-risk mRCC who will show rapid disease progression. The subset of mccRCC patients managed with WW is not often described in the literature. While WW is an acknowledged treatment strategy in the current international guidelines, there are also no well-validated criteria to identify mccRCC patients for this strategy . The time from the initial RCC diagnosis to the development of metastatic disease, a factor considered to be an indicator of poor prognosis in patients on systemic treatment, was not prognostic in patients on WW . In all prospective studies on WW, patients with a good or intermediate prognosis according to the IMDC score could be considered for a WW-strategy. The large diversity in the duration of WW underlines that the IMDC criteria alone is unable to distinguish between patients with an expected long or short period of WW . It is encouraging that two studies have shown that the identification of patients eligible for WW can be improved with the knowledge on the disease extent at diagnosis (number of involved organs). However, the number of patients in these trials limit the impact of these findings. Exploring other non-invasive biomarkers to distinguish indolent from more aggressive disease is therefore desired. As such, several other researchers have investigated the prognostic value of tumor signal presence in cfDNA, also referred to as circulating tumor DNA (ctDNA), in patients with RCC. There is some evidence suggesting that the ctDNA level prior to surgery in the earlier stages of disease is associated with higher recurrence rates . Alternatively, the presence of ctDNA has also been associated with an inferior outcome in the treatment of metastatic disease . These were, however, only small cohorts of patients, and solid data on the prognostic value of ctDNA presence is still lacking. In addition, various methods have been used for the detection of ctDNA, varying from somatic mutations to untargeted methods such as copy number alterations . As renal cell carcinoma is known to be characterized mainly by epigenetic alterations, methylation profiling therefore poses a sensible approach for the detection of tumor DNA in blood . Therefore, more recent work has focused on methylation approaches for ctDNA detection in RCC patients. The advantages of methylation analysis have been demonstrated in more recent work showing that methylation profiling identified the presence of ctDNA at the earlier stages of disease . For our study, it is remarkable that only fifteen percent of the patients in our study that included only patients with stage IV disease, were methylation positive using our approach. This might be explained by the fact that mccRCC patients were previously found to have low ctDNA amounts in general . Moreover, we used serum and not plasma as a source for our cfDNA analyses for availability reasons, which likely complicated the detection of tumor specific methylation patterns, as serum is known to contain lower tumor fractions due to a higher contamination of DNA originating from leukocytes . Other studies investigating cfDNA in stage IV disease found larger proportions of patients to have detectable ctDNA, ranging between around 30% and 100% . The fact that we used serum and not plasma is a limitation of our study and was related to the availability of materials. In addition, the sample size of our study is relatively small, although the majority of patients had serum samples available. The lack of a large series of RCC in patients in the literature probably reflects the challenge of performing clinical studies in patients with RCC. To our knowledge, however, this is the first series that has related cfDNA methylation patterns to clinical outcome in mccRCC patients. Moreover, it is important to note that we found the methylation score to be significantly associated with the PFS, but not with the WW time. As per the protocol, some patients who progressed according to RECIST 1.1 did not start with systemic treatment directly, explaining the difference between the observed PFS and WW times. The main reasons not to start treatment were the progression of overall small sized metastatic lesions or lesions growth not leading to overt symptoms; the decision not to start treatment in these cases reflects the current daily practice. This raises the question of whether a biomarker that is better correlated with radiological progression than with WW time will ever be clinically relevant in this setting, even if it outperforms the IMDC score. 5. Conclusions Our study demonstrates that RCC-specific DNA methylation is detectable in the blood of part of the patients and appears to be associated with disease progression. Together, these results provide a proof-of-concept that cfDNA methylation analysis could be used as a prognostic tool in RCC patients. For the WW time, we only observed a non-significant trend towards an association with the methylation score. Although this study did not find definite proof that cfDNA methylation patterns are associated with WW time, this should be validated in larger studies, in which plasma should be collected prospectively. Furthermore, its additional value to the IMDC score remains to be elucidated. Acknowledgments The authors would like to express gratitude to the team members from the IMPACT studies for participating in the execution of the trial. In addition, the authors would like to thank Soumya Zacharia and Matthew Freedman for kindly providing us with the sequencing data from the stage IV RCC patients and HBDs described in their paper (Nuzzo, P.V., Berchuck, J.E., Korthauer, K. et al. Detection of renal cell carcinoma using plasma and urine cell-free DNA methylomes. Nat Med 26, 1041-1043, 2020). Supplementary Materials The following supporting information can be downloaded at: Figure S1: Flow chart of study samples. File S1: Supplementary methods. Click here for additional data file. Author Contributions M.K.B., S.R.V., C.M.L.v.H., S.S. and S.M.W. were involved in the conceptualization and design of the analyses. S.R.V., S.F.O., W.C.M.-v.d.H.v.O., J.W.M.M. and C.M.L.v.H. were involved in the patient inclusion and data collection. M.K.B., S.R.V., R.G.B., J.B.B., J.G. and S.M.W. were involved in the data analyses. M.K.B., S.R.V. and S.M.W. wrote the manuscript, which was reviewed and edited by all of the authors. All of the authors read and approved the final manuscript. The work reported in the paper has been performed by the authors, unless clearly specified in the text. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of all participating centers (METC-2013-283 or NL44748.091.13). This trial is registered with the Netherlands Trial Register, number NCT02228954. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The sequencing data are available in the NCBI Sequence Read Archive (SRA; accessed on 15 February 2023) under Accession code PRJNA895206. The clinical metadata are available from the corresponding author upon request. The sequencing data of 32 stage IV RCC patients and 32 HBDs described by Nuzzo et al. were requested from the corresponding author. Conflicts of Interest The authors declare no conflict of interest of financial interests except for R.B, J.B. and J.G. who report being shareholders in Methylomics B.V., a commercial company that applies MeD-seq to develop methylation markers for cancer staging. Figure 1 Analytical approach. cfMEDIP; Cell-free methylated DNA immunoprecipitation, cfMeD-seq; Methylated DNA sequencing, HBD; Healthy blood donor, PD; progressive disease, RCC; Renal cell carcinoma, qMSP; quantitative methylation specific PCR. Figure 2 RCC-specific methylation score for healthy blood donors (HBDs) and patients. For HBDs, the mean and standard deviation is indicated. The cut-off for methylation positivity, represented by the upper limit of the 95% confidence interval of the methylation score in HBDs, is indicated by a dotted line. The patients are ordered by WW time. Figure 3 Kaplan Meier analyses for WW time (left) and PFS time (right). Patients are divided based on RCC-specific methylation score. A log-rank test was performed to compare the curves. cancers-15-01374-t001_Table 1 Table 1 Patient demographics and clinical characteristics. Characteristics Patients in IMPACT-RCC Study (n = 42) Patients with Serum Samples Successfully Analyzed (n = 33) p-Value Gender Male, n (%) 31 (74%) 26 (76%) Female, n (%) 11 (26%) 7 (21%) 0.786 Median age in years (range) 66 (44-86) 66 (44-86) 0.790 Nephrectomy Yes, n (%) 36 (86%) 28 (85%) No, n (%) 6 (14%) 5 (15%) 1 Histology Pure clear cell, n (%) 32 (76%) 27 (79%) Mixed, n (%) 10 (24%) 6 (18%) 0.586 Time from diagnosis to first metastasis <1 year, n (%) 23 (55%) 19 (58%) >=1 year, n (%) 19 (45%) 14 (42%) 0.820 IMDC risk factors 0 (favorable) 14 (33%) 10 (30%) 1 (intermediate) 13 (31%) 10 (30%) 2 (intermediate) 15 (36%) 13 (39%) 0.941 cancers-15-01374-t002_Table 2 Table 2 Cox regression analysis. The tables were adopted from the main paper and updated according to the patients included within the biomarker analysis. The methylation scores resulting from the biomarker analysis were added as variables. Variables HR 95% CI HR p-Value (a) Cox proportional hazards regression analysis results for watchful waiting time. Clinical Age 1.00 0.96-1.024 0.99 IMDC criteria, per unit increase in score 2.01 1.19-3.41 0.01 Number of affected organ sites per patient 1.43 0.96-2.11 0.07 Sum of CT measured diameter of lesions per patient (cm) 1.02 1.00-1.05 0.08 Molecular RCC-specific methylation score 2.18 0.81-5.87 0.12 (b). Cox proportional hazards regression analysis results for time until RECIST-defined PD Clinical Age 1.01 0.96-1.05 0.82 IMDC criteria, per unit increase in score 1.35 0.86-2.10 0.19 Number of affected organ sites per patient 1.07 0.73-1.57 0.74 Sum of CT measured diameter of lesions per patient (cm) 1.01 0.99-1.04 0.30 Molecular RCC-specific methylation score 4.45 1.32-15.05 0.02 HR; Hazard ratio, CI; Confidence interval, IMDC; International Metastatic Renal Cell Carcinoma Database Consortium, CT; computed tomography. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000043

The study aimed to investigate the mastitis' emerging causative agents and their antimicrobial sensitivity, in addition to the hematological, biochemical indicators, oxidative biomarkers, acute phase protein (APP), and inflammatory cytokine changes in dairy farms in Gamasa, Dakahlia Governorate, Egypt. One hundred Holstein Friesian dairy cattle with clinical and subclinical mastitis were investigated and were allocated into three groups based on a thorough clinical examination. Escherichia coli and Staphylococcus aureus were found responsible for the clinical and subclinical mastitis in dairy farms, respectively. Multiple drug resistance (MDR) was detected in 100%, and 94.74% of E. coli and S. aureus isolates, respectively. Significantly low RBCs count, Hb, and PCV values were detected in mastitic cows compared with both subclinical mastitic and control groups; moreover, WBCs, lymphocytes, and neutrophil counts were significantly diminished in mastitic cows compared to the controls. Significantly higher levels of AST, LDH, total protein, and globulin were noticed in both mastitic and subclinical mastitic cows. The haptoglobin, fibrinogen, amyloid A, ceruloplasmin, TNF-a, IL-1b, and IL-6 levels were statistically increased in mastitic cows compared to the controls. Higher MDA levels and reduction of TAC and catalase were identified in all the mastitic cases compared to the controls. Overall, the findings suggested potential public health hazards due to antimicrobial resistance emergence. Meanwhile, the APP and cytokines, along with antioxidant markers can be used as early indicators of mastitis. mastitis E. coli S. aureus oxidative/antioxidant molecules APP inflammatory cytokines Princess Nourah bint Abdulrahman UniversityPNURSP2023R23 This study was supported by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R23), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. pmc1. Introduction In veterinary medicine, bovine mastitis is considered one of the most common and economically important diseases affecting dairy herds worldwide. It mostly causes major economic losses . Hence, the economic impact of mastitis is usually due to decreased milk production, increased costs of veterinary treatment, and premature culling of infected animals ; in addition, it causes alterations in the milk and udder physically, chemically, and pathologically . Staphylococcus aureus is one of the major common pathogens causing mastitis in dairy animals . The ability of the organism to cause infections is probably due to the expression of various toxins, virulence factors, and cell wall adhesion proteins. The bacterium can survive phagocytosis in the udder and often causes chronic inflammation . These infections frequently do not respond to routine therapy. However, other microorganisms may be attributed to mastitic infections like Escherichia coli, Salmonella spp., and Streptococci or Listeria spp. . Bovine mastitis poses a great one-health concern and zoonotic danger to humans due to the capability of this food-borne pathogen to be transmitted through milk or even through workers to consumers The use of antimicrobial agents on cattle farms is often useful but the excessive use of it to control or prevent the mastitic infections may be contributing to the global antimicrobial resistance development . Besides, antimicrobial residue leads to a decrease in the quality of milk . Bovine mastitis is classified into two categories: clinical mastitis and subclinical mastitis; clinical mastitis is characterized by observable clinical signs of the udder and signs of inflammation with general health disturbance ; subclinical mastitis is difficult to be detected at once due to the lack of visible signs. It takes days or weeks to be observed , producing huge losses in the milk production due to the long-time persistence of infection and alterations of physical and chemical properties of the milk . Usually, subclinical mastitis is more common than clinical mastitis . The number of Somatic cells in the milk may be considered a diagnostic marker for subclinical mastitis ; these somatic cells consist of macrophages, neutrophils, lymphocytes, and some mammary epithelial cells. In the case of the healthy udder, macrophages are the predominant cell, while during early mastitis neutrophils become the predominant cells . Host-pathogen interactions lead to the activation of the innate immune response to recognize infected microorganisms or rechange immune components by increasing the number of macrophages and releasing cytokines, which recruit leukocytes at the site of infection and trigger the local and systemic acute phase response . Furthermore, acute phase proteins such as haptoglobin, cathelicidins, and peptidoglycan recognition protein were recorded at higher rates during infection ; similarly, high levels of IL-1b, IL-8, and TNF-a were expressed . The pathogenesis of udder bacterial infection and bovine immune response must be studied to establish reliable biomarkers which can be detected at the early stage of the infection. Thus, these reliable biomarkers were measured in our study for accurate detection of the changes assigned with the bacteriological agent invasion and accompanied changes in biochemical and immunological parameters related to both clinical and sub-clinical mastitis. Meanwhile, the antimicrobial resistance of our strains was assessed to measure the current danger of multidrug resistance strains affecting public health. 2. Materials and Methods 2.1. Animal Population The present study was conducted in January 2021 on 100 dairy cows: healthy cows (n = 20), clinical mastitis-infected cows (n = 30), and subclinical mastitis-infected cows (n = 50) from two different farms located on the road of Gamasa city, Dakahlia Governorate, Egypt. Their ages ranged between 4 and 7 years. All dairy cattle diagnosed in our study were Holstein Friesian. The study was performed on cows that had not received any medication in the week before sampling. On the farm, modern management techniques and good hygiene standards were applied. Only automatic milking machines were used at both farms. All investigated cows were subjected to a thorough clinical examination. After milking the diseased cows, the size, the conformation of the udder, and the relative size of all quarters were inspected. The udder tissue and supramammary lymph nodes were also thoroughly examined. Cows with clinical mastitis were identified if one or more of the following signs were observed: cardinal signs of inflammation in one or more of an udder's quarters, signs of systemic reactions (fever, depression, and disturbed appetite), or abnormal physical character of milk (clot formation, discoloration, and alterations in viscosity, aberrant smell, and the presence of blood). Due to the absence of observable clinical signs in cows with subclinical mastitis, the presumptive diagnosis was based on the laboratory diagnostic tests of milk samples, the California mastitis test (CMT), and somatic cell count (SCC). Cows with positive CMT or having SCC > 200,000 cells/mL and lacking clinical signs were considered affected with subclinical mastitis. Briefly, the California mastitis test (CMT), was performed as follows: a plastic vessel with 4 shallow wells was used for collecting approximately 2 mL of milk from each udder quarter. Then, an equal amount of alkali reagent (kerbl(r) reagent) was added. A gentle circular motion was applied to the mixtures in a horizontal plane for 5 s and the different degrees of gel were recorded, according to the system used in the Nordic countries as the scoring is made from 1-5. Meanwhile, all the milk samples were examined automatically for somatic cell count by using The Nucleo Counter(r) SCC-100TM. The sample was warmed in a water bath at 35 degC for 5 min and then mixed automatically before reading . All procedures were performed following the principles and specific guidelines presented in the Mansoura University Animal care and approved by its Ethical Committee. 2.2. Sample Collection Milk and blood samples were collected from each individual dairy cow (n = 100), including healthy cows (n = 20), clinical (n = 30), and subclinical mastitis cows (n = 50). The teats were wiped using alcohol for sterilization and all samples were taken in sterilized vials. After milk samples were taken aseptically, they were transported in coolers (4 to 8 degC) for subsequent bacteriological analyses within 6 hrs. All milk samples were sent to the Department of Bacteriology, Mycology and Immunology lab, Faculty of Veterinary Medicine, Mansoura University, Egypt. For blood samples, the halter was used to position the animal's head in a slightly elevated manner to expose the jugular vein with minimal restraint to get the blood samples without causing injury. One blood sample was collected with an anticoagulant (EDTA) for complete blood counting. The second blood sample was collected in a heparinized tube which was rapidly centrifuged at 3000x g for 10 min for separation of blood plasma. The collected plasma was used for the estimation of fibrinogen. The third blood sample was collected in plain test tubes, left for 15 min to clot, and centrifuged at 3000 rpm (4 degC) for serum separation. The separated serum was stored at -80 degC for further determination of biochemical, inflammatory, and oxidative stress/antioxidant parameters. 2.3. Bacterial Identification All collected milk samples were centrifuged for 10 min. A drop from the sediment was cultivated onto Baird-Parker agar, 5% sheep blood agar, MacConkey's agar, and Mannitol salt agar, (Oxoid, Ltd., Basingstoke, UK) at 37 degC for 24-48 h. Bacterial colonies were classified according to their phenotypic characters on the culture media. All milk samples that give more than two bacterial species on bacterial culture plates were considered contaminated samples and were discarded. Presumptive characterization of the isolated bacteria was carried out based on Gram's stain and biochemical characteristics according to Quinn and his group . 2.4. Molecular Confirmation of S. aureus and E. coli Isolates 2.4.1. DNA Extraction The DNA was extracted from all suspected isolates by the boiling method . Two to three colonies from suspected S. aureus and E. coli isolates were suspended in 200 uL deionized free water and boiled for 10 min, and then centrifugated for 10 min. The supernatants were transferred to a new sterile Eppendorf tube and stored at -20 degC as a DNA sample. 2.4.2. Molecular Confirmation Using Polymerase Chain Reaction (PCR) for S. aureus and E. coli Isolates All the isolates suspected to be S. aureus by biochemical typing were confirmed by amplification of species-specific nuc gene (encoding for the S. aureus-specific thermonuclease) using the primers: nuc-F:(GCGATTGATGGTGATACGGTT) and nuc-R: (AGCCAAGCCTTGACGAACTAA AGC) by using PCR amplification technique according to Sallam et al. . In brief, A 25 mL PCR reaction mixture contained 5 mL of DNA template, 12.5 mL of 2X PCR master mix (Thermo Scientific, United States), 6.5 mL of deionized nuclease-free water, and 1 mL of each primer. PCR conditions were 94 degC for 2 min for initial denaturation, followed by 35 cycles of 98 degC for 10 s, 58 degC for 30 s, and 68 degC for 60 s, then 68 degC for 7 min as a final extension step; this cyclic reaction was run using a 96 well Applied Biosystem, 2720 thermal cycler. The PCR products were visualized using 1% agarose gel using a UV transilluminator and a Gel Documentation System (cleaver scientific ltd UV gel documentation system, USA). S. aureus strain from a previous study was used as a positive control . Furthermore, all the biochemically characterized E. coli isolates were subjected to PCR amplification for encoding of the Genus-specific primer 16S-rRNA gene using the following primer pair: 16S-rRNA-F-GCGGACGGGTGAGTAATGT and 16S-rRNA-R-TCATCCTCTCAGACCAGCTA (Table 1). PCR protocol was performed as per Teichmann et al. . The reaction was performed in a total volume of 25 mL with 5 mL DNA template, 12.5 mL 2X PCR master mix (Thermo Scientific, United States), 6.5 mL deionized nuclease-free water, and 1 mL of each primer. A 96-well Applied Biosystem and a 2720 thermal cycler were used for PCR amplification. The following PCR amplification reaction was applied: initial denaturation for 5 min at 94 degC; 30 cycles of 94 degC for 15 s, 69 degC for 30 s, and 72 degC for 30 s; final extension for 7 min at 72 degC. PCR products were run on 1% agarose gel and were visualized using a UV transilluminator and a Gel Documentation System (cleaver scientific ltd UV gel documentation system, USA). The strain used as PCR positive control was supplied from a previous study . 2.5. Antimicrobial Sensitivity Test for S. aureus and E. coli Strains All the confirmed bacterial isolates were characterized for their antimicrobial sensitivity tests on Muller Hinton agar (Oxoid, Ltd., Basingstoke, UK) by using the disc diffusion technique according to CLSI . Firstly, the twenty-one E. coli isolates were tested for antimicrobial sensitivity against 12 different antimicrobial compounds by disc diffusion test. The following antimicrobial discs (Oxoid, Ltd., Basingstoke, UK) were used: penicillin (10 mg), cefuroxime (30 mg), cefoperazone (30 mg), amikacin (30 mg), streptomycin (15 mg), neomycin (5 mg), azithromycin (15 mg), nalidixic acid (30 mg), trimethoprim-Sulfamethazole (25 mg), gentamycin (10 mg), chloramphenicol (30 mg), and rifamycin (5 mg) (Table 2). In addition, thirty-eight S. aureus isolates were examined against 10 antimicrobial agents for their antimicrobial sensitivity using a disc diffusion test on Muller Hinton agar (Oxoid, Ltd., Basingstoke, UK). These antimicrobial discs (Oxoid, Ltd., Basingstoke, UK) were ampicillin (10 mg), oxacillin (15 mg), ceftazidime (30 mg), kanamycin (30 mg), streptomycin (15 mg), norfloxacin (10 mg), ciprofloxacin (5 mg), chloramphenicol (30 mg), tetracycline (30 mg), and gentamycin (10 mg) (Table 2). The results were interpreted according to CLSI . Resistance to more than two antibiotics from different antimicrobial classes was recorded as MDR . A multiple antibiotic resistance (MAR) index was calculated according to Krumperman . 2.6. Complete Blood Cell Analysis Red blood cell count (RBCs), hemoglobin (Hb), hematocrit value (PCV), and total and differential leukocytic count were analyzed according to Morar et al. . 2.7. Biochemical Markers, Acute Phase Protein, and Inflammatory Cytokines Analysis The serum activity of aspartate aminotransferase (AST, catalog No.; AS101) was estimated using kits obtained from the Randox company (Kearneysville, VA, USA). Lactate dehydrogenase (LDH, catalog No.; TK41214), total protein (Catalog No.; MD1001291), and albumin (Catalog No.; MX1001020) were assessed using kits obtained from the Spinreact company (Santa Coloma, Spain) according to the described methods of its manufacturer's instructions. Serum levels of acute-phase protein (APP) including haptoglobin (Catalog No.; ab137977), amyloid A (Catalog No.; ab274407), fibrinogen (Catalog No.; ab108842), ceruloplasmin (Catalog No.; ab108818), and ferritin (Catalog No.; ab108698) were estimated using kits obtained from the Abcam company (Cambridge, UK) according to the standard protocol of their specific pamphlets. Serum tumor necrosis factor-alpha (TNF-a, Catalog No.; MBS2609886), interleukins, IL-1b, IL-6, (Catalog No.; # ESS0027, and # RBOIL6I) (Invitrogen-Thermo Fisher Co., Waltham, MA, USA) and IL-10, (Catalog No.; ab277386) (Abcam Co., Cambridge, UK) levels were estimated using specific bovine ELISA commercial kits according to the methodology of each enclosed pamphlet. All parameters were measured spectrophotometrically using a 5010 Photometer (ROBERT RIELE GmbH & Co KG, Berlin, Germany). 2.8. Oxidative Stress/Antioxidant Parameters The serum levels of malondialdehyde, total antioxidant capacity (TAC) superoxide dismutase (SOD), and catalase were estimated spectrophotometrically following the illustrated approaches of Ohkawa et al. , Benzie and Strain , Nishikimi et al. , and Aebi , respectively. 2.9. Statistical Analysis Data analysis was carried out using a statistical software program (SPSS for Windows, Version 21, Cary, NC, USA). The Kolmogorov-Smirnov test was selected to assess the normal distribution of the data. The assessed data were normally distributed; therefore, the means and standard mean of error (SME) for each variable were statistically analyzed and presented. The post hoc test with ANOVA (Tukey's test) was used to assess statistical differences between the two groups. In all statistical analyses, the results were considered significant at p < 0.05. 3. Results 3.1. Characterization of E. coli and S. aureus among Clinical and Sub-Clinical Mastitic Milk Out of all investigated mastitic milk samples, a total of 70% (21/30) E. coli isolates and 76% (38/50) S. aureus isolates were identified from clinical mastitis milk and sub-clinical mastitis milk, respectively, using PCR assay. All the other samples were discarded and counted as contaminated samples. A total of twenty milk samples from healthy cows were negative for bacterial culture. Due to the absence of observable clinical signs in animals infected with subclinical mastitis, a presumptive diagnosis was done based on laboratory diagnostic tests of milk samples using the CMT and SCC. Cows with positive CMT or those having SCC > 200,000 cells/mL but lacking clinical signs were considered affected with subclinical mastitis. 3.2. Antimicrobial Susceptibility Testing for E. coli and S. aureus among Clinical and Sub-Clinical Mastitic Milk The results of antimicrobial susceptibility testing of E. coli isolates (n = 21) are listed in Table 2. E. coli isolates were resistant to penicillin, cefuroxime, cefoperazone, azithromycin, nalidixic acid, trimethoprim-sulfamethoxazole, rifamycin, and gentamycin. They displayed intermediate resistance to neomycin (61.9%), amikacin (52.4%), and chloramphenicol (47.6%). E. coli isolates were more sensitive to streptomycin (38.1%). Multiple drug resistance (MDR) was detected in all tested E. coli isolates (resistant to >=3 antimicrobial class) and the most prevalent antimicrobial pattern was ''C, P, DA, STX, RA, N, NA, CXM, CEP, AZM'' (Table 3). Moreover, the antimicrobial susceptibility testing of S. aureus isolates (n = 38) is listed in Table 2. S. aureus isolates were highly resistant to ampicillin, oxacillin, and gentamycin (76.3%, 76.3%, and 73.7%), respectively, and they displayed intermediate resistance to ceftazidime (52.6%), kanamycin (47.4%), and streptomycin (50%). S. aureus was more sensitive to norfloxacin (34.2%), ciprofloxacin (36.8%), chloramphenicol (26.3%), and tetracycline (23.7%). Multiple drug resistance (MDR) was detected in 94.74% of the total tested isolates (resistant to >=3 antimicrobial classes). Only two S. aureus strains were found to be sensitive to all antimicrobials tested. The most prevalent antimicrobial pattern was K, NOR DA, TE, OX, CIP, CAZ, AMP, and S (Table 3). 3.3. Hematological and Serum Biochemical Parameters of Dairy Cows with Subclinical and Clinical Mastitis As presented in Table 4, the obtained results showed significantly lower RBCs count (p < 0.01), Hb (p < 0.001), and PCV (p < 0.01) values in mastitic cows compared with both subclinical mastitic and control cows. Moreover, WBCs (p < 0.01), and neutrophil (p < 0.001) counts were significantly diminished in clinical and subclinical mastitic cows compared to the controls. Significantly higher levels of AST (p < 0.001, p < 0.01), LDH (p < 0.001, p < 0.01), total protein (p < 0.001, p < 0.05), and globulin (p < 0.001, p < 0.05) in both mastitic and subclinical mastitic cows unlike that of the controls. However, the serum level of albumin (p < 0.05) statistically declined in mastitic cows only compared to other groups. 3.4. Serum Acute Phase Protein of Dairy Cows with Subclinical and Clinical Mastitis To assess the mechanisms involved in the progression and damage of mammary gland tissue during mastitis, serum acute-phase proteins (APP) and inflammatory cytokines were estimated in our study. The haptoglobin (p < 0.001), fibrinogen (p < 0.01), and Amyloid A (p < 0.001, p < 0.01) levels were found to be highly elevated in the serum of both mastitic and subclinical mastitic cows unlike that of the controls . Moreover, ceruloplasmin (p < 0.05) levels were significantly raised in the serum of mastitic cows compared to the healthy controls . No significantly valuable differences in serum ferritin levels of all the groups were identified . 3.5. Serum Inflammatory Cytokines of Dairy Cows with Subclinical and Clinical Mastitis TNF-a (p < 0.001), IL-1b (p < 0.001, p < 0.01), and IL-6 (p < 0.001, p < 0.01) were statistically increased in mastitic and subclinical mastitic cows compared to the healthy controls . Meanwhile, the IL-10 (p < 0.01) decreased only in mastitic cows relative to the remaining groups . 3.6. Serum Antioxidant\Oxidative Stress Parameters of Dairy Cows with Subclinical and Clinical Mastitis During the progression of mastitis, the bacterial infection caused the generation of an accentuated reactive oxygen species (ROS) and impairment of antioxidant molecules confirmed in our results by higher malondialdehyde (MDA) levels (p < 0.001), along with a reduction of total antioxidant capacity (TAC) (p < 0.05, p < 0.001) and catalase (p < 0.01, p < 0.001) in mastitic and subclinical mastitic cows compared to the control non-infected one . Superoxide dismutase (SOD) (p < 0.05) was statistically diminished only in mastitic cows compared to the controls . 4. Discussion Mastitis is a mammary gland inflammatory illness that causes huge, enormous economic losses in the dairy industry . It occurs when a pathogenic microbe enters the mammary gland, usually by disrupting physical barriers like the teat canal . Once the barrier is breached, a prompt and effective defense response is required to prevent the spread of pathogenic organisms and additional injury to mammary gland tissue . Mastitis, in its clinical and subclinical forms, is considered one of the most devastating diseases that affect dairy herds and represents 21% of reported diseases in dairy cattle, with an annual incidence of 37% . To our knowledge, there were no studies applied to clinical or subclinical mastitic cows that directly link the bacterial causative agent, and serum biochemical, antioxidant, and inflammatory markers (APP & cytokines) with mastitis risk. That is why the goal of this study was to investigate the bacterial cause and detect serum biochemical, antioxidant, and inflammatory markers in dairy cows that were linked to clinical and subclinical mastitis susceptibility. Mastitis is a complex disease with varied etiological causes, contagious bacterial, environmental, and opportunist . In our study, the bacterial agent was detected in high proportion 73.75% (59/80) of all mastitic milk samples. This confirms previous studies which mentioned bacterial mastitis as the most common etiology . Furthermore, our findings revealed that E. coli and S. aureus were the causative agents responsible for our mastitic cases which are in accordance with previous studies that described these two microorganisms to be the most common and significant bacterial agents responsible for mastitis . E. coli is an environmental agent capable of invading the cow's teat through ascending infection . E. coli was recovered from 70% (21/30) of our clinical mastitis cases. Higher results were obtained in France and Egypt; investigating more than 80% of the clinical mastitic cases were caused by E. coli . In Egypt, a previous report showed a slightly low occurrence (about 31%) of clinical mastitic cases where E. coli was the causative agent . Ahmed et al. recovered a low incidence (9.1%) of E. coli-related mastitic cases. S. aureus is a contagious microorganism that invades the udder and infects all the quarters. In our study, S. aureus recovered at a rate of 76% (38/50) from the subclinical cases which came following other studies in Egypt . From Egypt, another study was found to have higher results than ours; about 91.48% of the samples were identified as S. aureus . Antimicrobial resistance is an important hazard that is mentioned as the silent tsunami facing modern medicine. Nowadays, MDR has been a serious challenge facing scientists . Examination of our isolates from farms that did not receive any previous medication the last week relived a dangerous result for MDR. Our E. coli mastitic strains showed extensive resistance against seven antimicrobial classes, b-lactam, cephalosporin, macrolide, quinolone, sulphonamide, lincosamide, and rifamycin. Previous reports discussed E. coli raised antimicrobial resistance against ampicillin, streptomycin, and sulfonamides ; in addition, extended-spectrum b -lactamases resistance was investigated in other reports . These results might be attributed to the broad-spectrum antimicrobials, trimethoprim-sulfonamides, oxytetracycline, fluoroquinolones, cefquinome, and ceftiofur which are used in the farms for the systematic treatment of mastitic coliform . Thus, E. coli have developed resistance to these extensively used antimicrobial classes. In this study, the isolated Staphylococcus aureus strains showed slightly lower antimicrobial resistance results than the E. coli strain. Our study exhibited higher resistance against ampicillin which came in accordance with previous studies . S. aureus produces penicillinase enzyme which makes the treatment by b-lactamase antimicrobial class more problematic. Third-generation cephalosporin was considered for treatment of the mastitis cases caused by S. aureus. It was revealed that about half of the strains were ceftazidime resistant which was the same as other previous studies . We observed that about 73% of the strains developed resistance to gentamicin which was higher than in other studies . Reports from China showed that antimicrobial agents such as penicillin, ampicillin, streptomycin, gentamicin, ciprofloxacin, and sulfamethoxazole-trimethoprim were usually applied for mastitis in dairy farms ; these antimicrobial agents act by inhibiting protein synthesis . Resistance against penicillin, ampicillin, erythromycin, tetracycline, and clindamycin was reported in different countries . In this study, the low resistance against tetracycline and chloramphenicol was reasonable. Since tetracycline resistance is usually acquired by horizontal gene transfer; besides, chloramphenicol is not used anymore in the veterinary field. The proportion of MDR S. aureus in our study was 94.74%. These results are similar to previous studies from Malaysia and Brazil . The inappropriate use of antimicrobials in farms may be involved in the emergence or spread of antimicrobial-resistant strains; these strains could be transmitted to humans via direct contact with animals or via ingestion of infected food . Thus, misuse led to the environmental release of the antimicrobial resistance genes and resistant bacterium. In this case, the environment and the animals will serve as an end reservoir of the antimicrobial resistance genes . This limited the options used for the therapy against these agents . Periodical surveillance strategies must be taken to control this dramatic development of MDR bacterial strains . Nowadays, the emergence of novel strategies such as the usage of antimicrobial combination, phage therapy, and peptide therapy had been studied . Changes in hemato-biochemical parameters and leukocyte counts can be employed as crucial indications of the animal's physiological or pathological status (mastitis). Following our results, the mastitis-affected cows showed a significant decrease in Hb, PCV, and RBCs counts, with an increase in TLC as compared to healthy cows . These findings were also consistent with a prior study that found a significant fall in RBC, Hb, and PCV values in mastitis-affected animals, resulting in anemia . Earlier studies also found that mastitic cows had a higher leukocyte, granulocyte count, lymphopenia, and anemia . TLC levels were recorded to be increased in affected animals, as well as monocyte, neutrophil, and eosinophil count . Various immunomodulatory actions may be responsible for the considerable increase in WBC and neutrophilic counts in mastitic cows . Furthermore, Abba et al. attributed those neutrophils enhanced the chemotactic molecules generated by infectious pathogens, as well as other immune system components that activate neutrophil recruitment to infection sites. Compared to healthy cows, the activities of AST and LDH exhibited a considerable increase in mastitic cows, which could be attributed to stressful conditions. Our findings were consistent with prior research . In terms of total protein findings, our results revealed a significant increase in serum total protein and globulin with lower albumin levels in mastitic groups when compared to healthy ones. These findings agreed with Ali et al. and Garba et al. who found a significant increase in total protein levels with significantly lower albumin levels in cows with clinical and subclinical mastitis. Similarly, previous reports recorded high levels of globulin and total protein in mastitic cows . The immunological response is the main cause of the decrease in albumin levels that are linked to udder infection . As well, hypoalbuminemia may be interpreted as the stress that occurs during mastitis, which increases protein catabolism . Increased serum globulin levels could be related to the development of antibodies in the form of gamma-globulin, which is responsible for neutralizing the invading microorganism's effect . Alternatively, Sarvesha et al. , and Krishnappa et al. recorded the mastitic group had a significantly lower level of total proteins in mastitic in indigenous cows, crossbred cattle, and buffaloes. To identify cows with subclinical mastitis, laboratory diagnostics is crucial. Mastitis control is primarily dependent on determining SCC and the CMT, which aim to detect the number of cells in the milk sample. The other useful diagnostic tool is microbial culture, which complements SCC and CMT . However, all mentioned diagnostic methods have their limitations and therefore novel biomarkers of subclinical mastitis are highly desired. These sensitive indicators include cytokines and acute phase protein measurements, particularly haptoglobin, fibrinogen, and amyloid A which could be determined in cow serum and/or milk, and in the future may become useful in early mastitis diagnostics as well as a preventive tool. This may contribute to increase the detection of mammary gland inflammation in cows, especially in subclinical form, and consequently improve milk quality and quantity. Over the last few decades, research has revealed that acute-phase protein quantitation (APP) e.g., ceruloplasmin, fibrinogen, haptoglobin, and amyloid A, can be found in blood plasma or serum and it can be used for disease diagnosis and prognosis detection, as well as to assess the degree of inflammation . In the current investigation, we found that ill cows had significantly higher levels of haptoglobin, fibrinogen, amyloid A, and ceruloplasmin in subclinical and clinical mastitic animals than in the healthy cows. The production of proinflammatory cytokines like TNF-a, which exacerbate the inflammatory process and stimulate neutrophil phagocytic activity, could be to blame for the increased amounts of APPs . The elevated levels of APP are in accordance with those obtained by Winter et al. in cows with Listeria monocytogenes-induced mastitis, as well as in clinical and subclinical mastitic dairy cows . Data about subclinical and clinical mastitis demonstrate inflammatory responses to the intramammary infection driven by IL-1b, IL-6, and TNF-a. Moreover, the host defense response in mastitis is characterized by the continuation or resolution of initial inflammation . Additionally, our aim of the study was to determine inflammatory and regulatory cytokines in the serum of dairy cows with subclinical and clinical mastitis and to help in the diagnosis of subclinical or clinical mastitis. Knowledge about the inflammatory and regulatory cytokines in naturally occurring mastitis is lacking as most studies focus on pathogen-induced mastitis. In naturally occurring mastitis, the study of cytokines is a potential tool for early and timely diagnosis and in prospective pathogenesis-based treatment. Our finding recorded statistically increased TNF-a, IL-1b, and IL-6 in mastitic and subclinical mastitic cows compared to the healthy controls, while the IL-10 was decreased only in mastitic cows relative to the other groups. Similarly, it has been shown that pro-inflammatory cytokines are important in fighting the original infection. Increased levels of TNF-a and IL-1b in subclinical mastitis were discovered in this study, suggesting that they play a role in the early stages of mastitis development . The pro-inflammatory cytokines IL-12, IL-6, TNF-a, and IL-1b were found to be significantly elevated in lactating cows suffering from clinical mastitis . Variations in these proinflammatory concentrations corresponded to the onset of illness signs. As previously reported, increased pH and significant decreases in fat, SNF, protein, and lactose were identified in the mammary gland tissue of these cows . In contrast to our findings, previous research showed a non-significant rise in IL-2 and IL-6 markers in cows with subclinical mastitis . All the living organisms, particularly dairy cows, produce free radicals because of their active metabolism. Oxidative stress occurs when homeostasis is disrupted, which is mostly caused by the generation and accumulation of free radicals, which can lead to mastitis in dairy cows . Antioxidants protect the body from oxidative damage caused by free radicals by scavenging them directly or by inhibiting the action of oxidizing enzymes . In the present study, there was a significant increase in MDA, along with a reduction of TAC, SOD, and catalase in subclinical and clinical mastitic animals compared to the control non-infected ones. The considerable increase in MDA levels, as well as the significant drop in TAC and catalase activity, such findings could imply an increase in free radical activity, which would reflect the state of oxidative stress that happens in such situations . Previously similar findings have been reported by Sharma et al., and Jhambh et al. . Increased consumption to neutralize ROS generated by the inflamed gland could explain the decreased antioxidant enzymatic activity, indicating a weakened antioxidant defense mechanism . Furthermore, these changes could be due to the high demand for high SOD, catalase, and GPx activities for elevated levels of oxidant damage caused by inflammatory reactions in the mammary gland tissue or insufficient nutrition, which has a significant impact on the level of blood lipid peroxidation and lack of energy increases blood plasma levels of MDA in clinical mastitis animals . Nonetheless, this study has limitations to discuss. our baseline scenario assumed that blood parameters were only affected by the mastitis inflammation caused by the bacterial infection, while the bacterial-caused mastitis might result in a decrease in the immune system efficiency which would permit the entrance of secondary invaders or decrease cows' food intake causing a nutritional deficiency sign. Thus, to solve those limitations a future study on broad aspects should be done where a large scale of mastitic cows in different geographical regions inside Egypt examined for different microbiological, environmental, and nutritional disorders besides, performing a correlation between the revealed data to a better understanding the linkage of mastitis inflammation and microbes. This can be accomplished with the help of new computational biology techniques or through sophisticated technology and artificial intelligence algorithms. Moreover, the study emphasizes the necessity to evaluate additional cytokines to the already studied, including Interleukin-1 alpha (IL-1a) Interleukin-4 (IL-4), Interleukin-17 (IL-17), Interleukin-13 (IL-13), cathelicidin LL37, nuclear factor kappa B (NF-kB), and transforming growth factor-beta 1 (TGF-b1). Together would explain their relationships in detailed interrelations by correlation analysis to substantiate the knowledge of the broad cytokine participating in both host immune defense and mastitis inflammation. Finally, it might be the simplest way to promote the pasteurization of milk so that humans will not get exposed to resistant bacteria. 5. Conclusions In conclusion, the current findings show that dairy cow mastitis is related to significant immunological, and oxidative changes, including hematological, biochemical indicators, oxidative acute phase protein (APP), and inflammatory cytokine with the predisposing factors for mastitis resistance/susceptibility highlighted by our findings. These findings suggest that variations in these biomarkers could be utilized to diagnose such illnesses. The variable pattern of antioxidant, APP, and inflammatory cytokines in subclinical and clinical mastitic dairy cows could be a biomarker for bovine immune status which not only predicts the most susceptible risk time for disease occurrence but also builds up an effective management protocol to improve health through proper breeding and vaccination regimens. Author Contributions A.S. Conceptualization and Visualization; A.S., A.M.M.F. and G.E.E., Validation and Methodology; A.S. and G.E.E., Resources and Data analysis; A.S., A.M.M.F. and G.E.E. Writing-original draft, Writing--Editing and Reviewing; D.E., A.A., N.A., M.F.E.-k. and E.K.E. drafted and revised the manuscript, A.S. and G.E.E. Preparing the manuscript for publication. The authors declare that all data were generated in-house and that no paper mill was used. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The present study was conducted according to the Animal Ethics Procedures and Guidelines of Mansoura University of Egypt and approved by the Animal Administration and Ethics Committee of Lanzhou Institute of Faculty of Veterinary Medicine (Permit No. R/109). The farm owner gave an informed consent form in writing. Informed Consent Statement Not applicable. Data Availability Statement All the data generated or analyzed during this study are available from the corresponding author upon reasonable request. Conflicts of Interest The authors declare no conflict of interest. Abbreviations E. coli, Escherichia coli; S. aureus, Staphylococcus aureus; SCC, somatic cell count; CMT, California mastitis test; MDR, multidrug resistance; MAR, multiple antibiotic resistance index; RBCs, Red blood cell count; Hb, Hemoglobin; PCV, hematocrit value; WBCs, Total leukocyte count; AST, Aspartate aminotransferase; LDH, lactate dehydrogenase; MDA, Malondialdehyde; TAC, Total antioxidant capacity; SOD, Superoxide dismutase; APP, Acute phase proteins; TNF-a, tumor necrosis factor-alpha; IL1B, Interleukin-1b; IL-6, Interleukin-6; IL-10, Interleukin-10; IL-1a, Interleukin-1 alpha; IL-4, Interleukin-4; IL-17, Interleukin-17; IL-13, Interleukin-13; NF-kB, nuclear factor kappa B; TGF-b1, transforming growth factor-beta 1. Figure 1 Serum Acute phase protein of dairy cows with subclinical and clinical mastitis. Data were represented as Mean +- SME. This means in with different superscripts are significantly different (p < 0.05). a significantly different from b and c, a for the high value, b for intermediate value between a and c, while c for the lower value. Haptoglobin (A), fibrinogen (B), Amyloid A (C), ceruloplasmin (D), and ferritin (E). Figure 2 Serum inflammatory Cytokines of dairy cows with subclinical and clinical mastitis. Data were represented as Mean +- SME. This means in with different superscripts are significantly different (p < 0.05). a significantly different from b and c, a for the high value, b for intermediate value between a and c, while c for the lower value.TNF-a (A), IL-1B (B), IL-6 (C) and IL-10 (D). Figure 3 Serum antioxidant/oxidative stress parameters of dairy cows with subclinical and clinical mastitis. Data were represented as Mean +- SME. This means in with different superscripts are significantly different (p < 0.05). a significantly different from b and c, a for the high value, b for intermediate value between a and c, while c for the lower value. Malondialdehyde (MDA, A), total antioxidant capacity (TAC, B), Superoxide dismutase (SOD, C), and catalase (D). animals-13-00892-t001_Table 1 Table 1 Primers were used for amplification in this study. Gene Bacteria Gene Primer Sequences (5' to 3') bp Reference nuc S. aureus Nuc-F F-GTGCTGGCATATGTATGGCAATTG 660 Nuc-R R-CTGAATCAGCGTGTCTTCGCTCCAA 16s-rRNA E. coli 16S rRNA-F F-GCGGACGGGTGAGTAATGT 200 16S rRNA-R R-TCATCCTCTCAGACCAGCTA animals-13-00892-t002_Table 2 Table 2 Antimicrobial sensitivity detected in different Escherichia coli and Staphylococcus aureus isolates from clinical and sub-clinical mastitic milk. Bacteria Antimicrobial Agent Family CPD Resistance No/% Intermediate No (%) Sensitive No/% E. coli Penicillin b-lactam 10 21 (100%) - - Cefuroxime Cephalosporin 30 21 (100%) - - Cefoperazone 30 21 (100%) - - Amikacin Aminoglycoside 30 11 (52.4%) 8 (38.1%) 2 (9.5%) Streptomycin 15 8 (38.1%) 8 (38.1%) 5 (23.8%) Neomycin 5 13 (61.9%) 3 5 (23.8%) Azithromycin Macrolide 15 21 (100%) - - Nalidixic acid Quinolone 30 21 (100%) - - Trimethoprim- Sulfamethazole Sulphonamide 25 21 (100%) - - Gentamycin Lincosamide 10 21 (100%) - - Chloramphenicol Phenicols 30 10 (47.6%) 8 (38.1%) 3 (14.29%) Rifamycin Rifamycin 5 21 (100%) - - S. aureus Ampicillin b-lactam 10 29 (76.3%) 5 (13.2%) 4 (10.5%) Oxacillin 15 29 (76.3%) 5 (13.2%) 4 (10.5%) Ceftazidime Cephalosporin 30 20 (52.6%) 10 (26.3%) 8 (21.1%) Kanamycin Aminoglycoside 30 18 (47.4%) 19 (50%) 1 (2.6%) Streptomycin 15 19 (50%) 13 (34.2%) 6 (15.8%) Norfloxacin Quinolone 10 13 (34.2%) 5 (13.2%) 20 (52.6%) Ciprofloxacin Fluoroquinolone 5 14 (36.8%) 14 (36.8%) 10 (26.3%) Gentamycin Lincosamide 10 28 (73.7%) 10 (26.3%) - Chloramphenicol Phenicols 30 10 (26.3%) 14 (36.8%) 14 (36.8%) Tetracycline Tetracycline 30 9 (23.7%) 9 (23.7%) 20 (52.6%) animals-13-00892-t003_Table 3 Table 3 Antimicrobial sensitivity pattern and multiple antimicrobial resistance detected in different Escherichia coli and Staphylococcus aureus isolates from clinical and sub-clinical mastitic milk. Bacteria Antibiotypes Resistance Pattern Isolates No (%) MAR Index E. coli I C, P, DA, STX, RA, AK, NA, CXM, CEP 1(4.8%) 0.75 II C, P, DA, STX, RA, NA, CXM, CEP, AZM 1(4.8%) 0.75 III P, DA, STX, RA, N, NA, CXM, CEP, AZM 3(14.3%) 0.75 IV C, P, DA, STX, RA, AK, NA, CXM, CEP, AZM 3(14.3%) 0.83 V C, P, DA, STX, RA, N, NA, CXM, CEP, AZM 5(23.8%) 0.83 VI P, S, DA, STX, RA, N, NA, CXM, CEP, AZM 1(4.8%) 0.83 VII P, S, DA, STX, RA, N, AK, NA, CXM, CEP, AZM 4(19.04%) 0.92 VIII P, S, DA, STX, RA, AK, NA, CXM, CEP, AZM 3(14.3%) 0.83 S. aureus I OX, CAZ, AMP 2(5.3%) 0.3 II K, DA, CAZ 4(10.5%) 0.3 III NOR, DA, OX, AMP 1(2.6%) 0.4 IV OX, CLP, CAZ, AMP 1(2.6%) 0.4 V NOR, OX, CAZ, AMP 3(13.2%) 0.4 VI K, C, DA, OX, S 5(13.2%) 0.5 VII C, DA, CAZ, AMP, S 1(2.6%) 0.5 VIII K, OX, CIP, CAZ, AMP 4(13.2%) 0.5 IX DA, OX, CAZ, AMP, S 4(10.5%) 0.5 X NOR, DA, OX, CIP, AMP 4(13.2%) 0.5 animals-13-00892-t004_Table 4 Table 4 Hematological and serum biochemical parameters of dairy cows with subclinical and clinical mastitis. Parameters Control Subclinical Mastitis Clinical Mastitis RBCs (106/mL) 7.41 +- 0.24 a 6.81 +- 0.33 a 5.45 +- 0.19 b Hb (g/dL) 10.13 +- 0.15 a 9.39 +- 0.10 a 8.21 +- 0.07 b PCV (%) 31.51 +- 0.70 a 29.02 +- 0.71 a 26.38 +- 0.13 b WBCs (103/mL) 10.32 +- 0.37 c 13.44 +- 0.09 b 18.05 +- 0.71 a Lymphocyte (103/mL) 7.56 +- 0.16 8.07 +- 0.05 8.34 +- 0.66 Neutrophil (103/mL) 2.32 +- 0.29 c 4.46 +- 0.06 b 9.08 +- 0.36 a Monocyte (103/mL) 0.44 +- 0.18 0.91 +- 0.05 0.64 +- 0.26 AST (U/L) 46.17 +- 2.24 c 66.45 +- 1.22 b 94.57 +- 2.08 a LDH (U/L) 261.21 +- 14.88 c 479.77 +- 9.56 b 766.23 +- 10.81 a T. protein (g/dL) 7.43 +- 0.09 c 8.37 +- 0.08 b 9.02 +- 0.24 a Albumin (g/dL) 3.47 +- 0.15 a 3.20 +- 0.36 a 2.48 +- 0.11 b Globulin (g/dL) 3.97 +- 0.22 c 5.17 +- 0.68 b 6.54 +- 0.31 a Data were represented as Mean +- SME. Means in the same row with different superscripts is significantly different (p < 0.05). AST, Aspartate aminotransferase; LDH, lactate dehydrogenase. 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PMC10000044

The aim of this study was to investigate the effect of low-protein diets supplemented with rumen-protected lysine (RPLys) and methionine (RPMet) on growth performance, rumen fermentation, blood biochemical parameters, nitrogen metabolism, and gene expression related to N metabolism in the liver of Holstein bulls. Thirty-six healthy and disease-free Holstein bulls with a similar body weight (BW) (424 +- 15 kg, 13 months old) were selected. According to their BW, they were randomly divided into three groups with 12 bulls in each group in a completely randomized design. The control group (D1) was fed with a high-protein basal diet (CP13%), while bulls in two low-protein groups were supplied a diet with 11% crude protein and RPLys 34 g/d*head + RPMet 2 g/d*head (low protein with low RPAA, T2) or RPLys 55 g/d*head + RPMet 9 g/d*head (low protein with high RPAA, T3). At the end of the experiment, the feces and urine of dairy bulls were collected for three consecutive days. Blood and rumen fluid were collected before morning feeding, and liver samples were collected after slaughtering. The results showed that the average daily gain (ADG) of bulls in the T3 group was higher than those in D1 (p < 0.05). Compared with D1, a significantly higher nitrogen utilization rate (p < 0.05) and serum IGF-1 content (p < 0.05) were observed in both T2 and T3 groups; however, blood urea nitrogen (BUN) content was significantly lower in the T2 and T3 groups (p < 0.05). The content of acetic acid in the rumen of the T3 group was significantly higher than that of the D1 group. No significant differences were observed among the different groups (p > 0.05) in relation to the alpha diversity. Compared with D1, the relative abundance of Christensenellaceae_R-7_group in T3 was higher (p < 0.05), while that of Prevotellaceae _YAB2003_group and Succinivibrio were lower (p < 0.05). Compared with D1 and T2 group, the T3 group showed an expression of messenger ribonucleic acid (mRNA) that is associated with (CPS-1, ASS1, OTC, ARG) and (N-AGS, S6K1, eIF4B, mTORC1) in liver; moreover, the T3 group was significantly enhanced (p < 0.05). Overall, our results indicated that low dietary protein (11%) levels added with RPAA (RPLys 55 g/d +RPMet 9 g/d) can benefit the growth performance of Holstein bulls by reducing nitrogen excretion and enhancing nitrogen efficiency in the liver. rumen-protected amino acids growth performance nitrogen metabolism Holstein bulls China Agriculture Research System of MOF and MARACARS-37 Hebei Beef cattle Innovation teamHBCT2018130202 This study is Supported by China Agriculture Research System of MOF and MARA; F (CARS-37); Funded by Hebei Beef cattle Innovation team (HBCT2018130202). pmc1. Introduction Protein, as typically the most expensive macronutrient of diets, plays critical roles in the health, growth, production, and reproduction of animals. However, protein ingredient shortages and nitrogen pollution challenge the livestock farming worldwide, albeit these problems have been alleviated in recent decades due to an increase in demand for animal source food from a fast-growing population with rising incomes . Therefore, enhancing the utilization efficiency of dietary protein and reducing excretory losses would be alternative strategies to solve these problems . Low-protein diets have been proven to enhance nitrogen utilization . However, restricting N intake also sacrificed the growth performance and productivity of animals , which has been attributed to limiting amino acid deficiency in low-protein diets . Lysine (Lys) and methionine (Met) are the top two limiting amino acids (LAA) for ruminants . Adding rumen-protected Lys and Met in low-protein diets was considered an efficient way to the meet animal amino acids requirement, as they could escape from rumen degradation and increase the supply of amino acids to the intestines, thus improving the N utilization . Incorporating rumen-protected Lys and (or) Met into low-protein diets was reported to increase dry matter intake in transition cows . Previous studies also suggested that rumen-protected Lys and (or) Met in low-protein diets promoted milk protein yield in high-producing dairy cows and maintained milk production and milk protein yield while reducing the N losses in urine in dairy cows . The question of how to reduce nitrogen emissions of ruminants without affecting their production performance has always been the focus of scholars, and the research in this area has mostly been focused on dairy cows; however, there have been few studies conducted on Holstein bulls. Nitrogen recycling contributes to effective N utilization in ruminants , and ruminal microbiota and the liver play important roles in this nitrogen metabolism . Therefore, the aim of this study was to investigate the effect of low-protein diets supplemented with rumen-protected lysine (RPLys) and methionine (RPMet) on growth performance, rumen fermentation, blood biochemical parameters, nitrogen metabolism, and gene expression related to N metabolism in the livers of Holstein bulls. 2. Materials and Methods This study was conducted between March 2016 and June 2016 at Hongda an animal husbandry in Baoding, P. R. China. The experimental protocol (YXG 1711) was approved by the Institutional Animal Care and Use Committee of Hebei Agricultural University. 2.1. Animals, Experimental Design, and Diets Thirty-six healthy and disease-free Holstein bulls with a similar body weight (BW; 424 +- 15 kg, aged 14 months old) were selected. According to their BW, they were randomly divided into 3 groups with 12 bulls in each group in a completely randomized design. The control group (D1) was fed with a high-protein basal diet (CP13%), while bulls in two low protein groups were supplied diet with 11% crude protein and RPLys 34 g/d*head + RPMet 2 g/d*head (low protein with low RPAA, T2) or RPLys 55 g/d*head + RPMet 9 g/d*head (low protein with high RPAA, T3). Basic diets were prepared according to Japanese feeding standard (2008) for beef cattle (Table 1). The RPAA (Hangzhou Kangdequan Feed Limited Company, Hangzhou, Zhejiang, China) feed was used with a rumen protection rate of 60.0% and was premixed with 100 g of grounded corn which, was used as a carrier for the supplement and was the same amount of grounded corn as that supplied to bulls in the D1 group. All animals were fed ad libitum the basic diets and with free access to clean water. All the experimental animals were housed in tie stalls according to the groups and were fed twice daily at 06:00 and 18:00 h following the removal of the feed refusals before morning feeding. The experiment consisted of 3 periods: a 14-day adaptation period, a 2-month feeding period, and a 7-day sample collection period. Holstein bulls were weighted before morning feeding at the beginning and end of every feeding period. 2.2. Sample Collection The diet offered and refused for individual bulls was weighed every day throughout the trial to average daily dry matter intake (ADMI). Samples of individual feed ingredients, orts, and diets were collected weekly during the experimental period and stored at -20 degC . At the beginning of the experiment, all Holstein bulls were weighed before feeding in the morning to obtain their initial weight. Similarly, at the end of the trial, all Holstein bulls were weighed before morning feeding to obtain the final weight, and the average daily gain (ADG) was calculated as (final weight-initial weight)/test days. Based on the ADMI and ADG, the feed weight ratio (F/G) was calculated. At the end of the feeding period, four Holstein bulls in each group were randomly selected, and a 10-mL blood sample was collected via jugular venipuncture from each bull before morning feeding. The samples were immediately centrifuged at 3000 rpm for 15 min, and the serum samples were collected and stored at -20 degC for further analysis. After 2 h of morning feeding at the end of the feeding period, the ruminal fluid samples of four bulls were collected via an oral stomach tube equipped with a vacuum pump. We discarded the first 100 to 200 mL of fluid collected to reduce the chance that the stomach tube rumen samples were contaminated with saliva. Once again, approximately 200 mL of rumen fluid was collected, and about 20 mL was taken, filtrated with four layers of sterile cheesecloth, and then transferred to 2-mL sterile tubes and stored in liquid nitrogen for further analysis. Three bulls in each group were randomly selected and euthanized at the end of the feeding experiment after 2 h of morning feeding. The middle part of liver tissue was immediately collected after animal sacrifice and cut into 5-mm fragments; the tissue sample was then placed into sterile tubes and stored in liquid nitrogen for further analysis. Another three bulls in each group were randomly selected after the feeding period and were transferred to metabolic cages. After a 5-day adaption period, feces and urine were collected during the next 3 days. Total feces and urine were respectively collected daily before morning feeding. The feces of each bull were weighted, mixed, subsampled (100 g/kg), and stored at -20 degC. Each bull fecal sample was evenly divided into two parts, one with 10% (10:1) sulfuric acid solution and the other without acid, before being dried, crushed, sifted, and stored at room temperature for the determination of nutrient content. The urine of each bull was collected using a plastic container with 10 mL of 10% sulfuric acid to prevent the loss of ammonia; then, after the volume was measured, the urine was filtered with four layers of gauze filter, and subsamples (100 mL/individual) were stored at -20 degC for urine nitrogen measurement. 2.3. Laboratory Analysis Offered and refused feed and feces were dried at 55 degC for 48 h, ground to pass through a 1-mm screen (Wiley mill, Arthur H. Thomas, Philadelphia, PA, USA), and stored at 4 degC for analysis of chemical composition. The dry matter (DM, method 934.01), ash (method 938.08), crude protein (CP, method 954.01), ether extract (EE, method 920.39), Ca (method 927.02), and P (method 965.17) contents of the samples were determined according to the procedures of the AOAC , and NDF (amylase) and ADF content was analyzed using the methods of Van Soest et al. . Lysine and methionine content in the feed was analyzed using an automatic AA analyzer (Hitachi 835, Tokyo, Japan). Serum alanine transferase (ALT), aspartate transferase (AST), albumin (ALB), total protein (TP), glucose (GLU), and blood urea nitrogen (BUN) were analyzed using an automatic biochemical analyzer (Hitachi 7020, Tokyo, Japan). Serum growth hormone (GH) and insulin-like growth factor-1 (IGF-1) contents were measured with enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's specifications (HZ Bio.CO., Shanghai, China). The pH value of the rumen fluid was measured immediately by using a digital pH analyzer (PHS-3C, Shanghai, China), and ammonia nitrogen (NH3-N) and microbial protein (MCP) were determined following recommendations provided in previous studies . Volatile fatty acid (VFA) concentrations in rumen fluid were analyzed using gas chromatography (TP-2060F, Tianpu. Co., Ltd., Beijing, China). The DNA in rumen fluid was extracted using the CTAB method using a commercial kit (Omega Bio-Tek, Norcross, GA, USA), and, after DNA was purified with 1% agarose gel electrophoresis, the library was constructed using a TruSeq(r) DNA PCR-Free Sample Preparation Kit (Illumina, Inc., San Diego, CA, USA). Then, the constructed library was quantified using HiSeq2500 PE250 (Illumina, Inc., San Diego, CA, USA). Sequences data were analyzed using the QIIME2 pipeline according to a previous study and submitted to NCBI with project ID P2016030502-S2-3-1. The primer of target genes (Table 2) was designed according to the bovine gene sequences reported in NCBI and synthesized by the Shanghai Biotedchnology Technology Corporation Limited Company. The total amount of ribonucleic acid (RNA) was extracted from the liver tissue of Holstein bulls with a miRNeasy kit (Qiagen, Hilden, Germany); then, RNA quality was determined using NanoDrop 2000 (NanoDrop Tec, Rockland, DE) with OD260/OD280 ranging between 1.9 and 2.1. Real-time polymerase chain reaction (PCR) was performed to quantify the expression of target genes, using an SYBR Green PCR Master mix (Takara bio-Co., Shiga, Japan) and following the manufacturer's protocols. The gene expression of liver tissue was calculated using the method of 2-DDCt, where the expression of ACTB was used as referenced D1. 2.4. Statistical Analysis The data management was performed using a spreadsheet program with Excel, and statistical analysis was carried out using R software (version 3.6.3, R Foundation for Statistical Computing, Vienna, Austria.) with a one-way analysis of variance (ANOVA) model: Y = a + Xi + ei, where Y is the observed parameters, a is the overall mean, Xi is the ith treatment effect, and ei is the residual error. All data were shown using least squares means, and significant differences among treatments were declared at p < 0.05 and a tendency if 0.05 < p <= 0.10. 3. Results 3.1. Growth Performance There was no significant difference (p > 0.05) in ADG, ADMI, and F/G among different groups; however, the F/G in the T2 and T3 groups decreased by 8.45% and 6.67%, respectively, compared with D1 (Table 3). 3.2. Nitrogen Metabolism Compared with the D1 group, the intake of nitrogen and the amount of nitrogen excretion by feces and urine were significantly lower in the T2 and T3 groups (p < 0.05). The ratio of nitrogen excretion by feces and nitrogen intake (FN/IN) was lower in T3 compared with the D1 and T2 groups, while the ratio of nitrogen excretion by urine and nitrogen intake (UN/IN) was lower in the T2 and T3 groups compared to the D1 group. Thus, a significantly higher nitrogen utilization rate was observed in both T2 and T3 groups compared with the D1 group (p < 0.05; Table 4). 3.3. Serum Biochemical Index Low-protein diet with RPAA supplementation had no effect on concentrations of ALT, AST, ALB, TP, GLU, and GH in serum (p > 0.05). Concentration of serum BUN significantly decreased; however, the concentration of serum IGF-1 significantly increased in the T3 group compared with the D1 group (p < 0.05; Table 5). 3.4. Rumen Fermentation No significant difference was detected in the rumen pH, concentration of NH3-N, MCP, propionate, and butyrate, and in the ratio of acetate/propionate among different groups (p > 0.05). The concentration of acetate in the T3 group was significantly higher than that in D1 and T2 (p < 0.05; Table 6). 3.5. Rumen Microbiota No significant difference was observed in alpha diversity among the different groups (p > 0.05; Table 7). The relative abundance of the highest 16 abundant bacteria at the genus level was compared among the different groups. However, the relative abundance of Ruminococcaceae_NK4A214 in the T3 group was lower than that in the D1 group (p < 0.05), and the abundance of Christensenellaceae_R-7_group in the T3 group was lower than that in both D1 and T2 groups (p < 0.05). Meanwhile, the relative abundance of Prevotellaceae_YAB2003_group in T3 was higher than that in the D1 group (p < 0.05), and the relative abundance of Succinivibrio in T3 was higher than that in both the D1 and T2 groups (p < 0.05; Table 8). 3.6. Gene Expression in Liver Tissue The expression of the CPS-1, ASS, ARG, OTC, and N-AGS genes, which relate to nitrogen metabolism or urea metabolism in liver tissue, are shown in Figure 1. The expression of CPS-1, ARG, and N-AGS was significantly upregulated in the T3 group (p < 0.05), although no significant difference was observed between the rT2 and D1 groups (p > 0.05). The expression of CPS-1, ARG, and N-AGS increased by 25%, 18%, and 13% in the T2 group compared with D1. The expression of ASS and OTC was upregulated in both the T2 and T3 groups compared with D1 (p < 0.05). The expression of the SLC3A2, IRS1, PDK, P13K, TSC1, TSC2, mTORC1, eIF4EBP1, S6K1, and eIF4B genes, which are related to the nitrogen metabolism in liver tissue, are shown in Figure 2. The low-protein diet with RPAA supplementation did not affect gene expression of SLC3A2, P13K, TSC2, and eIF4EBP1 (p > 0.05); however, the expression of IRS1, PDK, S6K1, and eIF4B genes in liver tissue increased significantly (p < 0.05), and the expression of the mTORC1 gene also increased (p = 0.09), while the expression of TSC1 gene decreased significantly (p < 0.05). 4. Discussion Protein is one major factor that affects the health, growth, and production of ruminants. Moreover, although people tend to formulate high-protein diets to achieve a better production of ruminants, the global protein shortage is increasing , and high-protein diets overload the environment by increasing nitrogen (N) excretion through urine and feces , which is harmful for the sustainability of the livestock industry. By providing bulls with a low-protein diet (11% CP) supplemented with rumen-protected lysine and methionine, our findings indicate that, compared with a high-protein diet (13% CP) group which followed the recommended Japanese feeding standard for beef cattle , our low-protein diet supplemented with RPAA increased ADG and N utilization and decreased N excretion through urine and feces. These findings were comparable with previous studies in which the feeding of rumen-protected Lys and (or) Met to castrated cattle increased daily gain and reduced urinary nitrogen and urea nitrogen in urine . The World Health Organization (WHO) proved that the addition of RPAA to a low-protein diet increases N utilization, reduces N emission and environmental pollution, and promotes the growth performance of dairy cows . Blood biochemical parameters are sensitive to animal health and nutrient condition . The serum content of ALT, AST, ALB, TP, GLU, BUN, GH, and IGF-1 was used to assess the nutrient condition of bulls with different treatment groups. From this, we observed that BUN content decreased, and IGF-1 content increased, in bulls provided with a low-protein diet supplemented with RPAA, while other indexes were not affected. The serum BUN content reflects the nitrogen balance of ruminants and negatively correlated with N utilization . When ruminants were provided with low-dietary protein with a higher N utilization, serum BUN decreased . The main function of IGF-1 relates to the inhibiting of protein degradation and the promoting of protein synthesis to maintain nitrogen balance and to improve the growth performance of animals . These observations further explained the improvement in N utilization and growth performance of bulls on a low-protein diet supplemented with RPAA. When cattle are fed with low-protein diets, urea N recycling can be considered a high-priority metabolic function because a continuous N supply for microbial growth in the rumen is a strategy for animal survival . The abundance of the microflora reflects its ability to adapt to a particular environment and compete for available nutrients; moreover, it indicates its importance to the overall function of the microbiome as a whole . The ACE (reflecting the richness of bacteria in the sample), Shannon, and PD-whole-tree (reflecting the microbial diversity in feces) indexes were used to assess the alpha diversity of rumen microbiota. Previous studies have demonstrated that rumen fermentation and microbiota are sensitive to protein levels or feed ingredients in ruminants, which were also sensitive biomarkers of N utilization . By monitoring the rumen fermentation and microbiota, we observed an increase in the acetate content of rumen; however, other parameters including NH3-N and MCP content were not significant affected, which is similar to the results of a study by Martin et al. . The addition of methionine analogue 2-hydroxy-4-methylthiobutyric acid (HMB) and esterified 2-hydroxy-4-methylthiobutyric acid (HMBi) to the diet of dairy cows significantly increased the content of rumen total volatile fatty acids (TVFAs) (37). Some studies have shown that methionine hydroxy analogue (MHA) can increase the ratio of acetic acid and butyric acid in rumen content . Research has showed that 0.52% of methionine could increase the content of butyric acid in rumen, while 0.26% methionine did not affect the content of VFA . The above results show that the effect of methionine on rumen VFA content is unpredictable. The alpha diversity of microbiota in rumen was not affected by treatment, and only a small portion of bacteria at the genus level (~5% in abundance) was determined to be significantly different between groups with a decreased relative abundance of Ruminococcaceae_NK4A214_group and Christensenellaceae_R-7_group and increased Prevotellaceae_YAB2003_group and Succinivibrio in bulls on a low-protein diet supplemented with RPAA. These findings hinted that bulls on a low-protein diet supplemented with RPAA would maintain the rumen fermentation and maintain ruminal microbiota homeostasis compared with that from D1. The liver plays important roles in the utilization efficiency of recycled N. The excess nitrogen in the rumen is usually inhaled into the animal's blood in the form of ammonia, which is then metabolized by the liver to synthesize urea. All the urea synthesized by the liver, some of which is secreted via saliva into the rumen and intestines of animals, are reused by bacteria, protozoa, and other microorganisms; the other part is filtered by the kidneys and excreted with the urine . The urea cycle plays a key role in maintaining a positive balance of nitrogen in anima, especially at low dietary nitrogen levels. S6K1 and eIF4EBP1 are genes that regulate protein translation downstream of mTORC1. The S6K1 gene can promote protein translation by stimulating the phosphorylation of downstream eIF-4B, RPS6, eIF-2, and PAPB , and the SLC3A2, IRS1, PDK, P13K, TSC1, TSC2, mTORC1, eIF4EBP1, S6K1, and eIF4B genes are related to nitrogen metabolism in the liver; moreover, these genes would become overexpressed when blood ammonia increased to increase urea synthesis and balance the blood ammonia . However, unexpected results were observed in the current experiment: when feeding bulls with a low-protein diet supplemented with RPAA, we observed that the serum BUN decreased but the expression of genes associated with urea synthesis in liver increased. This finding can explain why the low-protein diet supplemented with RPAA induced an increase in N efficiency; however, the mechanism behind these upregulated genes in the liver was unclear. Previous studies have demonstrated that AA in diets not only provide animal nutrition but also act as a functional regulator and have ability to stimulate expression altering in multiple tissue cells such as mammary tissue , polymorphonuclear cells , and adipose tissue , as well as liver tissue . The influence of RPLys and RPMet on liver genes' expression requires further study. As the number of samples selected in this study is limited, it is necessary to further test the current data in the future research. 5. Conclusions In summary, providing low dietary protein (11%) with RPLys (55 g/d) and RPMet (9 g/d) to bulls could increase their nitrogen utilization rate, serum IGF-1 content, ruminal acetate content, and expression genes associated with urine metabolism and nitrogen metabolism in liver compared to that with high protein (13%). Our findings indicate that providing a low-protein diet supplemented with RPAA could benefit bulls mainly by increasing liver nitrogen metabolism and utilization; however, the RPAA's affecting of liver gene expression at a nutrition level or as a signal molecule still requires further study. Author Contributions Conceptualization, S.Z., B.L., Y.L. and Q.L.; methodology, S.Z.; software, S.Z.; validation, S.J. and M.W.; formal analysis, S.Z.; investigation, S.Z. and Y.L.; resources, Q.L.; data curation, S.Z., H.X., B.L. and Y.L.; writing--original draft preparation, S.Z. and S.J.; writing--review and editing, H.X., M.W. and Q.L.; visualization, S.Z.; supervision, Y.S., Y.G., J.L., Y.C. and Q.L.; project administration, Y.G., J.L., Y.C. and Q.L.; funding acquisition, Y.C. and Q.L. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by Institutional Animal Care and Use Committee of Hebei Agricultural University (YXG 1711). Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors have declared that they have no competing interests. Figure 1 The relative expression levels of CPS-1, ASS, ARG, OTC, and N-AGS mRNA in liver. Control group (D1), low protein with low RPAA (T2), and low protein with high RPAA (T3). CPS-1 = Carbamoyl-phosphate synthase 1; ASS = Argininosuccinate synthase; ARG = Arginase; OTC = Ornithine carbamoyltransferase; N-AGS = N-acetylglutamate synthase. *: The difference is significant. **: The difference is extremely significant. Figure 2 The relative expression levels of SLC3A2, IRS1, PDK, P13K, mTORC1, eIF4EBP1, S6K1, and eIF4B mRNA in liver. Control group (D1), low protein with low RPAA (T2), low protein with high RPAA (T3). SLC3A2 = Solute carrier family 3 (amino acid transporter heavy chain), member 2; IRS1 = Insulin receptor substrate 1; PDK = Phosphoinositide-dependent protein kinase; P13K = Phosphoinositol 3-kinase; TSC1 = Tuberous sclerosis complex 1; TSC2 = Tuberous sclerosis complex 2; mTORC1 = Mammalian target of rapamycin complex 1; S6K1 = Ribosomal protein S6 kinases; eIF-4B = Eukaryotic initiation factor. *: The difference is significant. **: The difference is extremely significant. animals-13-00843-t001_Table 1 Table 1 Ingredients and chemical compositions of experimental diets. Items 2 Treatment 1 D1 T2 T3 Ingredient, % of DM Flaked corn (%) 35.72 37.7 37.7 Bran (%) 0 3.72 3.72 Soybean meal (%) 4.2 1 1 Cottonseed meal (%) 7.5 3.8 3.8 DDGS (%) 4 5.2 5.2 NaHCO3 (%) 0.83 0.83 0.83 Premix 2 (%) 2.75 2.75 2.75 RPLys (g/d) 34 55 RPMet (g/d) 2 9 Corn straw silage (%) 36 36 36 Millet straw (%) 9 9 9 Total (%) 100 100 100 Chemical composition CP (%) 13.00 11.00 11.00 Ca (%) 0.73 0.73 0.73 P (%) 0.36 0.35 0.35 NDF (%) 38.59 39.25 39.25 ADF (%) 23.11 22.82 22.82 Lys (%) 0.24 0.40 0.51 Met (%) 0.14 0.14 0.18 NEmf 3 (MJ/kg) 6.42 6.42 6.42 MP (%) 10.03 9.09 9.09 1 (1) control group (D1), (2) low protein with low RPAA (T2), (3) low protein with high RPAA (T3). 2 The premix provided the following per kg of feed: VA 8 800 IU, VD3 2 035 IU, VE 82.5 mg, D-biotin 1.1 mg, Nicotinamide 55 mg, b-carotene 3.3 mg, Cu 18.7 mg, Mn 49.5 mg, Zn 82.5 mg, Mg 880 mg, Co 0.66 mg, I 1.1 mg, Se 0.55 mg, Ethoxyquin 13.75 mg. 3 NEmf is a calculated value, while the other nutrient levels are measured values. animals-13-00843-t002_Table 2 Table 2 Primer of target genes in liver. Gene Name Accession Number Primer and Probe Sequences Amplification Products Mammalian target of rapamycin complex 1 (mTORC1) XM_002694043.5 Forward: AAGCCGCGCGAACCT 177 Reverse: CTCCATGGTGACGTAGTGCT Insulin receptor substrate 1 (IRS1) XM_003585773.4 Forward: CTCCAGCGAGGATCTAAGCG 100 Reverse: TAGGTCTTCATTCTGCTGTGATGT Phosphoinositol 3-kinase (P13K) NM_001206047.1 Forward: CTTATTTCAAGAGCCCCGTGGT 115 Reverse: TGGAGCCATCAGACGCTATC Phosphoinositide-dependent protein kinase (PDK) NM_001205957.1 Forward: TGAGAGCAACGATGGAGCAT 107 Reverse: TCCTCGGTCACTCATCTTCAC Tuberous sclerosis complex 1 (TSC1) XM_005213449.3 Forward: CCTGTCTACACAGACAACCCC 157 Reverse: ATGGCCGCTTCTTCTTTATCCT Tuberous sclerosis complex 2 (TSC2) XM_010819198.2 Forward: AGAAGAGCTGGCGGACTTT 120 Reverse: GATGCGATGTACTCATCAAGGT Eukaryotic initiation factor (eIF-4B) XM_005206207.1 Forward: CTGGAAGGGGATGTTTCAACC 116 Reverse: ATATTGGGTTCCCGAGCAGC Eukaryotic initiation factor 4E binding protein 1 (eIF-4EBP1) NM_001077893.2 Forward: CGGGGTCACTAGCCCTACA 105 Reverse: AACTGTGACTCTTCACCGCCTG Ribosomal protein S6 kinases (S6K1) NM_205816.1 Forward: CAACCAGGTCTTTCTGGGTT 121 Reverse: TGTTCGTGGGCTGCCAATAA Solute carrier family 3 (amino acid transporter heavy chain), member 2 (SLC3A2) NM_001024488.2 Forward: GCAACCTAGCGGACCTAAAGGA 109 Reverse: GTCTCTGTGAGGTCATCCTCC Carbamoyl-phosphate synthase 1 (CPS-1) XM_015458817.1 Forward: TGGGATTAAGGTTGCAGGTTTG 100 Reverse: CTTTTCTTCCCGTAGCCACT N-acetylglutamate synthase (N-AGS) XM_002696039.4 Forward: CTCTTCAGCAACAGGGGTTC 141 Reverse: GTAGTCGTCCCGGAGCTTTT Ornithine carbamoyltransferase (OTC) NM_177487.2 Forward: AGTGCGGCTAAATTCGGGAT 113 Reverse: AGCTTGGTACCGTTCTCCTTG Argininosuccinate synthase (ASS) NM_173892.4 Forward: CCACAGGAAAGGGGAACGAC 100 Reverse: TAGAACTCGGGCATCCTCCA Arginase (ARG) XM_005210924.3 Forward: AGAACTAGAGTGTGATGTGAAAGA 122 Reverse: CAGCCAGCTTTTCACTTGCT animals-13-00843-t003_Table 3 Table 3 Effects of low-protein dietary inclusion of RPLys and RPMet on average daily gain, average daily feed intake, and feed/gain ratio of Holstein bulls. Items 2 Treatment 1 p-Value D1 T2 T3 ADG (kg) 1.37 +- 0.22 1.42 +- 0.06 1.55 +- 0.07 0.055 ADMI (kg) 10.09 +- 1.20 10.17 +- 0.83 10.09 +- 0.81 0.967 F/G 7.34 +- 1.05 7.20 +- 0.80 6.72 +- 0.75 0.157 1 (1) Control group (D1), (2) low protein with low RPAA (T2), (3) low protein with high RPAA (T3). 2 ADG = average daily gain; DMI = dry matter intake; ADMI = daily dry matter intake; F/G = feed weight ratio. animals-13-00843-t004_Table 4 Table 4 Effects of low-protein dietary inclusion of RPLys and RPMet on nitrogen metabolism of Holstein bulls. Items 2 Treatment 1 P-Value D1 T2 T3 Intake of Nitrogen (IN) (g/d/*head) 216.76 +- 3.49 a 181.81 +- 0.40 b 179.69 +- 1.16 b <0.001 Fecal Nitrogen(FN) (g/d*head) 71.10 +- 0.42 a 59.98 +- 0.76 b 57.50 +- 0.23 c <0.001 Urinary Nitrogen (g/d*head) 62.76 +- 1.20 a 45.74 +- 2.89 b 43.31 +- 0.38 b <0.001 FN/IN (%) 32.81 +- 0.48 a 32.99 +- 0.39 a 32.00 +- 0.17 b 0.037 UN/IN (%) 28.95 +- 0.33 a 25.16 +- 1.59 b 24.10 +- 0.17 b 0.002 Nitrogen Utilization Rate (%) 38.24 +- 0.70 b 41.85 +- 1.97 a 43.90 +- 0.31 a 0.004 Nitrogen Metabolic Rate (%) 56.91 +- 0.69 b 62.45 +- 0.31 a 64.55 +- 0.31 a 0.002 1 (1) Control group (D1), (2) low protein with low RPAA (T2), (3) low protein with high RPAA (T3). 2 Note: nitrogen utilization rate (%) = 100 x (IN - FN - UN)/IN; nitrogen metabolic rate (%) = 100 x digestion nitrogen rate/nitrogen utilization rate. a, b, c Means within a row with different superscripts differ (p <= 0.05). animals-13-00843-t005_Table 5 Table 5 Effects of low-protein dietary inclusion of RPLys and RPMet on blood biochemistry of Holstein bulls. Items 2 Treatment 1 p-Value D1 T2 T3 ALT(U/L) 23.95 +- 1.30 24.68 +- 0.39 25.49 +- 6.03 0.833 AST(U/L) 70.78 +- 3.80 71.01 +- 2.34 73.00 +- 6.50 0.758 ALB(g/L) 23.79 +- 0.68 23.89 +- 1.50 24.32 +- 1.52 0.872 TP(g/L) 62.94 +- 1.27 62.97 +- 1.23 64.47 +- 1.24 0.196 GLU(mmol/L) 3.35 +- 0.14 3.44 +- 0.14 3.49 +- 0.25 0.542 BUN(mmol/L) 2.80 +- 0.50 a 2.31 +- 0.24 ab 1.81 +- 0.09 b 0.026 GH(mg/L) 22.66 +- 4.53 22.99 +- 1.84 23.16 +- 2.14 0.980 IGF-1(mg/L) 142.91 +- 1.74 b 144.47 +- 3.36 ab 149.32 +- 2.14 a 0.047 1 (1) Control group (D1), (2) low protein with low RPAA (T2), (3) low protein with high RPAA (T3). 2 ALT = serum alanine transferase; AST = aspartate transferase; ALB = albumin; TP = total protein; GLU = glucose; BUN = blood urea nitrogen; GH = serum growth hormone; IGF-1 = insulin-like growth factor-1. a, b Means within a row with different superscripts differ (p <= 0.05). animals-13-00843-t006_Table 6 Table 6 Effects of low-protein dietary inclusion of RPLys and RPMet on rumen fermentation of Holstein bulls. Items 2 Treatment 1 p-Value D1 T2 T3 pH 6.50 +- 0.21 6.39 +- 0.05 6.44 +- 0.13 0.598 NH3-N(mg/dL) 12.67 +- 1.34 12.29 +- 3.03 12.23 +- 3.35 0.978 MCP(mg/mL) 4.59 +- 1.37 4.91 +- 0.41 4.95 +- 0.77 0.843 Acetate(mmol/L) 86.35 +- 6.65 b 87.56 +- 0.94 b 95.54 +- 1.30 a 0.026 Propionate(mmol/L) 36.92 +- 2.52 37.84 +- 2.91 38.01 +- 0.36 0.818 Butyrate(mmol/L) 14.13 +- 1.85 14.28 +- 0.43 13.32 +- 0.01 0.546 Acetate/Propionate (A/P) 2.41 +- 0.11 2.32 +- 0.20 2.40 +- 0.18 0.801 1 (1) Control group (D1), (2) low protein with low RPAA (T2), (3) low protein with high RPAA (T3). 2 NH3-N = ammonia nitrogen; MCP = microbial protein; VFA = volatile fatty acid (VFA). a, b Means within a row with different superscripts differ (p <= 0.05). animals-13-00843-t007_Table 7 Table 7 The effects of RPLys and RPMet on the alpha diversity analysis index of Holstein bulls. Items Treatment 1 p-Value D1 T2 T3 Richness index Shannon 8.37 +- 0.57 8.53 +- 0.37 8.09 +- 0.49 0.462 Diversity index ACE 2178.57 +- 281.67 2063.77 +- 81.19 1942.29 +- 90.40 0.224 Phylogenetic diversity PD-whole-tree 94.83 +- 5.33 95.79 +- 5.48 88.50 +- 6.73 0.218 1 (1) Control group (D1), (2) low protein with low RPAA (T2), (3) low protein with high RPAA (T3). animals-13-00843-t008_Table 8 Table 8 Effects of low-protein dietary inclusion of RPLys and RPMet on bacterial genus in the rumen fluids of Holstein bulls. Items Treatment 1 p-Value D1 T2 T3 Prevotella_1 23.78 +- 0.11 23.16 +- 7.20 27.72 +- 6.56 0.589 Rikenellaceae_RC9_gut_group 5.39 +- 0.22 4.87 +- 0.89 3.87 +- 1.33 0.209 Succinivibrionaceae_UCG-002 4.59 +- 2.09 3.92 +- 0.98 3.87 +- 1.08 0.805 unidentified_Bacteroidales_RF16_group 3.88 +- 1.19 2.67 +- 0.65 3.28 +- 1.49 0.380 Ruminococcaceae_NK4A214_group 2.61 +- 0.42 a 2.22 +- 0.63 ab 1.40 +- 0.52 b 0.030 Ruminococcaceae_UCG-014 2.48 +- 0.51 2.50 +- 0.38 2.44 +- 0.78 0.990 Ruminobacter 2.28 +- 0.66 3.35 +- 2.64 2.04 +- 0.47 0.495 Christensenellaceae_R-7_group 2.04 +- 0.36 a 1.85 +- 0.53 a 1.02 +- 0.41 b 0.022 UGG003 Prevotellaceae_UCG-003 1.80 +- 0.44 2.52 +- 0.98 1.78 +- 0.53 0.275 Treponema_2 1.52 +- 0.13 1.64 +- 0.31 1.93 +- 0.38 0.290 Fibrobacter 1.42 +- 0.44 1.68 +- 0.35 1.23 +- 0.24 0.360 Ruminococcus_1 0.76 +- 0.13 0.70 +- 0.20 0.57 +- 0.1 0.301 Selenomonas_1 0.55 +- 0.11 0.53 +- 0.27 0.57 +- 0.07 0.958 Succinimonas 0.55 +- 0.15 0.48 +- 0.16 0.35 +- 0.03 0.236 Prevotellaceae_YAB2003_group 0.27 +- 0.02 b 0.38 +- 0.14 ab 0.59 +- 0.05 a 0.011 Succinivibrio 0.20 +- 0.04 b 0.26 +- 0.03 b 0.53 +- 0.08 a 0.001 1 (1) Control group (D1), (2) low protein with low RPAA (T2), (3) low protein with high RPAA (T3). a, b Means within a row with different superscripts differ (p <= 0.05). 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PMC10000045

Clinical, electrocardiographic and echocardiographic parameters in Pega donkeys are scarce in the literature; hence, this study was performed to describe the echocardiographic and electrocardiographic measurements in Pega breed donkeys. The objectives of this study were to describe and illustrate the clinical, electrocardiographic, and echocardiographic parameters in Pega donkeys used for reproduction. Fifty Pega breed donkeys were evaluated, with an average age of 3.4 years and with 20 males and 30 females. In each animal, the electrocardiographic examination at rest was performed using the TEB(r) computerized system, and the echocardiographic examination was performed using an ultrasound device with a Doppler function multifrequency sectorial transducer in 2D mode (Sonosite(r) M turbo). Standardizing the electrocardiographic and echocardiographic parameters for the Pega breed donkey can contribute to future assessments regarding possible changes that excessive effort can promote in these parameters to a management engrossed on animal welfare. heart equine electrocardiogram echocardiogram donkeys This research did not receive external funding. pmc1. Introduction Currently, donkeys are animals of great economic interest and as companion animals, a fact that has increased awareness of the welfare and care of these animals, increasing the demand for specialized veterinary services . Anatomical and physiological differences have been reported between donkeys and horses; therefore, the use of clinical data, treatments, and diagnostic protocols from horses to donkeys can trigger diagnostic errors and inappropriate therapeutic administration . There are a variety of differences in behavior and social organization between donkeys and horses, although they are often housed as companion animals or as a homogeneous group. However, this does not mean that the nature of social relationships between different species of equids is the same as that between their own species . Brazil has the largest herd of horses in Latin America and the third largest in the world. There are 8 million donkeys and horses, with transactions of R$ 7.3 billion only through the production of horses . The main breeds of donkeys used to obtain mules are the Paulista or Brasileiro, the Pega, and the Northeastern donkey. The Pega is a national donkey breed with economic value throughout the national territory, and it is raised for the production of new breeders and for marching mules. It is a versatile animal used for pack animals, in preparing the soil, herding cattle, horseback riding, functional tests, walking contests among many modalities, and is currently the most used in the training of saddle-type. The animals of the Pega breed are strictly gaiters and have great value in the production of hybrids (mules). The magpie donkey market is focused on breeding. The Pega donkey is medium-sized with a minimum height at the withers of 125 cm for males and 120 cm for females. Male animals of this breed weigh approximately 300 kg, and females weigh 240 kg . A suspicion of potential heart disease may appear after routine examinations or during the prepurchase evaluation of an animal. On the other hand, the care of an equine may be requested due to a specific complaint of cardiovascular signs; however, this assessment is not as widespread in donkeys . The clinical symptoms or the absence of them are important factors in the assessment of a possible cardiac study for reflection on the potential differential diagnoses . Heart disease is rare in horses compared to other domestic species due to the large cardiac reserve, and overt clinical signs are often seen only when there is severe dysfunction or during forceful exercise. Although murmurs and arrhythmias are commonly detected in equines, they are often of physiological origin and have no pathological significance . Arrhythmias during or immediately after exercise are common occurrences in equine athletes. The spectrum of these rhythm variations covers clinically irrelevant arrhythmias, arrhythmias that can cause poor performance, and rhythms with risk of death . A standard base-apex electrocardiogram at rest should be performed in all horses with arrhythmia not attributable to second-degree atrioventricular block . Digital electrocardiogram telemetry systems are readily portable and can be used to obtain real-time digital monitoring and recording at rest or during exercise . In donkeys, there is little information in the literature regarding arrhythmic events that may occur during exercise, and there is no standardized information about possible arrhythmias that can be considered common in the species . Exercise-induced cardiac fatigue and cardiac dysrhythmias are well-described conditions identified in high-level human athletes that increase in frequency with intensity and duration of exercise . Thus, since electrocardiographic and echocardiographic parameters in Pega donkeys are scarce in the literature, as well as the description of possible arrhythmic events that may be considered common in the species and the description of cardiovascular diseases, the objectives of this study were to describe and illustrate the clinical, electrocardiographic at rest and echocardiographic parameters in Pega donkeys, which may contribute to future studies aiming to assess the influence of exercise on such parameters as well as the activity of the autonomic nervous system in these animals during overexertion since they are already adapted for such efforts. The description of these parameters in Pega donkeys can contribute to a better understanding of specific changes that may occur in donkeys, where evaluations in horses are often used as a reference for these animals, which can lead to erroneous conclusions, and the particularities of different categories and breeds of animals must be respected. 2. Materials and Methods 2.1. Animals and Study Site The present study was carried out according to animal welfare standards and approved by the Ethics Committee on the Use of Animals (CEUA) of the Faculty of Veterinary Medicine and Animal Science of the Sao Paulo State University "Julio de Mesquita Filho", Botucatu Campus, under the protocol CEUA-0029/2021. The present study was carried out on 50 clinically healthy adult donkeys aged under 13 years, 20 males (stallions) and 30 females. Before enrollment in the study, all donkeys underwent complete physical examination to exclude systemic conditions (colic, respiratory diseases, orthopedic diseases). The animals were kept in paddocks and individual pens and were fed with Tifton hay produced on the property and pelleted feed with 15% protein; they were offered a total of 3 kg of daily feed divided into two portions, and commercial mineral salt was added for horses at will. The study was carried out at the stud farm Criatorio Ximbo, located in the district of Maristela-SP, belonging to the city of Laranjal Paulista-SP, with latitude 23deg02'9'' south and longitude 47deg50'12'' west. For this purpose, 50 donkeys were evaluated, with an average age of 3.4 years; there were 20 males and 30 females, all from the same establishment. The average height at the withers of the animals was 1.30 m, and the average weight was 230 kg (the males had a height at the withers of approximately 1.35 m and the females 1.20 m, with the weight of the males being approximately 260 kg and the females 230 kg). The animals in the present study were mainly used for reproduction. Figure 1 illustrates a donkey, Pega breed, 8 years old, male. The study was carried out after approval from the animal use ethics committee in accordance with the animal welfare standards upon signature of the owner's informed consent form. 2.2. Analysis Groups The animals were evaluated according to their clinical parameters, such as heart rate (HR--beats per minute--bpm), respiratory rate (RR--movements per minute--mpm), mucosal color, rectal temperature (degC), and capillary filling time (CFT). As there are no specific heart rate (HR) parameters for donkeys, the HR values were divided according to the reference for horses (28-60) and similar to the division into groups according to HR adopted by Guccione and colleagues . Thus, the division of the study according to HR by class was as follows: class 1: HR <= 30 bpm, class 2: HR 30-60 bpm, and class 3 > 60 bpm. 2.3. Electrocardiographic Examination The electrocardiographic examination was performed at rest using the TEB(r) computerized system (Brazilian Electronic Technology, Sao Paulo-SP, Brazil), and the electrocardiographic tracings were recorded with a sensitivity of 1 mV = 1 cm and at a speed of 25 mm/s, compiling the bipolar I, II, and III lead and unipolar amplified aVR, aVF, and aVL. The electrodes were attached to the skin using "alligator" clips soaked with alcohol. Recordings were performed as described by Loon and colleagues (2010) , placing the positive electrode on the left side above the heart apex (green), just behind the olecranon; the negative electrode on the right side, cranial to the scapula, close to the jugular vein (red); and the ground electrode attached to the animal's withers (black). For each electrocardiographic record, HR, rhythm, P wave duration and amplitude, PR interval duration, QRS complex duration, R wave amplitude, S wave amplitude, QT interval, and QTc duration were analyzed, as well as the duration and amplitude of the T wave. 2.4. Echocardiographic Examination To obtain the echocardiographic evaluation, the animals were kept in a station, manually contained, without any type of sedation for the examination. Trichotomy and cleaning of the right and left thoracic regions were performed 10 cm above the height of the olecranon, with the right thoracic limb carefully placed forward. The echocardiographic examination was performed using an ultrasound device (M-turbo Sonosite model, Fujifilm do Brasil Ltd.a., Sao Paulo-SP, Brazil) with a Doppler function and a 2-8 MHz multifrequency sectorial transducer in 2D mode. The transducer was positioned between the 4th and 5th intercostal space. The measurements were always performed by the same operator, obtaining three measurements from each assessment. Through the right parasternal window, in cross-section, M-mode, at the height of the papillary plane, in diastole were measured: interventricular septal thickening (IVS), left ventricular internal diameter (LVID), left ventricular free wall thickness LVFW, and right ventricular diameter in diastole (RIVDd). In systole, interventricular septal thickening (IVS), left ventricular internal diameter (LVID), and left ventricular free wall thickness (LVFW) were analyzed. The fractional shortening of the left ventricle (FS) was obtained with the Teichholz method. To calculate the FS (%), the following formula was used: (LVIDd-LVIDs/LVIDd) x 100, and the left ventricular ejection fraction (EF) was also recorded. The diameter of the left atrium (LA) and the aorta on the same plane was measured, and subsequently, the left atrium/aorta ratio was calculated (LA/Ao) . Pulmonary flow velocity (pulmonary velocity) and pressure gradient between the right ventricle and pulmonary artery at the level of the pulmonary plane was also measured. Pulmonary diameter and aortic diameter were also measured, and the pulmonary/aorta ratio (pul/ao) was obtained from the cross-section of the base. Aortic flow velocity (Ao) was measured. The pressure gradient between the left ventricle and aorta artery was also compiled by the left parasternal window to optimize the outflow tract view. 2.5. Statistical Analysis The results are illustrated with the mean, standard deviation, and minimum and maximum values. To analyze the parameters, the normality test used was the Shapiro-Wilks test; to compare the proposed moments, the Mann-Whitney test was used. All discussions were carried out at a 5% significance level. 3. Results The mean, standard deviation, and minimum and maximum values of the clinical parameters in the Pega donkey are represented in Table 1. The average age of the animals was 3.4 years, with an HR of 67 bpm and an RR of 35 mpm. The mean values and standard deviation of echocardiographic measurements and parameters of the healthy Pega breed donkeys are represented in Table 2. The mean values and standard deviation of electrocardiographic measurements and parameters of the 50 healthy Pega breed donkeys are tabulated in Table 3. Table 3 shows the mean HR of 50 donkeys (65 bpm). According to the class division approved in the study, no animal presented sinus bradycardia and an HR below 35 bpm (class 1 = 0), 22 animals (44%) had an HR within class 2 (30-60 bpm), and 28 animals (56%) presented an HR compatible with class 3 (>60 bpm). In view of the horse reference, the predominant rhythm was sinus tachycardia. Bifid P waves were observed in the animals in the present study. There was a predominance of the QRS complex pattern of the rS-type . The amplitude of the T wave varied, with 45% (22 animals) showing positive T waves, 28% (14 animals) showing negative T waves, and 28% showing biphasic T waves. Arrhythmic events were not detected in the animals in the present study. 4. Discussion The present study represented the normal reference range of echocardiographic and electrocardiographic measurements and parameters in healthy Pega donkeys. The mean HR results in this study from a clinical examination (67 bpm) and electrocardiogram (ECG) (65 bpm) were higher than those reported by Guccione and colleagues in donkeys (47 bpm). The authors evaluated the HR in 15 donkeys with the Holter examination during the day and night. The mean HR in our study and that reported by the mentioned authors in horses are superior, indicating that there are differences in the activity of the autonomic nervous system (ANS) between donkeys, asses, and horses. There is a need for studies explaining and comparing ANS activity between them. The different acquisition techniques of these parameters should also be considered since in our study, the HR recording was immediate to manipulation, which may have contributed to higher values. Escudero and colleagues carried out a study to evaluate the electrocardiographic parameters in both in Zamorano-Leones using the conventional method. In their study, most animals presented a bifid P wave similar to what occurred in our study. In our study, there was a predominance of the QRS complex pattern of the rS-type, differing from the study by the aforementioned authors, who found predominance of the QR pattern. We believe that these differences may be due to different breeds and sexes, as the authors mention that they found a QS pattern only in females. A negative shape was present for the T wave in lead II, which is in agreement with the results obtained by Escudero and colleagues. The method used for the electrocardiographic assessment and the breed may have influenced the different results for the duration and amplitude of the electrocardiographic waves. Al-Haidar and colleagues demonstrated in horse studies that breed has an influence on echocardiographic parameters. The scarce information in the literature on electrocardiographic parameters in donkeys requires studies aimed at standardizing clinical and electrocardiographic parameters with a focus on breed differences and not only in relation to the difference between species. Just as in horses, there are differences in echocardiographic parameters between breeds; in donkeys, there may be differences in electrocardiographic parameters. The echocardiographic parameters obtained in the M mode in the present study were higher when compared with the donkey parameters originated by Roberts and colleagues , and LVFWd for the Pega breed was similar to those of the aforementioned authors for males with an average of 1.68 cm. Horse heart size and dimensions increase in relation to the animal's height and weight, but according to Farag and colleagues , a study with donkeys did not find an association between echocardiographic parameters in both the B mode and the M mode with age and body weight. In our study, we did not divide the animals according to weight, but the parameters analyzed as a whole are for males and females. In horses, age has a great effect on the inner diameter of the aortic and pulmonary arteries. In addition, body weight also has a significant effect on all echocardiographic dimensions, but sex has no effect on any of them . In humans, the aortic diameter may increase with age, and collagen changes and systemic arterial hypertension may contribute to aortic artery aneurysm . Weight, as already proven in allometric scales, influences echocardiographic parameters. Therefore, studies dividing Pega breed donkeys according to different age groups and body weights are needed. Scarce data in the literature and the need for racial distinction emphasize the need to standardize electrocardiographic and echocardiographic parameters in donkeys. Because the animals do not show obvious clinical signs, cardiovascular diseases in this species can be underdiagnosed. The early diagnosis of changes in heart rate and cardiovascular structure can contribute to the use of these animals in modalities that require excessive effort and reduce the number of conditions that go undiagnosed . Cardiovascular disease can be seen in donkeys, presumably with a prevalence similar to horses. Available information about these conditions is uncommon in this species, which does not make the diseases rare. However, a large proportion of cardiovascular diseases are described in horses that lead to reduced performance. Considering that donkeys and assess are not commonly used as riding animals and their athletic attitude is limited, these diseases could easily be underdiagnosed . According to Reef and colleagues , a regular sinus rhythm with a range HR at rest from 24 bpm to 44 bpm is the most common rhythm detected in horses. The adult horse has high vagal tone present at rest, especially in good physical conditions, resulting in low resting heart rates and rhythm disturbances that disappear with excitement, exercise, or any intervention that increases the sympathetic tone. Thus, according to reports by Reef and colleagues and with the mean heart rates found in the animals in this study, we observed that donkeys have high HR compared to horses, which may lead us to infer that reference values for horses should not be used to assess normality parameters for donkeys. Studies using HRV analysis in this context are necessary since, as illustrated in this study, donkeys seem to have a higher sympathetic tone when compared to horses, which have a predominance of vagal tone. Several studies describe cardiovascular affections in horses, as well as electrocardiographic patterns ; echocardiographic patterns in the species and in foals are also related to weight, sex, and age, as well as common changes in the category of athlete animals . According to the echocardiographic parameters obtained in the present study, donkeys seem to have lower values for these parameters in horses, as described in the literature. Because Pega donkeys are widely used in work modalities that require physical effort , we highly recommend echocardiographic and electrocardiographic parameter examinations. In recent years, in human and veterinary medicine, modern Doppler echocardiography techniques have been introduced to assess myocardial function by measuring cardiac muscle velocities and deformations. Studies in marathon runners on the effect of training on myocardial deformation parameters demonstrated that longitudinal myocardial contractility and high early diastolic velocities (E wave) were directly correlated with increased left ventricular diastolic internal diameters . Future studies in donkeys can also be used to assess myocardial function to establish the impact that high workloads can have on the cardiovascular system. Finally, several studies have illustrated the effect of estrogen and testosterone on ventricular function. Castration studies also suggest gender-specific differences in cardiac structure and function. Castrated male rats showed reduced heart weight with a significantly lower ejection fraction and cardiac output, hypocontractility, and delayed cardiomyocyte relaxation, whereas exercise attenuated and testosterone replacement completely reversed these effects. Male rats showed decreased contractile reserve and a faster transition to heart failure with left ventricular dilatation, loss of concentric remodeling, and diastolic dysfunction compared to their female counterparts. In addition, estradiol is positively associated with right ventricular ejection fraction while in men and women, testosterone was positively associated with right ventricle mass and volume . We believe that the effects of estrogen may have influenced our results, and studies also focusing on hormone dosages could contribute in greater magnitude to illustrate the effect of hormones in donkeys on electrocardiographic and echocardiographic parameters. Heart rate variability corresponds to the fluctuation in the interbeat intervals and can measure the functioning of the ANS. Actually, in horses, as well as in other animals, HRV analysis methodologies and data interpretation are still debated. Therefore, it is interesting to investigate the use of HR and HRV in donkey clinics and research, especially in relation to animal welfare, where noninvasive and reliable indicators are urgently needed . Our study may contribute to future studies aiming to evaluate ANS activity in donkeys. Our study has some limitations. The age of the animals may have influenced the results since the groups included animals aged 13 years and 1 year. We used animals used only for reproduction. Studies using other categories of animals are needed. We believe that gender influenced our results, but studies using hormone levels could provide better information. We did not divide the animals in the group into different types of weight, which may have influenced the results because males tend to have higher weight than females, and we used animals of both sexes in our group. 5. Conclusions Echocardiographic and electrocardiographic evaluation is feasible in donkeys, and there are parameter similarities described in the equine species. For the analysis and investigation of cardiovascular diseases in the species, it is necessary to evaluate specific parameters for donkeys. The standardization of such parameters can contribute to future studies aiming to evaluate the influence of exercise on the parameters of this species since, in horses, the changes induced by exercise in cardiac electrical conduction are described in the literature, as well as contributing to studies evaluating the heart rate variability in the species. Acknowledgments The authors want to thank farm Criatorio Ximbo, Maristela-SP, Brazil, and Jose Carlos Mendonca (Calu), who allowed us to carry out the study on his stud farm. Author Contributions All authors took part in designing the study. M.L.G.L. contributed with conceptualization; A.S.C.A. and K.C.d.O. contributed with data curation; A.S.C.A. contributed with formal analysis; L.V.d.O.F. and R.K.S.C. contributed with investigation; S.B.C. contributed with methodology; M.L.G.L. and S.B.C. contributed with project administration; D.A.C.Q. contributed with resources and software; M.H.T. contributed with statistical analysis; M.L.G.L. contributed with supervision and validation; K.C.d.O. and L.V.d.O.F. contributed with visualization; A.S.C.A. contributed with roles/writing--original draft, writing--review and editing. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The present study was carried out according to animal welfare standards and approved by the Ethics Committee on the Use of Animals (CEUA) of the Faculty of Veterinary Medicine and Animal Science of the Sao Paulo State University "Julio de Mesquita Filho", Botucatu Campus, under the protocol CEUA-0029/2021. Informed Consent Statement Not applicable. Data Availability Statement The datasets are available from the corresponding author on reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 A donkey Pega breed, 8 years old, male. Figure 2 Image obtained by the two-dimensional echocardiogram of a donkey, male, 6 years old, right parasternal window, cross-section, aortic plane, left atrium/aorta ratio (LA/Ao). Figure 3 Electrocardiogram, donkey, male, 6 years old, base-apex plane, rS pattern and bifid P waves. animals-13-00861-t001_Table 1 Table 1 Clinical parameters (mean, standard deviation, minimum and maximum) in Pega breed donkeys. Parameter Mean +- SD Minimum Maximum Age (years) 3.46 +- 3.08 1.00 13.00 HR (bpm) 67.20 +- 16.38 40.00 120.00 RR (mpm) 35.60 +- 9.08 20.00 60.00 T degC 37.82 +- 0.63 36.60 39.10 animals-13-00861-t002_Table 2 Table 2 Echocardiographic parameters in Pega breed donkeys from the M-mode of the LV and the short axis view of the LA and aorta (mean, standard deviation, minimum and maximum). Parameter Mean +- SD Minimum Maximum IVSd (cm) 1.74 +- 0.34 1.26 2.59 LVIDd (cm) 6.80 +- 1.08 4.60 9.17 LVFWd (cm) 1.73 +- 0.33 1.17 2.67 IVSs (cm) 3.05 +- 0.55 1.83 4.59 LVIDs (cm) 4.07 +- 0.71 2.45 5.47 LVFWs (cm) 2.59 +- 0.46 1.77 3.84 RIVDd (cm) 1.81 +- 0,61 1.0 3.74 EF (%) 68.82 +- 7.09 53.00 86.00 FS (%) 39.92 +- 5.99 27.90 55.80 LA (cm) 5.27 +- 0.64 4.03 6.51 Ao (cm) 4.03 +- 0.59 2.67 5.14 LA/Ao 1.31 +- 0.14 1.02 1.67 Pulmonary diameter 3.28 +- 0.45 2.25 4.30 Aortic diameter 4.04 +- 0.59 2.70 5.17 Pul./Ao 0.82 +- 0.10 0.66 1.35 Pul.Veloc. (cm/s) 91 +- 14.71 64.60 135.00 Pul.Gr. Pres.(mmHg) 3.39 +- 1.11 1.67 7.29 Aortic Veloc. (cm/s) 87.60 +- 14.95 60.30 130.60 Ao.Gr. Pres. (mmHg) 3.13 +- 1.06 1.45 6.82 The level of significance was defined as p < 0.05. IVSd: interventricular septal thickening in diastole; LVIDd: left ventricular internal diameter in diastole; LVFWd: left ventricular free wall thickness in diastole; IVSs: interventricular septal thickening in systole; LVIDs: left ventricular internal diameter in systole; LVFWs: left ventricular free wall thickness in systole; RIVDd: right ventricular diameter in diastole; EF: ejection fraction; FS: fractional shortening of the left ventricle; LA: left atrial diameter; Ao: internal diameter of the aortic root; LA/Ao: left atrium and aorta diameter ratio; Pul./Ao: pulmonary and aorta diameter ratio; Pul. Veloc.: Pulmonary velocity; Pres. Gr. Pul.: Pressure gradient between right ventricle and pulmonary artery; Aortic Veloc.: Aortic velocity; Ao. Pres. Gr.: Pressure gradient between the left ventricle and aorta. animals-13-00861-t003_Table 3 Table 3 Electrocardiographic parameters in Pega breed donkeys (mean, standard deviation, minimum, maximum), males (n = 20) and females (n = 30). Parameter Mean +- SD Minimum Maximum HR (bpm) 65.00 +- 16.50 35.00 95.00 P (ms) 111.00 +- 18.38 73.00 160.00 P (mV) 0.31 +- 0.05 0.18 0.46 PR (ms) 230.80 +- 41.57 150.00 337.00 QRS (ms) 112.36 +- 15.84 87.00 177.00 R (mV) 0.10 +- 0.02 0.04 0.20 S (mV) 1.90 +- 0.45 0.93 3.03 QT (ms) 432.74 +- 61.12 320.00 573.00 QTc (ms) 442.00 +- 32.90 349.00 538.00 T (ms) 137.58 +- 32.20 80.00 213.00 T (mV) 0.46 +- 0.17 0.23 0.90 RR (ms) 974.36 +- 277.47 630.00 1757.00 The level of significance was defined as p < 0.05. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000046

Epithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties. EMT has been closely associated with cancer cell aggressiveness. The aim of this study was to evaluate the mRNA and protein expression of EMT-associated markers in mammary tumors of humans (HBC), dogs (CMT), and cats (FMT). Real-time qPCR for SNAIL, TWIST, and ZEB, and immunohistochemistry for E-cadherin, vimentin, CD44, estrogen receptor (ER), progesterone receptor (PR), ERBB2, Ki-67, cytokeratin (CK) 8/18, CK5/6, and CK14 were performed. Overall, SNAIL, TWIST, and ZEB mRNA was lower in tumors than in healthy tissues. Vimentin was higher in triple-negative HBC (TNBC) and FMTs than in ER+ HBC and CMTs (p < 0.001). Membranous E-cadherin was higher in ER+ than in TNBCs (p < 0.001), whereas cytoplasmic E-cadherin was higher in TNBCs when compared with ER+ HBC (p < 0.001). A negative correlation between membranous and cytoplasmic E-cadherin was found in all three species. Ki-67 was higher in FMTs than in CMTs (p < 0.001), whereas CD44 was higher in CMTs than in FMTs (p < 0.001). These results confirmed a potential role of some markers as indicators of EMT, and suggested similarities between ER+ HBC and CMTs, and between TNBC and FMTs. E-cadherin epithelial-to-mesenchymal transition mammary tumors SNAIL TWIST ZEB This research received no external funding. pmc1. Introduction Mammary gland cancer is the most common tumor in women and in female dogs , and the third most common neoplasia in cats . Human breast cancer (HBC) is classified into four main subtypes according to the expression of estrogen receptor (ER), progesterone receptor (PR), and epidermal growth factor receptor ERBB2, as follows: (i) Luminal A tumors (ER+ and/or PR+, ERBB2-); (ii) Luminal B tumors (ER+ and/or PR+, ERBB2+); (iii) ERBB2-overexpressing tumors (ER-, PR-, ERBB2+); and (iv) triple-negative (ER-, PR-, ERBB2-) breast cancer (TNBC) . TNBCs are typically high-grade carcinomas characterized by an aggressive behavior and a poor prognosis, with high risk of distant metastasis and death . Canine mammary tumors (CMTs) are classified based on morphologic features . Fifty per cent of CMTs are malignant with a 20% risk of metastasis . The majority (80-90%) of feline mammary tumors (FMTs) are characterized by a highly aggressive behavior that leads to rapid progression and distant metastasis development . Typically, FMTs lack the expression of ER, PR, and ERBB2, and have been considered a remarkable spontaneous model for TNBC . In all three species, mammary tumors exhibit both inter- and intra-tumor heterogeneity as a consequence of genetic and non-genetic aberrations . Over the past 20 years, the investigation of cell differentiation/phenotypic markers has been used in both human and veterinary medicine, primarily to improve our knowledge of the histogenesis of mammary tumors . In the normal human, canine, and feline mammary gland, two cell subpopulations are present: luminal epithelial cells, positive for cytokeratin (CK) 7, CK8, CK18, and CK19; and basal/myoepithelial cells, variably positive for CK5, CK6, CK14, CK17, SMA, calponin, vimentin, and p63 . In HBC, the evaluation of cell differentiation proteins is frequently performed in association with routine diagnostic markers (ER, PR, ERBB2, and Ki-67) to better classify this tumor. The identification of HBC subtypes has a diagnostic, prognostic, and therapeutic value, and is associated with the cell differentiation and epithelial-to-mesenchymal transition (EMT) status of the neoplastic population according to a hierarchical model . EMT is a key event that neoplastic epithelial cells use to acquire a mesenchymal phenotype . As a result, tumor cells obtain the ability to detach from the primary tumor mass, invade the surrounding tissue, migrate throughout the body, and eventually give rise to metastases in distant organs . The classical EMT is characterized by a decreased expression of epithelial markers and a complementary upregulation of mesenchymal markers. Classical EMT transcription factors, namely snail family transcription repressor 1/2 (SNAIL), TWIST, and zinc-finger-enhancer binding protein 1/2 (ZEB) are known to orchestrate EMT by regulating cell adhesion, migration, and invasion, also interacting with different signaling pathways and microRNAs . Although this is a well-de-scribed process that promotes metastasis formation, accumulating evidence suggests the existence of an intermediate state called partial EMT or hybrid E/M, whereby both epithelial and mesenchymal markers are co-expressed in cancer cells . The aim of this study was to investigate the mRNA expression of classical EMT-related transcription factors SNAIL, TWIST, and ZEB in human, canine, and feline mammary tumors. Additionally, we studied the expression of key proteins involved in the EMT process, including E-cadherin and vimentin, and of proteins related to the tumor phenotype, such as ER, PR, ERBB2, Ki-67, cytokeratin (CK) 8/18, CK5/6, CK14, and CD44. 2. Materials and Methods 2.1. Tissue Collection Human samples were collected from the Istituto Oncologico Veneto (IOV, Padua, Italy), whereas canine and feline samples were collected from local veterinary clinics. The human sample collection was approved by the IOV Ethics Committee. All patients or patients' owners provided informed, written consent to use their samples for this study. Specifically, samples from 5 healthy human mammary gland tissues (MGTs), 5 ER+ HBCs, 5 TNBCs, 4 healthy canine MGTs, 10 canine mammary tumors (CMTs) (5 grade I and 5 grade II), 6 healthy feline MGTs, and 6 grade III FMTs were collected. In this study, to avoid contaminations with other tumor cell subpopulations, we selected only simple tubular carcinomas (STC), which are composed of only one tumor cell subpopulation (luminal epithelial cells) . Healthy MGTs were collected from tumor-bearing patients during the therapeutic/diagnostic surgical procedures, with no additional sampling performed only for the study. Sampling was performed by surgeons. At the time of sampling, most of the tissue was fixed in 4% formaldehyde for histopathology and immunohistochemistry, whereas a peripheral small portion of tumor and normal tissues (approx. 0.5 cm2 each) was collected and preserved in RNALater (Ambion, Austin, TX, USA), according to manufacturer's instructions. In the lab, before RNA extraction, a small portion of each RNALater-preserved sample was fixed in 4% formaldehyde and embedded in paraffin to check the content of the samples themselves. Four-mm tissue sections were stained with hematoxylin and eosin, and slides were visualized under the microscope to further confirm the presence of healthy tissue in the samples labelled as "healthy" and of tumor tissue in the samples labelled as "tumor". 2.2. RNA Extraction and Real-Time Polymerase Chain Reaction For gene expression analysis, a small portion of each tissue sample preserved in RNALater was used for RNA extraction using Trizol Reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer's protocol. The extracted RNA was treated with RNAse-free DNAse I (New England Biolabs, Ipswich, MA, USA). Five-hundred ng of total RNA from each sample was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (Invitrogen). The cDNA was then used as a template for quantitative real-time PCR using the ABI 7500 Real-Time PCR System (Applied Biosystem) to evaluate the mRNA expression of the following EMT-related genes: SNAIL1, SNAIL2, TWIST1, TWIST2, ZEB1, ZEB2. All the samples were tested in triplicate. ACTB was used as a house-keeping gene. The primer sequences are reported in Table 1. The primers were designed using NCBI Primer-BLAST. To examine primer specificity, the dissociation curves of qPCR products were assessed to confirm a single amplification peak. The qPCR reactions were then purified using the ExoSAP-IT PCR product cleanup (Applied Biosystems) and sequenced at the BMR Genomics (Padua, Italy). The sequences were then verified using the NCBI BLAST database. For data analysis for each sample, the DDCt value was calculated and expressed as a relative fold change (2-DDCt), as described in . Real-time PCR efficiency was calculated by performing a dilution series experiment and applying the following formula to the standard curve: efficiency = 10(-1/slope) - 1 . Real-time PCR efficiency was between 90 and 100% for all the samples. 2.3. Immunohistochemistry Immunohistochemistry (IHC) was performed on the above-mentioned samples as well as on additional human breast tissue samples from the Division of Anatomic Pathology archive of the University of Padua Hospital, and on additional canine and feline mammary tissue samples from the anatomic pathology archive of the Department of Comparative Biomedicine and Food Science of the University of Padua. Specifically, IHC was per-formed on the following tissue samples: 10 ER+ HBC, 11 TNBCs, 11 CMTs grade I, 11 CMTs grade II, 12 FMTs grade III. Sections (4 mm) were processed with an automatic immunostainer (BenchMark XT, Ventana Medical Systems), as previously described . Briefly, the automated protocol included the following steps: a high-temperature antigen unmasking (CC1 reagent, 60 min), primary antibody incubation (1 h at RT, see below for dilutions), an ultrablock (antibody diluent, 4 min), hematoxylin counterstain (8 min), dehydration, and mounting. Negative controls omitted the primary antibody, whereas adnexa, epidermis, and non-tumor mammary gland, when present, were used as positive controls for CK8/18, CK5/6, CK14, E-cadherin, vimentin, and Ki-67. For ERBB2, an additional technical external positive control was used (ERBB2 3+ HBC), whereas the species-specific cross-reactivity was previously tested in dogs and cats . For ER and PR, feline and canine uterus as well as ovary were also stained as positive controls. For CD44, the lymph node was used as positive control. Positive control tissues, typically collected from necropsies, were derived from the same archive as the canine and feline mammary tumor samples. The following antibodies were tested: anti-ER alpha (anti-ERa) (NCL-ER-6F11 1:40, Novocastra in human and feline species--NCL-ER-LH2 1:25, Novocastra in canine species); anti-PR (NCL-PGR-312 1:80, Novocastra in human and feline species); an-ti-ERBB2 (A0485 1:250, Dako in canine and feline species); anti-CK8/18 (NCL-L-5D3 1:30, Novocastra); anti-CK5/6 (D5/16 B4 1:50, Dako); anti-CK14 (NCL-LL 002 1:20, Novocastra); anti-E-cadherin (610182 1:120, BD Biosciences); anti-CD44 (550538 1:100, BD Biosciences); anti-vimentin (M0725 1:150, Dako); and anti-Ki-67 (M7240 1:50, Dako). In the human species, ERBB2 immunolabeling was performed with Bond Oracle HER2 IHC System for BOND-MAX (Leica Biosystems), containing the anti-ERBB2 antibody (clone CB11, ready-to-use). IHC positivity was semi-quantitatively and separately evaluated by ECVP-boarded (V.Z.) and experienced (L.C.) pathologists. Specifically, cytoplasmic and nuclear positivity were measured as a percentage of positive cells for all markers (100 cells per field in 10 high-power fields were counted). ERBB2 was scored as 0, 1+, 2+, and 3+ according to the American Society of Clinical Oncology (ASCO) 2018 recommendations (10% cut-off), with 2+ and 3+ cases considered weakly and strongly positive for complete membrane immunolabeling, respectively. The protein expression of the studied markers was evaluated in the epithelial/luminal component. Additionally, immunolabeling was observed in healthy/hyperplastic adjacent mammary tissue, and in this case normal basal/myoepithelial cells were also evaluated. 2.4. Statistical Analysis Statistical analyses were performed using Prism version 9.3.1 (GraphPad Software, San Diego, CA, USA). To verify mean differences among groups, either the Student's t-test or the one-way ANOVA with Tukey's multiple comparison test was used, when values were normally distributed. A Mann-Whitney test or Kruskal-Wallis test were used when values were not normally distributed. Normality was tested using the Shapiro-Wilk test. The Spearman's rank correlation analysis was used to analyze associations between variables. The level of significance was set at p < 0.05. 3. Results 3.1. Gene Expression We sought to investigate the mRNA expression of the EMT transcription factors SNAIL, TWIST, and ZEB in mammary tumors compared with healthy tissue. In HBC , SNAIL1 showed a higher mRNA expression in TNBCs when compared with ER+ (p < 0.05). Conversely, the mRNA expression of TWIST1, TWIST2, and ZEB1 in ER+ and TNBCs was significantly lower than in healthy MGTs (p < 0.05). Additionally, TNBCs had a significantly lower mRNA expression of SNAIL2 and ZEB2 when compared with healthy MGTs (p < 0.05). In CMTs , SNAIL1 showed a higher mRNA expression in STC II when compared with healthy MGTs (p < 0.01) and STC I (p < 0.001). The mRNA expression of SNAIL2, ZEB1, and ZEB2 was lower in tumors than healthy MGTs, although not statistically significant. In FMTs , tumors showed a lower mRNA expression of SNAIL1, SNAIL2, TWIST1, TWIST2, ZEB1, and ZEB2 when compared with healthy MGTs, which was significant only for ZEB1 (p < 0.05). 3.2. Immunohistochemistry Next, we aimed to study the expression of key proteins involved in the EMT process. The expression of the studied markers was evaluated in the tumor epithelial luminal cell population. CD44 and ERBB2 staining was membranous, whereas CK8/18, CK5/6, CK14, and vimentin staining was cytoplasmic. E-cadherin staining was present in either or both membrane and cytoplasm and it was separately evaluated. Ki-67, ER, and PR staining was nuclear. As expected, epithelial luminal cells of healthy MGT in all three species were diffusely positive for CK8/18, membranous E-cadherin, ER, PR, and occasionally positive for CK5/6, CK14, and CD44. The basal/myoepithelial cells of healthy MGT in all three species were diffusely positive for CK5/6, CK14, CD44, and vimentin, and occasionally also positive for ER and PR. Results for the human, canine, and feline mammary tumors are summarized in Table 2, Table S1 and are graphically represented in Figure 4. In HBC , ER+ tumors had a high protein expression (roughly 100%) of CK8/18, whereas they were negative for basal cytokeratins CK5/6 and CK14. In TNBCs, the protein expression of CK8/18, although fairly heterogeneous, was lower than in ER+ (p < 0.001) and the protein expression of CK5/6 was higher than in ER+ (p < 0.05). In ER+ tumors the protein expression of E-cadherin was predominantly membranous , whereas in TNBCs E-cadherin protein expression was often lost from the membrane and pre-dominantly cytoplasmic . Membranous E-cadherin protein expression was higher in ER+ than in TNBCs (p < 0.001), whereas cytoplasmic E-cadherin protein ex-pression was higher in TNBCs when compared with ER+ (p < 0.001) . Overall, the expression of this protein was quite heterogeneous across the samples. Interestingly, a strong negative correlation between membranous and cytoplasmic E-cadherin protein expression was found in ER+ (r = -1, p < 0.001) and in TNBCs (r = -0.9, p < 0.001) . CD44 protein expression was lower in ER+ than in TNBCs , although not statistically significant. Notably, in TNBCs, a strong positive correlation between CK5/6 and CK14 expression (r = 0.8, p < 0.01), and a moderate positive correlation between CD44 and vimentin (r = 0.6, p = 0.05), were found. All CMTs were positive (>1%) for ER and, therefore, classified as ER+. ER protein expression was lower in STC II than in STC I (p < 0.01). The protein expression of E-cadherin was quite heterogeneous across the samples. As in HBC, a strong negative correlation between membranous and cytoplasmic E-cadherin protein expression was found in the CMTs (r = -0.974, p < 0.001) . In addition, in STC II, a strong positive correlation between CK8/18 and membranous E-cadherin (r = 0.8, p < 0.01) and a strong negative correlation between CK8/18 and cytoplasmic E-cadherin (r = -0.8, p < 0.01) were found. Interestingly, in STC II, Ki-67 expression was positively correlated with CK8/18 (r = 0.7, p < 0.05) and membranous E-cadherin (r = 0.8, p < 0.01) expression, and negatively correlated with cytoplasmic E-cadherin expression (r = -0.7, p < 0.05). All FMTs were negative for ER (<1%), PR (<1%), and ERBB2 (either 0 or 1+), and were therefore classified as triple negative. E-cadherin protein expression was quite heterogeneous. As in the HBCs and CMTs, a strong negative correlation between membranous and cytoplasmic E-cadherin protein expression was found (r = -0.984, p < 0.001) . In addition, a strong negative correlation between CK5/6 and vimentin expression was found (r = 0.8, p < 0.01). CD44 protein expression was higher in the CMTs than in the FMTs (p < 0.001) . Vimentin and Ki-67 protein expression was lower in the CMTs than in the FMTs (p < 0.001) . The expression of the studied markers was not associated with other histopathological features, such as vascular invasion or regional lymph node metastases (data not shown). Moreover, no significant correlations were found between gene and protein expression of the analyzed markers. 4. Discussion In this study, we investigated the expression of genes and proteins involved in one of the processes thought to play a major role in cancer progression: epithelial-to-mesenchymal transition . EMT is an evolutionally conserved morphogenetic program during which epithelial cells undergo a series of changes allowing them to acquire a mesenchymal phenotype . During classical EMT, epithelial cells lose the expression of tight junction molecules such as membranous E-cadherin and acquire mesenchymal properties such as migration, invasiveness, and elevated resistance to apoptosis. Transcription factors like SNAIL, TWIST, and ZEB regulate this process and are activated by a variety of signaling pathways, including TGF-a, Notch, and Wnt/b-catenin . SNAIL is a classical regulator of EMT that represses E-cadherin transcription in both mouse and human cell lines . In HBC, it has been associated with tumor recurrence and metastasis , and with poor patient prognosis . In contrast to the findings of other authors , we found that the mRNA expression of SNAIL2 was significantly lower in TNBCs than in healthy MGTs. In CMTs, SNAIL1 expression was higher in STC II when compared with healthy MGTs and STC I, indicating a possible association of EMT with a higher aggressiveness of these tumors. SNAIL2 in CMTs did not show any difference between healthy MGT and tumor tissue, confirming what other authors have also found . Conversely, in FMTs, there was a trend such that STC III had a lower mRNA expression of SNAIL1 and SNAIL2 when compared with healthy MGTs. To the best of our knowledge, SNAIL has never been investigated in feline tumors. It is believed that TWIST plays an essential role in cancer metastasis . In HBCs and FMTs, the mRNA expression of TWIST1 and TWIST2 was lower in tumors than in healthy MGTs, which differs from what some authors have found in HBC , but is similar to what other authors have found in HBC and in FMTs . ZEB1 has been implicated in carcinogenesis in breast tissue because it enhances tumor cell migration and invasion . In our samples, ZEB1 mRNA expression was lower in tumor than in healthy MGTs, as previously reported by other authors in HBC . Although one study examined the expression of ZEB1 and ZEB2 in five canine mammary carcinoma cell lines , to the best of our knowledge, ZEB mRNA expression has never been studied in CMT and FMT tissues. Overall, our data suggest that these transcriptional factors are often downregulated in tumors compared with healthy MGTs, except for SNAIL1 in TNBCs and in CMTs STC II. The RNA isolated from healthy tissues came from the whole mammary gland, which is composed of different cell populations, namely epithelial cells, connective tissue, and fat. Although these transcription factors are barely detectable in normal mesenchymal cells of adult tissues , adipose tissue expresses these genes variably . As a result, the mRNA levels of these genes in healthy samples can be dramatically influenced by the presence of non-mammary gland tissues, such as fat. Moreover, it is possible that the number of cells undergoing classical EMT is low when compared with the tumor bulk, which is known to be characterized by a remarkable intra-tumor heterogeneity . Furthermore, some authors believe that these genes are regulated post-transcriptionally . Furthermore, accumulating evidence suggests the existence of cell populations with a hybrid E/M state, which exhibit increased plasticity and metastatic potential, characterized by the co-expression of epithelial and mesenchymal markers . However, the expression of some of these markers may be associated with a complete EMT status, whereas others may be associated with a partial EMT status. For example, it is believed that SNAIL1 is a stronger inducer of complete EMT than SNAIL2, which is rather associated with a hybrid E/M state . This suggests that the choice of the markers to be analyzed is fundamental and may help in identifying intermediate EMT states more precisely. In addition, in order to study the EMT process, it would be interesting in the future to investigate the expression of these markers at a single cell level, using single-cell omics approaches such as Laser Capture Microdissection or single-cell RNA sequencing. In the present study, we also assessed the protein expression of several phenotypic as well as EMT-related markers, such as ER, PR, ERBB2, CK8/18, CK5/6, CK14, E-cadherin, CD44, vimentin, and Ki-67, in a subset of HBCs, CMTs, and FMTs. The HBC ER+ samples showed a high expression of luminal CK8/18, and a negative expression of basal CK5/6 and CK14, confirming the strong association between ER+ tumors and highly differentiated glandular cells (CK8/18+), as well as null expression of basal CKs (CK5/6, CK14). In the TNBCs, the protein expression of CK8/18 was highly heterogeneous, whereas the expression of CK5/6 and CK14 was low in most of the samples. This result, in concordance with another study , supports the idea that the terms "basal-like cancer" and "triple-negative breast cancer" are not interchangeable. Indeed, only a small percentage of TNBCs are basal-like . The CMTs were positive for ER, whereas the FMTs were negative for ER, PR, and ERBB2. Despite only a few samples being analyzed, these data suggest, as already proposed by other authors , a similarity between CMTs and HBC ER+ and between FMTs and TNBCs. In CMTs and FMTs, the protein expression of CK8/18, CK5/6, and CK14 was highly heterogeneous, confirming the high inter- and intra-tumor heterogeneity . Basal CK14 protein expression was higher in FMTs than in CMTs, confirming that FMTs are more "basal-like" when compared with CMTs . E-cadherin is a cellular adhesion molecule, and its disruption may contribute to the enhanced migration and proliferation of tumor cells, leading to invasion and metastasis . In our samples, E-cadherin protein expression was evaluated in the membrane and in the cytoplasm of tumor cells, separately. Overall, the expression of E-cadherin was highly heterogeneous across the samples of the three species, confirming once more the high inter-tumor heterogeneity of mammary cancer in the three species. In human ER+ tumors, E-cadherin protein expression was predominantly membranous, whereas in TNBCs it was predominantly cytoplasmic, confirming that the delocalization of the protein is associated with increased tumor aggressiveness . These results confirm that it is not only the loss of E-cadherin that correlates with increased tumor aggressiveness, but also the protein translocation from the membrane to the cytoplasm, as already described . Together with E-cadherin, CD44 has been extensively studied in tumor cell differentiation, invasion, and metastasis, and is thought to be involved in the EMT process in HBC . Although a few studies on HBC have shown that protein overexpression of CD44 is associated with poor prognosis and metastasis , others have shown that downreg-ulation of its expression is correlated with an adverse outcome . For this reason, the role of CD44 in the behavior and prognosis of HBC is controversial . In our study, CD44 expression was heterogeneous and lower overall in ER+ tumors compared with TNBCs. This trend agrees with study findings by Klingbeil and collaborators, who found high levels of CD44 expression in tumors with a basal-like or triple-negative phenotype, suggesting an association of this protein with an aggressive phenotype in HBC . CD44 was highly expressed (roughly 85%) in our CMT samples, regardless of the tumor grading, as well as in the healthy mammary gland tissues. Moreover, other authors found no differences between benign CMTs, malignant CMTs, and normal mammary gland tissues, suggesting that CD44 is not associated with aggressiveness in canine mammary tumors . In FMTs, the expression of CD44 was low overall (approximately 5%). Sarli and collaborators evaluated the intramammary/intratumoral and extramammary/extratumoral expression of CD44 in feline normal mammary tissues, benign tumors, and malignant tumors in relationship to lymphangiogenesis . They found that CD44 had a significantly higher expression in intramammary/intratumor areas compared with extramammary/extratumor areas in both benign and malignant tumors. Additionally, no statistically significant differences in CD44 expression between normal mammary gland, benign tumors, and malignant tumors were found. To the best of our knowledge, no other studies on CD44 expression in FMT tissues are present within the literature. These data, together with our findings, suggest that CD44 is not a useful marker of malignancy in cats. Another protein that is well-studied and plays a central role in the EMT process, and therefore in tumor invasion and metastasis, is vimentin . Vimentin is one of the major intermediate filament proteins and is ubiquitously expressed in normal mesenchymal cells . Recent studies have reported that vimentin knockdown causes a decrease in genes linked to HBC metastasis, such as the receptor tyrosine kinase Axl . In our study, we also evaluated the expression of vimentin in HBCs, CMTs, and FMTs. We found a higher expression of vimentin in TNBCs compared with ER+, although not statistically significant. This result suggests that vimentin expression is associated with the triple-negative subtype, aggressive behavior, and a poor prognosis of HBC, as previously reported by many authors . In CMTs, vimentin expression is low (approximately 15%), con-firming the low aggressiveness of mammary tumors in dogs, which is in concordance with the findings of other authors . Conversely, in FMTs, the expression of vimentin, although heterogeneous, was quite high (approximately 70%), suggesting the high aggressiveness of mammary tumors in this species , as well as their similarities with TNBCs . Unfortunately, as a limitation of this study, only grade I and II CMTs were included. No RNALater-sampled canine tumors were diagnosed as grade III. For possible IHC analyses in our archive of paraffin-embedded tissues, a very limited number of grade III simple CMTs were found (14 cases over five years) that were often already vascular/lymph node invasive (10/14). This study would not benefit much from adding only IHC analysis of grade III CMTs that already have invaded the vascular system or with metastases. We still believe that the study allowed the collection of some new data on the most frequent FMTs and CMTs in comparison with HBC samples assessing both gene and protein expression. 5. Conclusions In summary, this study showed that most of the classical EMT-related transcription factors SNAIL, TWIST, and ZEB are downregulated in tumor tissues compared with healthy tissues, although additional analyses should be performed to better investigate them in neoplastic clones and in a larger set of samples. IHC analyses indicated a potential role of some markers, namely vimentin and E-cadherin, but not of others (i.e., CD44) as indicators of EMT (including loss of cell differentiation and increased malignancies). Moreover, all the IHC data seem to support the already proposed similarities between FMTs (grade III) and TNBCs, as well as between CMTs (grade I and II) and ER+ HBCs. The two species are widely discussed as potential spontaneous models of specific HBC subtypes . Supplementary Materials The following supporting information can be downloaded at: Table S1: Immunohistochemical expression of estrogen (ER) and progesterone (PR) receptors and ERBB2 in the samples. Click here for additional data file. Author Contributions Conceptualization, V.Z. and A.S.; methodology, A.S., G.B. and C.G.; formal analysis, L.C., S.F., A.S., G.B., C.G. and S.M.; investigation, A.S.; resources, V.Z., M.P., E.O. and S.M.; data curation, A.S. and V.Z.; writing--original draft preparation, A.S., writing--review and editing, V.Z.; supervision, V.Z. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Ethics Committee of Istituto Oncologico Veneto (IOV, Padua, Italy). Ethical review and approval were waived for the animal samples in this study since as per Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010, regarding the protection of animals used for scientific purposes, the Italian legislature (D. Lgs. n. 26/2014) does not require approval from ethical committees for the use of stored samples in retrospective studies or when using samples submitted for diagnosis where owners signed an informed consent. Informed Consent Statement Informed consent was obtained from all subjects involved in the study and written informed consent was obtained from the owner of the animals. Data Availability Statement The data presented in this study are available upon request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 mRNA expression of SNAIL1, SNAIL2, TWIST1, TWIST2, ZEB1, and ZEB2 in human breast cancer. SNAIL1 showed a higher mRNA expression in TNBCs when compared with ER+ and healthy MGTs. TWIST1, TWIST2, and ZEB1 mRNA expression was lower in estrogen receptor-positive (ER+) and triple-negative breast cancer (TNBCs) than in healthy tissues. SNAIL2 and ZEB2 mRNA expression was lower in TNBCs than in healthy tissues. MGT, mammary gland tissue. * p < 0.05. Figure 2 mRNA expression of SNAIL1, SNAIL2, ZEB1, and ZEB2 in canine mammary tumors. SNAIL1 mRNA expression was higher in simple tubular carcinomas grade II when compared with healthy tissues. SNAIL2, ZEB1, and ZEB2 mRNA expression was lower in tumors when compared with healthy tissues. MGT, mammary gland tissue; STC I, simple tubular carcinoma grade I; STC II, simple tubular carcinoma grade II. ** p < 0.01, *** p < 0.001. Figure 3 mRNA expression of SNAIL1, SNAIL2, TWIST1, TWIST2, ZEB1, and ZEB2 in feline mammary tumors. SNAIL1, SNAIL2, TWIST1, TWIST2, ZEB1, and ZEB2 mRNA expression was lower in simple tubular carcinomas (STC) grade III than in healthy tissues. MGT, mammary gland tissue. * p < 0.05. Figure 4 Immunohistochemistry results for cytokeratin (CK) 8/18, CK5/6, CK14, membranous E-cadherin (E-cad M), cytoplasmic E-cadherin (E-cad C), CD44, vimentin in estrogen receptor-positive (ER+) and in triple-negative breast cancers (TNBCs) (A). CK8/18 and membranous E-cadherin expression was lower in TNBCs than in ER+. CK5/6 and cytoplasmic E-cadherin expression was higher in TNBCs than in ER+. A strong negative correlation between membranous and cytoplasmic E-cadherin expression was found in ER+ (B) and in TNBCs (C). Immunohistochemistry results for ER, CK8/18, CK5/6, CK14, E-cad M, E-cad C, CD44, vimentin, and Ki-67 in canine (CMTs) and feline mammary tumors (FMTs) (D). ER and CD44 protein expression was lower in FMTs than in CMTs. Vimentin and Ki-67 expression was higher in FMTs than in CMTs. A strong negative correlation between membranous and cytoplasmic E-cadherin expression was found in CMTs (E) and FMTs (F). * p < 0.05; *** p < 0.001. Figure 5 Carcinoma, mammary gland. Immunohistochemistry for E-cadherin (A,B) and CD44 (C-F). 20x magnification. (A) Human. Invasive ductal carcinoma, grade I. In estrogen receptor-positive (ER+) human breast cancer, immunolabeling for E-cadherin is mainly membranous. Inset: higher magnification. (B) Human. Invasive ductal carcinoma, grade III. In triple-negative breast cancer (TNBC), immunolabeling for E-cadherin is sometimes partially lost from the cell membrane and often present within the cytoplasm. Inset: higher magnification. (C) Human. Invasive ductal carcinoma, grade III. CD44 expression is rarely positive in ER+ tumors. (D) Human. Invasive ductal carcinoma, grade III. Membranous CD44 immunolabeling is diffusely expressed in TNBCs. I Canine. Simple carcinoma grade II. The immunolabeling for CD44 is membranous and diffusely evident. (F) Feline. Simple carcinoma, grade III. CD44 expression is negative in tumor cells. Figure 6 Carcinoma, mammary gland. Immunohistochemistry for vimentin (A,B) and Ki-67 (C,D). 20x magnification. (A) Canine. Simple carcinoma, grade II. In canine mammary tumors (CMTs), vimentin expression is negative in luminal cells (asterisk) and positive in basal/myoepithelial cells (arrow). (B) Feline. Simple carcinoma, grade III. In feline mammary tumors (FMTs), vimentin expression is evident in approximately 90% of tumor cells, including luminal cells. (C) Canine. Simple carcinoma, grade I. In CMTs, Ki-67 expression is detectable in approximately 10% of tumor cells. (D) Feline. Simple carcinoma, grade III. In FMTs, Ki-67 expression is evident in approximately 40% of tumor cells. animals-13-00878-t001_Table 1 Table 1 Primer sequences used in this study. HUMAN DOG CAT SNAIL1 F: 5'-TTCTCACTGCCATGGAATTCC-3' R: 5'-GCAGAGGACACAGAACCAGAAA-3' F: 5'-ACTGCAGCCGTGCCTTTG-3' R: 5'-AAGGTTCGGGAACAGGTCTTG-3' F: 5'-CACCTGTTTCATGGGCAATTT-3' R: 5'-CATCGGTCAGGCTGAAGCA-3' SNAIL2 F: 5'-GCACACTGAGTGACGCAATCA-3' R: 5'-AGCACAGGAGAAAATGCCTTTG-3' F: 5'-TTTTCTGGGCTGGCCAAA-3' R: 5'-CGCCCAGGCTCACGTATT-3' F: 5'-TGCAGACCCATTCGGATGT-3' R: 5'-CAGCAGCCAGATTCCTCATGT-3' TWIST1 F: 5'-GTCTAGAGACTCTGGAGCTGGATAACT-3' R: 5'-CGCCCTGTTTCTTTGAATTTG-3' - F: 5'-TTAGAAGAGCAGAACCCAAAT-3' R: 5'-CTGCCCGTCTGGGAATCA-3' TWIST2 F: 5'-AGGACGGTCCCCACATAGG-3' R: 5'-ACATAAGACCCAGAAGAAAAATCCA-3' F: 5'-CAGACACGGTCCCCACACA-3' R: 5'-AACCCAGAAGAAAAGATCCAAACA-3' F: 5'-GGAAACGCGACGCTGAGT-3' R: 5'-GGAAGCCACAGATGCACTTTG-3' ZEB1 F: 5'-GATGATGAATGCGAGTCAGATGC-3' R: 5'-ACAGCAGTGTCTTGTTGTTGT-3' F: 5'-AAAATGAGCAAAACCATGATCCTAA-3' R: 5'-CCCTGCCTCTGGTCCTCTTC-3' F: 5'-CCCACACGACCACAGATAAGG-3' R: 5'-TGAATTCATAATCCACAGGTTCA-3' ZEB2 F: 5'-CCAGCTCGAGCGGCATA-3' R: 5'-GCCACACTCTGTGCATTTGAA-3' F: 5'-TTACCCAGGTCGCCCGTAA-3' R: 5'-TTAGCCTGAGCGGAGGATCA-3' F: 5'-CACGATCCAGACCGCAGTTA-3' R: 5'-GTCGCGTTCCTCCAGTTTTC-3' ACTB F: 5'-TGGCACCACACCTTCTACAA-3' R: 5'-CCAGAGGCGTACAGGGATAG-3' F: 5'-TGGCACCACACCTTCTACAA-3' R: 5'-CCAGAGGCGTACAGGGATAG-3' F: 5'-TGGCACCACACCTTCTACAA-3' R: 5'-CCAGAGGCGTACAGGGATAG-3' animals-13-00878-t002_Table 2 Table 2 Immunohistochemistry results in human (HBC), canine (CMT), and feline (FMT) mammary cancer represented as mean +- standard deviation (SD) of percentage of positive cells. Statistics refers to the comparison of the expression of a specific marker between groups of the same species. IHC (Mean +- SD) HBC ER+ HBC TNBC CMT STC I CMT STC II FMT STC III ER 81.7 +- 18.4 negative 30.4 +- 17.4 ** 13.1 +- 16.4 ** negative PR 57.5 +- 42.6 negative NP NP negative ERBB2 0 or 1+ # negative negative negative negative CK8/18 99.5 +- 1.6 *** 53.0 +- 36.9 *** 91.7 +- 10.4 70.8 +- 38.0 73.0 +- 19.9 CK5/6 Negative * 14.2 +- 26.22 * 54.9 +- 32.9 53.6 +- 37.5 26.4 +- 31.8 CK14 0.4 +- 0.8 3.2 +- 6.3 9.6 +- 11.7 13.1 +- 25.4 51.3 +- 37.9 CD44 20.7 +- 25.8 48.9 +- 38.6 85.7 +- 14.5 86.8 +- 12.4 4.7 +- 5.4 E-cad M 77.8 +- 20.5 *** 24.7 +- 15.7 *** 58.9 +- 18.8 66.9 +- 14.8 57.0 +- 23.3 E-cad C 16.4 +- 19.1 *** 62.7 +- 19.3 *** 35.1 +- 15.7 25.8 +- 13.6 37.9 +- 22.3 Vimentin 8.9 +- 26.7 23.8 +- 30.1 4.2 +- 9.3 2.5 +- 3.1 68.9 +- 34.3 Ki-67 NP NP 14.0 +- 7.0 18.4 +- 10.4 49.6 +- 13.9 ER, estrogen receptor; PR, progesterone receptor; E-cad, E-cadherin; M, membrane; C, cytoplasm; TNBC, triple-negative breast cancer; STC I, simple tubular carcinoma grade I; STC II, simple tubular carcinoma grade II; STC III, simple tubular carcinoma grade III. #, ERBB2 was scored as 0, 1+, 2+, and 3+ according to the American Society of Clinical Oncology (ASCO) 2018 recommendations (10% cut-off). None of the samples was scored as 2+ or 3+. * p < 0.05; ** p < 0.01; *** p < 0.001. NP, not performed. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000047

The reason for undertaking this study was to investigate soft tissue response to high-intensity laser therapy (HILT) by measuring changes in skin surface temperature and longissimus dorsi muscle tone in the thoracolumbar back area in Thoroughbreds with back pain and diagnosed with and without Kissing Spines Syndrome (KSS). Thoroughbreds aged 3-4 years with clinically presented back pain underwent a radiological examination (to assess a lack or presence of KSS) and longissimus dorsi muscle palpation (to assess muscle tone and pain degree). The subjects were divided into two groups, those with KSS (n = 10) and those without KSS (n = 10). A single HILT treatment on the longissimus dorsi muscle, on the left side, was performed. Thermographic examination and palpation were repeated before and after HILT to assess changes in skin surface temperature and muscle pain response. In both groups, HILT caused a significant increase in skin surface temperature of 2.5 degC on average and a palpation score reduction of 1.5 degrees on average (p = 0.005 for both measurements), without differences in any outcome measures between the groups. Furthermore, the correlation between changes in the average skin surface temperature and the average palpation scores in horses with and without KSS were negative (rho = 0.071 and r = -0.180, respectively; p > 0.05). The results of the present study are encouraging, but further studies with larger samples, a longer follow-up period and comparisons with placebo control groups are needed to draw a more valid conclusion. high-intensity laser therapy Kissing Spines Syndrome back pain thermography thoroughbreds racehorse This research received no external funding. pmc1. Introduction Thoracolumbar back pain is an important factor causing poor performance in ridden horses . A horse's back protects and stabilizes the body frame and is composed of both soft tissue, such as the muscles and ligaments, and the rigid skeletal elements, which create functional consistency. Equine back disorder may be the result of their individual conformation. Soft tissue injuries are often found in long-backed horses, while osseus lesions are diagnosed in short-backed animals . The longissimus dorsi muscle and supraspinous ligament are commonly found to be the soft tissue cause of thoracolumbar pain . The main cause of epaxial muscle and ligament pain is improper saddle and girth fitting , the animal's emotional tension or physiological stress , and the rider's load on the horse's back . One of the most common osseus causes of back pain is dorsal spinous process impingement, which is a part of overriding dorsal spinous processes, often referred to as "Kissing Spines Syndrome" (KSS) . KSS can be diagnosed in all breeds, at any age, and any sex , but racing Thoroughbreds have been shown to have a higher prevalence when compared with other breeds . KSS is mostly localised between the T10-T18 vertebrae, but it can also affect the lumbar spine . One of the major clinical sings of KSS is chronic longissimus dorsi muscle soreness and increased muscle tone. The pathway of secondary epaxial muscle pain, associated with primary osseus lesions in horses, is not understood in detail. One possible explanation, based on human medicinal knowledge, is that spinal vertebrae diseases cause local ipsilateral multifidus muscle atrophy, resulting in spinal instability. The musculoskeletal system then compensates for the disability by epaxial muscle tension and shortening . The clinical signs of back pain are highly variable , but orthopedic evaluation of muscle pain in horses is traditionally assessed by palpation . Deep palpation allows for a general evaluation of back health, based on spinal mobilization and palpable muscle hypertonicity trigger points . Pain is detected based on the evaluation of "pain reactions", which are measured in terms of several scoring systems and scales used by both scientists and practitioners . Longissimus dorsi pain, whether of primary or secondary origin, reduces thoracolumbar spine flexibility and, thus, disrupts its proper biomechanics . The elimination of epaxial muscle spasms and pain is therefore an important part of treating back diseases in horses. Several treatment modalities are available for treating epaxial muscle pain in horses. Medical therapies include the use of general administered non-steroidal anti-inflammatory drugs or local corticosteroid injections . Alternative treatments include manual therapy such as massage, stretching or chiropractic, and physical therapies like magnetic field therapy, extracorporeal shock wave therapy and hydrotherapy . Currently, laser therapies such as high-intensity laser therapy (HILT) are very popular . HILT is a non-invasive and safe therapy based on the application of focused light generated by class IV (power > 0.5 W) laser devices . It utilises high peak power (1-3 kW), infrared wavelengths (600-1000 nm) and short single pulse durations (>150 ms). As such, HILT can reach deep tissue without excessive thermal effects or cellular damage and can be used in the treatment of bigger joints and large muscle groups, which are difficult to reach with low power lasers . Only a few equine studies have demonstrated the positive results of HILT in the treatment of musculoskeletal disorders. HILT is effective in the treatment of soft tissue injuries, such as tendinopathies and desmopathies . In an earlier article, we described eleven clinical cases of horses with tarsal osteoarthritis, treated with HILT as a monotherapy, where we found that HILT reduced joint pain and lameness grade, but poorly limited joint discomfort after a flexion test in short-term outcomes . Studies describing the impact of laser therapy on muscle pain and tension have been conducted in both experimental animals and humans. Lopes-Martins et al. have found that 0.5 J/cm2 and 1 J/cm2 (655 nm and 2.5 mW) doses of low-level laser therapy prevent the development of muscle fatigue in rats after repeated tetanic contractions. Ramos et al. have reported that 3 J doses of low-level laser therapy at 810 nm and 100 mW are effective for improving functional outcomes in the early phase following tibialis anterior muscle strain injury in rats. In humans, studies have shown that the photothermal effects of HILT may lead to improved muscle relaxation which, thus, reduces pain . Moreover, HILT (808 nm wavelength, continuous wave mode, average power of 4.28 W/cm2) has resulted in positive effects in relieving muscle tension in patients with hemiplegia . The only research which evaluated the clinical effectiveness of laser therapy in treating thoracolumbar pain in horses was performed by Haussler et al. . They found that laser treatment produced significant reductions in back pain, epaxial muscle hypertonocity and trunk stiffness in 61 competitive western performance horses. However, this research involved the use of low-level laser therapy. A review of the published literature was carried out using the following databases: Scopus, Web of Science and Google Scholar. Additionally manual searches were performed in the indices of the Equine Veterinary Journal and Journal Equine Veterinary Science. The key words for the literature search included "HILT", "High Intensity Laser Therapy", "musculoskeletal pain", "muscle pain", "Kissing Spines Syndrome", and "Thoroughbreds". To the best of our knowledge, this study is the first clinical examination of the effectiveness of HILT in treating muscle tissue in horses with back muscle pain. The objectives of the study were to evaluate and compare soft tissue responses to HILT by measuring changes in skin surface temperature and longissimus dorsi muscle tone in the thoracolumbar back area in Thoroughbreds presenting back pain (diagnosed with or without KSS). It was hypothesised that HILT would increases skin surface temperature and reduce longissimus dorsi muscle tone in both groups of horses but that better effects would be achieved in horses without KSS. 2. Materials and Methods The Animal Welfare Advisory Team at Wroclaw University of Environmental and Life Sciences approved the study design, which is in compliance with Polish and European Union legislation on animal experimentation (no 1/2023). The procedures used in this study were deemed not to cause pain, suffering, distress or lasting harm equivalent to or higher than that caused by the introduction of a needle (article 1.5f EU directive 2010/63/EU). Ethical approval was granted without a formal application. Written consent was obtained from Partynice Racecourse in Wroclaw for all the racehorses participating in this study. 2.1. Horses and Inclusion Criteria The horses were selected from the Partynice Racecourse in Wroclaw (Poland) in September 2021. A total of 20 3-4 year-old racehorses (11 stallions and 9 mares) were selected after fulfilling the inclusion criteria. The criteria were as follows: (1) present longissimus dorsi muscle pain in thoracolumbar region palpation; (2) be healthy according to basic clinical examination and to have no clinical lameness in walking and trotting on hard ground in a straight line; (3) maintain the same race training programme with the same trainer; (4) be free from any systemic and local administration of anti-inflammatory drugs or analgetic drugs during the 8 weeks before the study; (5) be free from any manual or physical therapy, including acupuncture, massage, osteopathy or magnetotherapy in the 4 weeks prior to the study; (6) have the presence or lack of radiographic signs of KSS (radiological assessment and KSS criteria are described below); (7) have the presence of pigmented skin in the thoracolumbar back area (black, bay or chestnut coat color). 2.2. Study Design The horses were divided into two groups, those with KSS (n = 10) and those without KSS (n = 10). On the examination day, each horse underwent the same examination protocol. The horses were inspected early in the morning and before any activity. All the measurements were conducted with the animal standing equally on all weight bearing limbs in a stable corridor. Thermographic examination was performed to determine the skin surface temperature of the thoracolumbar back, followed by longissimus dorsi muscle palpation, to assess tone and pain degree. A single laser treatment of the longissimus dorsi muscle was then performed. The thermographic examination followed by palpation were then repeated immediately after HILT to assess changes in skin surface temperature and muscle pain response. 2.3. Radiographic Examination and Palpation Laterolateral projections of the thoracolumbar spine were performed for the purposes of radiological KSS assessment. The horses stood square and weightbearing on hard ground, with the head and neck in a natural position to avoid false changes in the distance between the dorsal spinous processes . The images were blindly and retrospectively evaluated by one veterinarian (P.Z.). According to Turner , a diagnosis of KSS can be made when two or more vertebrae touch or overlap. Non-instrumental palpation, i.e., conventional palpation, was performed unilaterally (left side) on the longissimus dorsi muscle in the area between the fifteenth thoracic and the second lumbar vertebrae. A palpation scoring system for horse muscle tone and pain reaction was used, according to Varcoe-Cocks et al. , which is as follows: (0) soft, with low muscle tone; (1) normal tone; (2) stiff muscle but not painful; (3) stiff and/or painful muscle with slight associated spasms but without horse movement; (4) painful muscle with associated spasms and local horse movement, i.e., pelvic tilt and extension response; (5) very painful muscle with spasm and behavioural response, i.e., ears flat back and kicking. The horses were examined and scored subjectively for longissimus dorsi muscle pain by a qualified equine physiotherapist (M.S.D.) 2.4. Thermographic Examination The thermographic examination was performed with a VarioCam HR infrared camera (uncooled microbolometer focal plane array; resolution, 640 x 480 pixels; spectral range, 7.5-14 mm; accuracy +-1 degC, sensitivity 0.02 degC InfraTec, Dresden, Germany). Prior to the study, the thermal camera was quality assured using an Isotech 988 blackbody calibration source (Isothermal Technology, Stockport, United Kingdom). The examination protocol was the same as previously described by Soroko et al. . To reduce the effect of environmental factors, like air draughts and sunlight, the images were obtained within an enclosed stable with closed windows. Horses were examined with the acclimatization period of 30 min prior the imaging and had a brushed back area, approximately 1 h before examination. The distance between the horse's back and the equipment was set at 1.5 m for all images, with the emissivity set at 1 for all measurements . The average ambient temperature in the stable was 19 degC and humidity 45% at the time that the images were taken, as measured by a TES 1314 thermometer (TES, Taipei, Taiwan). Regions of interest (ROIs) were determined for each thermographic image. The average skin surface temperature of the square treatment area (10 x 10 cm2) in the region under investigation was determined using IRBIS 3 Professional software (InfraTec, Dresden, Germany) . The thermographic examination was performed by the same therapist (M.S.D.). 2.5. High Intensity Laser Therapy The laser therapy was performed with a class IV Polaris HPS laser (Astar, Bielsko-Biala, Poland), a commercial laser light source used in human physiotherapy and in veterinary medicine. The Polaris HPS has two synchronised sources of different wavelength laser light in the near-infrared spectrum. The first wavelength is 808 nm (AlGaAs laser with 8 W of output power), while the second is 980 nm (InGaAs/AlGaAs laser with 10 W of output power). The two wavelengths are emitted simultaneously with the propagation axes of the two laser beams being coincident. The same parameters were used at both wavelengths. The energy density was 20 J/cm2, power was 5 W, frequency was 100 Hz and duty cycle was 80%. The total energy delivered over a period of 500 s was 2000 J. The square treatment area was the same as the region previously identified as an ROI in the thermographic examination and was localised over the longissimus dorsi muscle in the thoracolumbar junction on the horse's left side (in the area between the fifteenth thoracic and the second lumbar vertebrae). The treatment area was not shaved, and no other skin preparation was performed. The laser treatment was administered using a handpiece held in firm contact with the tissue and manually moved during treatment while maintaining even irradiation of the treatment surface. The handpiece spot size was 5 cm2. Both the person holding the horse and the therapist wore laser goggles. The laser scanning in all horses was performed by the same veterinarian (P.Z.). 2.6. Statistical Analysis Statistical analysis of the results obtained was performed using STATISTICA v. 13.3 (TIBCO Software Inc., Palo Alto, CA, USA) and a MS EXCEL template (Microsoft, Redmond, Washington, USA). The data were first plotted for normal distribution analysis using the Shapiro-Wilk test. The significance level was set at p < 0.05. For the quantitative continuous and discrete variables, the following basic descriptive statistics were estimated: medians (Me), lower (Q1) and upper (Q3) quartiles, extreme lowest values (Min) and extreme highest values (Max). Depending on the test results, the non-parametric Wilcoxon signed-rank test was used to compare baseline and post-treatment within a group, while Mann-Withney's U test was used for comparison between the groups. Correlations between differences in skin surface temperatures and palpation score after the application of HILT in the two groups were calculated using Spearman's rank correlation coefficient (rho). 3. Results The average skin surface temperatures and average palpation scores in horses diagnosed with and without KSS, both before and after HILT, did not differ significantly (p > 0.05; Table 1). In both groups, HILT treatment caused average skin surface temperature to increase by 2.5 degC and a palpation score reduction of 1.5, which gave a highly significant difference . There was no correlation between changes in the average skin surface temperature and average palpation scores in either group (rho = 0.071 for group without KSS and r = -0.180 for with KSS, p > 0.05; Table 2). 4. Discussion Horses are predisposed to general back pain and back disorders because of the type and intensity of their work . Furthermore, a study carried out on 572 horses showed that Thoroughbreds have a 76% greater prevalence as well as a higher KSS grading system than other breeds . This is probably because the tips of their spinous processes are close to each other . The elimination of epaxial muscle spasms and pain (whether it is primary or secondary) plays an important role in the welfare of racehorses. In this small trial, the hypothesis that HILT increases skin surface temperature and reduces longissimus dorsi muscle sensitivity in horses with and without KSS was confirmed, but the groups did not differ in terms of the parameters measured, as we expected. Moreover, there was no correlation between changes in the average skin surface temperature and the average palpation score in either group. Similar findings, regarding skin surface temperature increases after HILT, have been reported in our previous study performed on clinically healthy tissue. A single HILT irradiation of the dorsomedial aspect of the tarsal joint in 16 racing Thoroughbreds caused a significant increase in skin surface temperature in the treatment area . In both this study and the previous one, the mean temperature increase was 2.5 degC. The laser parameters used in our previous study were lower compared to those used in the current study. A possible explanation for the similar thermal effect in both studies might be found in regard to the preparation of the irradiated area. In our previous research, the treatment area was additionally clipped. It is known that when laser light passes through a coat it can cause reflection, absorption and a scattering of photons, thus reducing photon penetration in the target tissue . The photothermal effect of HILT induces mild local hyperthermia sufficient enough to accelerate cellular activity and vasodilatation, thus leading to enhanced local blood circulation in irradiated tissue . This results in a hastened removal of inflammatory cytokines, improvement in the mitochondrial oxidation process, production of adenosine triphosphate, and more efficient absorption of tissue swelling . In the current study, we did not control the presence and degree of vasodilatation, although our previous research confirmed vessel diameter increase in irradiated tissue immediately after HILT . Mild inflammation is reduced when tissue temperature increases by more than 1 degC, and an analgesic effect and muscle relaxation is obtained when it increases by 2-3 degC. Changes in tissue extensibility can be reached with a temperature increase of 3-4 degC . Muscle sensitivity is one of the main causative factors in decreased muscle flexibility in horses diagnosed without KSS. Conversely, both bone tissue and inflamed muscles with the connective tissue are the main targets in the treatment of horses diagnosed with KSS. The present study showed a palpation score reduction in the longissimus dorsi muscle of 1 to 2 degrees in both groups of horses. In their prospective study, Alayat et al. also confirmed that the photothermal effect leads to improved fascia extensibility and a muscle-relaxing effect and, thus, a reduction in muscle sensitivity. Similar results have been found in human hemiplegic patients, where HILT rapidly decreased muscle tension during laser irradiation, with low muscle tension maintained following treatment . Muscle relaxation and muscle sensitivity reduction are also the outcome of the photomechanical effect of HILT . A particular waveform with regular peaks of elevated values of amplitude and distances (in time) between them can rapidly induce in the deep tissue a photomechanical effect . HILT can provide extremely brief beats at a maximum repetition rate, thus creating real pressure. Heat waves travel through the tissue, stimulating the free nerve endings and causing their inhibition . A possible explanation for the lack of differences between the groups in longissimus dorsi muscle sensitivity reduction is the short-term HILT efficacy control. It can be assumed that, in horses without KSS, the myorelaxation effect can be longer than in horses with KSS, which (as a chronic disease) causes constant tissue irritation. Lee et al. have pointed out that in humans with hemiplegia, treatment efficacy decreases exponentially as a function of illness duration. Moreover, many other studies have shown that the effectiveness of treatment decreases with the duration of the disease . In our study, we did not clarify the underlying cause of pain in horses without KSS. Furthermore, in the horses with KSS, we have not established the real cause of back pain. Further studies are needed to clarify this matter. None of the horses included in the study experienced skin burns, swelling or pain reactions during or after HILT application. Moreover, all horses had no interruption in their daily race training due to complications after HILT. The maximum temperature reached after HILT was 34.5 degC, while superficial thermal injuries are noted after topical application of heat at 50 degC . The use of different HILT parameters, with different protocols, may be necessary in future research investigating the maximum positive effects of laser therapy, without tissue overheating. The main limitation of this study was the lack of long-term assessment, especially in horses diagnosed with KSS. The other limitation included a lack of control or laser-sham group, and a blinding test was not included for the post-treatment data collection. There is need for follow-up data to investigate the number and frequency of treatments needed to achieve the best therapeutic effects. The relatively small sample size in both groups of horses was also a study limitation. It would be ideal to have a larger number of Thoroughbreds presenting with back pain, regardless of whether they have KSS. However, from a clinical point of view, it was possible to indicate the positive impact of HILT in the case of treatment performed on a large muscle, such as the longissimus dorsi muscle. Automatic laser scanning is also recommended in future research to overcome the limitations of manual scanning, although the same laser operator was used in all applications. 5. Conclusions In conclusion, HILT is a safe and supportive treatment method for longissimus dorsi muscle pain and discomfort in Thoroughbreds under the conditions of this study. Furthermore, the photothermal and muscle-relaxing effects were similar in horses suffering from longissimus dorsi muscle pain, regardless of the presence of KSS. The results of the present study are encouraging, but further blinded studies with larger samples, longer follow-up periods and possible comparisons with placebo control groups are needed to make a more valid conclusion. Author Contributions Conceptualization, P.Z. and M.S.-D.; methodology, P.Z. and M.S.-D.; validation, P.Z. and M.S.-D.; formal analysis, K.D.; investigation, P.Z.; resources, P.Z.; writing-original draft preparation, P.Z.; visualization, K.D.; supervision, M.S.-D. and I.S.R.-G.; project administration, P.Z.; All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The Animal Welfare Advisory Team in Wroclaw University of Environmental and Life Science approved the study design in compliance with Polish and European Union legislation on animal experimentation (no 1/2023). The procedures used in this study are deemed not to cause pain, suffering, distress or lasting harm equivalent to or higher than that caused by the introduction of a needle (article 1.5f EU directive 2010/63/EU), ethical approval was granted without a formal application. Informed Consent Statement Written consent was obtained from Partynice Racecourse in Wroclaw for all racehorses participating in this study. Data Availability Statement Data is contained within that article. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Thermographic images of the thoracolumbar back region. The square area (ROI) indicates the skin surface temperature before HILT (left), with an average temperature of 29.8 degC, and immediately after (right), with an average temperature of 32.9 degC. Figure 2 Differences in skin surface temperatures after HILT application in both groups (with/without KSS) and the Wilcoxon signed-rank test results. Tavg refers to the average skin surface temperature after HILT. Figure 3 Differences in palpation scores after HILT application in both groups (with/without KSS) and the Wilcoxon signed-rank test results. animals-13-00794-t001_Table 1 Table 1 Skin surface temperature, palpation score and differences in skin surface temperature and palpation score (median and IQR) before and after HILT treatment for horses diagnosed with and without KSS. Parameters Without KSS (n = 10) With KSS (n = 10) Without KSS vs. With KSS (p-Value) Power 1 - b Tavg before HILT (degC) 30.9 (29.8 to 31.1) 31.5 (29.9 to 31.9) 0.140 0.974 Min-Max 29.1-31.2 28.9-32.5 PS before HILT (score) 2.5 (2 to 3) 2.5 (2 to 3) 0.969 0.051 Min-Max 1-4 1-4 Tavg after HILT (degC) 33.1 (32.8 to 33.8) 33.2 (33.0 to 33.3) 0.734 0.562 Min-Max 32.2-34.3 32.4-34.5 PS after HILT (score) 1 (0 to 2) 1 (1 to 2) 0.601 0.796 Min-Max 0-2 0-2 DTavg (degC) 2.6 (2.3 to 3.1) 2.4 (1.3 to 3.3) 0.307 0.863 Min-Max 1.4-3.8 0.7-3.5 DPS (score) -2 (-2 to -1) -1 (-2 to -1) 0.322 0.999 Min-Max -2--1 -3--1 Abbreviations: HILT, high intensity laser therapy; KSS, kissing spines syndrome, Tavg, average skin surface temperature; PS, palpation scores; DTavg, differences in values of average skin surface temperature after HILT; DPS, differences in values of palpation scores after HILT. animals-13-00794-t002_Table 2 Table 2 Correlation between changes in average skin surface temperatures (DTavg) and changes in palpation scores (DPS) after application of high-intensity laser therapy (HILT) in groups with Kissing Spines Syndrome (KSS) and without KSS and results of Spearman's rank correlation coefficient (rho). Statistics Without KSS (n = 10) With KSS (n = 10) Without KSS and With KSS (n = 20) Rho (95% CI) 0.071 (-0.585 to 0.671) -0.180 (-0.727 to 0.507) -0.064 (-0.493 to 0.389) p-value 0.831 0.590 0.780 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000048

Animals (Basel) Animals (Basel) animals Animals : an Open Access Journal from MDPI 2076-2615 MDPI 10.3390/ani13050805 animals-13-00805 Editorial Editorial: Role of Non-Coding RNAs in Animals Do Duy Ngoc 12* Suravajhala Prashanth 34* 1 Faculty of Veterinary Medicine, Viet Nam National University of Agriculture, Hanoi 100000, Vietnam 2 Department of Animal Science and Aquaculture, Dalhousie University, Truro, NS B2N 5E3, Canada 3 Bioclues.org, Hyderabad 500072, India 4 Amrita School of Biotechnology, Amrita Vishwa Vidyapeetham, Clappana 690525, India * Correspondence: [email protected] (D.N.D.); [email protected] (P.S.) 23 2 2023 3 2023 13 5 80526 12 2022 10 1 2023 21 2 2023 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). pmcThe importance of non-coding RNAs (ncRNAs), such as microRNAs (miRNA), long non-coding RNAs (lncRNA), and circular RNAs (circRNA), in gene regulation is increasingly being appreciated in many species. Thanks to the next generation of sequencing methods, thousands of ncRNAs have been identified in different livestock species and their functions are undergoing characterization. Clearly, ncRNAs are involved in many gene regulation processes and pathways related to economically important traits in livestock species . In addition, ncRNAs are also considered to be potential biomarkers for infectious diseases in different farm animal species . This Special Issue contains 13 articles summarizing the diverse roles of ncRNAs in livestock . In particular, we report on different species of animal that have been used to study the functions of ncRNAs. These include chicken , pigs , goats , sheep , cattle , mice , and insects . Exploring the expression of ncRNAs in different biological conditions is an important step in understanding their function. Bo et al. identified 20,269 lincRNAs, including 16,931 novel lncRNAs, expressed in the testes of Yiling goats. The authors also suggested that ENSCHIT00000000777, ENSCHIT00000002069, and ENSCHIT00000005076 were the key lincRNAs in the process of testis development, a fact derived via co-expression analyses. Gan et al. used RNA sequencing to identify 302 miRNAs expressed in pig umbilical venous blood (UVB) and umbilical arterial blood (UAB). As a result, 106 and 22 miRNAs were specifically expressed in the UVB and UAB, respectively. The authors also suggested that miR-423 and miR-122-5p can be used as characteristic miRNAs of UVB and UAB, respectively. Hao et al. performed RNA-Seq to study the roles of lncRNAs in the mammary glands of lactating Small-Tailed Han (STH) ewes and Gansu Alpine Merino (GAM) ewes. The authors identified 1894 lncRNAs that were differentially expressed, among which 31 and 37 lncRNAs were up and downregulated respectively, when comparing STH ewes with GAM ewes. In addition, the authors also found that the development and proliferation of mammary epithelial cells via the enrichment of the target genes, followed by the morphogenesis of the mammary gland, ErbB signaling pathway, and Wnt signaling pathway could be important for mammary gland and milk yield regulation. To explore the function of lncRNAs in pig spleens at different stages of development, Li et al. profiled the systematic characters of mRNA and lncRNA repertoires in three groups of spleens from nine Yorkshire pigs, with three aged seven days, 90 days, and 180 days, respectively. The authors identified 19,647 genes in addition to 219 known and 3219 putative lncRNA transcripts; 1729 genes and 64 lncRNAs therein were found to express differentially. The authors also found that differentially expressed genes and the potential target genes of differentially expressed lncRNAs both performed the crucial roles of up-regulation in immune activation and hematopoiesis, and down-regulation in cell replication and division, in 90- and 180-day-old pigs compared to seven-day-old pigs. MiRNAs are the most well-studied molecules in the ncRNAs. Several studies contained within this Special Issue attempt to validate their functions. Guo et al. indicated the potential roles of miR-149-5b in the regulation of bovine adipocyte differentiation . Xu et al. show that, by targeting PTEN, MicroRNA-148a can regulate the proliferation and differentiation of ovine preadipocytes. Qiao et al. provided evidence of the role of miR-F4-C12 50 in the regulation of adipose accumulation, finding that it performed this role by targeting PIK3R1 in castrated male pigs . Wang et al. identified 852 known miRNAs and 179 novel miRNAs in female ICR mice mammary glands at the virgin stage, day 16 of pregnancy, day 12 of lactation, day 1 of forced weaning, and day 3 of forced weaning. The authors also discovered a novel miRNA (novel-mmu-miR424-5p) involved in regulating the expression of the CSN2 gene. Other studies also validated the function of circRNAs and lncRNAs . Regarding the circRNA function, Wang et al. indicated that circEZH2 can sponge miR-378b to regulate milk fat metabolism , while Shen et al. showed that circular PPP1R13B can target miR-9-5p to promote chicken skeletal muscle satellite cell proliferation and differentiation . Lastly, via RNA sequencing and the follow-up validation, Jian et al. confirmed that the roles of lncPGC in the regulation of primordial germ cell formation in chickens were mediated by TCF7L2. This Special Issue focused not only on livestock species but also on the roles of lncRNAs in insects. Choudhary et al. provided an updated review of the functions and molecular mechanisms of the mode of action of different insect lncRNAs. Taken together, these contributions enlighten the research community on contemporary breakthroughs and suggest approaches for performing future functional studies of the regulatory roles of ncRNAs in animals. We anticipate that both expert scientists and readers can benefit from the state-of-the-art studies addressing the roles of ncRNAs contained within this Special Issue. Acknowledgments We would like to thank all the authors who contributed their papers to this Special Issue. Author Contributions D.N.D. and P.S. wrote and edited the original draft of the manuscript. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest The authors declare no conflict of interest. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References 1. Williams A. Functional aspects of animal microRNAs Cell. Mol. Life Sci. 2008 65 545 562 10.1007/s00018-007-7355-9 17965831 2. Weikard R. Demasius W. Kuehn C. Mining long noncoding RNA in livestock Anim. Genet. 2017 48 3 18 10.1111/age.12493 27615279 3. Miretti S. Lecchi C. Ceciliani F. Baratta M. MicroRNAs as biomarkers for animal health and welfare in livestock Front. Vet. Sci. 2020 7 578193 10.3389/fvets.2020.578193 33392281 4. Yang Z. He T. Chen Q. The roles of circRNAs in regulating muscle development of livestock animals Front. Cell Dev. Biol. 2021 9 619329 10.3389/fcell.2021.619329 33748107 5. Raza S.H.A. Wijayanti D. Pant S. Abdelnour S.A. Hashem N.M. Amin A. Wani A.K. Prakash A. Dawood M.A. Zan L. Exploring the physiological roles of circular RNAs in livestock animals Res. Vet. Sci. 2022 152 726 735 10.1016/j.rvsc.2022.09.036 36270182 6. Hu G. Do D.N. Davoudi P. Miar Y. Emerging roles of non-coding RNAs in the feed efficiency of livestock species Genes 2022 13 297 10.3390/genes13020297 35205343 7. Do D.N. Dudemaine P.-L. Mathur M. Suravajhala P. Zhao X. Ibeagha-Awemu E.M. MiRNA regulatory functions in farm animal diseases, and biomarker potentials for effective therapies Int. J. Mol. Sci. 2021 22 3080 10.3390/ijms22063080 33802936 8. Oyelami F.O. Usman T. Suravajhala P. Ali N. Do D.N. Emerging Roles of Noncoding RNAs in Bovine Mastitis Diseases Pathogens 2022 11 1009 10.3390/pathogens11091009 36145441 9. Bo D. Jiang X. Liu G. Hu R. Chong Y. RNA-Seq Implies Divergent Regulation Patterns of LincRNA on Spermatogenesis and Testis Growth in Goats Animals 2021 11 625 10.3390/ani11030625 33653002 10. Choudhary C. Sharma S. Meghwanshi K.K. Patel S. Mehta P. Shukla N. Do D.N. Rajpurohit S. Suravajhala P. Shukla J.N. Long Non-Coding RNAs in Insects Animals 2021 11 1118 10.3390/ani11041118 33919662 11. Gan M. Liu L. Zhang S. Guo Z. Tan Y. Luo J. Yang Q. Pan H. Li X. Wang J. Expression Characteristics of microRNA in Pig Umbilical Venous Blood and Umbilical Arterial Blood Animals 2021 11 1563 10.3390/ani11061563 34071966 12. Gao W. Zhang C. Jin K. Zhang Y. Zuo Q. Li B. Analysis of lncRNA Expression Profile during the Formation of Male Germ Cells in Chickens Animals 2020 10 1850 10.3390/ani10101850 33050652 13. Guo H. Khan R. Abbas Raza S.H. Suhail S.M. Khan H. Khan S.B. Abd El-Aziz A.H. Zan L. RNA-Seq Reveals Function of Bta-miR-149-5p in the Regulation of Bovine Adipocyte Differentiation Animals 2021 11 1207 10.3390/ani11051207 33922274 14. Hao Z. Luo Y. Wang J. Hu J. Liu X. Li S. Jin X. Ke N. Zhao M. Hu L. RNA-Seq Reveals the Expression Profiles of Long Non-Coding RNAs in Lactating Mammary Gland from Two Sheep Breeds with Divergent Milk Phenotype Animals 2020 10 1565 10.3390/ani10091565 32899158 15. Jiang J. Chen C. Cheng S. Yuan X. Jin J. Zhang C. Sun X. Song J. Zuo Q. Zhang Y. Long Noncoding RNA LncPGCR Mediated by TCF7L2 Regulates Primordial Germ Cell Formation in Chickens Animals 2021 11 292 10.3390/ani11020292 33498947 16. Jin X. Hao Z. Zhao M. Shen J. Ke N. Song Y. Qiao L. Lu Y. Hu L. Wu X. MicroRNA-148a Regulates the Proliferation and Differentiation of Ovine Preadipocytes by Targeting PTEN Animals 2021 11 820 10.3390/ani11030820 33803986 17. Li X. Han X. Sun C. Li G. Wang K. Li X. Qiao R. Analysis of mRNA and Long Non-Coding RNA Expression Profiles in Developing Yorkshire Pig Spleens Animals 2021 11 2768 10.3390/ani11102768 34679790 18. Shen X. Wei Y. You G. Liu W. Amevor F.K. Zhang Y. He H. Ma M. Zhang Y. Li D. Circular PPP1R13B RNA Promotes Chicken Skeletal Muscle Satellite Cell Proliferation and Differentiation via Targeting miR-9-5p Animals 2021 11 2396 10.3390/ani11082396 34438852 19. Wang D. Zhao Z. Shi Y. Luo J. Chen T. Xi Q. Zhang Y. Sun J. CircEZH2 Regulates Milk Fat Metabolism through miR-378b Sponge Activity Animals 2022 12 718 10.3390/ani12060718 35327115 20. Wang W. Zang X. Liu Y. Liang Y. Cai G. Wu Z. Li Z. Dynamic miRNA Landscape Links Mammary Gland Development to the Regulation of Milk Protein Expression in Mice Animals 2022 12 727 10.3390/ani12060727 35327124 21. Xu Q. Chen J. Liu X. Luo Y. Wang T. Fang M. miR-F4-C12 Functions on the Regulation of Adipose Accumulation by Targeting PIK3R1 in Castrated Male Pigs Animals 2021 11 3053 10.3390/ani11113053 34827785

PMC10000049

Global climate change has become a trend and is one of the main factors affecting biodiversity patterns and species distributions. Many wild animals adapt to the changing living environment caused by climate change by changing their habitats. Birds are highly sensitive to climate change. Understanding the suitable wintering habitat of the Eurasian Spoonbill (Platalea leucorodia leucorodia) and its response to future climatic change is essential for its protection. In China, it was listed as national grade II key protected wild animal in the adjusted State List of key protected wild animals in 2021, in Near Threatened status. Few studies on the distribution of the wintering Eurasian Spoonbill have been carried out in China. In this study, we simulated the suitable habitat under the current period and modeled the distribution dynamics of the wintering Eurasian Spoonbill in response to climate change under different periods by using the MaxEnt model. Our results showed that the current suitable wintering habitats for the Eurasian Spoonbill are mainly concentrated in the middle and lower reaches of the Yangtze River. Distance from the water, precipitation of the driest quarter, altitude, and mean temperature of the driest quarter contributed the most to the distribution model for the wintering Eurasian Spoonbill, with a cumulative contribution of 85%. Future modeling showed that the suitable distribution of the wintering Eurasian Spoonbill extends to the north as a whole, and the suitable area shows an increasing trend. Our simulation results are helpful in understanding the distribution of the wintering Eurasian Spoonbill under different periods in China and support species conservation. waterbird maximum entropy wintering habitat species' range shift Second National Survey on Terrestrial Wildlife Resources in ChinaNational Waterbird Simultaneous Survey of ChinaThis work was supported by the National Waterbird Simultaneous Survey of China in 2016 (20 April 2017) and the Second National Survey on Terrestrial Wildlife Resources in China (21 April 2017). pmc1. Introduction The Intergovernmental Panel on Climate Change (IPCC) report showed that global warming temperature may be 1.5 degC higher than the preindustrial level by 2040. Moreover, it is projected to increase by nearly 0.2 degC every ten years . Climate change is considered one of the crucial factors affecting biodiversity patterns and species distribution; it is also a major issue for conservationists . Thus far, the distribution and population of species have significantly changed, even related to species extinction . Previous studies have shown that as the climate warms, some species change their current suitable habitats and mainly migrate to high altitudes and high latitudes to adapt to the changing environment, whereas others lose their existing suitable habitats . As sensitive species, birds are particularly affected by climate change because they are highly sensitive to climate and weather . Shifts in bird distribution have already been linked to climate change . Moreover, some studies have suggested that global bird and mammal populations have decreased considerably in regions with high temperatures . Besides knowledge of the natural history and biology of a species, an understanding of the suitable habitat of a species is essential for species conservation and conservation planning . Comparing the current and future predicted distributions of species enables conservationists to develop protection and project plans . Therefore, scientifically understanding the impact of climate change on species distribution is crucial to adequately protect species in the context of climate change . Species distribution models (SDMs), also known as ecological niche models, are essential tools in basic ecology and biogeography and are widely used in analyzing the impacts of climate change on species . SDMs are one method to correlate species distribution information and relevant environmental variables and obtain the potential distribution of species through multiple algorithms based on niche theory . The development of SDMs originated from the BIOCLIM model . Then, many niche models, such as the generalized linear model (GLM), genetic algorithm for rule set production (GARP), random forest (RF), and maximum entropy (MaxEnt) were developed to predict species distributions. MaxEnt is widely used because it only needs species occurrence data to make high stability and high precision predictions under incomplete data and small sample size conditions . It is also used to predict suitable habitats for birds , biodiversity loss , species conservation, and options for nature-protected areas . The Eurasian Spoonbill is a common species and belongs to the Least Concern category under the IUCN Red List . In China, it was listed as national grade II key protected wild animal in the adjusted State List of key protected wild animals in 2021, in Near Threatened status . There are three subspecies, namely: Platalea leucorodia leucorodia, Platalea leucorodia archeri, and Platalea leucorodia balsaci. Among them, Platalea leucorodia leucorodia is distributed in China. The global population of the Eurasian Spoonbill (Platalea leucorodia) is approximately 63,000-65,000 . Most countries have only a few hundred breeding pairs with a decreasing trend . The Eurasian Spoonbill likes to live near wetlands, such as the banks of rivers, lakes, and reservoirs in open plains and mountainous and hilly areas, swamp wetlands, and offshore and coastal areas. It mainly winters in the lower reaches of the Yangtze River, South China, and southeast coastal areas . As a migratory species, the Eurasian Spoonbill is more vulnerable to a series of specific threats (such as habitat loss/fragmentation, overexploitation, and climate change) than nonmigratory species, thereby hindering the sustainable existence of its population . Climate change is one of the main factors determining bird distribution and geographic range change . Studies on the European and African Eurasian Spoonbill showed that climate change alters the wintering migration distance, the distribution of suitable habitats, and impacts the protection network . Most of the previous studies regarding the Eurasian Spoonbill have focused on breeding behavior, geographical distribution, living habitats and habitat selection, overwintering ecology, and distribution in local areas . However, studies on the distribution of wintering habitats and the response and adaptation to climate change of the Eurasian Spoonbill in China are limited. Thus, relevant research is necessary to develop the protection work appropriately. In this study, we used the MaxEnt model to simulate the potential suitable wintering habitat of the Eurasian Spoonbill. We also predicted the future influences on suitable wintering habitats under climate change scenarios for the 2050s and 2070s. In particular, we aimed to address the following questions: (1) Where is the current potential wintering distribution of the Eurasian Spoonbill? (2) What environmental factors affect the wintering distribution? (3) What is the impact of climate change on the current suitable wintering habitats? Our study will provide guidance for future field surveys and make conservation workers aware of the potential threat of climate change to this important species and other endangered species, thereby allowing suitable conservation and management work. 2. Materials and Methods 2.1. Source of Species Occurrence Data The Eurasian Spoonbill occurrence data used in this study were derived from the record during the Winter National Waterbird Simultaneous Survey of China in 2016 . In this survey, 846 survey areas and 5255 observation points were determined based on the collection of historical data and literature, combined with the investigation and research on the migration and overwintering of migratory birds across the country, from 8 to 17 January 2016. The investigators conducted the survey synchronously using the unlimited distance point count method at the survey point with monocular (binocular) telescopes. The observation range is the longest distance that can be distinguished by a monocular telescope. Meanwhile, GPS devices were used to record the species' locations. The synchronous survey here means that investigators used the same technical methods to conduct the investigation within 4 to 10 days . A total of 105 sites of the Eurasian Spoonbills were found, involving 55 counties in 15 provinces. We selected only sites located at least 2 km from each to avoid spatial autocorrelation caused by clustered occurrences . Finally, 87 locations for Eurasian Spoonbill occurrence points in China were obtained . 2.2. Environmental Variables According to previous research reports, vegetation type, topography (altitude), climate, water distance, land-use type, and two human interferences (distance to road and villages) may affect the choice of wintering habitats for the Eurasian Spoonbill . Therefore, we selected six categories of environmental datasets with 25 environmental variables, including 19 bioclimatic variables, 1 topographic variable (altitude), vegetation type variable, water distance variable, land-use type variable, and 2 human interference variables (distance to road and villages). Among these environmental variables, 19 bioclimatic layers were acquired from the WorldClim dataset under the current and future periods, with a resolution of 30'' (approximately 1 km2 on the ground) . The future climate data for the 2050s (average for 2041-2060) and 2070s (average for 2061-2080) were downloaded from the Beijing Climate Center Climate System Modeling version 1.1 (BCC-CSM 1.1) for future model building . In addition, we used four IPCC-CMIP5 representative concentration pathways (RCPs): one low mitigation gas emission scenario (RCP2.6), two moderate gas emission scenarios (RCP4.5 and RCP6.0), and one high greenhouse gas emission scenario (RCP8.5), which were labeled after the possible pathways of radiative forcing values (measured as +2.6, +4.5, +6.0, and +8.5 W/m2, respectively) in 2100 . The vegetation type variable and land-use type were downloaded from the Resource and Environment Science and Data Center . We downloaded the topographic variable (altitude) from the WorldClim dataset . The water distance variables and human interference variables (distance to road and villages) were acquired from the 1:1 million National Catalogue Service for Geographic Information dataset . Because land-use and distance to water are influenced by multiple socio-economic drivers, it is not easy to simply estimate the future condition of these two factors. Hence, we used the current datasets for land-use and distance to water as conservative prognoses for the future. Including dynamic and static variables can limit any extra uncertainty and improve the performance of the model under future climate scenarios . In this study, all of these environmental variables were transformed into American Standard Code for Information Interchange (ASII) file format using ArcGIS 10.2 Conversion Tools. They were resampled at a 30'' spatial resolution. We used SPSS software 19 to reduce multicollinearity among these environmental variables by establishing the correlation coefficient matrix and eliminating highly correlated variables (r > 0.8) . Ultimately, 13 environmental variables were selected (Table 1). 2.3. MaxEnt Model Prediction The 87 occurrence site data and filtered environmental variable data were imported into MaxEnt software 3.3.3 . In our model, we randomly selected 25% of the occurrence data for model testing, and the remaining 75% of the data were used for model training . In addition, the number of iterations was set to 500 and 10,000 background points to reduce uncertainty. Other values were kept as default. The average value of 15 repeated modelings was selected as the final prediction result. The jackknife approach was used to analyze the contribution of each environmental variable to the model based on how significantly each environmental variable occupied the species distribution . Meanwhile, we used the receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC) to verify the accuracy of the model. The AUC value is not affected by the threshold, and the evaluation results are relatively objective. Therefore, it is widely used for the accuracy evaluation of SDMs . The accuracy of model prediction is directly proportional to the AUC value, which ranges from 0 to 1. An AUC value of 0.50-0.60 indicates failure, 0.60-0.70 represents poor performance, 0.70-0.80 indicates moderate performance, 0.80-0.90 means the modeling results can be adopted, and a value >0.9 indicates high performance . The results of the modeled species distribution are continuous values that vary between 0 and 1. We imported the predicted result of the model into ArcGIS10.2. We reclassified it to obtain four potential habitat categories: highly suitable habitat (p >= 0.6), moderately suitable habitat (0.4 <= p < 0.6), poorly suitable habitat (0.2 <= p < 0.4), and unsuitable habitat (p < 0.2). The area of each partition was also calculated. 2.4. Habitat Distribution Change and the Core Distributional Shifts After using the current climate data to model the spatial extent of suitable habitat for the Eurasian Spoonbill, we obtained the future distribution of the species by performing model projections with eight future scenarios (RCP2.6-2050, RCP2.6-2070, RCP4.5-2050, RCP4.5-2070, RCP6.0-2050, RCP6.0-2070, RCP8.0-2050, and RCP8.5-2070). The change of suitable distribution area was obtained by comparing the distribution areas in the present and future periods through ArcGIS10.2. The results were classified into four categories: habitat loss, consistently unsuitable habitat, unchanged suitable habitat, and becoming suitable habitat. Afterward, we calculated the centroid of the suitable habitat in the current and future scenarios using the SDM toolbox, a Python-based GIS software10.7, to evaluate the trend of change in the suitable area . 3. Results 3.1. Model Performance and Potential Distribution of Current Wintering Habitat The AUC value of the model showed that the MaxEnt model performed well (AUC = 0.985) . This finding indicates that the MaxEnt model exhibited a good performance that can be used to predict the potential wintering habitat for the Eurasian Spoonbill. The current suitable habitat total area was 106,621 km2. The highly suitable area, moderately suitable area, and poorly suitable area were 22,197, 30,185, and 54,239 km2, representing 21%, 28%, and 51% of the suitable habitat area, respectively. The suitable habitat of white spoonbills is wetlands, including natural wetlands such as offshore and coastal areas, rivers, lakes, and marshes, as well as artificial wetlands such as reservoirs, rice paddies, salt farms, and fish farms. The suitable habitats are distributed in 21 provinces (autonomous regions and municipalities) in China, mainly located in Hubei, Hunan, Jiangxi, and Anhui provinces; a few are scattered in Jiangsu, Henan, Shaanxi, Shandong, and Xinjiang provinces; very few are scattered in Yunnan, Guangdong, Zhejiang, Shanghai, Chongqing, and Sichuan provinces . 3.2. Major Environmental Factors Affecting the Distribution of Wintering Spoonbills The MaxEnt model's internal jackknife test of factor importance showed that water_distance (52.2% contribution rate), precipitation of the driest quarter (Bio17, 15.3% contribution rate), altitude (Alt, 10.0% contribution rate), and mean temperature of the driest quarter (Bio9, 8.0% contribution rate) made the greatest contribution to the distribution model for the Eurasian Spoonbill. The cumulative contributions of these factors reached values as high as 85.5% (Table 2). Figure 4 showed that the suitable altitude ranged from -100 m to 100 m. The highest suitability occurred when the area was near water. For hydrothermal conditions, the suitable range and optimal value of the precipitation of the driest quarter (Bio17) were from 110 mm to 190 mm. The suitable range of the mean temperature of the driest quarter (Bio9) was from 4 degC to 10 degC. 3.3. Future Changes in Suitable Habitat Area The MaxEnt model predicted a dramatic range shift in the distribution of the suitable wintering habitat for Eurasian Spoonbill under four climate scenarios. The suitable distribution would expand to the northwest, while the suitable wintering habitat in the south would decrease. Compared with the current distribution, the suitable areas of northeastern Hunan, southeastern Hubei, Jiangxi, Jiangsu, and Anhui provinces would be lost. In contrast, the suitable areas of Shandong, Tianjin, Shaanxi, and the border with Henan Shanxi provinces greatly increase . The suitability of the Eurasian Spoonbill distribution range would improve with climate change (Table 3). Especially in the RCP6.0-2070s, the suitable wintering habitat was expected to increase by 77.28% (Table 4). In general, the suitable area showed a trend of increasing area and expanding northeast in the future. 3.4. The Shift of the Core Suitable Habitat Center under Different Climate Scenarios The current habitat centroid of the Eurasian Spoonbill is at 114deg43' E and 31deg12' N in Hubei Province. The centroid of suitable habitat shifts to 114deg48' E and 32deg43' N in southeast Henan under RCP2.6-2050s. Moreover, it shifts to 113deg59' E and 33deg17' N in south central Henan under RCP2.6-2070s. Under RCP4.5-2050s, the centroid of suitable habitat is at 113deg36' E and 32deg56' N in southwest Henan. However, the centroid of suitable habitat shifts to the southeast (114deg58' E, 32deg26' N) under RCP4.5-2070s. The Eurasian Spoonbill migrates from southeast Henan (114deg52' E, 32deg24' N) under RCP6.0-2050s to west central Henan (113deg32' E, 33deg14' N) in the 2070s. The centroid of suitable habitat is at 114deg6' E, 33deg53' N in central Henan under RCP8.5-2050s. Finally, it shifts to northwest Henan (113deg16', E 34deg39' N) in the 2070s. Overall, the distribution center of the suitable habitat of the Eurasian Spoonbill generally moves from northeast Hubei to Henan from the present to the future. However, there was no significant correlation of the centroid shift observed between the different concentrations of greenhouse gas emissions . 4. Discussion Our study provides the first look at the current and future species potential wintering distribution of the Eurasian Spoonbill in China. It shows that the suitable range changes dramatically under future climate scenarios, where the suitable habitat area would increase and shift to the northwest as a whole. In this study, we found that the distance to water represents the environmental variable that contributes the most to the suitable wintering habitat and spatial distribution of the Eurasian Spoonbill. However, its effect on the probability of occurrence of the species is nearly constant. Only places close to water are suitable for the Eurasian Spoonbill. This finding is not very surprising because the Eurasian Spoonbill is a waterbird living and feeding in wetlands during winter . The Eurasian Spoonbill is a specialist waterbird in shallow wetlands because it can only feed in water not deeper than 40 cm . Furthermore, our results indicated that the suitable range of the precipitation of the driest quarter is from 80 mm to 230 mm. Thus, the precipitation of the driest quarter may limit the habitat selection of the Eurasian Spoonbill. Moreover, the mean temperature of the driest quarter may determine whether the temperature is suitable for the wintering Eurasian Spoonbill. For example, Poyang Lake and Dongting Lake are the main wintering areas for the Eurasian Spoonbill. The average temperature in January of Poyang Lake is 4.5 degC . The average January temperature of Dongting Lake is 4.1 degC to 4.5 degC . This finding is consistent with our results. Furthermore, this species prefers lower altitudes; this finding is also consistent with the results from a previous study . It lives in wetlands below 100 m above sea level, and any significant change in temperature and precipitation may affect the potential distributions of the species, as shown in other research for other avian geographical distributions . Climate change is one of the main factors affecting the distribution of birds . The WWF (World Wide Fund for Nature) reported that global climate change threatens the survival of most birds . Biologists need to predict the effect of climate change on species distribution, which has important implications for species conservation . In our study, under a full dispersal assumption, we found that compared with the current situation, the average suitable wintering habitat area showed an increasing trend in the north. However, current suitable habitats will be lost, and some areas will experience a reduction in Eurasian Spoonbill habitat by up to 37.77% by 2070 These results are consistent with other studies; for example, Wu et al. reported that during the period from 1951 to 2010, more than 50% of the 14 species of birds in China migrated to the north, whereas Li et al. found that more than 40% of 108 species of birds in China will migrate approximately 90 km to the north by 2080 . Birds are influenced by changes in temperature and precipitation . Studies have shown that the average annual precipitation in China will increase (0%-20%), particularly in northern China and northwest China , and wetlands will provide more food and habitat for waterbirds . In addition, climate warming will change the distribution pattern of birds and expand their distribution range . This is consistent with our research results. At the same time, some studies have indicated that suitable habitats for some waterbirds decrease as the climate warms . In extreme cases, the temperature in China is predicted to rise in the range of 2.5-5 degC by the end of the 21st century . This scenario is likely to be one of the main contributors to the current loss of suitable habitat. We quantified the changes in the suitable wintering habitat of the Eurasian Spoonbill under four climate scenarios. We found that the area increases in comparison to the current distribution in the 2050s and 2070s, except in RCP4.5-2070s. No certain regular change is determined in the habitat area, probably because the relationship between temperature and precipitation in different climate scenarios is complex and nonlinear; this finding is similar to the findings of other studies , and the intrinsic reasons deserve further study in the future. Our results indicated that some areas suitable for the Eurasian Spoonbill wintering at present (Jiangxi, Hunan Zhejiang, and Jiangsu) may not be suitable in the future because of climatic factors, such as dryness or high temperature. At the same time, in the future there will be some new suitable areas in the north and northwest (Shaanxi, north Shandong, Tianjin, the junction of Shaanxi, Shanxi, and Henan). Hence, climate change is an important factor affecting the overwintering distribution of birds . When protecting the Eurasian Spoonbill and other waterbirds, we should pay attention to these areas and the phenomenon. A large proportion of the population of the wintering Eurasian Spoonbill is mainly concentrated in the middle and lower reaches of the Yangtze River . However, only some habitats are located in the protected area. These areas are under great pressure from human activities and habitat destruction. For instance, the wetland landscape pattern and ecological functions of Poyang Lake and Dongting Lake changed because of the expansion of water conservancy projects, agriculture, and aquaculture . These changes may cause the Eurasian Spoonbill to move to smaller areas. Establishing protected areas is one of the effective means of protecting biodiversity and habitats. Therefore, some efforts, such as protecting the existing protected areas and establishing new reserves, should be undertaken in the areas identified as suitable habitats for Eurasian Spoonbills. At the same time, long-term surveys and monitoring in these areas can be considered in order to thoroughly understand the population status. Climate change is an important factor, and bird species have responded to climate change . It has also been shown that if the impact of climate change is not considered when establishing protected areas, some species may move out in the future . Moreover, conservationists believe that it is very meaningful to consider the impact of short-term or long-term extreme weather when protecting and managing species in the context of climate change . Therefore, we suggest integrating the current situation and future climate change effects when designing nature reserves and making management plans for the Eurasian Spoonbill or other waterbirds throughout China. Many factors, such as interspecific relationships (competition and predation), land-use change, and other limited factors may affect the distribution of species. It is hard to model all impact factors. Although the MaxEnt model is widely used, the prediction results of this single model still have errors. Hence, in future research, we suggest that more factors affecting the distribution of species should be considered comprehensively, and the best model should be selected through comparative analysis of multiple models. 5. Conclusions In this study, we predicted the distribution pattern and dynamic changes of the wintering Eurasian Spoonbill in response to climate change by using the MaxEnt model. Our results indicated that water distance, precipitation of the driest quarter, altitude, and mean temperature of the driest quarter were important factors shaping the distribution of the Eurasian Spoonbill. At present, suitable wintering habitats are mainly distributed in the middle and lower reaches of the Yangtze River in China. With climate change, the suitable wintering area showed an increasing trend in the future, and its suitable distribution would shift to the northwest as a whole. This suggests that species distribution could be affected by climate change. We suggest that the current situation and future impacts of climate change should be taken into account in nature reserve design and management planning for the Eurasian Spoonbill and other endangered waterbirds throughout China. Acknowledgments This work was supported by the National Waterbird Simultaneous Survey of China in 2016 and the Second National Survey on Terrestrial Wildlife Resources in China. We thank the Academy of Inventory and Planning, National Forestry and Grassland Administration for supporting us in using the second national terrestrial wildlife resources survey winter waterbird simultaneous survey of China in 2016 data for our study. Furthermore, we are grateful to all the staff who conducted the investigation. We also would like to thank Majda and Chao Zhang for their valuable comments and linguistic assistance on the manuscript. Author Contributions Conceptualization, A.F. and X.L.; data curation, A.F. and E.G.; formal analysis, A.F.; funding acquisition, X.T. and Z.L.; investigation, X.T., Z.L. and E.G.; methodology, A.F.; writing--original draft, A.F.; supervision, X.L.; validation, X.L. and F.H.; visualization, A.F., J.W. and Z.Z.; writing--review and editing, A.F. and X.L. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest No conflict of interest exits in the submission of this manuscript, and the manuscript is approved by all authors for publication. Figure 1 Eurasian Spoonbill occurrence sites in the National Waterbird Simultaneous Survey of China in 2016. Figure 2 ROC curve and AUC value (note: ROC represents receiver operating characteristic; AUC represents the area under the ROC curve). Figure 3 The current potential distribution of suitable areas for the Eurasian Spoonbill. Figure 4 Response curves of probability of presence (based on the logistic output of the MaxEnt model) for the Eurasian Spoonbill (vertical axis) to Distance to water (a), Precipitation of the driest quarter (b), Altitude (c), and Mean temperature of the driest quarter (d); the curves are shown as means (red lines) with standard deviation (SD, blue buffers). Figure 5 Potential wintering habitat of Eurasian Spoonbill under different climate change scenarios. (a) Climate scenario RCP2.6 in 2050; (b) Climate scenario RCP2.6 in 2070; (c) Climate scenario RCP4.5 in 2050; (d) Climate scenario RCP4.5 in 2070; (e) Climate scenario RCP6.0 in 2050; (f) Climate scenario RCP6.0 in 2070; (g) Climate scenario RCP8.5 in 2050; (h) Climate scenario RCP8.5 in 2070. Figure 6 Spatial shifts for the Eurasian Spoonbill under different climate change scenarios. (a) Climate scenario RCP2.6 in 2050; (b) Climate scenario RCP2.6 in 2070; (c) Climate scenario RCP4.5 in 2050; (d) Climate scenario RCP4.5 in 2070; (e) Climate scenario RCP6.0 in 2050; (f) Climate scenario RCP6.0 in 2070; (g) Climate scenario RCP8.5 in 2050; (h) Climate scenario RCP8.5 in 2070. Figure 7 The centroid distributional shifts under different climate scenarios for the Eurasian Spoonbill. animals-13-00856-t001_Table 1 Table 1 Environmental variables used to drive the MaxEnt model in this study. Variables Description Unit Bio2 Monthly mean diurnal temperature range degC x 10 Bio3 Isothermality Dimensionless Bio6 Min. temperature of the coldest month degC x 10 Bio9 Mean temperature of the driest quarter degC x 10 Bio10 Mean temperature of the warmest quarter degC x 10 Bio15 CV of precipitation Dimensionless Bio17 Precipitation of the driest quarter mm Alt Altitude m Veg Vegetation type Dimensionless LUCC Land-use type Dimensionless Water_distance Distance to water m Road_distance Distance to road m Village_distance Distance to village m animals-13-00856-t002_Table 2 Table 2 Relative contributions of the environmental variables to the MaxEnt model. Variable Contribution Rate (%) Variable Contribution Rate (%) Water_distance 52.2 CV of precipitation 1.8 Precipitation of the driest quarter 15.3 Land-use 0.7 Altitude 10 Vegetation type 0.5 Mean temperature of the driest quarter 8 Village_distance 0.3 Min. temperature of the coldest month 6.8 Road_distance 0.1 Mean temperature of the warmest quarter 2.5 Isothermality 0.1 Monthly mean diurnal temperature range 1.8 animals-13-00856-t003_Table 3 Table 3 Suitable distribution areas for Eurasian Spoonbill under different climate scenarios (note: RCP, representative concentration pathway. RCP2.6 represents the minimum greenhouse gas emission scenario, RCP4.5 represents the medium greenhouse gas emission scenario, RCP6.0 represents the high greenhouse gas emission scenario, RCP8.5 represents the highest greenhouse gas emission scenario). Period Climate Scenario Highly Suitable Moderately Suitable Poorly Suitable Suitable Area Area km2 Percentage % Area km2 Percentage % Area km2 percentage % Area km2 Current 22,197 21 30,185 28 54,239 51 106,621 2050s RCP2.6 22,658 18 39,725 32 62,191 50 124,574 RCP4.5 17,939 16 31,966 29 59,669 55 109,574 RCP6.0 23,486 21 29,409 26 59,735 53 112,630 RCP8.5 39,491 23 48,611 28 83,625 49 171,727 2070s RCP2.6 22,121 19 32,063 27 63,053 54 117,237 RCP4.5 19,037 18 32,616 31 53,157 51 104,810 RCP6.0 45,243 24 48,433 26 95,499 50 189,175 RCP8.5 20,556 17 31,597 25 72,137 58 124,290 animals-13-00856-t004_Table 4 Table 4 Dynamics of changes in suitable wintering habitat area for the Eurasian Spoonbill under different climate scenarios (note: RCP, representative concentration pathway. RCP2.6 represents the minimum greenhouse gas emission scenario, RCP4.5 represents the medium greenhouse gas emission scenario, RCP6.0 represents the high greenhouse gas emission scenario, RCP8.5 represents the highest greenhouse gas emission scenario). Climate Scenario Year Area (km2) Proportion of Area (%) Gain Loss Stable Total Change Gain Loss Stable Total Change Representative concentration Pathway (RCP)2.6 2050 40,320 21,680 85,737 18,640 37.82 20.33 80.41 17.49 2070 46,764 35,805 71,565 10,959 43.86 33.58 67.12 10.28 Representative concentration Pathway (RCP)4.5 2050 31,611 27,459 79,735 4152 29.65 25.75 74.78 3.9 2070 30,041 30,759 76,379 -718 28.18 28.85 71.64 -0.67 Representative concentration Pathway (RCP)6.0 2050 30,090 23,801 83,424 6289 28.22 22.32 78.24 5.9 2070 91,603 9200 98,679 82,403 85.91 8.63 92.55 77.28 Representative concentration Pathway (RCP)8.5 2050 85,274 19,821 87,741 65,453 79.98 18.60 82.30 61.38 2070 58,911 40,269 66,821 18,642 55.25 37.77 62.67 17.48 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000050

The aim of this research has been to assess the effect of the dietary protein level on piglet growth and post-weaning diarrhea (PWD) incidence. Piglet fecal microbiota and feces composition were also assessed. The experiment was carried out on 144 weaned piglets (Duroc x Large White; 72 piglets per treatment) and lasted from weaning (at 25 days of age) until the end of the post-weaning phase (at 95 days). Two dietary protein levels were compared: high (HP; 17.5% crude protein on average, during the experiment) and low (LP; 15.5% on average). Lower (p < 0.01) average daily gain and feed conversion ratio were observed in LP piglets in the first growth phase. However, at the end of the post-weaning period, the growth parameters were not significantly different in the two diets. Diarrhea scores were lower in piglets fed LP diets than in piglets fed HP diets (28.6% of the total vs. 71.4% in the HP piglets). Fibrobacteres, Proteobacteria, and Spirochaetes were more abundant in the feces of the piglets fed LP diets. Feces nitrogen content was lower in piglets fed LP diets. In conclusion, low protein levels in the diet can reduce the incidence of PWD while only marginally affecting growth parameters. piglet post-weaning diarrhea dietary protein fecal microbiota feces composition ASSOCIAZIONE NAZIONALE ALLEVATORI SUINIThis research was funded by the ASSOCIAZIONE NAZIONALE ALLEVATORI SUINI (www.anas.it; accessed on 19 February 2023) within the framework of an agreement between ANAS and CREA to evaluate the effect of different dietary protein levels on piglets from weaning to 30 kg. pmc1. Introduction The period between weaning (i.e., removal from the mother) and reaching the bodyweight for transfer to the fattening boxes (at about three months of age and 35-40 kg of body weight) is more delicate from the point of view of health and functioning of the piglet digestive system. Among the numerous factors that can intervene to destabilize the delicate balance of the piglet's intestine, linked to an acceleration phase of both morphological and enzymatic maturation, diet-linked factors undoubtedly play a fundamental role . Among the dietary factors, the quantity of protein fed to piglets plays a leading role. In fact, in this phase, the coverage of nutritional needs requires a protein level higher than the digestive potential of the piglet. According to Kim et al. , a protein level of between 21.5% and 24% is required for modern fast-growing lines, a level that, in fact, is higher than the piglet's digestive capacity; these authors suggest a protein level not higher than 18% in the first days after weaning and with a consistent addition of synthetic amino acids. De Lange et al. pointed out that low protein levels are beneficial for the gut health of piglets because the presence of undigested proteins, as it can occur with high dietary protein levels, allows the proliferation of a bacterial flora producing toxins capable of altering the intestinal barrier. This alteration implies: the colonization of the intestinal epithelium by pathogenic microorganisms; the acceleration of the production of enterocytes that, being immature, have an exudative rather than absorbent attitude; greater ease of crossing the cellular barrier by specific bacterial toxins (edema disease). Zhang et al. highlighted that high-protein diets increase the microbial fermentation of proteins, peptides, and amino acids. According to Gao et al. , high protein levels favor the production of ammonia and branched-chain-fatty acids and, therefore, the proliferation of pathogenic bacterial flora, while low protein levels favor the production of short-chain fatty acids (SCFAs), primarily butyric acid, which favors the proliferation of beneficial bacterial flora. The same authors, comparing two protein levels (17% vs. 30%), both obtained exclusively with casein, highlighted that with a high protein level, the bacterial diversity of the microbiota is reduced. The increase in ammonia can negatively affect the formation of intestinal epithelial cells . The reduction of beneficial Lactobacilli that accompanies the maturation of the pig's intestine and the variations in the buffering effect of pH due to protein fermentation can make the intestinal environment more susceptible to the emergence of opportunistic pathogens, such as Bacteroides and Clostridium species . Opapeju et al. compared four diets administered to piglets with an initial weight of about 6.5 kg: control feed with 21% crude protein (CP); feed with 19% CP and deficient in isoleucine; 19% raw protein feed supplemented with synthetic isoleucine to reach the isoleucine level of the control feed; 17% raw protein feed supplemented with isoleucine and valine to reach the ratio indicated by the ideal protein. The control group showed better production performance in terms of growth and conversion index, although they showed softer feces, a greater amount of ammonia in the feces, and a greater depth of the crypts of the intestinal mucosa, indicating an acceleration of production of enterocytes; essentially, better production performance, but greater susceptibility to the onset of a syndrome affecting the gastrointestinal system. This situation of precarious equilibrium could be easily kept under control only with targeted antibiotic prophylaxis, which, however, is no longer allowed, and even the spaces for metaphylaxis, albeit careful, become increasingly restricted. Therefore, it is necessary to identify feeding strategies that reduce the risk of the appearance of alterations in the gastrointestinal function of the pig and reserve the use of antibiotics for clinically overt pathological situations. Knowledge of the relationship between pig microbiota and diet can be used to orient the intestinal microbial dynamics in the desired direction by diet manipulation . The microbiota of healthy piglets susceptible to post-weaning diarrhea (PWD) has been the subject of numerous investigations , which highlighted how the state of health of the pig and its susceptibility to diseases, such as PWD, can be related to the change in the composition of the microbiota during the early stages of growth. All factors affecting PWD susceptibility also affect microbiota composition. Among these, pig feeding plays a primary role. Heo et al. observed a reduction in PWD in piglets challenged with an enterotoxigenic strain of Escherichia coli, when fed with lower protein levels. Rist et al. suggested reducing proteins and increasing fermentable carbohydrates in the diet to reduce harmful protein fermentations. Luise et al. showed that lower dietary protein levels could reduce the intestinal fermentation of undigested proteins and the consequent risk of diarrhea. The aim of this research has been to assess the effect of the dietary protein level on piglet growth and PWD incidence. Piglet fecal microbiota and feces composition were also assessed to support the understanding of the results. 2. Materials and Methods 2.1. Animal Ethics All animal procedures were performed in strict accordance with the Code of Ethics of the World Medical Association Accessed on 19 February 2023). 2.2. Experimental Design The experiment was carried out at our experimental pig farm in San Cesario sul Panaro (Modena, Italy), on Duroc Italiana x Large White Italiana crossing lines. A total of 144 weaned piglets was used, half barrows and half females. The experiment lasted from weaning, when the age of the piglets was 25 +- 1.5 days, until the end of the post-weaning phase, corresponding to 70 days of trial and 95 days of age of the animals (Table 1). Two dietary protein levels were compared (Table 2): high (HP) and low (LP), in two growth phases characterized by different diet compositions. The diet composition (Table 3) was changed 25 days after the start of the experiment, at the expected piglet body weight of 15 kg (and actual weight of 17 kg). Synthetic amino acids were also supplemented to ensure a balanced feed formulation. Specifically, total lysine levels in the first and second feeding periods were set at 1.40% and 1.20% of the feed, respectively. The percentages of methionine, cystine, threonine, and tryptophan were balanced according to the proportion of the ideal protein with the addition of synthetic amino acids. After separation from the mother, the piglets were housed in cages of 12 individuals each, distributed as evenly as possible within each cage by body weight, age, and litter of origin. Since it was not possible to accommodate 12 cages (3.3 m2 each) within the same room, the males were housed in one room (6 cages) and the females in another (6 cages). In this way, it was necessary to accept that we were dealing with a confused effect (room and sex), while the factor of interest of the experiment (protein level) was homogeneously represented in both rooms. Each cage was equipped with a hard-plastic floor. The complete feed was administered ad libitum in a hopper feeder with 4 places to ensure sufficient access to feed for all piglets; in each cage, there was a nipple drinker. The temperature of the air in the rooms was 23 degC +- 1 degC, whereas the humidity was not controlled. Forty-six days after weaning, the piglets were moved to larger pens in the fattening area and housed in 12 pens (9 m2 each) of 12 piglets each. The floor was thermally insulated concrete. The pens were arranged in 2 rows of 6 pens (3 adjacent pens for males and 3 adjacent pens for females), one row for each protein level. The animals were given 1 kg of feed per animal and per day in two daily meals. In this phase, the administration of feed was limited to reduce the risk of diarrhea related to the stress of the change of housing. In the first two days, the meal was dry and distributed on the ground, gradually passing to a wet meal in the trough over the next three days. In each pen, the water was still available through a nipple drinker. The assignment of the treatment to the pens followed the criterion of minimizing the possibility of mixing feces from pigs fed different protein levels. In the fattening area, 21 degC was always ensured during the experiment. The effects of dietary protein level on piglet growth, health status, and feces microbiota were considered for the following periods (Table 1):- Period I: From the start of the experiment until the change of feed; - Period II: From the change of feed until the change of housing; - Period III: From the change of housing to the end of the post-weaning period (end of the experiment). 2.3. Growth Parameters The animals were weighed individually at the start of the experiment, at the day of diet change, at the day of change in housing, and at the end of the experiment. For each period, average daily gain (ADG = [body weight at the end of the period--body weight at the beginning of the period]/day), average daily feed intake (ADFI = feed consumption in the period/day), and feed conversion rate (FCR = ADFI/ADG) were calculated. Since feed consumption was known only at the cage/pen level, the values of the ADFI and FCR variables were not known at the individual level and thus were calculated at the cage/pen level. For homogeneity, the ADG values, although individually known, were also processed at the cage/pen level, with six replicates in total for each treatment. 2.4. Diarrhea Scores and Corrective Interventions The health of the piglets was monitored daily. A score was assigned to each cage/pen based on the number of cases and the extent (mild, medium, severe) of diarrheal phenomena in piglets, visually assessed from the consistency of the feces (Table 4). For each growth period, individual diarrhea scores were summed for each treatment and related to the period's total diarrhea score. Piglets suffering from diarrhea before the change of diet (Table S1) were treated (Table S2) parenterally with the antibiotics enrofloxacin or marbofloxacin. No antibiotics were administered between the time of the diet change and the date of transfer to the pens because there were no cases of diarrhea. After the transfer, all individuals were treated orally with colistin sulfate, starting 5 days after the transfer and for 8 days. It was decided to resort to oral mass therapy because the appearance of overt diarrhea (score 4 or higher) occurred five days after the moving, in four out of six boxes of the HP treatment and in one out of six boxes of the LP treatment (Table S3). No treatment was applied in the last 12 days before the third and final sampling of feces. 2.5. Feces Chemical Characterization Feces samples were collected at the end of each growth period by piglet rectal ampoule stimulation. For each cage/pen, individual samples were pooled into a composite sample. Samples were immediately frozen and stored at -20 degC until analysis. Dry matter, organic matter, total (Kjeldahl) nitrogen and ammonium nitrogen contents, and pH, were determined according to the APHA methods . Crude fiber (CF) and fiber fractions were determined on fecal samples dried at 60 degC. Crude fiber was determined according to . Hemicellulose and cellulose concentrations were estimated by determining neutral-detergent (NDF) and acid-detergent (ADF) fiber fractions and acid-detergent lignin (ADL) according to . The difference between NDF and ADF is an estimate of the hemicellulose content; that between ADF and ADL of the cellulose content. For volatile fatty acid determination, 1 g of the sample was diluted with 3 mL distilled water, then centrifuged at 4000 rpm for 15 min. The supernatant was used for the analyses. Half mL of sample supernatant was added to 0.25 mL 4% H3PO4 and 0.25 mL internal standard to a final volume of 1 mL. A microliter of this mixture was injected in the injection port of the gas-chromatograph (Shimadzu GC 2010 Pro), equipped with a NukolTM capillary column (Supelco, cat. no. 24107), 30 m x 0.25 mm internal diameter, 0.25 mm film thickness. Total volatile fatty acid content (mg L-1) was calculated as the sum of the individual concentrations of acetic, propionic, butyric and iso-butyric, valeric and iso-valeric, and caproic and iso-caproic acids. 2.6. DNA Extraction, Library Construction, and Sequencing Total DNA was extracted from the fecal samples after thawing using the QIAamp PowerFecal Pro DNA Kit (QIAGEN, The Netherlands) according to the manufacturer's instructions. The V3-V4 regions of the 16S rRNA gene were amplified using the 341F (5'-CCTACGGGNGGCWGCAG-3') and 805Rmod (5'-GACTACNVGGGTWTCTAATCC-3') (based on , with degenerate bases) primers. Library construction and sequencing were performed at the Sequencing Platform, Fondazione Edmund Mach, Italy. More in detail: each sample was amplified by PCR using a 25 mL reaction mixture with 1 mM of each primer. More in detail, 12.5 mL of 2x KAPA HiFi HotStart ReadyMix and 10 mL forward and reverse primers, were used in combination with 2.5 mL of template DNA (5 ng/mL). The PCR reactions were carried out by GeneAmp PCR System 9700 (Thermo Fisher Scientific) and the following cycling conditions: initial denaturation step at 95 degC for 3 min (one cycle); 25 cycles at 95 degC for 30 s, 55 degC for 30 s, 72 degC for 30 s; final extension step at 72 degC for 5 min (1 cycle). The amplification products were checked on 1.5% agarose gel and purified using the CleanNGS beads (CleanNA, Waddinxveen, The Netherlands) following the manufacturer's instructions. Afterward, a second PCR was used to apply dual indices and Illumina sequencing adapters Nextera XT Index Primer (Illumina), by 7 cycles PCR (16S Metagenomic Sequencing Library Preparation, Illumina). The amplicon libraries were purified using the CleanNGS beads (CleanNA, The Netherlands), and the quality control was performed on a Typestation 2200 platform (Agilent Technologies, Santa Clara, CA, USA). Finally, all barcoded libraries were pooled in an equimolar way and sequenced on an Illumina(r) MiSeq (PE300) platform (MiSeq Control Software 2.5.0.5 and Real-Time Analysis software 1.18.54.0). A total of 3.093.548 raw reads were detected across the samples by the Illumina MiSeq sequencing platform (PE300) (Illumina, Santa Monica, CA, USA). 2.7. Statistical Analysis The estimate of the effect of the protein level in the diet on growth performance was carried out by means of a one-factor analysis of variance. The effect of the sex/room factor was included in the block effect. Values of F with p > 0.05 were considered not significant (NS). The analysis of variance (ANOVA) for the effect of diet on diarrhea incidence was applied to the sum of the scores of the period for each treatment replication. Since in the second period the sum of the scores was very low, for the purposes of ANOVA, the first two periods were merged into a single period. Two-way ANOVA (fixed sources of variation: time of sampling, protein level, time of sampling x protein level) was applied to operational taxonomic units (OTU) percentages to estimate the effect of diet and growth period on the relative abundance of phyla, families, and genera in the analyzed samples, using the procedure MIXED, SAS language . A threshold equal to 0.1% of the total reads was adopted to include the phylum, family, or genus in the analysis. Mean multiple comparisons were performed using the statement LSMEANS and the Tukey HSD test. The same two-way ANOVA design was applied to the statistical analysis of feces composition. 2.8. Bioinformatic Analyses Data were pre-processed using the MICCA v. 1.7 pipeline and rarefied to an equal depth of 45.225 reads per sample. OTUs were created de novo by clustering sequences with 97% sequence identity and classified using the RDP software version 2.11. The alpha diversity of the populations, the relative abundance (%) of microbial components down to the family and sex level, and their grouping based on the sources of variation were estimated. Alpha diversity is an index of the richness (number) and diversity (relative abundance) of OTUs in a population. The richness of species is indicated by the total number of OTUs ("Observed") in the microbial community: the higher the number, the more species are present. The CHAO1 index estimates the richness of species, giving more weight to the less abundant ones. The value of CHAO1 is at least equal to "Observed" and increases as the number of rarer species increases. It can be calculated as follows:CHAO1 = Sobs + F1(F1 - 1)/(2 x (F2 + 1))(1) where Sobs is the number of observed species and F1 and F2 are the count of singletons and doubletons, respectively. The Shannon index is calculated as follows:Shannon Index = -(pi ln(pi))(2) where: S is the summation from 1 to the total number of OTUs, and pi is the proportion of the community represented by the OTU i. It increases with increasing species richness, uniformity, and uncertainty of the estimate. The samples were grouped for compositional similarity (beta diversity) using Principal Coordinate Analysis (PcoA), which is a multivariate method of data analysis used to explore and to visualize similarities or dissimilarities of data . 3. Results 3.1. Growth Performance The piglets fed HP level showed a greater ADG (p < 0.01) than those fed LP (Table 5) in the first 25 days of the experiment until the change of diet, while the increases were the same for the two protein levels in the period from the change of diet to the change of housing. The difference remained significant (p < 0.05) during the entire phase in the cages. The FCR value was also significantly better in HP. Conversely, ADFI was not significantly different in piglets fed different protein levels. In the pen housing, no differences were detected between the two treatments as the feed was rationed. The body weight of the piglets at the end of the post-weaning period was not different in the two diets. 3.2. Health Status Diarrhea cases started to occur in the early post-weaning period (Table S1). They were concentrated in the period between weaning and change of feed (Period I; score summation: 107 out of 234, i.e., 45.7% of the total; Table 6) and in the period between change of housing and the conclusion of the post-weaning period (Period III; 121 out of 234: 51.7% of the total). In Period II, overall, feces had normal consistency, as well as in the first and last part of Period III (Tables S1 and S3). The overall incidence of diarrhea in the LP treatment was 28.6% of the total (67 out of 234 scores) and 71.4% in the HP treatment. Specifically, from weaning until feed change (Period I), the diarrhea scores in the LP treatment were 15.0% of the total (16 out of 107); after feed change (Period II), they were 16.7% of the total (1 out of 6), while after the change of housing (Period III) they rose to 41.3% (50 out of 121). The effect of the dietary protein level on diarrhea score summations was highly significant (p < 0.01) until change of housing (Period I + Period II), whereas it was not significant (p = 0.140) from the change of housing until the end of the experiment (Period III). Poor feces consistency was noted in all the boxes in the HP treatment, starting from the twelfth day of the experiment (Table S1). The phenomenon initially extended to the whole pen in three out of six pens and then progressively became reduced, presumably due to therapeutic interventions (Table S2), until it disappeared two days after the change of feed. 3.3. Composition of the Fecal Microbiota The alfa diversity of the microbiota (intra-sample diversity) increased after the change of feed and further increased after the change of housing , while it was not influenced by the protein level in the diet . The analysis of beta diversity (inter-sample diversity) using the PCoA tool allowed the clustering of the fecal samples into three groups, corresponding to the three times of sampling . The protein level in the diet allowed only partial separation of the treatments. The most represented phyla in the piglet fecal samples (Table 7) were Bacteroidetes and Firmicutes, which together accounted for 90.6% of reads. The relative abundance of phyla changed in relation to the sampling date: at the first sampling, 25 days after weaning, Firmicutes and Actinobacteria were more abundant than in the two subsequent samplings, whereas the other classified phyla: Bacteroidetes, Spirochaetes, Proteobacteria, and Fibrobacteres were more abundant in the two samplings following the first, without significant differences between the second sampling, at the end of a period characterized by a change of feed, and the third sampling, at the end of a period that had begun with the change of housing. Fibrobacteres, Proteobacteria, and Spirocheaetes were, on average, more represented in the feces of piglets on a low-protein diet, whereas Firmicutes were more abundant in the feces of piglets on a high-protein diet. Overall, the most represented bacterial families were: Prevotellaceae, Lachnospiraceae, Ruminococcaceae, and Porphyromonadaceae (65.1% of the total reads). While the influence of the sampling time was evident, the same cannot be said for that of the treatment (Table S4). Low protein levels in the diet were associated with a higher abundance of Fibrobacteraceae, Succinivibrionaceae, Sutterellaceae, and Spirochetaceae, whereas Firmicutes-belonging families prevailed in the HP fecal samples: Lachnospiraceae and Eubacteriaceae were more abundant along all the post-weaning period, whereas Erisipelotrichaceae, Clostridiaceae 1, and Peptostreptococcaceae were more abundant only in the first sampling event. Lactobacillaceae were the only Firmicutes more abundant in the feces of piglets on a low-protein diet and only in the first sampling event (significant interaction effect: Sampling time x Protein level). A few classified families were little or not at all influenced by both the period of growth and the protein level; among these, Enterobacteriaceae. Among the classified genera, the most abundant were Prevotella (27.2%), followed by Clostridium sensu strictu (3.9%), Lactobacillus (3.3%), Alloprevotella (3.2%), Treponema (3.1%) (Table S5). Selected genera were more represented in the families significantly influenced by the dietary protein level, alone or in interaction with the sampling time (Table 8). Lactobacillus and Treponema were more represented in the LP fecal microbiota, whereas Roseburia, Blautia, and some Clostridium genera were more abundant in HP. 3.4. Feces Composition Almost all the traits analyzed varied significantly over time (Table 9). More specifically, the concentration of dry matter, organic matter, crude fiber, and fiber fractions increased over time, whereas total and ammonium nitrogen concentrations decreased. The pH slightly increased after the feed change. The dietary protein level also affected the feces composition. Piglets fed low protein had feces that were richer in dry and organic matter and lower in total and ammonium nitrogen all over the experiment period. Even though crude fiber concentration was not affected by the dietary protein level, however, the fiber fractions were. In fact, hemicellulose and cellulose concentrations were higher in the LP fecal samples. As for volatile fatty acids, the concentrations of propionic, valeric, and especially isovaleric acid slightly increased after the change of feeding. No effect on the protein level was detected, except for the isovaleric acid concentration, which tended to be lower in low-protein diets. 4. Discussion 4.1. Dietary Protein Level, Growth Performance, and Susceptibility to PWD In our experiment, the low protein level negatively affected the growth of the piglets (ADG and FCR) only in the first post-weaning period (up to the change of diet), whereas it had a clear positive impact on diarrhea intensity reduction. Literature on the matter reports contrasting results, as also highlighted by Wang et al. . Heo et al. reported a higher incidence of PWD when feeding weaned piglets at a high (24.3%) compared with a low (17.3%) CP diet. Wen et al. observed a higher incidence of PWD for piglets fed higher protein levels (up to 23%). Rattigan et al. obtained contrasting results by working in sanitary vs. unsanitary conditions. Reducing the dietary CP level did not affect growth performance; however, in sanitary conditions, it increased the Enterobacteriaceae abundance in the colon and the incidence of diarrhea occurrence, whereas the opposite occurred in unsanitary conditions. Limbach tested the effect on growth and PWD incidence in piglets fed soybean-maize diets at three protein levels (22% AA-balanced, 19% AA-balanced, and 16% protein AA-unbalanced) and concluded that low CP diets may be used for the initial post-weaning period to reduce piglet susceptibility to PWD without largely impacting growth performance. Lynegaard et al. overall reduced CP in the first weaning phase (6-9 kg) from 19.1% to 16.6 and 14%, and from 18.4% to 16.2-17.4%, in the second phase (9-15 kg), depending on the treatment, while they left the protein almost constant in the third phase (15-30 kg) of growth. Under these conditions, they observed a decrease in PWD incidence for treatments with reduced dietary protein, as well as lower ADG and FCR values in piglets fed lower protein levels up to 15 kg body weight. The disadvantage, especially in terms of ADG, also remained in the following period. In their experiment, however, the reduction of CP in the early post-weaning growth period was more pronounced than in our experiment. According to , CP levels too low (i.e., 3% below average: 17%) are considered harmful in consideration of maladaptive changes to small intestinal morphology and pepsin activity in weaned piglets. 4.2. Piglet Fecal Microbiota in the Post-Weaning Period The observed increase in the microbiota's alfa diversity during piglet growth has also been reported by other authors , and it can be linked to changes in diet as well as to the progressive maturation of the intestinal system . On the contrary, dietary protein levels have been reported not to influence alpha diversity in pigs . Our results confirm these findings. The most represented phyla (Table 7) are Bacteroidetes, Firmicutes, Spirochaetes, and Proteobacteria (96.9% relative abundance), and the most abundant bacterial families: Prevotellaceae, Lachnospiraceae, Ruminococcaceae, and Porphyromonadaceae in the piglet fecal samples of this experiment are those recurring in the feces of healthy piglets . Clostridium sensu stricto, Roseburia, Paraprevotella, Clostridium XIVa, and Blautia have been reported as major representative genera after weaning . In general, the results in the literature refer to piglets stressed on purpose or younger than those in this experiment. The considered microbiota is more often that of the intestinal system, different from the fecal one . In our experiment, we considered the microbiota of the feces of piglets raised in protected conditions, which should be the most likely or, in any case, desired in real farms. This protection has been applied both at the environmental level (attention to the absence of causes of stress) and at the health level (preventive interventions with drugs). This may explain the low presence of Enterobacteriaceae, which is normally associated with stressful conditions. 4.3. Dietary Protein Level, Composition of the Fecal Microbiota, and PWD Susceptibility The effect of dietary protein level on the composition of gut microbiota has mainly been studied for finishing pigs. Moderate protein levels in the diet have been found to modify the gut microbiota composition and to improve the ileal barrier function . On the contrary, information on piglets is scarce, especially when referring to the composition of the fecal microbiota. In our experiment, differences in the composition of the fecal microbiota due to the protein level in the diet were found above all in the first sampling, that is, at the end of the first period after weaning and before the change of feed, just when the effect of protein intake on the incidence of diarrhea phenomena was most evident. Given this coincidence, we can think of associating the higher susceptibility of the piglets to PWD with the greater presence of Firmicutes-belonging families (with the exclusion of Lactobacillaceae), which were more abundant in T1 in the fecal microbiota of piglets fed HP diets. Conversely, a lower incidence of PWD can be associated with the prevailing abundance in LP diets of families belonging to Lactobacillaceae (gen. Lactobacillus), Fibrobacteraceae (gen. Fibrobacter), Succinivibrionaceae, and Spirochetaceae (gen. Treponema). Yang et al. compared the fecal microbiota of healthy piglets and that of diarrheal piglets in three stages of growth: lactation, intermediate stage, and weaning (solid diet for piglets) and noted that, with the transition to solid feeding, the incidence of Lactobacillus and E. coli decreased while that of Prevotella increased. They related reduced numbers of Bacteroides, Ruminococcus, Bulleidia, and Treponema, which are responsible for the digestion of solid foods, to the onset of post-weaning piglet diarrhea. The genera prevalent in the fecal microbiota of piglets fed HP diets, such as Roseburia, Blautia, Eubacterium, and selected Clostridium species, are commonly found in piglet fecal microbiota and are all fermentative. Their greater abundance in the HP feces can be the consequence of the incomplete digestion of complex fermentable substrates because less digestible feed components are more available for microbial activities in the gut terminal tract. The greater presence of Clostridia in the microbiota of piglets fed HP diets can be related to their ability to metabolize amino acids . Conversely, the increased presence of Fibrobacter in low-protein diets can be related to the availability of higher amounts of undigested cellulose in the final intestinal tract. In fact, Fibrobacter is a fibrolytic bacterial species commonly present in the pig intestinal microbiota , although it is a typical colonizer of the rumen. Fibrobacter uses glucose, cellulose, and cellobiose as carbon and energy sources with the production of succinate, acetate, and formate . Fibrobacter and Treponema have been reported as more abundant in diets of ruminants fed with higher amounts of lignocellulosic components . 4.4. Dietary Protein Level and Feces Composition Feces composition clearly depends on the type of food ingested and on the use that the animal can make of the food. The less digestible the food, and the worse the digestive abilities of the animal, the higher the quantity of feed that remains undigested in the feces. Higher crude fiber contents accompanying the change of feed can be the reason for worse digestibility: a higher crude fiber content was, in fact, found in the piglet feces at the second and third sampling (i.e., at the change of housing and at the end of the post-weaning period). An important side effect of the reduction in dietary protein was the lower concentration of nitrogen in the feces, possibly because a greater quantity of ingested nitrogen was assimilated instead of wasted. This result has been reported by several authors. Zhao et al. found a significant reduction in N excretion in 90-day-old pigs fed protein diets 3.5% lower than standard. Yang et al. observed a linear decrease in fecal total nitrogen for decreasing dietary protein levels in the diet of growing pigs. Other components in the diet may interfere with nitrogen metabolism. In fact, in maize, the nitrogen-free extractives consist of starch, whereas in soybeans, the starch is less than 1%, and soluble carbohydrates are mainly sucrose, raffinose, and stachyose . High amounts of raffinose and stachyose from soybean in the diet are supposed to reduce the digestibility of nitrogen and amino acids in growing pigs . Therefore, in HP-fed piglets, the greater nitrogen excretion can also derive from a lower digestibility due to the higher percentages of soybean oligosaccharides. Interestingly, Zhang et al. showed a higher incidence of diarrhea cases in piglets fed with soybean flour as is or with added stachyose compared to the control consisting of corn with concentrated soybean protein. Short-chain fatty acids are final metabolites of the intestinal microbiota, produced mainly in the large intestine, where they are used by mono-gastrics as a source of energy. The energy contribution of the SCFA in pig metabolism is important. Weaning has been reported to affect the concentrations of SCFA in the intestine . In our experiment, the dietary protein level did not affect the SCFA content, apart from the case of isovaleric acid, which was less abundant in LP diets. Some confirmations and some interesting insights emerge from this experience. The first of these is whether the body weight differences found in the experiment cancel out during the subsequent stages of growth of the animals and, once this has been established, how far one can go with the reduction of the protein content in particularly critical phases (first 15 days after weaning, sudden changes in the environment) without compromising the subsequent productive career. The second interesting point is to investigate the evolution of the microbiota to identify moments in which an analysis of this can serve as an indicator of the evolution of the state of health of the gastrointestinal system. From our results, it appears that the phase immediately following weaning is the most subject to changes in the fecal microbiota composition and most suitable for earlier identification of possible stress conditions. Everything must be understood in terms of optimizing individual and mass therapeutic interventions in order both to improve the profitability of breeding and to reduce the risk of the appearance of antibiotic-resistance phenomena. 5. Conclusions This research confirms that a reduction in the protein content of feed can reduce the appearance and severity of gastrointestinal syndromes in piglets in particularly stressful stages of rearing (removal from the mother, change of housing) while only marginally affecting growth performance. Low-protein diets, resulting in excreta with lower quantities of nitrogen than those of standard diets, may allow for potential environmental benefits. The variations in the microbiota are largely determined by the growth phase, which in turn is accompanied by an evolution of the diet. The different protein levels at the same age caused slight but significant variations in some components of the intestinal microbiota. Further research is needed to ascertain whether microorganisms found in low-protein diets can be considered indicators of lower susceptibility to diarrhea in piglets. Acknowledgments Authors wish to thank Antonio Marino, Ciro Vasmara, Gianni Marchetto, and Anna Orsi for their valuable technical support. The activity of Rosa Marchetti was carried out in the context of her association with CREA-ZA. Supplementary Materials The following supporting information can be downloaded at: Table S1: Number of interventions with parenteral therapy on piglets, from the onset of diarrhea to change of housing; Table S2: Diarrhea scores from the start of the experiment until the change of housing; Table S3: Diarrhea scores from the change of housing until the end of the experiment; Table S4. Bacterial families with relative abundance greater than 0.1% in the piglet fecal microbiota, depending on sampling time and protein level; Table S5: More abundant (>1% reads) bacterial genera in the fecal microbiota of weaning piglets. Click here for additional data file. Author Contributions Conceptualization, M.G., L.B. and G.D.C.; methodology, G.D.C. and L.B.; validation, G.D.C. and D.B.; formal analysis, R.M. and M.P.; investigation, V.F.; resources, M.G. and L.B.; data curation, V.F. and R.M.; writing--original draft preparation, G.D.C., R.M. and L.B.; writing--review and editing, R.M. and G.D.C.; visualization, R.M.; supervision, L.B.; project administration, V.F.; funding acquisition, L.B. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethical review and approval were waived for this study due to the fact that no practices were applied likely to cause pain, suffering, distress or prolonged damage equivalent to or greater than that caused by inserting a needle. In fact, our trial consisted in comparing diets with different levels of protein, within a range that well represents the normal uses in Italian pig farming. The only practice different from those normally used on farms was the sampling of feces from the rectum. It is a minimally invasive and very fast manipulation, likely to cause less pain, suffering or distress than that caused by inserting a needle. No animals were slaughtered in the course of the trial. Informed Consent Statement Not applicable. Data Availability Statement Data are contained within the article or supplementary material. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Box plots of the alpha diversity indexes (observed, Chao1 and Shannon) as a function of (a) sampling time and (b) protein level (high, red dots; or low, blue dots). T1, T2, and T3 correspond to fecal samplings after weaning and before the change of feeding, before the change of housing, and at the end of the experiment (end of the post-weaning period), respectively. Figure 2 Principal coordinate analysis of the piglet fecal microbiota. Red dots: high protein level, blue dots: low protein level. Contour lines highlight the 3 sampling times, T1, T2, and T3, from left to right. animals-13-00795-t001_Table 1 Table 1 Time sequence of events from weaning to the end of the experiment. Growth Period Event (Initial and Final of the Period) Time from the Start of the Experiment (Days) Piglet Age (Days) Measurement of Piglet Body Weight Fecal Sampling Weaning 0 25 + - Period I Change of feed 25 50 + T1 Period II Change of housing 46 71 + T2 Period III End of post-weaning 70 95 + T3 + and - signs indicate that the actions have or have not been done, respectively. animals-13-00795-t002_Table 2 Table 2 Growth phases based on piglet body weight and overall crude protein percentage in the diet, depending on growth phase and protein level. Growth Phase Protein Level (% CP) High Low From the start of weaning to 15 kg (Period I and II) 18.5 16.5 From 15 kg to the end of the experiment (Period III) 16.5 14.5 animals-13-00795-t003_Table 3 Table 3 Feed ingredients, depending on the growth phase and dietary protein level. Protein Level Feed Composition, as Is 1 From 8 to 15 kg Body Weight From 15 to 30 kg Body Weight High Low High Low Ingredients Corn % 18.8 23.7 26.6 35.0 Barley % 20.0 20.0 15.0 15.0 Expanded wheat % 10.0 10.0 6.00 6.00 Soybean meal (48% CP 2) % 8.87 2.73 8.43 3.07 Wheat middlings, durum % 0.00 0.00 8.13 4.60 Bakery byproducts % 8.00 8.00 4.00 4.00 Whey powder, sweet % 6.00 6.00 0.00 0.00 Soybean protein concentrate (65% CP) % 5.00 5.00 2.00 2.00 Mineral vitamin premix % 5.00 5.00 2.50 2.50 Wheat bran, soft % 4.23 5.00 4.00 4.00 Wheat % 4.00 4.00 8.00 8.00 Wheat middlings, soft % 0.00 0.00 8.00 8.00 Fish meal (68% CP) % 3.00 3.00 2.00 2.00 Dextrose % 2.50 2.50 1.00 1.00 Monodicalcium phosphate % 1.57 1.64 0.00 0.21 Chicory pulp, dehydrated % 1.20 1.20 1.00 1.00 Coconut oil % 0.96 0.83 1.16 1.04 Soybean oil % 0.00 0.00 0.50 0.50 Acidifiers % 0.80 0.80 0.80 0.80 DL-Methionine % 0.05 0.12 0.18 0.25 L-Valine % 0.05 0.17 0.00 0.08 L-lysine HCl % 0.01 0.22 0.47 0.68 L-tryptophan % 0.01 0.04 0.05 0.09 L-threonine % 0.00 0.02 0.14 0.23 Calculated values Crude protein % 18.5 16.6 16.5 14.6 Crude fat % 5.00 5.00 4.50 4.50 Crude fiber % 2.96 2.85 3.90 3.50 Ash % 6.07 5.87 5.50 5.31 Digestible Energy kcal kg-1 3472 3450 3324 3320 Metabolizable Energy kcal kg-1 3289 3277 3176 3180 Net energy kcal kg-1 2480 2510 2401 2447 Lysine % 1.40 1.40 1.20 1.20 Methionine % 0.54 0.58 0.45 0.49 Methionine + Cystine % 0.84 0.84 0.72 0.72 Threonine % 0.92 0.84 0.72 0.72 Tryptophan % 0.28 0.28 0.24 0.24 Valine % 0.98 0.98 0.86 0.84 Isoleucine % 0.78 0.65 0.68 0.56 Calcium % 0.65 0.65 0.59 0.62 Phosphorus % 0.78 0.77 0.50 0.50 1 Amino acid and phosphorus composition is reported as total content. 2 Crude protein. animals-13-00795-t004_Table 4 Table 4 Scores for diarrhea incidence assigned to each cage/pen on the basis of the fraction of litter suffering from diarrhea and diarrhea intensity. Fraction of Litter 1 Suffering from Diarrhea Diarrhea Intensity Mild Medium Serious 0 0 0 0 1/3 1 4 7 2/3 2 5 8 3/3 3 6 9 1 12 piglets per cage/pen. animals-13-00795-t005_Table 5 Table 5 Average value of selected growth parameters during the experiment and significance of the difference between high-protein and low-protein diets (n = 6 per treatment). Protein Level Growth Parameter High Low Significance of the Difference Initial body weight (kg) 7.80 7.77 NS From weaning to change of housing Period I 1 17.2 15.6 NS Period II 2 29.8 28.0 NS Period I + Period II 23.5 21.8 NS Average daily gain (g) Period I 377 313 p < 0.01 Period II 599 593 NS Period I + Period II 479 440 p < 0.05 Average daily feed intake (g) Period I 585 536 NS Period II 1181 1138 NS Period I + Period II 857 810 NS Feed conversion ratio (-/-) Period I 1.55 1.72 p < 0.01 Period II 1.97 1.92 NS Period I + Period II 1.79 1.84 p < 0.05 From change of housing to the end of post-weaning (period III) 3 Average daily gain (g) 457 437 NS Average daily feed intake (g) 952 952 NS Feed conversion ratio (-/-) 2.11 2.18 NS Final body weight (kg) 40.7 38.5 NS 1 Period I: From the start of the experiment until the change of diet; 2 Period II: From the change of diet until the change of housing; Period I + Period II: From the start of the experiment until the change of housing; 3 Period III: From the change of housing until the end of the experiment. animals-13-00795-t006_Table 6 Table 6 Influence of dietary protein level on the diarrhea score summations. The percentages of the scores are shown in parentheses with respect to the total score, referring both to the protein level in the period and to the whole period. Diarrhea Score Summations 1 Protein Level Significance of the Difference Growth period High Low Total in the Period Period I 91 (85.0) 16 (15.0) 107 (45.7) Period II 5 (83.3) 1 (16.7) 6 (2.6) Total (Period I + Period II) 96 (85.0) 17 (15.0) 113 (48.3) p < 0.01 Period III 71 (58.7) 50 (41.3) 121 (51.7) NS Total score 167 (71.4) 67 (28.6) 234 1 Scores were calculated as reported in Table 4. NS: not significant. animals-13-00795-t007_Table 7 Table 7 Relative abundance of bacterial phyla in the piglet fecal microbiota depending on sampling time and protein level 1,2. Phylum Sampling Time and Protein Level Protein Level Significance Level 3 T1 T2 T3 High Low Mean High Low Mean High Low Mean High Low Overall Mean Sampling Time Protein Level Sampling Time x Protein Level Actinobacteria 0.50 0.37 0.43A 0.23 0.18 0.21B 0.20 0.20 0.20B 0.31A 0.25A 0.28 ** NS NS Bacteroidetes 40.7 41.9 41.3B 49.4 50.7 50.1A 46.6 48.6 47.6A 45.6A 47.0A 46.3 ** NS NS Fibrobacteres 0.15 0.28 0.22B 0.38 0.72 0.55A 0.35 0.52 0.43A 0.29B 0.51A 0.38 ** ** NS Firmicutes 54.4 50.6 52.5A 40.6 37.2 38.9B 43.0 39.7 41.4B 46.0A 42.5B 44.3 ** * NS Proteobacteria 1.55 2.40 1.97B 3.02 3.27 3.14A 2.52 3.15 2.83A 2.36B 2.94A 2.67 ** * NS Spirochaetes 0.90 2.43 1.67B 3.93 5.38 4.66A 4.25 4.83 4.54A 3.03B 4.27A 3.60 ** * NS Unclassified 1.63 1.78 1.71B 1.73 1.83 1.78AB 1.98 1.97 1.97A 1.78A 1.86A 1.82 * NS NS Verrucomicrobia 0.00 0.08 0.04C 0.25 0.35 0.30B 0.57 0.57 0.57A 0.27A 0.33A 0.32 ** NS NS 1 T1: sampling after weaning, before the change of feed, T2: sampling after the change of feed and before changing of housing, T3: sampling after the change of housing and before the end of the experiment (end of the post-weaning period). 2 Values of relative abundance greater than 0.1% were considered. 3 Means sharing common letters within the phylum are not significantly different. Capital letters refer to the main factor effects, sampling time, and protein level. Underscore letters for interaction effects are omitted. NS: not significant, *, p < 0.05, **, p < 0.01. animals-13-00795-t008_Table 8 Table 8 Bacterial genera differentially abundant in fecal samples of piglets fed low- or high-protein diets. The notation (T1) indicates that the effect was present only at the sampling time T1. Phylum Family Genus Protein Level of Prevalent Abundance Classes of Relative Abundance >1% 0.1-1% Low High Fibrobacteres Fibrobacteraceae Fibrobacter + Firmicutes Clostridiaceae 1 Clostridium sensu stricto + (T1) Firmicutes Eubacteriaceae Eubacterium + Firmicutes Lachnospiraceae Roseburia Blautia Clostridium XlVa Lachnospiracea_incertae_sedis, Coprococcus, Anaerostipes, Ruminococcus2, Dorea, Fusicatenibacter + Firmicutes Lactobacillaceae Lactobacillus + (T1) Firmicutes Peptostreptococcaceae Clostridium XI + (T1) Firmicutes Erysipelotrichaceae Catenibacterium, Turicibacter, Erysipelotrichaceae_incertae_sedis + (T1) Proteobacteria Succinivibrionaceae Succinivibrio + Spirochaetes Spirochaetaceae Treponema Sphaerochaeta + animals-13-00795-t009_Table 9 Table 9 Composition of the fecal samples depending on sampling time (T1, T2, and T3) and protein level in the diet 1. Parameter Sampling Time Protein Level Significance Level 2 T1 T2 T3 High Low Mean High Low Mean High Low Mean High Low Sampling Time Protein Level Sampling Time x Protein Level Physico-chemical parameters: Dry matter (DM) 24.2 26.1 25.2C 26.5 27.6 27.1B 27.8 29.4 28.6A 26.2B 27.7A ** ** NS Organic matter (% DM) 87.6 88.8 88.2B 89.1 89.1 89.1A 89.2 89.8 89.5A 88.7B 89.2A ** ** ** Total N (% DM) 4.53 4.08 4.30A 3.58 3.29 3.43B 3.01 2.94 2.98C 3.70A 3.44B ** ** NS Ammonium N (% DM) 0.61 0.56 0.59A 0.61 0.51 0.56A 0.49 0.45 0.47B 0.57A 0.51B ** ** NS pH 6.31 6.32 6.31B 6.59 6.55 6.57A 6.62 6.52 6.57A 6.51A 6.46A ** NS NS Crude fiber (% DM) 15.4 14.8 15.1B 16.4 17.1 16.7A 17.6 17.2 17.4A 16.5A 16.4A ** NS NS Hemicellulose 19.9 23.0 21.4C 25.0 27.9 26.1B 30.3 32.1 31.1A 25.0B 27.7A ** ** NS Cellulose 14.4 15.6 15.0B 16.5 16.6 16.6A 16.1 16.3 16.2A 15.7B 16.5A ** * NS Volatile fatty acids (mmol kg-1 DM): Acetic 183 197 190A 237 214 225A 181 189 185A 200A 200A NS NS NS Propionic 62.1 60.3 61.2B 76.7 75.3 76.0A 66.1 66.1 66.1AB 68.3A 67.2A * NS NS Iso-butyric 13.5 26.6 21.1A 29.7 22.6 26.1A 18.5 20.2 19.4A 21.2A 23.1A NS NS NS Butyric 51.3 43.4 47.3A 53.7 47.2 50.5A 40.7 42.7 41.7A 48.5A 44.4A NS NS NS Isovaleric 10.7 12.1 11.4B 17.2 14.1 15.6A 15.1 12.7 13.9A 14.3A 13.0B ** * * Valeric 10.1 11.3 10.7B 14.0 11.7 12.9A 10.9 10.3 10.6B 11.6A 11.1A * NS NS Isocaproic 4.38 6.13 5.26A 4.42 5.43 4.92A 3.92 4.80 4.36A 4.24A 5.46A NS NS NS Caproic 3.18 3.18 3.18A 4.10 2.48 3.29A 2.33 2.32 2.32A 3.21A 2.66A NS NS NS Total 341 361 351A 441 395 418A 344 358 351A 400A 367A NS NS NS 1 T1: sampling after weaning, before the change of feed, T2: sampling after the change of feed and before changing of housing, T3: sampling after the change of housing and before the end of the experiment (end of the post-weaning period). 2 Means sharing commons letters within the phylum are not significantly different. Capital letters refer to the main factor effects, sampling time, and protein level. Underscore letters for interaction effects are omitted. NS: not significant, *, p < 0.05, **, p < 0.01. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000051

Altered dystrophin expression was found in some tumors and recent studies identified a developmental onset of Duchenne muscular dystrophy (DMD). Given that embryogenesis and carcinogenesis share many mechanisms, we analyzed a broad spectrum of tumors to establish whether dystrophin alteration evokes related outcomes. Transcriptomic, proteomic, and mutation datasets from fifty tumor tissues and matching controls (10,894 samples) and 140 corresponding tumor cell lines were analyzed. Interestingly, dystrophin transcripts and protein expression were found widespread across healthy tissues and at housekeeping gene levels. In 80% of tumors, DMD expression was reduced due to transcriptional downregulation and not somatic mutations. The full-length transcript encoding Dp427 was decreased in 68% of tumors, while Dp71 variants showed variability of expression. Notably, low expression of dystrophins was associated with a more advanced stage, older age of onset, and reduced survival across different tumors. Hierarchical clustering analysis of DMD transcripts distinguished malignant from control tissues. Transcriptomes of primary tumors and tumor cell lines with low DMD expression showed enrichment of specific pathways in the differentially expressed genes. Pathways consistently identified: ECM-receptor interaction, calcium signaling, and PI3K-Akt are also altered in DMD muscle. Therefore, the importance of this largest known gene extends beyond its roles identified in DMD, and certainly into oncology. DMD duchenne muscular dystrophy dystrophin malignancy cancer sarcoma University of PortsmouthStrategic PhD studentship Funding from the University of Portsmouth (under the Strategic PhD studentships scheme) is gratefully acknowledged. pmc1. Introduction Duchenne muscular dystrophy (DMD) is a debilitating and lethal neuromuscular disorder. Diagnosis is made between the age of two and five, but transcriptomes from pre-symptomatic DMD patients reveal typical dystrophic abnormalities . In fact, studies of human fetuses and in various animal models revealed that the pathology starts during prenatal development and continues into adulthood. The first DMD defects are detectable in developing cells even before their differentiation into muscle . Given that muscle regeneration replicates processes occurring in muscle development, and that some developmental mechanisms are reactivated in tumors, it is intriguing that changes in DMD gene expression are increasingly being described in various malignancies . Moreover, DMD downregulation affects cell proliferation , adhesion, migration, and invasion --traits that are commonly associated with tumor development. Gaining some understanding of whether these alterations, in a range of diverse cells, have a common origin and lead to a related outcome would expand our knowledge of the role of the DMD gene and potentially open up new treatment avenues. Such analysis must take into consideration the complexity of DMD, the largest human gene known, with 79 exons and eight independent tissue-specific promoters driving the expression of distinct dystrophin isoforms. Three promoters control the expression of 14-kb full-length transcripts encoding 427 kDa isoforms (Dp427). The Dp427m is expressed in myofibers and muscle stem (satellite) cells. In myofibers, it interacts with the dystrophin-associated protein complex (DAPC) with structural and scaffolding roles and an involvement in the regulation of various signaling pathways . Yet, in satellite cells, Dp427m has a different interactome and it is essential for asymmetric cell divisions ; in myoblasts the loss of its expression results in abnormalities of cell proliferation and migration . Dp427c and Dp427p full-length dystrophins are expressed in various neurons , where their loss during development has been linked to the neuropsychological impairment in DMD. Moreover, intragenic promoters give rise to transcripts encoding truncated isoforms: Dp260 in the retina, Dp140 in the CNS, Dp116 in Schwann glia , and Dp71, which is the most ubiquitous DMD product. Alternative splicing adds further structural and functional diversity . DMD transcripts are summarized in Figure S1 of Supplementary File S1. Importantly, loss of Dp427 expression, which is both necessary and sufficient for the occurrence of Duchenne MD, was also associated with increased metastasis of tumors with myogenic programs and soft tissue sarcomas . In contrast to this potential tumor suppressor role of the full-length dystrophin, Dp71 expression was essential for myogenic tumor cell growth . Interestingly, a putative role for the DMD gene was also suggested in various non-myogenic cancers, including carcinomas , melanoma , leukemia , lymphoma , and CNS tumors . Yet, many of the tumors showing phenotypic changes associated with decreased DMD gene expression originate from healthy tissues that are not generally believed to express the full-length dystrophin protein. Seemingly paradoxical, this observation is not limited to tumors: myoblasts, lymphocytes, and endotheliocytes express 14-kb dystrophin transcripts but are not known to synthesize detectable levels of dystrophin protein. Nevertheless, DMD downregulation leads to significant functional abnormalities in these cells . We hypothesized that malignancy can be used as a model to investigate changes in DMD gene expression across normal tissues and corresponding tumors, and aid our understanding of the overall role of this gene, which clearly extends beyond Duchenne MD. Therefore, we studied DMD mRNA and protein expression across various normal tissues and matching tumors, and explored transcriptomic alterations in primary tumors and corresponding tumor cell lines with altered DMD expression to identify putative downstream molecular pathways that could be associated with DMD dysregulation across human tissues. We also examined the association of DMD gene expression with the onset and survival endpoints in tumor patients. 2. Methods 2.1. Gene, Protein, and Clinical Datasets The RNA-seq data for tumor (n = 7894) and control (n = 2714) tissue samples was obtained from the UCSC Xena Functional Genomics Browser accessed on 10 December 2021). The "RSEM norm_count" and "RSEM expected_count" datasets of the TCGA TARGET GTEx cohort were used to examine expression data at the gene and transcript level, respectively, for a variety of cancers in The Cancer Genome Atlas (TCGA) database accessed on 10 December 2021), different non-diseased tissues in the Genotype-Tissue Expression (GTEx) project accessed on 10 December 2021), and two pediatric hematological malignancies in the TARGET (Therapeutically Applicable Research To Generate Effective Treatments) database accessed on 20 July 2022). Samples from the TCGA, TARGET, and GTEx have been re-analyzed and processed by the same bioinformatic pipeline to eliminate batch effects resulting from different computational processing . The expression level of DMD gene/transcripts was compared between tumor tissues and histologically normal tissues adjacent to the tumors (NATs) from the TCGA (16 comparisons). When expression data for NATs was not available in the TCGA, a comparison was made between DMD gene/transcripts expression in tumor samples from the TCGA and healthy tissue samples in the GTEx database (nine comparisons). Database sources in each comparison are stated in Supplementary Table S1. The level of DMD transcripts was also compared between two TARGET pediatric malignancies and GTEx healthy samples. The Proteomics DB data resource accessed on 15 February 2022) was used to examine the expression of the full-length dystrophin protein in normal tissues. This database contains quantitative proteomics data assembled from liquid chromatography tandem-mass-spectrometry (LC-MS/MS) experiments in a variety of normal tissues . Tissue specificity (TS) scores (i.e., the enrichment) of dystrophin protein across a variety of normal tissues were visualized at accessed on 12 October 2022), as well as the correlation between DMD gene and protein expression in 32 different tissue types covering all major organs, including skeletal muscle . The database "PaxDb" (Protein Abundances Across Organisms) was used to examine dystrophin protein abundance data across different normal tissues accessed on 12 October 2022). This database covers a large number of protein abundance datasets in a variety of tissues. For a specific protein of interest, it provides a table of abundances in all available datasets, along with the rank of this protein in the entire detectable proteome . Dystrophin protein expression in normal tissues was also examined using the Human Protein Atlas accessed on 15 February 2022), which contains results from immunohistochemistry assays conducted in a variety of tissues and cell types . Mutation and somatic copy number alteration (SCNA) data for primary tumor samples was derived from the datasets "somatic mutation (SNP and INDEL)--MC3 public version" and "gene-level copy number (gistic2_thresholded)" of the TCGA Pan-cancer (PANCAN) cohort available at the UCSC Xena Browser. The SCNA data was generated by the GISTIC2 algorithm, which generates putative gene copy number specific calls . It should be noted that deep deletions and amplifications are considered as biologically relevant for individual genes by default. Due to purity and ploidy differences between samples, and because these calls are usually not manually reviewed, there may be false positives and false negatives . DMD gene expression and mutation data for tumor cell lines from the Cancer Cell Line Encyclopedia was obtained from cBioPortal website accessed on 19 December 2021). The log2 (norm_value + 1) miRNA mature strand expression data was obtained from the dataset "miRNA mature strand expression--Batch effects normalized miRNA data" of the TCGA PANCAN cohort at the UCSC Xena browser. Data for the American Joint Committee on Cancer (AJCC) pathologic tumor stage and the patient's age at the initial diagnosis was derived from the dataset "Phenotype--curated clinical data" of the TCGA PANCAN cohort. Tumor samples were allocated into the following age groups: 14-29 years, 30-39, 40-49, 50-59, 60-69, 70-79, and 80 and above years. These groups corresponded with the age groups for GTEx donors obtained from the GTEx portal with two exceptions. The GTEx data did not have any samples from patients older than 79 years old or younger than 20 years old. The normalized log-transformed RNA-seq data used to compare gene expression in primary tumor samples with low vs. high DMD expression in 15 TCGA primary tumors was obtained from the UCSC Xena Browser. Links to database sources can be found in Supplementary File S2. Expression data for the DMD gene in tumor cell lines was obtained from the "Expression 21Q3 Public" dataset using the Cancer Dependency Map (DepMap) portal accessed on 20 July 2022), which includes RNA-seq expression data for 1379 tumor cell lines from 37 lineages . Gene expression microarray data for skeletal muscle samples from DMD patients (n = 12) and healthy donors (n = 11) was obtained from the dataset GSE1004 available at the Gene Expression Omnibus (GEO) from the National Center for Biotechnology Information accessed on 23 May 2022). Survival data was derived from the dataset "Phenotype--curated clinical data" of the TCGA PANCAN cohort and the dataset "Phenotype--TARGET donor phenotype" of the TARGET PANCAN cohort available at the UCSC Xena Browser. The STRING database was used to construct dystrophin's protein-protein interaction (PPI) network . 2.2. DMD Transcript Identification and Classification The "RSEM expected_count" dataset of the TCGA TARGET GTEx cohort in the UCSC Xena Browser contains expression data for thirty DMD gene transcripts. The Ensembl database was used for transcript annotation and the identification of coding and non-coding transcripts, and protein products in the UniProt database. However, we note that estimates of transcript expression by RSEM may not be 100% accurate. Protein products were then aligned to known dystrophin isoforms in the UniProt database to identify the isoforms with the highest percentage of sequence identity to these protein products. The HMMER web server accessed on 23 February 2022) was used for the graphical representations of Pfam domains of different dystrophin isoforms. 2.3. Statistical Analysis of Differential DMD Gene and Transcript Expression between Tumor Tissues and Corresponding Controls Statistical analysis of the expression level of the DMD gene and transcripts was performed using the UCSC Xena Browser, using the two-tailed Welch's t-test. The p-Values were then adjusted for multiple testing using the Bonferroni correction; each of the obtained p-Values was multiplied by the total numbers of tests, and when this adjustment gave a value above 1, the corrected p-Values were set at 1. The Bonferroni-corrected p-Values were then compared with the overall level of a (a = 0.05). Log-transformed expression data was downloaded from the UCSC Xena Browser and the mean of the DMD gene and transcripts expression level for tumor (A) and control (B) samples was calculated. The following formula was then used to calculate Log Fold Change (LogFC): log (A/B) = log (A) - log (B). The UCSC Xena Browser's instructions can be found at accessed on 12 December 2021). 2.4. Hierarchical Clustering Analysis of DMD Transcripts Thirty DMD gene transcripts were ranked based on their mean abundance levels in primary tumors and corresponding control tissues, and the top ten highly expressed transcripts were identified. The expression values of the remaining twenty transcripts were collapsed into one value called "other transcripts". Expression values of the top ten highly expressed DMD transcripts as well as the "other transcripts" values were then standardized to a sum of 1 in order to compare the relative expression levels of these transcripts in different tumor and control tissues. An UPGMA hierarchical clustering analysis was then performed using the software R v4.0.4 on an Euclidean distance matrix with the hclust average method. The dendrogram was cut at different levels to yield 3-7 clusters (Supplementary Table S2). Six was chosen as the optimal number of clusters as it was the smallest number that provided more than two clusters with around 10 tissues, and higher numbers of clusters yielded additional smaller clusters with less than three tissues. The R script used to perform the analysis is available at accessed on 15 December 2022). Next, the expression levels of these ten transcripts were compared across three clusters that contained the majority of the analyzed tissues using the Kruskal-Wallis test and Dunn's multiple comparisons test. 2.5. Association between DMD Gene Expression and Mutation Status Tumor samples from 23 tumor types with available mutation and SCNAs data (n = 6751) were used to perform a univariate general linear model (GLM) analysis to determine whether there is a significant difference in DMD gene expression between samples with different mutation/SCNA types and samples with no mutations/SCNAs in the DMD gene locus using SPSS statistical software (v.28.0.0.0). Gender was selected as a random factor to account for gender differences between groups. Estimated Marginal (EM) means were calculated, and p-Values were adjusted using the Bonferroni correction. Next, primary tumor samples and tumor cell line samples (n = 921) were ranked according to their level of DMD expression, and those at the bottom 30% and top 30% were selected for further analysis. A Chi Square test was used to determine whether there is an association between DMD expression level and mutations in coding and non-coding regions, as well as SCNAs in tumor samples and between DMD mutations and expression level in tumor cell lines. 2.6. Association between DMD Gene Expression and Cancer Stage and Patient's Age at the Initial Diagnosis Tumor samples from 18 tumor types with available data for cancer stage, gender, and patient's age at the initial diagnosis (n = 6118) were classified into four groups based on the cancer stage: I, II, III and IV. A univariate GLM analysis was performed to determine whether there is a significant difference in DMD gene expression between these four stages. Gender and age were selected as random factors, and EM means were calculated for the four groups. The p-Values were adjusted using the Bonferroni correction. 2.7. Identification of Differentially Expressed Genes (DEGs) and miRNAs between Primary Tumor Samples with Low and High Levels of DMD Gene Expression Gene expression in primary tumor samples with low vs. high levels of DMD gene expression from 15 different primary tumors was compared. For each tumor, samples were divided into three groups: Group A (samples at the bottom 33.3% of DMD expression), Group B (samples at the top 33.3% of DMD expression) and Group C (the remaining samples with medium level of DMD expression). Using the software R v4.0.4, an Analysis of Variance (ANOVA) was preformed to identify the DEGs between the three groups, and p-Values were adjusted using the False Discovery Rate (FDR) correction. Next, Post-Hoc Pairwise T tests were performed to identify the DEGs between Groups A and B, and p-Values were adjusted using the Bonferroni correction. LogFC was calculated using the formula (LogFC): log (A/B) = log (A) - log (B). Genes with an FDR-corrected p-Value < 0.1, a Bonferroni-corrected p-Value < 0.05, and a |LogFC| value >= 0.7 were considered differentially expressed in Group A compared to Group B. The R script used to perform the analysis is available at accessed on 15 December 2022). Similarly, the differentially expressed miRNAs were identified. 2.8. Identification of DEGs in Tumor Cell Lines with Low and High Levels of DMD Gene Expression We performed two comparisons in tumor cell lines from two distinct categories, carcinoma and sarcoma. A two-class comparison analysis was conducted using the DepMap custom analysis tool accessed on 20 July 2022). The "Expression 21Q2 Public" was selected as the input dataset, and the cell lines with low DMD expression were used as the "in" group cell lines, while cell lines with high DMD expression were used as the "out" group cell lines. The analysis compared gene expression between the selected groups and generated estimates of effect size and corresponding q-values. The DEGs in each of these two comparisons (q-value < 0.05) were identified. 2.9. Identification of DEGs in Skeletal Muscle Samples from DMD Patients in Comparison to Healthy Skeletal Muscle The GEO2R tool at the NCBI website accessed on 23 May 2022) was used to identify the DEGs between 12 skeletal muscle samples collected from DMD patients and 11 samples collected from healthy donors. The default parameters of the GEO2R tool were used, and the NCBI-generated annotations were used to display the list of DEGs. The p-Values were adjusted using the FDR correction. Genes with an FDR-adjusted p-Value < 0.1 and a |LogFC| value >= 0.7 were considered differentially expressed between DMD and normal skeletal muscle samples. The R script generated by the GEO2R tool is available at accessed on 15 December 2022). 2.10. Identification of DEGs between Hematological Malignancies Samples with Low and High Levels of Dp71 Expression We compared gene expression in hematological malignancies samples with low vs. high levels of Dp71 expression from two TCGA and two TARGET studies. Samples (n = 286) were ranked based on the sum expression level of Dp71, Dp71b, and Dp71ab. Samples were then divided into three groups: Group A (samples at the bottom 33.3% of Dp71 expression), Group B (samples at the top 33.3% of Dp71 expression) and Group C (the remaining samples with medium level of Dp71 expression). Using the software R v4.0.4, an ANOVA was preformed to identify the DEGs between the three groups, and p-Values were adjusted using the FDR correction. Next, Post-Hoc Pairwise T tests were performed to identify the DEGs between Groups A and B, and p-Values were adjusted using the Bonferroni correction. Genes with an FDR-corrected p-Value < 0.1, a Bonferroni-corrected p-Value < 0.05, and a |LogFC| value >= 0.7 were considered differentially expressed in Group A compared to Group B. 2.11. Functional Enrichment Analysis The EnrichR tool was used to identify the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) Biological Process terms enriched in the DEGs in the previously mentioned comparisons. In comparisons where the number of DEGs was higher than 1000, only the top 1000 genes with the largest |LogFC| were used in the pathway and GO term enrichment analysis. 2.12. Survival Analysis Samples (n = 6931) from 15 different TCGA primary tumors were ranked based on their levels of DMD gene expression. Next, the following survival endpoints were compared between patients at the bottom 25% of DMD expression and those at the top 25% of DMD expression: overall survival (OS), progression-free interval (PFI), disease-specific survival (DSS), and disease-free interval (DFI). Samples (n = 271) from two TCGA and two TARGET studies of hematological malignancies were ranked based on the sum expression level of Dp71, Dp71b, and Dp71ab. Next, OS was compared between patients at the bottom 25% of Dp71 expression and those at the top 25% of Dp71 expression. Kaplan-Meier curves were generated in GraphPad and analyzed using the log-rank test. 3. Results 3.1. Significant Expression of Dystrophin Transcript and Protein in a Range of Healthy Tissues Expression data for 17 healthy tissues as well as skeletal muscle tissue from the GTEx database was examined. These were adrenal glands, bladder, breast, cervix, colon, esophagus, kidney, liver, lung, ovary, pancreas, prostate, skin, stomach, thyroid, uterus, and whole blood. DMD expression levels across this spectrum of healthy tissues were compared with the expression levels of two housekeeping genes (HKGs), PKG1 and HMBS, which were identified as HKGs across 32 tissues in the GTEx database . DMD expression averaged 79.3% of PKG1 (range between 30.8 and 94.6%) and 114.7% of HMBS (43.7 to 137.4%) expression levels with the lowest expression found in whole blood (Supplementary Table S3). In skeletal muscle, DMD expression was 94.6% of PKG1 and 138.2% of HMBS. DMD expression relative to PKG1 and HMBS was compared between skeletal muscle tissue and the previously mentioned 17 healthy tissues using a Kruskal-Wallis and Dunn's multiple comparisons test. The expression of DMD relative to PKG1 in skeletal muscle was comparable to that in the pancreas, uterus, and bladder (adjusted p-Value > 0.9999), and DMD expression relative to HMBS was comparable to that in the uterus, ovary, and bladder (adjusted p-Value > 0.9999). Given this widespread presence of considerable DMD transcript levels, we investigated whether it is accompanied by protein expression. We interrogated mass spectrometry (MS) protein expression datasets available at Proteomics DB. MS identified the full-length dystrophin protein in a variety of normal tissues . Moreover, quantitative profiling of the proteome of 201 samples from 32 tissues in the GTEx database identified dystrophin as a housekeeping protein, as it was present in all of the 32 tissues analyzed . Dystrophin protein expression was statistically significantly and positively correlated with DMD gene expression in those samples (Spearman correlation = 0.67, BH-adjusted p-Value < 0.1). Finally, in the Protein Abundance Database (PAXdb), dystrophin expression was ranked in the top 25% of MS-quantified proteins in a range of tissues, such as fallopian tubes, esophagus, uterus, bladder, colon, prostate, and rectum, in addition to the heart. Thus, data from three databases demonstrated significant expression of dystrophin protein in a range of healthy tissues. Therefore, we investigated whether alterations in DMD expression might occur in tumors that originate from tissues not commonly associated with dystrophin protein expression or function. 3.2. Downregulation of DMD Gene Expression across Malignancies We investigated RNA-seq expression data for 25 different types of primary tumors from the TCGA database (carcinomas, melanoma, lymphoma, and leukemia) and their corresponding NATs from the TCGA or healthy GTEx tissues. The analyzed primary tumors were acute myeloid leukemia (LAML), adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical and endocervical cancer (CESC), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), diffuse large B-cell lymphoma (DLBC), esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), kidney chromophobe cell carcinoma (KICH), kidney clear cell carcinoma (KIRC), kidney papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), rectal adenocarcinoma (READ), skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), thyroid carcinoma (THCA), uterine carcinosarcoma (UCS), and uterine corpus endometrioid carcinoma (UCEC). Global DMD gene expression was reduced in 20 out of 25 primary tumors in comparison to their corresponding control tissues . The largest difference in expression was found in primary breast invasive carcinoma (LogFC = -3.7), and the smallest in primary kidney papillary cell carcinoma (LogFC = -1). DMD gene expression was increased in primary tumors in two out of 25 comparisons, namely in primary thyroid carcinoma and diffuse large B-cell lymphoma, with LogFC values of 0.9 and 4.8, respectively. No significant expression changes were found in three comparisons (acute myeloid leukemia, kidney clear cell, and chromophobe cell carcinomas). To examine the impact of batch effects resulting from differences in processing between TCGA and GTEx samples in 13 comparisons, where DMD expression was compared between TCGA tumors and their corresponding NATs, we also compared DMD expression between these tumors and corresponding healthy tissues from the GTEx database. We found consistent results in 11 out of 13 comparisons, including BRCA, BLCA, UCEC, COAD, STAD, PRAD, LUSC, LIHC, LUAD, KICH, and THCA (Supplementary Table S4). In KIRP, DMD expression was reduced compared to kidney NAT in TCGA datasets (adjusted p-Value = 9.30 x 10-22), however, no statistically significant difference was found between DMD expression in KIRP and GTEx healthy kidney tissue samples (adjusted p-Value = 1). Moreover, there was no statistically significant difference in DMD expression between KIRC and paired kidney NAT samples (adjusted p-Value = 1), while DMD expression was found upregulated in KIRC samples compared to GTEx healthy kidney tissue (adjusted p-Value = 2.77 x 10-8). These inconsistencies might result from small sample sizes leading to a reduction in statistical power. Given the well-known multiplicity of transcripts originating from the DMD gene and the variability in their expression patterns, a hierarchical clustering analysis was performed to identify changes in DMD expression profiles at the transcript level across various control tissues and corresponding tumors. 3.3. Hierarchical Clustering Analysis of the Relative Expression of DMD Transcripts Distinguishes Tumor Tissues Of the top ten highly expressed transcripts (Supplementary Table S5), nine were predicted to be protein-coding . Three of these mRNAs were not canonical DMD gene transcripts. Of those, ENST00000358062.6 encoding H0Y304 protein is a poorly annotated transcript (Ensembl). The start of its CDS is unknown due to a 5' truncation of the available sequence. The 5' sequence upstream of the first exon (equivalent to exon 48 of the full-length transcript) is composed of the last 50 base pairs of the intron located between exons 47 and 48. Therefore, it is not clear whether this is a pre-mRNA sequence undergoing co-transcriptional splicing or a mature mRNA with this part of the intron spliced in. If the latter, the predicted protein encoded by such a transcript would have an N-terminus longer than Dp140 with valine as the putative initiation codon (UniProt). The transcript encoding H0Y864 appears to be a partial four-exon transcript, whose predicted protein sequence does not encode any functional domains . ENST00000475732.1 is a two-exon sequence not predicted to encode a protein. Alternative splicing is a discernible feature of DMD transcripts found in the analyzed samples, with the splice variant Dp71b lacking exon 78 having the highest mean abundance level of all DMD transcripts across the analyzed tumor and control tissues, followed by Dp427m. The mRNA encoding Dp140c differs from the canonical Dp140 transcript, as it lacks exons 28 to 31 (equivalent to exons 71 to 74 of the full-length transcript) . Hierarchical clustering analysis of these ten highly expressed transcripts in tumor and control tissues yielded six clusters. Supplementary Table S2 reports other dendrogram cut-offs. We focused mainly on three of these clusters that contained the majority of tumor and control tissues. The first cluster was composed of nine tumors and healthy whole blood and pancreas tissue. The second cluster was composed of 16 control tissues (including six healthy GTEx tissues and 10 NATs from the TCGA) as well as SKCM and PRAD tumor samples. Finally, the third cluster contained 12 tumors in addition to kidney and thyroid NATs from the TCGA. The relative expression of the transcript encoding Dp427m was significantly lower in the first and third clusters, which were composed mainly of tumors, compared to the second cluster with a majority of control tissues, while the relative expression of Dp71 variants was higher in the first and third clusters . Specific transcriptomic alterations associated with decreased DMD gene expression are described in Section 3.7. Transcripts ENST00000493412.1 and ENST00000475732.1 did not show significantly different levels of expression between any of the clusters , and the differential expression of ENST00000358062.6 was not followed due to its uncertain annotation. 3.4. Changes in the Expression of Dp427m and Dp71 Transcripts across Malignancies Given the importance of the full-length dystrophin, whose loss is responsible for Duchenne muscular dystrophy, and the predominance of the transcript encoding Dp427m (Supplementary Table S5), its expression patterns were analyzed in more detail. As Dp71 is the isoform most widely expressed across the body and its splice variants were among the top highly expressed DMD transcripts in tumor and control tissues (Supplementary Table S5), we also compared expression patterns of this transcript (Supplementary Table S6). Dp427m expression was decreased in primary tumors compared to control tissues in 17 out of 25 comparisons with LogFC values ranging from -7.2 to -2.7 in primary uterine carcinosarcoma and primary thyroid carcinoma, respectively . There was a statistically significant change in the expression of transcripts encoding Dp71 in 10 out of 25, Dp71ab in 13 out of 25, while Dp71b was altered in 20 out of 25 comparisons (a decrease in 17 and an increase in three) (Supplementary Table S6). Further analysis of these DMD transcripts showed that changes in overall DMD expression levels in two tumor categories were confounded by the opposing dysregulation of Dp427m and Dp71 variants. Specifically, while DMD expression was higher in primary thyroid carcinoma compared to thyroid NAT, Dp427m expression there was lower compared to thyroid NAT, and the increase in overall DMD gene expression resulted from the elevated expression of Dp71b . In contrast, in primary pancreatic adenocarcinoma, total DMD expression was lower compared to the healthy pancreas tissue, but this decrease resulted from the lower expression of transcripts encoding Dp71 variants, while Dp427m expression was higher in this tumor type (LogFC = 3.7 compared to healthy pancreas tissue) . 3.5. DMD Expression Downregulation Occurs Irrespective of Somatic Mutations within the DMD Locus Next, using datasets for samples from 23 of the previously mentioned TCGA tumors (LAML and SKCM samples were excluded as they did not have mutation and SCNA details available) we investigated the association between DMD expression and mutations in coding (CRs) and non-coding regions (NCRs) as well as SCNAs in the DMD gene. The majority of tumor samples had no identified CR mutations in the DMD gene locus (6201 out of 6751). A univariate GLM analysis was carried out to assess the effect of CR mutations and gender (to account for the X-chromosome localization of the DMD gene) as well as their interaction on DMD expression. The GLM indicated a significant effect for CR mutations on DMD expression (p = 0.007). There was no effect for gender (p = 0.703) or the interaction between CR mutations and gender (p = 0.648). Samples with missense and multiple mutations had significantly lower levels of DMD expression compared to samples with no CR mutations in the DMD locus (p < 0.001 and p = 0.043, respectively) . A Chi Square test revealed that there is an overrepresentation of DMD mutations in tumor samples with low DMD expression (X2(1) = 45.44, p < 0.0001). Specifically, 11.7% of samples in the low DMD group (237 out of 2025) had DMD CR mutations compared to 5.73% (116 out of 2025) in the high DMD group. However, in the low DMD group, 88.3% of samples (1788 out of 2025) did not have any detectable mutations in the CRs of the DMD locus. As for NCR mutations, 98.2% of tumor samples (6632 out 6751) had no mutations in the non-coding regions of the DMD gene. NCR mutations were found to have a significant effect on DMD expression (p = 0.031), but no effect was found for gender (p = 0.104) or the interaction between gender and NCR mutations (p = 0.698). Samples with intronic mutations had significantly lower DMD expression compared to samples without any DMD NCR mutations (p = 0.02) . A Chi Square test revealed that there is an overrepresentation of DMD NCR mutations in tumor samples with low DMD expression (X2(1) = 18.71, p < 0.0001). In the low DMD group, 3.01% of tumor samples (61 out of 2025) had DMD NCR mutations compared to 1.09% of samples (22 out of 2025) in the high DMD group. However, 96.99% of samples in the low DMD group (1964 out of 2025) did not have any NCR mutations in the DMD locus. Regarding SCNAs, 66.7% of samples had a normal copy number of the DMD locus (4503 out of 6751). Both SCNAs (p < 0.001) and gender (p = 0.006) had a significant effect on DMD expression. However, the interaction between the two factors did not have a significant effect. Samples with a normal DMD copy number had higher expression compared to samples with deep and shallow deletions and those with gains (p < 0.001). Additionally, samples with deep deletions had lower expression compared to those with amplifications (p = 0.002) . In samples from female patients, we found that there is an overrepresentation of DMD SCNAs in the low DMD group (X2(1) = 36.40, p < 0.0001), where 39.08% of samples (399 out of 1021) had SCNAs in the DMD locus compared to 26.54% in the high DMD group (271 out of 1021). In the low DMD group, 60.92% of samples (622 out of 1021) did not have any SCNAs. In samples from male patients, we also found that there is an overrepresentation of DMD SCNAs in the low DMD group (X2(1) = 69.82, p < 0.0001), where 44.18% of samples (444 out of 1005) had SCNAs in the DMD locus compared to 26.37% in the high DMD group (265 out of 1005). In the low DMD group, 55.82% of samples had no SCNAs. We further investigated the association between DMD expression and mutations using data from 921 tumor cell lines (cBioPortal). The majority of tumor cell lines had no identified mutations within the DMD gene region (n = 773), while the remaining had missense (n = 120), truncating (n = 11), splice (n = 4), and multiple mutations (n = 13). We found that there is an overrepresentation of DMD mutations in tumor cell lines with low levels of DMD expression (X2(1) = 9.727, p = 0.0018). However, only 23% of tumor cell lines with low DMD gene expression (63 out of 276) had DMD mutations, but the majority of cell lines (213 out of 276) had low levels of DMD expression without any detectable mutations. Therefore, downregulation of DMD expression in tumors cannot be simply attributed to somatic mutations or copy number alterations within the DMD locus, but rather involves a regulatory mechanism. 3.6. Association between DMD Expression and Cancer Stage and Patient's Age We investigated the association between DMD expression and stage in 18 different types of primary cancers with available data for stage, gender, and patient's age at the initial diagnosis. These tumors included ACC, BLCA, BRCA, CHOL, COAD, ESCA, HNSC, KICH, KIRC, KIRP, LIHC, LUAD, LUSC, PAAD, READ, SKCM, STAD, and THCA. The ages of patients ranged from 14 to 90 years old. A GLM analysis was performed to assess the effect of cancer stage on DMD expression while accounting for gender and age differences. Cancer stage was found to have an effect on DMD expression (p = 0.035). While no effect was identified for gender differences (p = 0.735), the interaction between cancer stage and gender was found to have an impact on DMD expression (p = 0.010). Age of patients was also found to have an impact on DMD gene expression (p < 0.001) as well as the interaction between age and cancer stage (p = 0.003). Samples from patients with stage I cancer had significantly higher levels of DMD gene expression compared to those with stage II (LogFC = 0.72, p < 0.001), stage III (LogFC = 0.50, p < 0.001), and stage IV cancer (LogFC = 0.69, p < 0.001) . Moreover, samples from younger patients had significantly higher DMD expression compared to samples from older patients . Twelve of the tumor tissues used in the previous analysis have corresponding healthy tissues in the GTEx database: adrenal glands, bladder, breast, colon, esophagus, kidney, liver, lung, pancreas, skin, stomach, and thyroid. We examined whether there is an association between DMD gene expression and age in these healthy tissue samples (n = 2863). The ages of donors ranged from 20 to 79 years old, and samples were grouped into six age groups: 20-29, 30-39, 40-49, 50-59, 60-69, and 70-79 years. There was no statistically significant effect for age (p = 0.484), gender (p = 0.647), or their interaction (p = 0.591) on DMD expression in these healthy samples. 3.7. Decreased DMD Gene Expression in Primary Tumors Is Associated with Specific Transcriptomic Alterations Expression of the DMD gene is significantly altered in tumors, with the majority having lower expression levels compared to their respective control tissues. This downregulation predominantly affects the transcript encoding the full-length dystrophin due to a regulatory alteration. To identify downstream molecular pathways that could be associated with such DMD downregulation (analogous to the impact of full-length dystrophin loss in muscle), we compared transcriptomes of primary tumor samples with low vs. high DMD gene expression from 15 different tumor types: BRCA, BLCA, UCEC, CESC, OV, COAD, STAD, PRAD, LUSC, ESCA, HNSC, LUAD, KIRP, and THCA, where Dp427m was found to be downregulated compared to control tissues. In addition, we included sarcoma (SARC) in this comparison because sarcomas originate from tissues (muscle and bone) known to express the DMD gene. For clarification, RNA-seq data for control tissues for sarcomas was not available in the TCGA TARGET GTEx cohort, and therefore DMD gene expression in sarcomas vs. control tissues could not be compared. In each tumor type, the DEGs between samples at the bottom 33.3% and top 33.3% of DMD expression were identified and used to perform a pathway enrichment analysis. Figure 6 shows the combined score values (-Log (p-Value) x odds ratio) for KEGG pathways that were found to be enriched in DEGs in more than 50% of the analyzed primary tumors (adjusted p-Value < 0.05). The differentially expressed transcripts in tumors with low DMD expression showed enrichment in the ECM-receptor interaction pathway in all 15 primary tumors analyzed. Calcium signaling and protein digestion and absorption were enriched in 13 tumors, cell adhesion molecules in 12, focal adhesion in 11, PI3K and cAMP signaling in 10, Wnt signaling in 9, and cGMP-PKG signaling and axon guidance were enriched in 8 comparisons. The specific GO Biological Process terms enriched in DEGs in primary tumors with low vs. high DMD expression were also identified . The GO terms enriched in more than 50% of tumors were: extracellular matrix organization (14 out of 15 tumors), axonogenesis (12), regulation of cell migration, synapse organization and nervous system development (11), skeletal system development, regulation of ERK1 and ERK2 cascade (10), and calcium ion transmembrane import into cytosol (8). Lists of DEGs in each comparison and results of the pathway and GO term analysis can be found in Supplementary File S2. In order to confirm that these transcriptomic changes between primary tumor samples with low vs. high DMD expression are associated with DMD downregulation and not a result of other factors, we repeated the analysis for each tumor type using three groups of tumor samples identical in size to the groups used in the previous analysis, but that were chosen at random. DMD gene expression was not statistically significantly different between the three random groups in all of the 15 tumors, and no statistically significant differences were found in the transcriptomes between these groups, confirming the specific gene expression alterations to be associated with DMD downregulation (Supplementary File S2). 3.8. Transcriptomic Alterations Associated with Decreased DMD Gene Expression in Tumor Cell Lines To further confirm that these transcriptomic changes are evoked by DMD downregulation specifically in cancer cells rather than originating from DMD expression in the stromal or infiltrating immune cells present in tumor tissue samples, we conducted a two-class comparison analysis using the DepMap portal to identify DEGs between cell lines of the same tumor origin (carcinoma and sarcoma) but with low vs. high level of DMD gene expression. While the DMD gene was not found to be essential for tumor cell line survival , its altered expression may have an important common effect across malignancies. One-hundred and forty tumor cell lines were grouped into four groups based on their level of DMD expression (low or high) and their origin (carcinoma or sarcoma) (Supplementary File S2). The ranges for DMD expression for these cell lines were as follows (Unit: log2 (TPM + 1)): carcinoma cell lines with high (3.3-7.4) and low (0-0.01) DMD expression, sarcoma cell lines with high (4.07-7.6) and low (0-0.06) DMD expression. The carcinoma cell lines used in this analysis originated from tissues where primary tumors were found to have a lower level of Dp427m transcript compared to control tissues. The two-class comparison analysis between cell lines with low vs. high DMD expression identified 998 DEGs for carcinoma, and 543 DEGs for sarcoma cell lines. Interestingly, in carcinoma cell lines with low DMD expression, the majority of DEGs were downregulated (n = 976 out of 998). Figure 6 shows the combined score values (-Log (p-Value) x odds ratio) for KEGG pathways that were found to be enriched in DEGs in these two comparisons (adjusted p-Value < 0.05). Pathway enrichment for the DEGs in carcinoma cell lines with low DMD expression suggested that DMD downregulation may affect the following KEGG pathways: ECM-receptor interaction (p = 0.037), protein digestion and absorption (p = 0.015), focal adhesion (p = 0.029), PI3K-Akt signaling (p = 0.021), cAMP signaling (p = 0.044), cGMP-PKG signaling (p = 0.024), and axon guidance (p = 0.002). Notably, these pathways were also enriched in the DEGs in more than 50% of primary tumors with low DMD expression. Similar to carcinoma cell lines, the following KEGG pathways were enriched in the DEGs in sarcoma cell lines with low DMD expression: ECM-receptor interaction (p = 0.048), PI3K-Akt signaling (p = 0.041), and cAMP signaling (p = 0.034). The calcium signaling pathway, which was enriched in the DEGs in more than 50% of primary tumors with low DMD expression, was also enriched in the DEGs in the comparison of sarcoma cell lines (p = 7.24 x 10-4). Moreover, GO Biological Process term analysis for DEGs in carcinoma cell lines with low vs. high DMD expression identified the following terms: extracellular matrix organization (p = 5.22 x 10-4), cell junction assembly (p = 0.012), and positive regulation of epithelial to mesenchymal transition (p = 0.038). Terms related to the development of the CNS and synaptic transmission: nervous system development (p = 8.39 x 10-5), neuron migration (p = 8.39 x 10-5), and regulation of neuron projection development (p = 1.04 x 10-5) were also found. The GO Biological Process term regulation of cell migration was enriched (p = 0.02) in DEGs in sarcoma cell lines with low vs. high DMD expression . Lists of the DEGs in each comparison and results of the pathway and GO term analysis can be found in Supplementary File S2. 3.9. Transcriptomic Alterations in Duchenne Skeletal Muscle Compared to Healthy Muscle Samples Next, we investigated whether the transcriptomic changes resulting from DMD downregulation in primary tumors and tumor cell lines are similar to those observed in skeletal muscles of DMD patients. We compared gene expression data from 12 DMD skeletal muscle and 11 healthy muscle samples. The GEO2R tool identified 1160 genes to be differentially expressed between DMD and healthy skeletal muscle. Figure 6 shows the combined score values (-Log (p-Value) x odds ratio) for the KEGG pathways that were found to be enriched in the top 1000 DEGs with the highest |LogFC| values in this comparison (adjusted p-Value < 0.05). Pathway enrichment analysis indicated DMD downregulation to be associated with changes in the following KEGG pathways: ECM receptor interaction (p = 7.82 x 10-11), calcium signaling pathway (p = 8.80 x 10-8), protein digestion and absorption (p = 3.67 x 10-8), cell adhesion molecules (p = 1.55 x 10-4), focal adhesion (p = 2.87 x 10-12), PI3K-Akt signaling (p = 7.82 x 10-11), cAMP signaling (p = 0.020), cGMP-PKG signaling (p = 0.007), and axon guidance pathways (p = 0.030). These very pathways were enriched in the DEGs in more than 50% of primary tumors with low DMD expression, and also enriched in DEGs in carcinoma and sarcoma cell lines with low vs. high DMD expression. GO Biological Process term analysis for DEGs in DMD skeletal muscle compared to healthy muscle identified the following terms: extracellular matrix organization (p = 6.25 x 10-17), axonogenesis (p = 9.35 x 10-5), regulation of cell migration (p = 3.39 x 10-12), synapse organization (p = 0.009), nervous system development (p = 1.87 x 10-4), skeletal system development (p = 2.22 x 10-4), regulation of ERK1 and ERK2 cascade (p = 5.15 x 10-8), and calcium ion transmembrane import into cytosol (p = 0.001). These GO terms were enriched in DEGs in more than 50% of primary tumors with low DMD expression. GO terms that were found to be in common with carcinoma cell lines with low DMD expression were: extracellular matrix organization, nervous system development, cell junction assembly (p = 0.004), regulation of neuron projection development (p = 0.005), neuron migration (p = 0.004), and positive regulation of epithelial to mesenchymal transition (p = 2.52 x 10-4). Finally, the GO term, regulation of cell migration, was found to be in common with sarcoma cell lines with low DMD expression. Furthermore, functional enrichment tests of the dystrophin protein-protein interaction network constructed using high confidence PPI information demonstrated that the enriched functional terms were consistent with the pathways found to be significantly enriched in the aforementioned comparisons of primary tumors, tumor cell lines, and DMD skeletal muscle: extracellular matrix organization (p = 1.10 x 10-18), axon guidance (p = 9.45 x 10-10), focal adhesion (p = 3.27 x 10-14), and PI3K-Akt signaling pathways (p = 9.27 x 10-9) . 3.10. Low DMD Gene Expression Is Associated with Poor Survival in Patients with 15 Different Primary Tumor Types Given these similarities between pathways altered in tumors and in the lethal muscle disease, we examined the association between DMD gene expression and patients' survival in the following tumor types: BRCA, BLCA, UCEC, CESC, OV, COAD, STAD, PRAD, LUSC, ESCA, HNSC, LUAD, KIRP, THCA, and SARC. We compared overall survival (OS), progression-free interval (PFI), disease-specific survival (DSS), and disease-free interval (DFI) endpoints between patients at the bottom 25% of DMD expression and those at the top 25% across all the aforementioned tumor types. OS was lower in the low DMD group (HR 1.33; 95% 1.17, 1.51; p < 0.0001) with 2417 days compared to 3253 for the high DMD group. PFI was also decreased in the low DMD group (HR 1.28; 95% 1.14, 1.45; p < 0.0001) with 2472 days compared to 3669 days, respectively. Finally, the low DMD group had lower DSS (HR 1.46; 95% 1.24, 1.72; p < 0.0001) and DFI (HR 1.30; 95% 1.06, 1.59; p = 0.012) compared to the high DMD group . In order to confirm that these changes in survival outcomes between patients with low vs. high DMD expression in their tumors are associated with DMD downregulation and not a result of other factors, we repeated the survival analysis using two groups of patients chosen at random, and no statistically significant differences in survival endpoints were found between the two. 3.11. Transcriptomic Alterations in Hematological Malignancies with Low vs. High Dp71 Expression The hierarchical clustering analysis revealed that the blood malignancies acute myeloid leukemia (LAML) and diffuse large B-cell lymphoma (DLBC) had a unique pattern of DMD transcripts, to the point that these two malignancies were classified as a separate cluster . While no changes in Dp427m expression were observed in both TCGA datasets for LAML and DLBC compared to healthy blood, Dp71 levels including its splice variants Dp71b and Dp71ab were higher in these tumors . Levels of Dp71 and its splice variants were also found higher in two TARGET datasets (acute myeloid leukemia and acute lymphoblastic leukemia) compared to GTEx healthy whole blood (Supplementary Table S7). However, we note that age differences between TARGET and GTEx donors might be a confounding factor when interpreting these results. Next, we compared gene expression between samples from the previously mentioned TCGA and TARGET datasets at the bottom 33.3% and top 33.3% of Dp71 expression across all tumor types. The pathways enriched in the top 1000 DEGs with the largest |LogFC| were: protein digestion and absorption (p = 8.59 x 10-5), ECM-receptor interaction (p = 3.87 x 10-4), focal adhesion (p = 0.003), and PI3K-Akt signaling pathway (p = 0.02). The identified GO Biological Process terms for these genes were extracellular matrix organization (p = 1.57 x 10-10), regulation of angiogenesis (p = 7.94 x 10-4), skeletal muscle development (p = 8.38 x 10-4), positive regulation of MAPK cascade (p = 0.005), regulation of ERK1 and ERK2 cascade (p = 0.006), regulation of cell migration (p = 0.01), and positive regulation of calcium ion import (p = 0.02) (Supplementary File S2). Thus, these pathways and processes were identical with those found in comparisons with low vs. high expression of Dp427m in completely unrelated malignancies. A comparison between random groups of samples from these hematological tumors did not reveal any statistically significant changes in gene expression. 3.12. Low Expression of Dp71 Is Associated with Poor Survival in Patients with Hematological Malignancies We compared OS between hematological malignancies patients with low and high expression of Dp71 and its splice variants. OS was lower in the low Dp71 group (HR 2.39; 95% 1.44, 3.99; p = 0.0003) with 792 days compared to 1992 for the high Dp71 group . For specificity testing, the survival analysis was conducted using two random groups of patients with these hematological malignancies and no statistically significant difference in overall survival was found. Thus, while specific dystrophins are differentially regulated across various tumors, the low expression of both full-length dystrophin and the Dp71 variants in the analyzed tumors is associated with analogous molecular alterations and significantly decreased patient survival. 4. Discussion We found a significant downregulation of DMD gene expression across diverse primary tumors. Both full-length and truncated dystrophin variants were differentially expressed, and hierarchical clustering of the top highly expressed transcripts distinguished tumors from corresponding control tissue samples. A similar trend for DMD downregulation across carcinomas has been described previously , and our analysis discriminating specific DMD transcripts showed that levels of Dp427m mRNA were statistically significantly decreased in the clusters composed mainly of tumor tissues , suggesting a specific impact of the loss of the full-length transcript. Indeed, pancreatic adenocarcinoma was the only primary tumor with higher levels of Dp427m transcripts compared to healthy pancreas tissue. In contrast, the relative expression of Dp71 splice variants was increased in tumor clusters. In contrast to the majority of primary tumor samples, in hematological malignancies, total DMD expression and the expression of the full-length and Dp71 transcripts was higher or unchanged when compared to normal blood, which also showed the lowest DMD expression relative to housekeeping genes of all the healthy tissues analyzed (Supplementary Table S3). However, human and mouse hematopoietic stem cells were found to express Dp71, and its expression was decreasing with cell differentiation (manuscript submitted), which agrees with whole blood showing the lowest DMD expression. Interestingly, while Dp71 expression is found upregulated in hematological malignancies, its low levels were found to be associated with the very same dystrophic molecular alterations in cancer cells. Despite the advantage that using NATs as control samples in cancer studies reduces individual and anatomical site-specific confounding factors and eliminates technical interlaboratory differences, it was found that these tissues are distinct from healthy and tumor tissues and represent a unique intermediate state between them . Although we showed that the results of comparing DMD expression between TCGA tumors and their corresponding NATs were consistent with those of comparing DMD expression between TCGA tumors and healthy GTEx tissues in 11 out of 13 comparisons, the unique transcriptomic profile of NATs might explain why thyroid NATs and kidney NATs from three different kidney TCGA tumors clustered with tumor tissues in the third cluster. The opposite was observed in the second cluster, where PRAD and SKCM samples clustered with control tissues. This might be the result of a high proportion of non-tumor cells in these tumor samples. Given some evidence of a causative link between DMD downregulation and phenotypic changes in tumor cells and that alterations in Duchenne, such as increased cell proliferation, abnormalities in adhesion, migration, and invasion are commonly associated with malignancy, transcriptomes of primary tumor samples as well as tumor cell lines with low vs. high levels of DMD gene expression were compared. While no causation can be confirmed at this stage, it is important to note that DMD dysregulation was associated with specific transcriptomic changes across 15 primary tumors and 140 various tumor cell lines . Functional enrichment analysis showed that the pathways and GO Biological Process terms significantly enriched in DEGs in primary tumors and cell lines with low vs. high DMD expression were consistent with the pathways and GO terms enriched in DEGs in DMD skeletal muscle as well as the functional dystrophin PPI network . Key pathways altered, including cell adhesion, ECM interactions, and PI3K-Akt signaling, correspond to alterations found in Duchenne patients' cells . The calcium signaling pathway enriched in DEGs in tumors samples with low DMD expression from 13 out of the 15 analyzed primary tumors, and well as in sarcoma cell lines with low DMD expression, agrees with the dysregulation of calcium signaling across a whole spectrum of dystrophic cells (reviewed in ), as do GO terms related to the regulation of the developmental mechanisms . Thus, the DMD gene may play similar roles in cancer and development, two processes showing biological and molecular similarities . The protein digestion and absorption amongst the top pathways identified is somewhat surprising, but it is also present in DMD muscles and must reflect the overlap between DEGs. For example, 17 genes in this pathway are shared with the ECM receptor interaction, focal adhesion, and PI3K-Akt, and six genes are shared with the cAMP and cGMP-PKG signaling pathways. Dystrophin in tumor cell lines did not correlate strongly with the presence of its established DAPC partners, suggesting that it may have a different role(s) than those in muscle cells (Supplementary Table S8). This is unsurprising, given that DAPs are known to differ in different tissues, with muscle and brain being the most notable examples. But even within muscle, the dystrophin interactome changes with differentiation, and functionally distinct DAPs exist in satellite cells and myofibers. The importance of the DMD gene in tumorigenesis is supported by the finding that low DMD expression was associated with poor survival outcomes in patients with 15 different types of tumors (14 carcinomas and sarcoma). The overall survival of cancer patients with decreased DMD expression in tumors was 27 months lower than that of patients with high DMD expression. However, since the low and high DMD groups used in this analysis were composed of 15 tumor types, the number of samples for each of those types is highly variable between the two groups, and this could possibly be a confounding factor when interpreting the results. In other studies, mutations in the DMD gene were associated with poor overall survival of patients of two out of 11 analyzed tumors, namely uterine corpus endometrioid carcinoma and breast invasive carcinoma . Dystrophin protein was also identified as a survival biomarker in upper gastrointestinal cancer patients, as poorer survival was observed in patients with low compared to high levels of dystrophin protein . In our analyses, the overall survival of patients suffering from hematological malignancies with decreased Dp71 expression was about 39 months lower than that of patients with high Dp71 expression. However, in low-grade glioma and B-cell chronic lymphocytic leukemia , high Dp71 expression was previously associated with poor patient survival. We found DMD expression to be associated with the tumor stage. Samples from patients with stage I had significantly higher levels of DMD expression compared to higher stages after controlling for age and gender differences. DMD expression was also found to decrease with the age of onset, as samples from younger patients had higher DMD expression compared to samples from older patients. This association between DMD expression and age was unique to tumor tissues, as no such association was found by us in the corresponding healthy tissues from the GTEx database, and also in a meta-analysis that identified genes with age-associated expression in human peripheral blood samples . It is worth noting that recent studies identified increased frequency of rhabdomyosarcomas in DMD patients which agrees with previous data on spontaneous rhabdomyosarcomas in dystrophic mice . Crucially, we demonstrated downregulation of DMD expression in tumors with incidence increasing with age. Therefore, with improved therapies, the risk of malignancy in DMD should be considered. As for hematological malignancies, which frequently affect children, poor survival was associated with Dp71 downregulation. Expression of this dystrophin is not affected in the vast majority of DMD patients. A further indication of the functional significance of the DMD gene in tumors is that DMD downregulation across various malignancies involves regulatory changes, not just results from somatic gene mutations. Although, as expected, the presence of some types of somatic mutations and SCNAs was associated with lower levels of DMD expression, in about 88% of tumor samples and 77% of cell lines, DMD downregulation could not be linked to mutations in the coding regions of the DMD gene. Moreover, DMD downregulation was not a result of SCNAs in about 61% of samples from female patients and about 56% of samples from male patients. While deletions were described as a causative factor for the downregulation of DMD gene expression in some tumors , significantly reduced expression was also found in the absence of deletions or nonsense mutations , in agreement with our comprehensive analysis across different malignancies. Moreover, in primary pancreatic adenocarcinoma, Dp427m transcript was increased while Dp71 expression was reduced . Given the DMD gene structure, such an expression pattern can only be explained by differential regulation. Targeted degradation of dystrophin transcripts was suggested before , and recently, an epigenetic mechanism responsible for reduced DMD transcript levels has been described . These data indicate that, rather than simply being an effect of random mutations, quite likely to occur in this large gene, DMD alterations in tumor cells may have a complex regulatory nature involving mechanisms such as transcriptional regulation, chromatin remodeling, or transcript degradation. Given that miRNAs might be responsible for the differential regulation of DMD expression in tumor samples, we investigated but did not find any differentially expressed miRNAs in tumor samples with low vs. high DMD expression that were common to all of the 15 primary tumor types analyzed (Supplementary Table S9). Thus, specific alterations in DMD gene expression are a common feature across a spectrum of malignancies, including those originating from tissues previously not associated with the expression of the full-length dystrophin. Yet, we found DMD transcript levels in these tissues to be comparable to the levels of housekeeping genes. Moreover, interrogation of proteomics datasets demonstrated a much wider distribution profile for the full-length dystrophin protein . Interestingly, according to the Human Protein Atlas , the antibody HPA002725, directed against amino acids 186-333 of the full-length dystrophin, detected moderate cytoplasmic and/or membrane staining in a range of normal tissues in addition to the expected staining in skeletal and cardiac muscle and the CNS. However, the antibody HPA023885, raised against amino acids 2843-2992 and therefore supposed to detect all dystrophin isoforms upstream of Dp71, showed staining in skeletal and cardiac muscle, while other tissues were negative. This staining pattern disagrees with the established expression of Dp260, Dp140, and Dp116, which is broader than that of Dp427 isoforms, and so it could not be accurate. Given the high sensitivity and specificity of the mass-spec , identification of the full-length dystrophin in a wide spectrum of normal tissues using this latter method is likely to represent the true expression status. Thus, dystrophin may be present in many tissues at low levels and/or in a tightly controlled spatiotemporal manner, which might be missed using standard detection methods. The molecular signature associated with decreased DMD expression in tumors and corresponding tumor cell lines is concordant with that found in Duchenne muscular dystrophy. The DMD gene encodes a spectrum of dystrophin isoforms, but significant expression of the majority of these appeared to be restricted to specific tissues. While loss of the full-length dystrophin results in Duchenne muscular dystrophy, mutations additionally disrupting other isoforms result in exacerbated phenotypes . In skeletal myofibers, cardiomyocytes, and neurons, which express the highest levels of the full-length dystrophin, this protein has been described to serve as a structural scaffold for proteins engaged in ECM and cell-cell interactions, and in intracellular signaling. However, more recent data demonstrate that the loss of DMD expression impacts a broader spectrum of cells than those affected in DMD. In myoblasts , lymphocytes , endotheliocytes , mesodermal , and myogenic cells , loss of DMD expression leads to significant abnormalities. Moreover, the same abnormalities can occur in very distinct cells, e.g., calcium dys-homeostasis was found across multiple cells , and the damaging purinergic phenotype affects myoblasts and lymphocytes . Some old findings, such as platelet abnormalities , have recently been vindicated . Yet, these defects cannot be clearly attributed to the loss of interaction between dystrophin and the known dystrophin-associated proteins. Indeed, these cell-autonomous defects appear to affect dystrophic cells, which, when healthy, express the 14-kb DMD transcript, but were not shown to produce detectable levels of full-length dystrophins. This phenomenon, where expression of the 14-kb DMD transcript in cells such as myoblasts and lymphocytes does not correlate with detectable dystrophin protein, was known for decades but just disregarded as an "illegitimate transcription" . Our data suggest that more attention should be given to the subtler DMD gene functions, beyond those causing the main symptoms of DMD. Such studies have a potential to identify new therapeutic targets for the treatment of this debilitating and still incurable disease. Moreover, given the poor survival rate of patients with tumors downregulating dystrophin, the DMD gene may be important in oncology. 5. Conclusions The data presented here indicate that the current view on the role of the DMD gene in health and disease requires significant re-evaluation. The functional significance of the DMD gene is far more widespread than its identified roles in Duchenne MD, and its downregulation in tumors is of particular importance as it was found to be associated with specific transcriptomic changes and reduced survival of patients across a variety of malignancies. Acknowledgments The authors thank Thomas Krag and Javier. F Novo for very helpful comments on the manuscript and many anonymous reviewers whose critique helped improving this work. Supplementary Materials The following supporting information can be downloaded at: Supplementary File S1: Figure S1. Chromosomal localization and transcripts encoded by the DMD gene. The vertical dashes indicate individual exons. The location of the first exons is indicated by the red boxes. The full-length dystrophins (Dp427) are encoded by transcripts consisting of 79 exons, with the M, C and P isoforms having unique N-termini encoded by specific first exons. Multiple splice variants have been indicated on the left. Figure S2. Full-length dystrophin protein expression in normal tissues and cells (Proteomics DB). Figure S3. The predicted domain structures encoded by DMD gene transcripts used in the hierarchical clustering analysis. The figure was obtained using the HMMER web server. Figure S4. Expression level of the top ten highly expressed DMD gene transcripts in three of the identified clusters. Expression data is represented as mean +- SEM (* p <0.05, ** p <0.01, *** p < 0.001, **** p < 0.0001). Figure S5. Total DMD gene expression and expression of transcripts encoding Dp427m, Dp71, Dp71b and Dp71ab in (A) primary thyroid carcinoma samples (n = 504) compared to thyroid NAT (n = 59) and (B) primary pancreatic adenocarcinoma samples (n = 178) and healthy pancreas tissue (n = 167). Red and blue violin plots represent tumor and control samples, respectively. Vertical black lines represent the median and quartiles. Comparisons where there is a statistically significant decrease in DMD gene/ transcript expression in tumor tissue compared to controls are highlighted in blue, while comparisons where there is a statistically significant increase in DMD gene/transcript expression in tumor tissue compared to controls are highlighted in red (**** p < 0.0001). Expression values for the DMD gene were derived from the "RSEM norm_count" dataset, while expression values for DMD transcripts were derived from the "RSEM expected_count" dataset of the TCGA TARGET GTEx cohort at the UCSC Xena Browser. The p-Values were calculated using the UCSC Xena Browser using a two-tailed Welch's t test and adjusted for multiple testing using the Bonferroni correction. Figure S6. STRING interaction diagram of dystrophin protein-protein interaction (PPI) network and the enriched functional terms according to "Reactome Pathways" and "WikiPathways". Each node depicts a gene, edges between nodes indicate interactions between the corresponding protein products. Table S1. DMD gene expression in 25 TCGA primary tumors and corresponding normal tissues adjacent to the tumors (NATs) from the TCGA or healthy tissues form the GTEx database. The p-Values were calculated using the UCSC Xena Browser using a two-tailed Welch's t test and adjusted for multiple testing using the Bonferroni correction. p-Values were multiplied by the total numbers of tests (n = 25) and compared with the overall level of a = 0.05. Statistically non-significant comparisons are highlighted in grey. Table S2. Number of tumor and control tissues in clusters generated by cutting the dendrogram of the hierarchical clustering analysis at different levels. Six is highlighted as it was chosen to be the optimal number of clusters. Table S3. DMD gene expression levels in 18 healthy tissues from the GTEx database represented as a percentage of two housekeeping genes (HKGs): PGK1 and HMBS. The percentage of DMD level of each HKG in healthy tissues divided by the percentage of DMD of each HKG in skeletal muscle is also displayed. Table S4. DMD gene expression in 13 TCGA primary tumors compared to NATs from the TCGA and healthy GTEx tissues. p-Values were calculated using the UCSC Xena Browser and adjusted for multiple testing using the Bonferroni correction. p-Values were multiplied by the total numbers of tests (n = 13) and compared with the overall level of a = 0.05. Statistically non-significant comparisons are highlighted in grey. Table S5. The top ten highly expressed DMD transcripts in the analyzed tumor and control tissues and their predicted protein products. The identity percentage of unknown vs. known dystrophin isoforms is shown. Source: Ensemble and UniProt. Table S6. Expression of four DMD gene transcripts in 25 TCGA primary tumors and corresponding NATs from the TCGA or healthy tissues form the GTEx database. p-Values were calculated using the UCSC Xena Browser using a two-tailed Welch's t test and adjusted for multiple testing using the Bonferroni correction. p-Values were multiplied by the total numbers of tests (n = 25) and compared with the overall level of a = 0.05. Statistically non-significant comparisons are highlighted in grey. Table S7. Expression of Dp427m and Dp71 splice variants in primary blood malignancies in the TARGET database compared to GTEx healthy whole blood. p-Values were calculated using the UCSC Xena Browser using a two-tailed Welch's t test and adjusted for multiple testing using the Bonferroni correction. p-Values were multiplied by the total numbers of tests (n = 2) and compared with the overall level of a= 0.05. Table S8. Values of Pearson's correlation between the expression of dystrophin protein and other members of the DAP complex (those encoded by SNTB2, DAG1, DTNBP1, DTNA, and SNTA1). Correlation values were calculated using the Data Explorer tool at the DepMap portal across all tumor cell lines using data from the "Proteomics" database. Table S9. Differentially expressed miRNAs between samples with low vs. high DMD gene expression that were common in five or more tumors out of 15 different primary tumor types (FDR-corrected p-Value < 0.1, Bonferroni-corrected p-Value < 0.05, |LogFC| value >= 0.5). Supplementary File S2: Additional information about the comparisons between the low and high DMD groups of primary tumors and tumor cell lines. Click here for additional data file. Author Contributions N.A. carried out all the analyses with support from M.B., G.T., M.K. and N.H. and wrote the initial manuscript. D.C.G. designed and supervised the project and wrote the final version of the manuscript with assistance from all co-authors. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement All data in this study came from the publicly available sources and no individual patient data requiring informed consent were used. Data Availability Statement All data are presented in the manuscript and Supplementary Materials. Conflicts of Interest Authors declare no conflict of interests. Figure 1 DMD gene expression level in primary tumors and corresponding control tissues. Red and blue violin plots represent tumor and control tissues, respectively. Vertical black lines represent the median and quartiles. Comparisons (n = 20) with a statistically significant downregulation of DMD expression in tumor samples compared to controls are highlighted in blue, and comparisons (n = 2) with a statistically significant upregulation of DMD expression in tumor samples compared to controls are highlighted in red (p < 0.05, Supplementary Table S1). The p-Values were calculated using the UCSC Xena Browser using a two-tailed Welch's t test and adjusted for multiple testing using the Bonferroni correction. Figure 2 Bar plots representing the relative expression levels of the top 10 highly expressed DMD transcripts in tumor (text in red) and control tissues (text in black) and a dendrogram displaying the clusters identified by the hierarchical clustering analysis. Abbreviations: CESC (cervical and endocervical cancer), ACC (adrenocortical carcinoma), LIHC (liver hepatocellular carcinoma), CHOL (cholangiocarcinoma), KIRP (kidney papillary cell carcinoma), BLCA (bladder urothelial carcinoma), UCEC (uterine corpus endometrioid carcinoma), THCA (thyroid carcinoma), KICH (kidney chromophobe cell carcinoma), DLBC (diffuse large B-cell lymphoma), LAML (acute myeloid leukemia), PRAD (prostate adenocarcinoma), HNSC (head and neck squamous cell carcinoma), LUSC (lung squamous cell carcinoma), LUAD (lung adenocarcinoma), SKCM (skin cutaneous melanoma), READ (rectal adenocarcinoma), COAD (colon adenocarcinoma), OV (ovarian serous cystadenocarcinoma), BRCA (breast invasive carcinoma), PAAD (pancreatic adenocarcinoma), USC (uterine carcinosarcoma), STAD (stomach adenocarcinoma), ESCA (esophageal carcinoma), KIRC (kidney clear cell carcinoma). Figure 3 Dp427m transcript expression level in primary tumors and corresponding control tissues. Red and blue violin plots represent tumor and control tissues, respectively. Vertical black lines represent the median and quartiles. Comparisons (n = 17) with a statistically significant downregulation of Dp427m expression in tumor samples compared to controls are highlighted in blue, and one comparison with a statistically significant upregulation of Dp427m expression in tumor samples compared to controls is highlighted in red (p < 0.05, Supplementary Table S6). The p-Values were calculated using the UCSC Xena Browser using a two-tailed Welch's t test and adjusted for multiple testing using the Bonferroni correction. Figure 4 Total DMD gene expression in tumor samples from 23 TCGA studies with/without (A) CR, (B) NCR mutations, and (C) SCNAs in the DMD gene. The number of samples in each group is indicated in brackets. A deep deletion indicates a deep loss (possibly a homozygous deletion). A shallow deletion indicates a shallow loss (possibly a heterozygous deletion). A gain indicates a low-level gain (a few additional copies, often broad). An amplification indicates a high-level amplification (more copies, often local). Expression data is represented as EM mean +- SEM. The p-Values were adjusted using the Bonferroni correction. Asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001). Figure 5 DMD gene expression in tumor samples from 18 TCGA tumors with different (A) cancer stages and (B) age groups. Expression data is represented as EM mean +- SEM. The number of samples in each group is indicated in brackets. The p-Values were adjusted using the Bonferroni correction (a: significant difference from 14-29 age group, b: significant difference from 30-39 age group, c: significant difference from 40-49 age group, d: significant difference from 50-59 age group, e: significant difference from 60-69 age group, f: significant difference from 70-79 age group, g: significant difference from above 80 age group, p * < 0.05, p ** < 0.01, p *** < 0.001). Patients aged between 14 and 29 years old had significantly higher DMD expression compared to patients in the 30-39 (LogFC = 0.84, p = 0.02), 40-49 (LogFC = 0.71, p = 0.02), 50-59 (LogFC = 0.93, p < 0.001), 60-69 (LogFC = 1.17, p < 0.001), 70-79 (LogFC = 1.30, p < 0.001), and above 80 age groups (LogFC = 1.54, p < 0.001). Patients aged between 30 and 39 years old had higher DMD expression compared to patients in the above 80 age group (LogFC = 0.70, p = 0.001). Patients in the 40-49 age groups had significantly higher DMD levels than patients in the 60-69 (LogFC = 0.45, p < 0.001), 70-79 (LogFC = 0.59, p < 0.001), and above 80 age groups (LogFC = 0.83, p < 0.001). Patients in the 50-59 age groups had significantly higher DMD levels than patients in the 60-69 group (LogFC = 0.24, p = 0.005), 70-79 (LogFC = 0.37, p < 0.001), and above 80 age groups (LogFC = 0.61, p < 0.001). Patients aged between 60-69 had significantly higher DMD levels than patients in the above 80 group (LogFC = 0.37, p = 0.002). Figure 6 KEGG pathways enriched in the DEGs in primary tumor samples and tumor cell lines with low vs. high DMD gene expression as well as in DMD skeletal muscle. Only pathways that were identified in more than 50% of primary tumor samples with low DMD expression are displayed (adjusted p-Value < 0.05). Data is represented as the combined score for the enrichment (-Log (p-Value) x odds ratio). Combined score values greater than 100 were given a value of 100. Figure 7 GO Biological Process terms enriched in the DEGs in primary tumor samples and tumor cell lines with low vs. high DMD gene expression as well as in DMD skeletal muscle (adjusted p-Value < 0.05). Data is represented as the combined score for the enrichment (-Log (p-Value) x odds ratio). Combined score values greater than 100 were given a value of 100. Figure 8 (A) Kaplan-Meier curves of overall survival, progression-free interval, disease-specific survival, and disease-free interval for patients with low and high levels of DMD gene expression. (B) Kaplan-Meier curve of overall survival for hematological malignancies patients with low and high levels of Dp71 expression. Numbers in brackets are overall survival times in days. Numbers of patients in each group and p-Values for the log-rank test are displayed in the figure. Figure 9 A schematic representation of the key pathways enriched in the DEGs in primary tumors with low DMD gene expression that are shared with DMD skeletal muscle. Top six pathways that were identified in more than 50% of primary tumor samples with low DMD expression are displayed schematically, including (1) ECM-receptor interaction, (2) Cell adhesion molecules, (3) Focal Adhesion, (4) Calcium Signaling, (5) PI3K-Akt signaling, (6) cAMP signaling. Arrows suggest the potential additional interactions between pathways. Dp427 only is shown. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000052

The purpose of this study was to provide a systematic meta-analysis on echocardiographic measurements in normal Thoroughbred and Standardbred horses. The current systematic meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). All the available published papers on the reference values of echocardiographic assessment via M-mode echocardiography were searched, and fifteen studies were finally selected for analysis. In both fixed and random effect, the confidence interval (CI) for the interventricular septum (IVS) was 2.8-3.1 and 4.7-7.5; for the left ventricular free-wall (LVFW) thickness, it was 2.9-3.2 and 4.2-6.7; and for the left ventricular internal diameter (LVID), it was -5.0-4.6 and -10.0--6.7, respectively. For IVS, the Q statistic, I-squared, and tau-squared were 925.3, 98.1, and 7.9, respectively. Similarly, for LVFW, all the effects were on the positive side of zero, with a range of 1.3-68.1. The CI indicated a significant variation among the studies (fixed, 2.9-3.2; random, 4.2-6.7). The z-values of LVFW for fixed and random effects were, respectively, 41.1 (p < 0.001) and 8.5 (p < 0.001). However, the Q statistic was 886.6 (p < 0.001). Moreover, the I-squared was 98.08, and the tau-squared was 6.6. By contrast, the effects of LVID fell on the negative side of zero, (2.8-83.9). The present meta-analysis provides an overview of the echocardiographic measurements of cardiac diameters in healthy Thoroughbred and Standardbred horses. The meta-analysis indicates variations in results among different studies. This result should be considered when evaluating a horse for heart disease and each case should be evaluated independently. cardiac diameters horses systematic review Deputyship for Research and Innovation, Ministry of Education, Saudi ArabiaINST093 This project was funded by the Deputyship for Research and Innovation, Ministry of Education, Saudi Arabia, through project number INST093. pmc1. Introduction Echocardiography is a non-invasive procedure that permits the thorough visualization of cardiac chambers as well as their valves and is helpful in the diagnosis and monitoring of animals with suspected or known heart diseases . Echocardiography provides an evaluation of cardiac output, ejection fraction, diastolic function, and fractional shortening . Moreover, it helps to diagnose cardiac diseases, including atrial septal defects, atrioventricular valve stenosis, aortocardiac fistula and hypertrophic cardiomyopathy, and dilated cardiomyopathy . M-mode echocardiography is used to obtain high-resolution real-time images of cardiac structures . It was the first applicable procedure in horses in the late 1970s . Via M-mode echocardiography, the heart chambers, myocardium, valves, pericardium, and great vessels can be easily visualized . M-mode echocardiography has a high sampling rate when compared with 2D-mode and is superior to real-time images in recording subtle changes in the wall and valve motion and is used for the assessment of the size and function of the left ventricle . In-depth reports on M-mode echocardiography in foals, adult horses, and ponies, including measures of heart size, have been provided . Echocardiography's ability to measure cardiac diameters is widely regarded as a pivotal technique for assessing the severity and prognosis of cardiac disease and the heart's reaction to physical exertion . Indeed, in horses, the effects of training , growth , body weight , animal's gender , and animal breed on echocardiographic measurements have been described. However, these effects have never been statistically tested. The majority of investigations to examine the intra- and inter-observer repeatability of equine echocardiographic measures were conducted in horses and donkeys . Meta-analysis is defined as a quantitative, epidemiological study design used to systematically assess previous research studies to reach conclusions about that body of research . Meta-analyses have attracted much attention from the general public and have become increasingly popular in the biomedical field . The medical and statistical literature contains numerous references to the theory and correct methodology for meta-analysis .In addition to obtaining definitive conclusions regarding the research topic at hand, meta-analysis estimates the real effect of a treatment or exposure on a certain outcome by combining data from many trials . Subsequently, a consolidated and quantitative review of such large and complex studies is obtained . The examination of variability or heterogeneity in the results obtained by previous studies is also a critical outcome of meta-analyses . Meta-analysis assists to overcome the lack of the statistical capacity to reach concrete conclusions in independent studies, as well as the failure to appropriately analyze the differences in the risk of extremely uncommon adverse events in large-scale studies . When the results of several studies, even if they are incompatible with one another, are systematically integrated, the real magnitude of the effect may be more precisely defined . In fact, extensive meta-analysis studies on echocardiographic measurements in humans have been carried out . However, in the veterinary field, limited studies were carried out on canine echocardiography . According to the authors' knowledge, there are no available studies in the literature on the meta-analysis of echocardiographic parameters in horses. Consequently, the objective of this systematic review was to present a meta-analysis on the measurements of the interventricular septum (IVS), the left ventricular free-wall (LVFW) thickness, and the left ventricular internal diameter (LVID), in healthy Standardbred and Thoroughbred horses. 2. Materials and Methods 2.1. Selected Studies The reference values for the echocardiographic evaluation of heart diameters in Thoroughbred and Standardbred horses were generated in this meta-analysis. 2.2. Types of Reference Individuals None of the horses investigated had any cardiac abnormalities or pathological conditions that might affect cardiac function or size. Any level of sample size was acceptable. 2.3. Inclusion and Exclusion Criteria 2.3.1. Inclusion Criteria - For papers written in a foreign language, the availability of the English version of the reports; - Measurements were obtained via M-mode echocardiography; - Measurements were obtained via the right parasternal short-axis view (RPSAX); - Measurements were obtained at end-systole and end-diastole. 2.3.2. Exclusion Criteria - Measurements taken from other different breeds; - Methods of examination other than RPSAXs; - Incomplete parameters of cardiac diameters; - Non-English published papers. 2.4. Search Strategy and Selection of Studies The goal was to find all published publications dating back to the early days of regular echocardiography. The authors searched the PubMed, Ovid, Sage, BESCO, CAB, Scopus, and ISI web of knowledge databases from the beginning of time until July 2018 using the search phrases ECHOCARDIOGRAPHY (title/abstract) AND ("NORMAL VALUES") (title/abstract) OR ("REFERENCE VALUES") (title/abstract). This method was supplemented by citation evaluation, Google Scholar searches, expert suggestions, and hand-searching. Using EndNote, a reference application, we integrated the database results (version X7; Thomson Reuters). The articles included in this study are shown in Figure 1. 2.5. Data Extraction and Analysis The following information was extracted using a data extraction form: the study's year, sample size, and provided summary statistics (i.e., the standard difference in means, standard error, variance, Hedges's g, and the difference in means), types of breed, and the measurements and methods of cardiac diameters during the cardiac cycle (Table 1). 2.6. Quality Assurance The current systematic meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . All the available published papers on the reference values of the echocardiographic assessment of cardiac diameters in normal healthy Thoroughbred and Standardbred horses were included, to minimize publication bias. 2.7. Statistical Analysis For statistical analyses, a commercial software program for meta-analysis was used (comprehensive meta-analysis version 2, USA). Descriptive statistics were applied to present the mean values of echocardiographic parameters in the finally selected papers. The variables meta-analysis used were fixed and random-effect models, 95% confidence intervals, effect size, heterogeneity, and weight. The effect size was determined using a standardized Z statistic and p-value . Heterogeneity was assessed using Cochrane's Q test with a significant value of p < 0.05, and the I2 statistic was used to define the % of true heterogeneity among the analyses. The I2 statistic was used to estimate the degree of heterogeneity, which refers to the total variation depending on the Q statistic and the number of trials (K). In fact, a negative value of I2 was considered equal to zero, and consequently, the I2 statistic ranged between 0% and 100%, and a value equal to or more than 50% was considered heterogeneous . With values of 25%, 50%, and 75%, low, moderate, and high degrees of heterogeneity were identified, respectively . Study weight was calculated as the base inverse square of the standard error of the effect of each trial. Forest plots were used to present the means and their confidence intervals in a graphic manner, and heterogeneous degrees were explored. Meta-regression was performed to study the effect of age, gender, and body weight on the echocardiographic measurements, and a result with a p-value < 0.05 was considered significant. 3. Results The final model of meta-analysis for the measurement of IVS, LVID, and LVFW in healthy Standardbred and Thoroughbred horses is presented in Table 2 and Figure 2, Figure 3 and Figure 4. The results revealed that all the effects of IVS were on the positive side of zero, in the range of 1.36-66.08. The 95% confidence interval (CI) supported this finding in both fixed and random effects, indicating significant variations among the studies . For the relative weight, the study by Al-Haidar et al. (2013) was assigned a relative weight of 0.01%, while that of Leadon et al. (1991) was assigned a relative weight of 57.97%. In our study, the test of the null hypothesis (two-tailed) showed that the z-values of IVS for fixed and random effects were 39.5 (p < 0.001) and 8.7 (p < 0.001), respectively. For heterogenicity, the Q statistic was 925.3, compared with the expected value of 17 (p < 0.001); the I-squared was 98.1, and the tau-squared was 7.9. On the other side, all the effects of LVID fell on the negative side of zero, in the range of 2.8-83.9. The 95% CI indicated a significant variation among the studies (fixed, 95% CI 5.08 to 4.6; random, 95% CI 10.08-6.7) . Under a fixed effect, the point estimate was -4.8, with a standard error of 0.102. By contrast, under a random effect, the point estimate was -8.3, with a standard error of 0.8. For the relative weight, the study by Al-Haidar et al. (2013) was assigned a relative weight of 0.01%, while that of Leadon et al. (1991) was assigned a relative weight of 51.57%. For the test of the null hypothesis (two-tailed), the z-values for the fixed and random effects were 48.09 (p < 0.001) and 9.7 (p < 0.001), respectively. For heterogenicity, the Q statistic was 844.949 (p < 0.001), and the I-squared and tau-squared values were 97.988 and 11.977, respectively. Similarly, for LVFW, all the effects were on the positive side of zero, in the range of 1.350 to 68.176. The 95% CI indicated a significant variation among studies . Under a fixed effect, the point estimate was 3.1, with a standard error of 0.07 and a 95% CI of 2.93.2. However, under a random effect, the point estimate was 5.4, with a standard error of 0.6 and a 95% CI of 4.2 to 6.7. For the test of the null hypothesis (two-tailed), the z-values for the fixed and random effects were 41.114 (p < 0.001) and 8.545 (p < 0.001), respectively. However, for heterogenicity, the Q statistic of LVFW was 886.648, compared with the expected value of 17 (p < 0.001), while the I-squared was 98.083, and the tau-squared was 6.680. 4. Discussion In the present study, we conducted a systematic meta-analysis on the echocardiographic measurements of IVS, LVID, and LVFW, in Thoroughbred and Standardbred horses. For IVS, LVID, and LVFW, considering both types of effects (fixed and random), there was a variation among the results of the measurements. It is speculated that the controversy about the reliability of studies is due to the precision of the technique used. This finding is consistent with that previously reported . In the present results, there was a non-significant effect of body weight, age, breed, and gender on the measurements. The combined effect of such factors with other unknown variables may be the cause of variations in measurements. It has also been reported that training and growth can affect echocardiographic measurements . For IVS, the confidence interval (CI) of 95% in the study of Al-Haidar et al. was about six times as wide as that in another study by Al-Haidar et al. , and the values in eleven other studies fell somewhere in between. Al-Haidar et al. showed that the effect of size was reduced to 2.9 in the fixed-effect model, in which the weights dominated in the current study. The overall effect estimate was accurate, with a tiny within-study error for IVS of 0.07. Currently, just 15 research were analyzed, and their impact sizes varied widely. Our standard error of IVS (0.6) was almost six times the fixed-effect value, indicating that our estimate of the mean impact was not exact. It has been reported that a within-study error is an accurate indicator to assess the mean impact . For LVID, the 95% CI reported by Pipers and Hamlin was about three times as wide as the one recorded by Brown et al. , while the values in fifteen other studies fell somewhere in between. Interestingly, the study of Brown et al. had a low effect size. Under a fixed effect, the effect size decreased to 4.8%. However, the 95% CI was 8.3 when random factors were considered. The LVID research had a slight within-study error due to a large number of individuals (15 in each group) (0.1). The standard error for the mean effect was 0.864, which was about three times the standard error for the fixed-effect value, indicating that the mean impact was not particularly precise. For LVFW, the 95% CI stated by Slater and Herrtage was about three times as wide as the one stated by Al-Haidar et al. . Under the fixed-effect model, the effect size was reduced to 3.144, but it was 5.498 under the random-effect model. The within-study error for LVFW was small (0.07), indicating an accurate estimate of the combined effect. The mean effect sizes varied, where the standard error was 0.643, which was about three times that of the fixed-effect value. The confidence interval (CI) of 95% for IVS supported these findings in both fixed and random effects, indicating a significant variation among the studies (fixed, 95% CI 2.8-3.1; random, 95% CI 4.7-7.5) and for LVID (fixed, 5.0 to 4.6; random, -10.0--6.7) and for LVFW (fixed, 2.9-3.2; random, 4.2-6.7). The measurements of the three variables varied among the studies, which may be due to the wide range of horse breeds and ages in most of the studies. The relative weight is the average of weights as a proportion of total weights, with all relative weights added up to 100% . For IVS, the study by Leadon et al. (22) was given 58% of the weight under the fixed-effect model but only 6% of the weight under the random-effect model despite having a high sample size (n = 600 for each group). Under the fixed-effect paradigm, we assumed that all the studies and the existing study had the same value. A more accurate estimate was found in the study by Leadon et al. . On the other hand, it was assumed that each study estimated a different impact in the random-effect model. The same study provided an accurate estimate of the IVS population, but because it was just one among several, we did not want it to dominate the analysis. As a result, we gave it 6% of the relative weight, which was more than the weight we gave it under fixed effects but lower than previous studies. For LVID, the study of Leadon et al. (1991) was assigned 52% of the weight under the fixed-effect model but only 6% of the weight under the random-effect model. The above-mentioned study provided an accurate estimate of its population. In our study, we assigned it 6% of the weight. This was more than the values in the other studies but not as dominant a weight as that we gave it under fixed effects. Similarly, for LVFW, the study by Leadon et al. was assigned 46% of the weight under the fixed-effect model but only 6% of the weight under the random-effect model. Regarding heterogeneity, under both fixed (common) and random (true) effects, the null hypothesis was that the effect would be zero. The null hypothesis was tested using the z-value, which was computed as Hedges's g/standard error (G/SE) for the corresponding model . In our study, the z-values for the fixed and random effects of IVS were 39.519 (p < 0.001) and 8.793 (p < 0.001), and for LVID, they were 48.090 (p < 0.001) and 9.719 (p < 0.001). For LVFW, the z-values were 41.114 (p < 0.001) and 8.545 (p < 0.001), respectively. In the present study, the Q statistic for IVS was 925.307, while for LVID, it was 844.949, and for LVFW, it was 886.648, compared with the expected value of 17 (p < 0.001). The Q statistic implied the observed dispersion, whereas the null hypothesis for heterogeneity argued that the studies assigned a common effect size. Therefore, the degrees of freedom were assumed to be equal to the Q statistic . While the Q statistic is used to test the null hypothesis that there is no dispersion across effect sizes, the I-squared and tau-squared are valuable parameters to evaluate . The I-squared of IVS was 98.163, while that of LVID was 97.988, and that of LVFW was 98.083, indicating that the real variations in effect sizes accounted for 90% of the apparent variance among the studies. On the basis of random error, only 10% of the observed variation may be predicted. However, the tau-squared of IVS was 7.905; that of LVID was 11.977; and that of LVFW was 6.680. This is the variance "between studies" that was used in computing the weights. Because they are derived using fixed-effect weights, the Q statistic and tau-squared are normally given in fixed-effect models but not in random-effect models. Q statistic solves the question of whether the fixed-effect model fits the data in the fixed-effect study (i.e., whether it is sufficient to suppose that the tau-squared is really zero). To give weights, however, the tau-squared is really set to zero . Unfortunately, the Q test is only employed in meta-analysts to inform on the presence or absence of heterogeneity; it does not reveal the degree of such heterogeneity. In a meta-analysis investigation, the I-squared index has been proposed to measure the degree of heterogeneity . In our study, the statistical differences between the studies may be attributed to the sample size, body weight, breed, and gender. The limitations of the present investigation should be acknowledged. First, like other meta-analyses reporting reference values, remarkable heterogeneity was an innate limitation, although we attempted to explore the source of heterogeneity and define reference values with detailed information. Second, not all variables were comparable, as only IVS, LVID, and LVFW were presented via M-mode echocardiography. These variables were selected for investigation in all the studies. However, in other meta-analytical studies in humans and animals, a wide range of cardiac diameters was included . Moreover, to minimize the limitations in our study, the checklist of PRISMA, which is considered a standard technique, was used. 5. Conclusions This is the first meta-analysis that summarizes the results of the currently available studies looking at the identification of normative values for IVS, LVID, and LVFW, as assessed via M-mode echocardiography in horses. All the variables of this meta-analysis indicated variations in the results among the different studies. This result should be considered when evaluating a horse for heart disease, and each case should be evaluated independently. Acknowledgments The authors extend their appreciation to the Deputyship for Research and Innovation Ministry of Education in Saudi Arabia, for funding this research work (project number INST093). Author Contributions Conceptualization, M.M., M.K., H.B. and S.E.-k.; methodology, M.M. and S.E.-k.; software, A.F., H.I. and M.E.-A.; validation, M.M., M.K., H.B., A.F., H.I. and S.E.-k.; formal analysis, M.E.-A.; investigation, A.F., H.I., M.E.-A., Y.A. and S.E.-k.; resources, M.M.; data curation, S.E.-k.; writing--original draft preparation, M.M., M.K., H.B., A.F., H.I. and S.E.-k.; writing--review and editing, M.M., M.K., K.M.A., A.F., H.I. and S.E.-k.; visualization, M.M.; supervision, K.M.A.; project administration, M.M., K.M.A., H.I. and S.E.-k.; funding acquisition, M.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Results of the literature search and disposition of echocardiographic articles screened for inclusion. Figure 2 Forest plot for interventricular septum (IVS) measurements in Thoroughbred and Standardbred horses. Studies and their respective citations: Al-Haidar et al., 2013 ; Al-Haidar et al., 2010 ; Bakos et al., 2002 ; Brown et al., 2003 ; Buhl et al., 2005 ; Collins et al., 2010 ; Grenacher and Schwarzwald, 2010 ; Kriz et al., 2000 ; Leadon et al., 1991 ; Long et al., 1992 ; Michima et al., 2004 ; Patteson et al.,1995 ; Pipers and Hamlin, 1977 ; Slater and Herrtage, 1995 ; Zucca et al., 2008 . Figure 3 Forest plot for left ventricular internal diameter (LVID) measurements in Thoroughbred and Standardbred horses. Studies and their respective citations: Al-Haidar et al., 2013 ; Al-Haidar et al., 2010 ; Bakos et al., 2002 ; Brown et al., 2003 ; Buhl et al., 2005 ; Collins et al., 2010 ; Grenacher and Schwarzwald, 2010 ; Kriz et al., 2000 ; Leadon et al., 1991 ; Long et al., 1992 ; Michima et al., 2004 ; Patteson et al.,1995 ; Pipers and Hamlin, 1977 ; Slater and Herrtage, 1995 ; Zucca et al., 2008 . Figure 4 Forest plot for left ventricular free-wall (LVFW) thickness measurements in Thoroughbred and Standardbred horses. Studies and their respective citations: Al-Haidar et al., 2013 ; Al-Haidar et al., 2010 ; Bakos et al., 2002 ; Brown et al., 2003 ; Buhl et al., 2005 ; Collins et al., 2010 ; Grenacher and Schwarzwald, 2010 ; Kriz et al., 2000 ; Leadon et al., 1991 ; Long et al., 1992 ; Michima et al., 2004 ; Patteson et al.,1995 ; Pipers and Hamlin, 1977 ; Slater and Herrtage, 1995 ; Zucca et al., 2008 . animals-13-00809-t001_Table 1 Table 1 Descriptive statistics for M-mode echocardiographic measurements of cardiac diameters in Thoroughbred and Standardbred horses. Study Name Sample Size Breed Age Range (Year) Gender Body Weight (Kg) Variables IVS LVID LVFW Systole Diastole Systole Diastole Systole Diastole 1 Al-Haidar et al., 2013 36 Thoroughbred 2-21 Y (5.24 +- 2.34) Mares Geldings Stallions 86-6 (452.9 +- 52.3) 3.99 +- 1.03 2.57 +- 1.03 6.20 +- 1.04 10.28 +- 1.02 3.16 +- 1.03 2.14 +- 1.03 28 Standardbred 1.7-21 Y (8.9 +- 5.24) Mares Geldings Stallions 370-541 (472.45 +- 36.31) 3.99 +- 1.02 2.57 +- 1.03 6.11 +- 1.03 9.98 +- 1.02 3.55 +- 1.03 2.14 +- 1.03 2 Al-Haidar et al., 2010 10 Standardbred 1.5-25.6 Y (10.10 +- 7.6) 5 Mares 5 Geldings 120-662 kg (459.8 +- 128.8) 4.05 +- 0.02 2.67 +- 0.02 6.25 +- 0.05 10.19 +- 0.05 3.48 +- 0.04 2.00 +- 0.02 3 Bakos et al., 2002 23 Standardbred 2-16 Y (Mean 6) 8 Mares 7 Stallions 8 Geldings 350-490 (Mean 427) 4.7 +- 0.3 3.0 +- 0.2 7.0 +- 0.6 10.7 +- 0.7 3.9 +- 0.4 2.7 +- 0.2 4 Brown et al., 2003 17 Thoroughbred 10.39 Males 450.7 5.26 +- 0.63 3.66 +- 0.54 8.65 +- 1.61 13.92 +- 1.97 5.29 +- 0.89 3.1 +- 0.42 5 Buhl et al., 2005 200 Standardbred 3-8 Mares 477 to 540 3.50 +- 0.02 2.56 +- 0.01 7.66 +- 0.04 11.47 +- 0.05 3.47 +- 0.02 2.39 +- 0.01 6 Collins et al., 2010 19 Thoroughbred Up to 4 months 11 Males 8 Females 190 (182.10 +- 15.16) 3.09 +- 0.29 1.96 +- 0.22 5.52 +- 0.67 7.78 +- 0.75 2.33 +- 0.32 1.57 +- 0.15 7 Grenacher and Schwarzwald, 2010 15 Standardbred >2 Y Geldings Females 539 +- 31 3.8 +- 0.55 2.5 +- 0.38 5.3 +- 0.89 10.2 +- 1.32 2.7 +- 0.43 1.7 +- 0.31 7 Thoroughbred >2 Y Geldings Females 548 +- 59 4 +- 0.52 3.0 +- 0.37 6.6 +- 0.87 10.9 +- 1.36 3.9 +- 0.47 2.2 +- 0.27 8 Kriz et al., 2000 13 Standardbred 3-4 Y Geldings 411 +- 10 4.32 +- 0.32 3.19 +- 0.33 8.07 +- 0.69 11.76 +- 0.66 3.73 +- 0.51 2.51 +- 0.33 9 Leadon et al., 1991 600 Thoroughbred Mean 19.3 +- 1.2 months 442 Males 158 Females 437 +- 35 2.0 +- 0.5 2.9 +- 0.4 6.3 +- 0.9 10.5 +- 0.9 3.8 +- 0.6 2.4 +- 0.4 10 Long et al., 1992 26 Thoroughbred 2-17 Y 3 Stallions 5 Mares 18 Geldings 432-648 (Mean 517) 4.55 +- 0.55 3.02 +- 0.39 7.35 +- 0.72 11.90 +- 0.71 3.96 +- 0.93 2.39 +- 0.26 11 Michima et al., 2004 35 Standardbred 5-18 Y Males Females 415.51 +- 36.76 4.17 +- 0.42 2.68 +- 0.29 5.94 +- 0.96 9.72 +- 0.72 4.23 +- 0.69 2.69 +- 0.32 12 Patteson et al., 1995 29 Thoroughbred 3-15 y (M 7.71 Y) 22 Females 16 Geldings 420-617 (M 517) 4.21 +- 046 2.85 +- 0.278 7.45 +- 0.615 11.92 +- 0.76 3.85 +- 0.414 2.32 +- 0.382 13 Pipers and Hamlin, 1977 25 Standardbred and Thoroughbred Mean 3.8 Y Males Females 300 kg 4.7 +- 0.3 3.0 +- 0.2 7.0 +- 0.6 10.7 +- 0.3 3.9 +- 0.4 2.7 +- 0.2 14 Slater and Herrtage, 1995 16 Thoroughbred 4-16 Y (mean 7 Y) 11 Mares 5 Geldings 450-620 (490) 4.6 +- 0.2 2.6 +- 0.2 7.3 +- 0.8 11.2 +- 0.8 3.8 +- 0.3 2.5 +- 0.3 15 Zucca et al., 2008 30 Standardbred 3-9 Y (mean 3.8 +- 1.6 Y) 11 Females 17 Male 2 Geldings 340-498 kg (435 +- 36 kg) 4.48 +- 0.36 3.10 +- 0.41 7.42 +- 1.05 11.64 +- 1.29 3.64 +- 0.52 2.55 +- 0.36 IVS = interventricular septum; LVID = left ventricular internal diameter; LVFW = left ventricular free wall; Y = year. animals-13-00809-t002_Table 2 Table 2 Final meta-analysis model for reference values of echocardiographic measurements in Thoroughbred and Standardbred horses. Variables Model Effect Size and 95% Confidence Intervals Test of Null (2-Tailed) Heterogeneity Tau-Squared Number Studies Point Estimate Standard Error Variance CI 95% Lower Upper Z-Value p-Value Q-Value Df (q) p-Value I-Squared IVS Fixed 15 2.9 0.1 0.01 2.8 3.1 39.5 <0.001 925.3 17 <0.001 98.1 7.9 Random 15 6.1 0.6 0.4 4.7 7.5 8.7 <0.001 - - - - - LVID Fixed 15 -4.8 0.1 0.01 -5.0 -4.6 -48.1 <0.001 844.9 17 <0.001 97.9 11.9 Random 15 -8.3 0.8 0.7 -10.0 -6.7 -9.7 <0.001 - - - - - LVFW Fixed 15 3.1 0.1 0.01 2.9 3.2 41.1 <0.001 866.6 17 <0.001 98.1 6.6 Random 15 5.4 0.6 0. 4 4.2 6.7 8.5 <0.001 - - - - - IVS = interventricular septum; LVID = left ventricular internal diameter; LVFW = left ventricular free wall. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000053

This study investigated the morphological, histological, and histochemical characteristics of the digestive tract of the marbled flounder (Pseudopleuronectes yokohamae). The relative length of the gut of the marbled flounder digestive tract was 1.54 +- 0.10 (n = 20), and it had a simple stomach and 6-9 pyloric caeca. The mucosal folds of the marbled flounder digestive tract exhibited a general branched morphology. The thickness and mucosal fold length of the intestinal muscularis externa showed similar aspects in all areas. The thickness of the intestinal muscularis externa was the thickest in the posterior intestine portion, and the length of mucosal folds was the longest in the anterior intestine portion. It was indicated that food digested by gastric acid in the stomach moves to the anterior portion (including pyloric caeca) and mid portion of the intestine, ensuring effective stimulation of cholecystokinin (CCK)-producing cells. In addition, the distribution pattern of CCK-producing cells in the intestine was very similar to that of mucus-secreting goblet cells. The CCK-producing cells and goblet cells in the marbled flounder were well-adapted to promote optimal control of the digestive process. Based on the morphological and histochemical studies, it was concluded that the marbled flounder displays a digestive tract comparable to that of fish species with carnivorous habits. marbled flounder digestive tract digestive physiology immunohistochemistry endocrine cells This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. pmc1. Introduction The digestive tract of fish is anatomically composed of a long hollow tube, and as in other vertebrates, it is functionally in charge of important processes such as digestion, absorption, and transportation of nutrients necessary for body growth from food . While the digestive tract of most fish is generally composed of a series of tubes including the mouth, pharynx, esophagus, stomach, intestine, rectum, and anus, the shape and structure vary depending on the species and feeding habits . In particular, due to the diversity of feeding and phylogenetic characteristics of the digestive tract of teleosts, many studies related to the morphological and structural characteristics and ontogenetic development of the digestive tract have been reported . The diversity of morphological and structural characteristics of fish digestive tracts indicates that each species has different dietary and digestive physiology characteristics . Therefore, to understand the diet and digestive physiology of fish, histological studies on digestive tract morphology and internal structure must be performed; and since the understanding of the fish digestive tract is related to fish nutrition, such studies can also contribute to fish management and conservation . The digestive system is the largest endocrine organ in vertebrates . Hormones and a wide range of signaling molecules secreted by various types of endocrine cells can rapidly and reversibly change the properties of the digestive system and other organ systems . Energy and nutrients are provided in a sequential process, where complex polymers are absorbed across the apical membrane of epithelial cells, transported to the systemic circulation, and hydrolyzed into smaller molecules . Digestive system function is regulated by various digestive hormones and substances. Among them, cholecystokinin (CCK) and mucus-secreting goblet cells play an important physiological role in the intestine of vertebrates, including fish. CCK, a gastrointestinal hormone, is a neurotransmitter peptide abundant in the brain and gut . CCK plays an important role in pancreatic enzyme secretion, gallbladder contraction, amino acid and sugar transport regulation, and intestinal peristalsis regulation . In addition, mucus-secreting goblet cells in the fish digestive tract produce lubricant on the surface of the mucous membrane, protecting the mucous membrane from damage caused by physical or chemical substances and digestive enzymes . In addition, mucus secreted from goblet cells of vertebrates, including fish, plays an important role in the digestion and absorption of substances . The marbled flounder (Pseudopleuronectes yokohamae), found throughout Korea, southern Hokkaido in Japan, and the East China Sea, is a fish species with high commercial value in both Korea and Japan and has been gaining attention as a new aquaculture target . Previous studies determined the marbled flounder to be a benthic fish that feeds on macrozoobenthos, such as Pelecypoda, Amphipoda, and Polychaete ; however, there have been few histological and histochemical studies concerning the digestive tract of species of the Pseudopleuronectes genus, including the marbled flounder. Interest in the digestive physiology of cultured fish is based on the expectation that the application of histological and histochemical knowledge to the development of aquaculture and husbandry prevents digestive disorders and helps in understanding the regulatory aspects of fish nutrition . Therefore, this study aims to provide basic knowledge for physiological and nutritional studies by describing the histological and histochemical characteristics of the marbled flounder digestive tract. 2. Materials and Methods Sample collection was carried out at the Aquafeed Research Center (Pohang, Korea) of the National Institute of Fisheries Science (NIFS) of Korea in accordance with the Guidelines for Experimental Animals of NIFS Institutional Animal Care Use Committee (2019-NIFS-IACUC-13). 2.1. Specimens Twenty marbled flounder juveniles, with average body length and body weight of 14.6 +- 0.5 cm and 56.6 +- 5.1 g, respectively, were obtained from the Aquafeed Research Center, Pohang, Korea. The first broodstock generation had been hatched in the Fisheries Resources Institute, Gyeongsangbuk-do, located near our research facility (ca. 45 km). Fish were kept in natural photoperiod and water temperature conditions. The collected specimens were anaesthetized with an overdose of 2-phenoxyethanol (150 ppm; Sigma-Aldrich, St. Louis, MO, USA) and their body lengths were measured. The body cavity was cut open through a mid-ventral incision and the digestive system, from the esophagus to the anus, along with the accessory organs, was removed through dissection from the body cavity. The length of the digestive tract was measured, and relative length of the gut (RLG) was calculated using the equation RLG = DL/SL, where DL is the digestive tract length (cm) and SL is the standard length (cm) (Table 1). 2.2. Histochemistry Samples from nine different regions of the digestive tract (esophagus, cardiac stomach portion, fundic stomach portion, pyloric stomach portion, pyloric caeca, anterior intestine portion, mid intestine portion, posterior intestine portion, and rectum) were fixed in Bouin's solution (Sigma-Aldrich, St. Louis, MO, USA), dehydrated through an ethanol-xylene series, and embedded in paraffin. Sagittal sections were cut at a thickness of 4 mm, and 10 sections per region of the digestive tract were prepared for staining. Sections were stained with Hansen's hematoxylin and 0.5% eosin (HE) for histological observation and were stained with Alcian blue (AB) at pH 2.5 and periodic acid-Schiff (PAS) for observations of mucus-secreting goblet cells . Microscopy of the length of the mucosal folds, thickness of the muscularis externa, and characteristics of the goblet cells from different regions of the digestive tract was carried out using a light microscope (Leica, DM3000 LED, Wetzlar, Germany) attached to a camera (Dhyana 400DC, Tucsen Photonics, Fuzhou, China) with a capture program (Tucsen Mosaic version 2.2, Fuzhou, China). 2.3. Immunohistochemistry Formalin-fixed tissue samples were embedded in paraffin and 4-mm sections were cut. For paraffin-embedded tissue immunohistochemistry staining, deparaffinized and rehydrated sections were incubated with 3% hydrogen peroxidase solution for 10 min at room temperature and stained with primary antibodies against cholecystokinin-8 (CCK-8) (1:20,000, Sigma-Aldrich, St. Louis, MO, USA). The stained sections were further incubated with peroxidase-conjugated secondary antibodies (EnVision+Rabbit, Dako) for 30 min, and Mayer's Hematoxylin was applied for counterstaining. The CCK-producing cells were observed using a light microscope (Leica, DM3000 LED, Wetzlar, Germany) attached to a camera (Dhyana 400DC, Tucsen Photonics, Fuzhou, China) with a capture program (Tucsen Mosaic version 2.2, Fuzhou, China). 2.4. Statistical Analysis All data were analyzed using the IBM SPSS 19 software package for Windows (SPSS Inc., Chicago, IL, USA). Data were evaluated for assumptions, including normality and homogeneity of variance, using the Shapiro-Wilk and Levene's tests, respectively, and no violation was detected (p > 0.05). Statistical analyses of data were conducted using one-way analysis of variance with a 95% significance level (p < 0.05). When a significant treatment effect was detected, the Tukey's honestly significant difference (HSD) test was used to assess significant differences among means. 3. Results 3.1. Histological Characteristics of the Digestive Tract The tissue of the digestive tract can be largely divided into four layers consisting of the serosa, muscularis externa, submucosa, and muscularis mucosae from outside to inside. The internal structure characteristics of each part are described as follows. Esophagus: The serosa of the marbled flounder esophagus is thin, and the muscularis externa is widely and well-developed. The submucosa between the muscularis externa and muscularis mucosae is thin, and the lamina propria is well-developed. The mucosal folds of the esophagus are thin, long, and are arranged to form regular branches towards the lumen. The average thickness of the muscularis externa and length of the mucosal fold were 485 and 1031 mm, respectively . Stomach: The stomach was investigated by dividing it into cardiac, fundic, and pyloric portions, and the mucosal folds of the stomach showed a regular shape without forming branches. The serosa is thin, similar to that in the esophagus, and the muscularis externa is widely and well-developed. In the center, the gastric glands with clear lumen formation are well-developed and are distributed primarily in the fundic portion. The thickness of the muscularis externa sequentially decreases along the pyloric portion, cardiac portion, and fundic portion (603, 245, and 150 mm, respectively); while the length of mucosal folds is reversed, sequentially increasing along the fundic portion, cardiac portion, and pyloric portion (1383, 1044, and 906 mm, respectively) . Pyloric caeca: There were six to nine pyloric caeca in marbled flounder, depending on the individual. The serosae of the pyloric caeca are so thin that it is difficult to differentiate them. The mucosal folds are irregular in shape and form branches. The degree of development of the muscularis externa is very weak compared to that of the esophagus and stomach (32 mm). The mucosal folds are shorter compared to those of the esophagus and stomach but longer than that of the intestine (669 mm) . Intestine: The intestine could be divided into the anterior portion, mid portion, and posterior portion according to the internal structures such as external shape, shape and length of mucosal folds, thickness of the muscularis externa, and degree of development of the epithelial layer. In the anterior portion, mucosal folds are irregular in shape and show clear formation of branches. Although the muscularis externa is very thin (57 mm), it is thicker than that of the pyloric caeca. The mucosal folds in the anterior portion are the longest (630 mm) among all intestine portions and are similar in length to those in the pyloric caeca. The mucosal folds of the mid portion are also irregular in shape, with clear formation of branches. While the muscularis externa is of a similar thickness (50.9 mm) to that of the anterior portion, the mucosal folds are shorter (486.6 mm). The mucosal folds of the posterior portion are irregular in shape and form branches. The muscularis externa is similar to those in other parts of the intestine but is thicker (70.4 mm). The mucosal folds are a similar length to those of the mid portion (458 mm) . Rectum: The rectum is well-developed, with distinct divisions in the serosa, muscularis externa, and submucosa, and the mucosal folds are irregular and complicated in shape compared to those in the intestine, forming branches. The muscularis externa is thicker (198 mm) and the mucosal folds are longer (859 mm) compared to those in the intestine and pyloric caeca . 3.2. Distribution and Characteristics of Goblet Cells The distribution and relative frequency of goblet cells differed according to their location in the digestive tract, as shown in Table 3 and Figure 3. Notably, no goblet cells were identified in the stomach of the marbled flounder. Mucus-secreting goblet cells were identified in the esophagus (5743 cells), pyloric caeca (300 cells), anterior intestine portion (650 cells), mid intestine portion (528 cells), posterior intestine portion (467 cells), and rectum (1943 cells). Among the digestive tract regions, goblet cells had significantly higher distribution in the esophagus, followed by the anus (p < 0.05). Goblet cells were more highly distributed in the intestine than in the pyloric caeca, and the number of goblet cells decreased when moving from the anterior to the posterior intestine portion. However, there was no significant difference in the number of goblet cells between the intestine and pyloric caeca (p > 0.05). Esophageal goblet cells were densely distributed and filled the lumen, portraying a spherical or oval shape. Goblet cells from the anterior intestine portion to the anus were distributed throughout the mucosal folds in a spherical or oval shape. 3.3. Distribution and Characteristics of CCK-Producing Cells CCK-producing cells are typical endocrine-like cells, characterized by a long spindle shape with a narrow apex pointing towards the intestinal lumen. Although marbled flounder CCK-producing cells were not found in the esophagus or stomach, they were found at various frequencies in the pyloric caeca and intestine, extending to the rectum. CCK-producing cells were identified in the pyloric caeca (65 cells), anterior intestine portion (62 cells), mid intestine portion (13 cells), posterior intestine portion (8 cells), and rectum (4 cells). They were primarily distributed in the pyloric caeca and anterior intestine portion, showing a lower frequency towards the rectum . 4. Discussion To clarify the physical and chemical characteristics of the marbled flounder digestive tract, this study conducted morphological, histological, and histochemical analyses. The length of the intestine in the digestive tract of teleosts is an important parameter to distinguish the dietary patterns of fish. Al-Hussaini (1947) divided fish by their feeding habit based on their RLG values: carnivorous fish, 0.5~2.4; omnivorous fish, 1.3~4.3; and herbivorous fish, 3.7~6.0. In addition, Dasgupta (2004) observed that the mean RLG values were 0.7 in carnivorous fish, 1.4 in omnivorous fish, 4.8 in herbivorous fish, and 3.7 in plankton feeder fish. The RLG value of marbled flounder in this study was found to be 1.54 +- 0.10, which was similar to that of omnivorous fish; however, the shape and anatomical structure of the digestive tract showed similar characteristics to those of carnivorous fish. According to the results of a recent study, the feeding habits of marbled flounder are in fact close to those of carnivorous fish, as their main prey is Polychaeta (45.3%) and Gastropoda (23.2%) . The esophagus of the marbled flounder showed a branched shape with mucosal folds along the longitudinal axis, similar to the esophagus of many teleosts , and a stratified epithelial layer consisting mainly of epithelial and secretory cells . This structure suggests that the esophagus of the marbled flounder can be expanded for the digestion and transportation of food. In the esophagus, the number of goblet cells was significantly higher than in other digestive tract tissues. Goblet cells secrete mucus to protect the mucosal epithelial layer and facilitate the transport of food substances and excretion into the digestive tract . Therefore, it is suggested that the presence of many goblet cells in the esophagus of the marbled flounder is for lubricating and protecting the esophagus. Digestion in the stomachs of teleosts can be divided into physical and chemical digestion. Physical digestion varies depending on the development of the muscularis externa and muscularis mucosa, and chemical digestion varies depending on the degree of gastric gland development . Fish with poorly developed muscularis mucosa but well-developed muscularis externa are suited for digesting a large amount of soft food. In contrast, fish with poorly developed muscularis externa but well-developed muscularis mucosa are suited for ingesting a small amount of hard food . In the case of the marbled flounder, the muscularis externa of the cardiac and fundic portions of the stomach has weak muscularis externa development compared to that of the pyloric portion, but the muscularis mucosa of the cardiac and fundic portions has well-developed gastric glands for chemical digestion. In addition, although there are no gastric glands in the pyloric portion, the muscularis externa is very well-developed, suggesting that the marbled flounder has a stomach with a structure suitable for eating a small amount of hard food. The pyloric caeca of fish are an evolutionary strategy to increase the total surface area of the digestive epithelium, without increasing the length or thickness of the intestine . Pyloric caeca are found in approximately 60% of teleosts, especially in carnivorous species . The number of pyloric caeca in the marbled flounder was found to be six to nine, and the morphological structure of the digestive tract was similar to those of carnivorous fish. However, since little research has been conducted on the correlation between the number of pyloric caeca and size of the teleost intestine and the relationship between the number of pyloric caeca and digestive physiology, additional research is needed. The shape of the mucosal folds of the intestine is more complex than that of the esophagus or stomach, and these structural characteristics increase the dwell time of food in the intestine and increase its surface area, improving digestion function and absorption of nutrients . In other words, the mucosal folds of the fish intestine are well developed (more complex and more branched) and have higher digestive activity . Mucosal folds in the anterior, mid, and posterior intestine portions of the marbled flounder showed similar branched patterns; however, the mucosal folds became shorter moving from the anterior intestine portion to the posterior intestine portion, suggesting that more digestion takes place in the anterior intestine portion than in the posterior intestine portion. Previous studies investigating the histochemical characteristics of mucus-secreting cells found that the shape, size, and distribution of goblet cells in the digestive tract differ depending on the fish species and digestive tract structure . The goblet cells in the digestive tract of marbled flounder showed a different shape and distribution compared to those of other fish. For marbled flounder, goblet cells were irregularly distributed without a regular arrangement from the upper part to the lower part of the mucosal folds, and the shape of goblet cells appeared to vary from spindle to oval. The number of goblet cells in the intestine of the marbled flounder decreased from the anterior to the posterior portion. Lee and Chin (1995) reported that the distribution of goblet cells in the gut of stomachless fish increased towards the posterior intestine portion, and Hur et al. (2013) reported that the distribution of goblet cells decreased towards the posterior intestine portion in stomach fish. Judging from the results of previous studies, marbled flounder displayed a digestive characteristic typical of stomach fish. The final absorption of water, ions, and proteins takes place in the teleost rectum and many mucin-containing epithelial cells exist in the rectum to contribute to the passage of feces, protection of the epithelium, and final absorption of substances . The histological and histochemical analysis results revealed that the marbled flounder rectum was composed of a complex columnar shape, similar to that of other teleost fish. In the epithelial lining, epithelial cells coated in secretions of neutral and acidic mucus and a number of goblet cells were observed, which were judged to contribute to the absorption of substances generally performed in the rectum. In fish, CCK cells are present among the endocrine cells of digestive tract and within the central and peripheral nervous system . These cells influence digestive processes by regulating food intake through peripheral satiety signals . Marbled flounder CCK cells were distributed throughout the digestive tract, including the pyloric caeca, intestine, and rectum, and CCK cell frequency was highest in the pyloric caeca and anterior portion of the intestine. CCK cells are found in various fish species, including flat fish species , and distribution patterns similar to that of the marbled flounder CCK cells were observed in the digestive tracts of hairychin goby Sagamia geneionema , blacktip grouper Epinephelus fasciatus , silver catfish Rhamdia quelen , dorado Salminus brasiliensis , and Neotropical freshwater fish Prochilodus lineatus . Considering the biological roles of CCK cells, their distribution pattern in marbled flounder suggests that the pyloric caeca and anterior intestine portion of the intestine play an important role in gallbladder contraction, pancreatic enzyme secretion, stimulation of gastrointestinal motility, and inhibition of gastric emptying functions . However, the distribution characteristics of neuroendocrine cells, including those that secrete CCK, in fish digestive tracts do not show common distribution characteristics among fish species and dietary patterns . 5. Conclusions The anatomical and functional structures of the marbled flounder digestive tract were investigated and considered to closely represent the morphology of the carnivorous fish digestive tract. Through investigating the physical and chemical characteristics of the digestive tract, basic information regarding the physical characteristics and nutritional studies of food suitable for the marbled flounder were provided. However, studies on the characteristics of goblet cells according to dyeing properties, absorptive cells, and endocrine cells distributed in the digestive tract should be conducted in more detail at the ultrastructural level. Acknowledgments This study was supported by grants from the National Institute of Fisheries Science, Republic of Korea (R2023025 and R2023029). Author Contributions J.-H.C.: conceptualization, methodology, data curation, writing--original draft, writing--review and editing, visualization. J.W.P.: investigation, validation, writing--review and editing. Y.-W.R.: investigation, validation, writing--review and editing. K.-W.K.: conceptualization, supervision. S.-W.H.: conceptualization, supervision, project administration, writing--review and editing. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All activities related to animal ethical considerations, such as euthanasia, were approved by the Institutional Animal Care and Use Committee of National Institute of Fisheries Science, Republic of Korea (2019-NIFS-IACUC-13) on 1 February 2019. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest We declare that we have no financial and personal relationship with other people or organizations that might inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service, and/or company that could be construed as influencing the content of this paper. Figure 1 Anatomical features of the marbled flounder Pseudopleuronectes yokohamae digestive tract divided into nine segments. Abbreviations: ES, esophagus; ST, stomach; AN, anus; es, esophagus portion; cs, cardiac stomach portion; fs, fundic stomach portion; ps, pyloric stomach portion; pc, pyloric caeca portion; ai, anterior intestine portion; mi, mid intestine portion; pi, posterior intestine portion; rt, rectum portion. Scale bar: 10 mm. Figure 2 Microphotographs of cross sections of the digestive tract of the marbled flounder Pseudopleuronectes yokohamae: (A) esophagus; (B) cardiac stomach portion; (C) fundic stomach portion; (D) pyloric stomach portion; (E) pyloric caeca; (F) anterior intestine portion; (G) mid intestine portion; (H) posterior intestine portion; (I) rectum. Abbreviations: lp, lamina propria; me, muscularis externa; mf, mucosal fold; gg, gastric gland. Scale bars indicate 100 mm. Figure 3 Microphotographs of mucus-secreting goblet cells in the digestive tract of marbled flounder Pseudopleuronectes yokohamae: (A) esophagus; (B) pyloric caeca; (C) anterior intestine portion; (D) mid intestine portion; (E) posterior intestine portion; (F) rectum. Abbreviation: Gc, goblet cell. Scale bars indicate 100 mm. Figure 4 Microphotographs of CCK-producing cells in the digestive tract of marbled flounder Pseudopleuronectes yokohamae. (A) pyloric caeca; (B) anterior intestine portion; (C) mid intestine portion; (D) posterior intestine portion; (E) rectum. Arrows indicate CCK-producing cells. Scale bars indicate 40 mm. animals-13-00936-t001_Table 1 Table 1 Body length, body weight, digestive tract length, and relative length of gut of the marbled flounder Pseudopleuronectes yokohamae. Marbled Flounder Sample number 20 Body length (cm) 14.6 +- 0.5 Body weight (g) 56.6 +- 5.1 Digestive tract length (cm) 22.5 +- 2.0 Relative length of the gut 1.54 +- 0.10 Values are the means +- standard deviation (n = 20). animals-13-00936-t002_Table 2 Table 2 Histological features of the digestive tract of the marbled flounder Pseudopleuronectes yokohamae. Muscularis Externa Thickness (mm) Mucosal Fold Length (mm) Esophagus 483.5 +- 23.1 1031.7 +- 26.0 Cardiac stomach portion 245.3 +- 31.8 1044.3 +- 90.4 Fundic stomach portion 150.1 +- 3.0 1383.3 +- 51.3 Pyloric stomach portion 603.5 +- 40.3 906.8 +- 45.0 Pyloric caeca 32.2 +- 1.2 669.4 +- 34.0 Anterior intestine portion 57.4 +- 8.6 630.7 +- 21.9 Mid intestine portion 50.9 +- 6.2 486.5 +- 20.2 Posterior intestine portion 70.4 +- 4.1 458.6 +- 34.1 Rectum 198.0 +- 10.8 859.5 +- 49.6 Values are the mean +- standard error (n = 20) animals-13-00936-t003_Table 3 Table 3 Numbers of goblet cells and CCK-producing cells in different digestive tract regions of the marbled flounder Pseudopleuronectes yokohamae. Numbers/Tissue Section Tissue Section Goblet Cells CCK-Producing Cells Esophagus 5743 +- 107 a None Cardiac stomach portion None None Fundic stomach portion None None Pyloric stomach portion None None Pyloric caeca 300 +- 32 c 65 +- 6 a Anterior intestine portion 650 +- 86 c 62 +- 3 a Mid intestine portion 528 +- 59 c 13 +- 1 b Posterior intestine portion 467 +- 72 c 8 +- 2 b Rectum 1943 +- 144 b 4 +- 1 b Values are the mean +- standard error (n = 20). Values followed by differing superscript letters are significantly different (Tukey's HSD test, p < 0.05). Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000054

The purpose of this study was to develop the potential of cottonseed protein concentrate (CPC) and Clostridium autoethanogenum protein (CAP) in the diet of rainbow trout (Oncorhynchus mykiss) by evaluating the effects of substituting fishmeal with a CPC and CAP mixture on growth performance, nutrient utilization, serum biochemical indices, intestinal and hepatopancreas histology. In a basal diet containing 200 g/kg fishmeal (Con), the mixture of CPC and CAP (1:1) was used to reduce dietary fishmeal to 150, 100, 50 and 0 g/kg, to form five diets with the same crude protein and crude lipid contents (CON, FM-15, FM-10, FM-5 and FM-0). Then, the five diets were fed to rainbow trout (35.00 +- 0.05 g) for 8 weeks. The weight gain (WG) and feed conversion ratio (FCR) of the five groups were 258.72%, 258.82%, 249.90%, 242.89%, 236.57%, and 1.19, 1.20, 1.24, 1.28, 1.31, respectively. FM-5 and FM-0 groups showed significantly lower WG and higher FCR than the CON group (p < 0.05). In terms of whole-body composition, such as moisture, crude ash, and crude protein, no significant difference was observed among all the groups (p > 0.05), except that significantly higher crude lipid content was detected in the FM-0 group than in the CON group (p < 0.05). In the FM-5 and FM-0 groups, protein efficiency, protein retention, intestinal protease activity and amylase activity were significantly lower than in the CON group (p < 0.05). Compared to the CON group, the serum contents of glucose and total cholesterol in the FM-0 group as well as MDA in the FM-5 and FM-0 groups were significantly increased, and catalase, superoxide dismutase, and total antioxidant capacity were decreased (p < 0.05). In intestine and hepatopancreas histology, the intestinal villus height in the FM-5 and FM-0 groups and villus width in the FM-0 group were decreased significantly (p < 0.05), while no significant difference in hepatopancreas morphology was observed among all the groups except that some vacuolization was observed in the FM-0 group (p > 0.05). In summary, the mixture of CPC and CAP can effectively replace 100 g/kg fishmeal in a diet containing 200 g/kg fishmeal without adverse effects on the growth performance, nutrient utilization, serum biochemical, or intestinal and hepatopancreas histology of rainbow trout. rainbow trout cottonseed protein concentrate Clostridium autoethanogenum protein growth performance nutrient utilization serum biochemical indices intestinal histology hepatopancreas histology National Key R&D Program of China2019YFD0900200 This work was supported by the National Key R&D Program of China (2019YFD0900200). pmc1. Introduction In aquaculture, more than 50% of production costs are incurred by feed , and fishmeal is the primary source of protein due to its balanced amino acid composition, high crude protein content, and unknown growth factors . Due to the shortage of marine resources and the growing demand for fishmeal in aquafeeds, fishmeal price has been rising continuously. Therefore, new protein sources must be sought to replace or decrease fishmeal inclusion in aquafeeds. Previous studies have shown that some plant protein sources can partly or completely replace dietary fishmeal, including soybean meal , cottonseed meal , peanut meal , canola protein isolate , and corn protein . However, the anti-nutrient factors in plant proteins may adversely affect the performance of fish . Therefore, exploring new plant protein sources with low anti-nutrient factors seems to be critical for their wide application in aquafeeds. Cottonseed protein concentrate (CPC) is derived from shelled cottonseed after oil extraction, dephenolization, and low-temperature drying. Generally, the crude protein content of CPC can reach 60% to 70% with plenty of arginine, histidine, and phenylalanine, and the free gossypol content is very low . CPC was reported to replace 385 g/kg fishmeal in a basal diet containing 700 g/kg fishmeal without adversely affecting the growth and flesh quality of largemouth bass (Micropterus salmoides) . The CPC prepared from glandless seeds completely replaced dietary fishmeal (400 g/kg) without adverse impacts on growth performance and feed utilization of black sea bass (Centropristis striata) . Moreover, CPC has also been reported to partially replace fishmeal in the diets of golden pompano (Trachinotus ovatus) , hybrid grouper (Epinephelus fuscoguttatus x E. lanceolatus) , and large yellow croaker (Larimichthys crocea) . As a new type of single-cell protein, Clostridium autoethanogenum protein (CAP) is obtained by liquid fermentation of Clostridium ethanolum with CO and ammonia as carbon and nitrogen sources. After the centrifugation and drying, the CAP product contains high levels of crude protein (80%) with high digestibility, high lysine content, and fewer anti-nutrient factors, but the arginine level is relatively low . The successful replacement of fishmeal with CAP has been reported in some aquaculture species. For example, no adverse effects on the growth performance, feed utilization, or intestinal histology were found in largemouth bass when CAP replaced 150 g/kg fishmeal in a basal diet with fishmeal inclusion of 350 g/kg . Similar results have also been reported in obscure pufferfish (Takifugu obscurus) , large yellow croakers , Pacific white shrimp (Litopenaeus vannamei) , juvenile turbot (Scophthalmus maximus L.) , and black sea bream (Acanthopagrus schlegelii) . Rainbow trout (Oncorhynchus mykiss) is a cold water fish, widely cultured throughout the world. Rainbow trout is a fast growing fish with tender flesh, no intermuscular spines, and plenty of DHA and EPA . Among salmon and trout, rainbow trout production is the second largest after Atlantic salmon (Salmo salar) . As a carnivorous fish, the commercial diet for rainbow trout usually contains a high level of fishmeal, thus, reducing the fishmeal inclusion in feed is of great significance to develop sustainable aquaculture of rainbow trout. To date, it has been reported that some plant protein sources can replace fishmeal in rainbow trout diets, such as enzymatical soybean meal , fermented soybean meal , corn protein concentrate , canola: pea . Recently, Zhao et al. reported that CPC successfully replaced 50% of dietary fishmeal without adverse impacts on growth performance or antioxidant capacity of rainbow trout. However, the control diet in that study contained a high level of fishmeal of up to 400 g/kg. Additionally, the inclusion of CAP in the diet has not yet been investigated in rainbow trout. In terms of amino acid composition, CPC contains much arginine and little lysine and methionine, while CAP contains plenty of lysine and methionine and relatively low arginine levels. From this point of view, the two protein sources may have complementary effects in amino acid composition. Therefore, we evaluated the effects of substituting fishmeal with a mixture of CPC and CAP (1:1) on the growth performance, nutrient utilization, serum biochemical indices, and intestinal and hepatopancreas histology in the present study. The findings will develop the potential of CPC and CAP in carnivorous fish feeds and provide a low fishmeal diet for rainbow trout. 2. Materials and Methods 2.1. Ethics Statement All the procedures for handling animals involved in this experiment are in accordance with the regulations of the Experimental Animal Ethics Committee and the Institutional Animal Care Committee of Shanghai Ocean University (Approval code: SFI 2020-23 of 20 May 2020). 2.2. Experimental Diets and Design First, a control diet was formulated to contain 200 g/kg fishmeal (CON). Then, the mixture of CPC and CAP (1:1) was used to reduce dietary fishmeal to 150, 100, 50 and 0 g/kg to form five diets with the same contents of crude protein (430 g/kg) and crude lipid (100 g/kg) (CON, FM-15, FM-10, FM-5 and FM-0). According to the method described by Xu et al. and Yao et al. , the protein ingredients such as fishmeal, CAP, CPC, etc., were ground and screened through a 60-mesh sieve, then mixed with liquid ingredients (fish oil, soybean oil, soybean lecithin). The five diets were prepared to form sinking pellets with a diameter of 3 mm using a single-screw extruder (LX-75 extruder, Longxiang Food Machinery Factory, Hebei, China). The extruding and air-drying temperatures were 90 +- 5 degC and 30 +- 2 degC, respectively. Then, all diets were stored at 4 degC until use. Table 1 shows the formulation and proximate composition of the diets. CPC and CAP were provided by Chengrun Jinlan Biotechnology Co., Ltd. (Xinjiang, China) and Shoulang New Energy Technology Co., Ltd. (Tangshan City, China), respectively. The crude protein content of CPC and CAP was 615.1 g/kg and 842.1 g/kg, respectively, and the crude lipid content was 23.6 g/kg and 1.9 g/kg, respectively. The free gossypol content in CPC was 172.8 mg/kg. Fishmeal was steam-dried fishmeal (TASA), and the crude protein and lipid contents were 682.1 g/kg and 90.0 g/kg, respectively. Table 2 shows the proximate composition and amino acids profile of the four ingredients. 2.3. Management of Experimental Fish and Feeding Rainbow trout were obtained from a local aquaculture farm in Sichuan (China). After transportation to the aquaculture station (Binhai, Shanghai, China), all fish were adapted to the environment and diets by feeding control diets for 2 weeks. A total of 300 juvenile rainbow trout (35.00 +- 0.05 g) were selected and randomly distributed into 15 indoor polyvinyl tanks with diameter, height, and water capacity of 1.0 m, 0.8 m, and 650 L, respectively. Three replicates (tanks) were designed for each treatment and each tank contained 20 fish. The system ran on the recirculation regime with a flowing rate of 10 L/min for each tank. During the feeding period, all the fish were fed at 09:00 and 16:00, and the feed intake was adjusted to ensure that no diet residue was found within 5 min when feeding was finished. The feces waste was siphoned out at the 2nd hour after feeding, and the cultured water was renewed (about 1/3) twice a week. During the feeding period of 56 days, the water quality was measured by measuring temperature (12-14 degC), dissolved oxygen content (6-8 mg/L), ammonia nitrogen content (<0.1 mg/L) and nitrite content (<0.1 mg/L). 2.4. Samples Collection At the beginning of the feeding trial, ten fish were sampled and stored at -20 degC to determine the whole-body composition of initial fish. At the end of the feeding trial, all the fish were deprived of diets for 24 h, and then counted and bulk weighed for each individual tank to calculate weight gain (WG), feed conversion ratio (FCR), specific growth rate (SGR), survival, and feed intake (FI). Six fish per tank were randomly selected, of which three fish were used for the measurement of final fish whole-body composition. For the other three fish, body length and weight were measured to calculate condition factor (K), and then the blood was collected from the cordial vein with a syringe. After centrifuging at 3500 rpm for 10 min (4 degC), the supernatant was stored at -80 degC to determine serum biochemical indices. Then, the three fish were dissected, and the weight of visceral and hepatopancreas were measured to calculate the viscerosomatic index (VSI) and hepatosomatic index (HSI). Then, hepatopancreas (1 cm x 0.5 cm x 0.5 cm) and foregut (1 cm) tissues were stored in Bouin's solution to observe tissue structure. The rest of the intestines were kept at -80 degC to determine digestive enzyme activity. 2.5. Measurement Indicators and Methods 2.5.1. Growth Performance and Physical Indices The growth performance and physical indices were calculated as follows:Survival (%) = 100 x (final number of fish/initial number of fish) WG (%) = 100 x [final weight (g) - initial weight (g)]/initial weight (g) FCR = feed consumption (g)/weight gain (g) SGR (%/day) = 100 x [ln final weight (g) - ln initial weight (g)]/days K (g/cm3) = 100 x [body weight (g)/body length3 (cm)3] VSI (%) = 100 x [visceral weight (g)/body weight (g)] HSI (%) = 100 x [hepatopancreas weight (g)/body weight (g)] FI (g/fish/d) = feed intake (g)/[(final fish number + initial fish number)/2]/days 2.5.2. The Diets and Whole-Body Composition AOAC method was used to measure moisture, crude ash, crude protein, and crude lipid contents in diets and whole-body composition . To determine the moisture content, samples were dried in an oven at 105 degC to constant weight. The combustion method, the auto Kjeldahl system (2300 Auto analyzer, Foss Tecator, AB, Hoganas, Sweden), and the chloroform-methanol method were used to determine the contents of crude ash, crude protein, and crude lipid, respectively. The dietary amino acid composition was determined according to the description by Xu et al. : The dried sample (70 mg) was added with 6 mol/L hydrochloric acid, then hydrolyzed at 110 degC for 24 h in a vacuum. After dilution (1:10), the amino acid content was determined by automatic analyzer (S-433D, Sykam, Munich, Germany). 2.5.3. Serum Biochemical Indices The serum activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and serum contents of malondialdehyde (MDA), triglyceride (TG), glucose (GLU), total cholesterol (TCHO), and total protein (TP) were determined by kits supplied by Nanjing Jiancheng Biological Co., Ltd., Nanjing, China. 2.5.4. Nutrient Retention The protein efficiency ratio (PER), protein retention (PR), and lipid retention (LR) were calculated as follows:PER (%) = 100 x [final body weight (g) - initial body weight (g)]/protein intake (g) PR (%) = 100 x [final body weight (g) x crude protein content of the final whole fish - initial body weight (g) x crude protein content of the initial whole fish]/protein intake (g) LR (%) = 100 x [final body weight (g) x crude lipid content of the final whole fish - initial body weight (g) x crude lipid content of the initial whole fish]/lipid intake (g) 2.5.5. Intestinal Digestive Enzyme Activity After thawing at 4 degC, intestinal samples (0.1 g) were homogenized with nine times volume of ice-cold normal saline (1:9 w/v), and then centrifuged at 3000 rpm for 10 min at 4 degC. The supernatant was stored at -20 degC for digestive enzyme activity. Protease activity was measured using the Folin phenol method , and the amount of enzyme that hydrolyzes casein and produces 1 mg tyrosine per minute (1 mg tissue protein) at pH 7.2 and 37 degC was defined as one unit (U/mgprot). Amylase activity was measured by iodine-starch colorimetry , and tissue protein (1 mg) reacted with the substrate at 37 degC for 30 min to hydrolyze 10 mg starch was defined as one unit (U/mgprot). The above indices were measured using kits supplied by Nanjing Jiancheng Biological Co., Ltd., Nanjing, China. 2.5.6. Intestinal and Hepatopancreas Histology For the embedding of foregut and hepatopancreas, we referred to the method described by Yang et al. . A slicer (Leica RM2235, Heidelberg, Germany) was used to cut the sections (7 mm), which was then stained by hematoxylin-eosin. The microscope (Nikon YS100, Melville, NY, USA) was used to observe and photograph morphology of intestine and hepatopancreas, and then the villus height, villus width and muscle thickness were measured with image analysis software (Image J14.0), referring to the description of Zhao et al. . 2.6. Statistical Analysis All data were presented as mean +- standard deviation (mean +- SD). Excel and SPSS 26.0 software were used to conduct a one-way ANOVA. Statistical significance among groups was determined using the Tukey's multirange test if significant differences were detected. 3. Results 3.1. Growth Performance and Physical Indices In Table 3, all the groups showed high survival, up to 100%. In the FM-15 and FM-10 groups, WG, FCR, and SGR were similar to those in the CON group (p > 0.05), while the FM-5 and FM-0 groups showed significantly lower WG and SGR (-6.12% and -3.51%, -8.56% and -4.82%), and higher FCR (+0.09, +0.12), than the CON group (p < 0.05). All the groups presented similar K, VSI, and HSI (p > 0.05). 3.2. Whole-Body Composition The whole-body composition is shown in Table 4. The crude lipid content in the FM-0 group was significantly higher than in the CON group (p < 0.05). There were no significant differences in the content of moisture, crude ash, and crude protein among all the groups (p > 0.05). 3.3. Nutrient Utilization and Intestinal Digestive Enzyme Activity In Table 5, the increasing replacement of fishmeal with the mixture of CPC and CAP tended to decrease the protease and amylase activity, and PER. The protease activity in the FM-10, FM-5, and FM-0 groups and the amylase activity in the FM-5 and FM-0 groups was significantly lower than in the CON group (p < 0.05). The FM-5 and FM-0 groups also showed significantly lower PER and PR than the CON group (p < 0.05). The LR of the FM-5 group was significantly lower than that of the other groups (p < 0.05). 3.4. Serum Biochemical Indices Table 6 shows the results of biochemical indices in serum. Serum contents of GLU and TCHO in the FM-0 group were significantly higher than those in the CON group (p < 0.05). Compared to the CON group, the FM-5 and FM-0 groups showed lower activities of SOD, T-AOC, and higher MDA content (p < 0.05). No significant difference in the activity of CAT, AST, ALT, or in the content of TG or TP was detected among all the groups (p > 0.05), but the increasing replacement of fishmeal with the CPC and CAP mixture tended to decrease the activity of CAT. 3.5. Intestinal Morphology Table 7 and Figure 1 depict the intestinal morphology. The villus height and muscle thickness in the FM-5 and FM-0 groups, as well as villus width in the FM-0 group, were significantly smaller than in the CON group (p < 0.05). 3.6. Hepatopancreas Morphology As shown in Figure 2, vacuolation and lipid droplets were observed in some hepatocytes in the FM-0 group, while the nucleus and cell structure of hepatocytes in the other groups showed no abnormality. 4. Discussion 4.1. Growth Performance, Whole-Body Composition and Nutrient Utilization The high replacement of fishmeal with plant protein sources may have adverse effects on the performance of aquatic animals . In basal diets with fishmeal inclusion of 250 g/kg, 450 g/kg, 452 g/kg, and 400 g/kg, fermented soybean meal , solvent-extracted cottonseed meal , corn protein , and CPC could reduce dietary fishmeal content to 150 g/kg, 225 g/kg, 338.1 g/kg, and 200 g/kg, respectively, without affecting the growth performance of rainbow trout. In the present study, the fishmeal content in the basal diet was only 200 g/kg, and the replaced fishmeal reached 100 g/kg. Both were much lower than the reported values in the above studies, realizing a low fishmeal diet for rainbow trout feed. It indicates that the CPC and CAP mixture is an excellent plant protein source to replace fishmeal in aquafeeds. However, the WG and PR of rainbow trout were significantly decreased, and FCR was significantly increased, when dietary fishmeal was reduced to 50 g/kg by inclusion of the plant mixture (Table 3). In largemouth bass, the WG and feed utilization were significantly decreased when CPC was used to substitute 70% of fishmeal in the basal diet containing 700 g/kg fishmeal . In Pacific white shrimp, the WG and PER were also decreased by the substitution of 45% of fishmeal with CPC in a diet containing 250 g/kg fishmeal . Similarly, the WG of largemouth bass was significantly decreased when dietary fishmeal was reduced from 350 g/kg to 150 g/kg by CAP inclusion . The substitution of 45% of dietary fishmeal with CAP also significantly decreased the WG and PER of Pacific white shrimp . There are several reasons connected with the decreased growth and nutrient utilization by the excessive replacement of fishmeal with the CPC and CAP mixture in the present study: (1) The mixture contains a lower level of methionine than fishmeal (Table 2), and the high substitution of fishmeal may lead to deficiency of methionine. Methionine is a precursor of protein synthesis, which can be converted into taurine to promote fish growth . (2) The high level of arginine in CPC may be another factor affecting the growth of rainbow trout. As excessive arginine causes antagonism with lysine, a high arginine level in the diet may negatively affect the lysine utilization and the growth of fish . (3) Fishmeal contains high levels of taurine, hydroxyproline, and some unknown growth factors that are essential for growth and intestinal health, while bacterial and plant proteins lack these active compounds . mTOR is a key signal molecule that regulates growth , and the efficiency of activating the mTOR signal pathway by fishmeal was found to be significantly higher than that by other protein sources such as CPC , corn gluten meal , and CAP . Taurine is a conditionally essential amino acid in fish, which can promote the growth of fish . The dietary supplementation of taurine reduced fishmeal inclusion from 240 g/kg to 160 g/kg without negative effects on the growth performance of largemouth bass . In a low fishmeal diet (70 g/kg), the supplementation of taurine and selenium yeast increased the growth and protein utilization of black sea bass . Hydroxyproline is a semi-essential amino acid in aquatic animals, which is a characteristic amino acid in collagen. In a turbot diet with high plant protein source inclusion, the supplementation of hydroxyproline (6 g/kg) increased the replaced ratio of fishmeal from 40% to 60% . Aksnes et al. also reported that the supplementation of hydroxyproline (2.9 g/kg) in a high plant protein source diet increased the WG of Atlantic salmon (Salmo salar L.) by 14%. (4) CAP and CPC have lower digestibility than fishmeal. The high fiber content in CPC and the presence of the cell wall in CAP may be an important factor affecting nutrient digestibility . Li et al. reported that the apparent digestibility coefficient of dry matter and crude protein of CPC were lower (-11.75% and -8.23%) than fishmeal in Pacific white shrimp, and high inclusion levels of CAP may reduce digestibility in animals. The present findings showed no significant difference in whole-body composition such as moisture, crude protein, and crude ash when CPC and CAP mixture was used to substitution fishmeal. Similar results were also reported in rainbow trout and black sea bream . However, the FM-0 group presented significantly higher crude lipid content than the CON group. Liu et al. found that the substitution of 75% of dietary fishmeal with CPC significantly increased the whole-body content of crude lipid in largemouth bass. Similar results were also described in southern flounder (Paralichthys lethostigma) . Due to the decrease in feed utilization (including protein utilization), the intake of protein can not be effectively utilized to synthesize body protein, but rather is converted into lipid for deposition. 4.2. Intestinal Morphology and Digestive Enzyme Activity The primary organ for digesting and absorbing nutrients of fish is the intestine, which impacts normal growth and development . Intestinal protease and amylase are the main digestive enzymes in the intestine, and the villus height and villus width determine the contact area between mucosal epithelial cells and chyme, while muscle thickness is beneficial to intestinal peristalsis and chyme propulsion . In the study, the intestinal protease and amylase activity tended to decrease with the decreasing inclusion of fishmeal, and the digestive enzyme activity, villus height, and muscle thickness in the FM-5 and FM-0 groups were significantly lower than those in the CON group. Wu et al. found that the replacement of 45% of dietary fishmeal with CAP significantly decreased intestinal protease and amylase activity, resulting in intestinal damage and inflammation in large yellow croaker. In Pacific white shrimp, the intestinal villus height was significantly decreased when 40% of dietary fishmeal was replaced with CAP, but the supplementation of phosphorus improved the intestinal health . Similar results were also observed in silver sillago (Sillago sihama Forsskal) and large yellow croaker when CPC replaced a high proportion of fishmeal. With the decrease in fishmeal inclusion, the bioactive substances such as taurine and trimethylamine oxide tend to decrease, which may reduce the ability of the pancreas and intestinal glands to secrete digestive enzymes. In juvenile cobia (Rachycentron canadum) and golden pompano , the supplementation of taurine in the diet was reported to increase the trypsin activity and improve the intestinal structure, respectively. 4.3. Serum Biochemical Indices AST and ALT are two important transaminases in amino acid metabolism, which exist in the heart and liver. When the liver is damaged, the cell membrane permeability of hepatocytes increases, and the two transaminases in the cytoplasm will be released into the blood, resulting in increased activity in the blood . No significant difference in serum activities of AST and ALT was observed in the present study, although the hepatocytes in the FM-0 group presented some vacuolation and lipid droplets, indicating lipid accumulation in the liver. Such a result was consistent with the increasing whole-body crude lipid content in this group. However, in hybrid grouper and largemouth bass , AST and ALT activity in serum were significantly increased when CPC and CAP replaced 36% and 50% of dietary fishmeal, respectively. It could be that the combination of CAP and CPC in the present study produced complementary effects and overcame their respective shortcomings. In addition, CPC is rich in arginine, and arginine could protect the liver by reducing the production of pro-inflammatory cytokines and free radicals . Physiologically, blood GLU reflects the health of fish, which was also measured as a stress marker to evaluate the stress response caused by dietary changes . The present study showed that blood GLU content was significantly increased only in the FM-0 group. In grass carp (Ctenopharyngodon idllus), the serum content of GLU was also significantly increased when 100 g/kg CAP was used to replace soybean meal . In general, a high blood GLU level would lead to a stress response and affect the growth performance of fish . Serum TCHO is an important indicator of body lipid metabolism. The content of TCHO in the FM-0 group was also significantly higher than that in the CON group, which was consistent with the increase in whole-body crude lipid content in this group. Generally, the production and scavenging of active oxides are in dynamic equilibrium, and excessive free radicals lead to lipid peroxidation . MDA is the toxic product of lipid peroxidation, and it can adversely affect health. SOD is the primary substance for organisms to scavenge oxygen free radicals, and CAT scavenge hydrogen peroxide to protect cells from toxicity, while T-AOC is usually used to evaluate the antioxidant capacity of animals . In the present study, the activities of SOD and T-AOC in the FM-5 and FM-0 groups were all significantly lower, while the MDA content was significantly higher than those in the CON group, indicating that the high substitution of fishmeal by CPC and CAP mixture decreased the antioxidant capacity and broke the dynamic balance of free radicals. It has been reported that the high substitution of fishmeal (36%, 57.1%) with either CPC or CAP significantly increases the serum content of MDA in hybrid grouper and largemouth bass . Dietary supplementation of taurine alleviated the oxidative damage, such as decreasing the content of MDA , which has been confirmed in rice field eel (Monopterus albus) and spotted knifejaw (Oplegnathus punctatus) . In addition, the free gossypol in CPC may adversely affect the antioxidant system, although the free gossypol level in CPC is much lower than that in cottonseed meal. Gossypol can easily bind to proteins in the electron transfer chain of mitochondria, interfere with the function of mitochondria, and lead to excessive production of reactive oxygen species, thereby inhibiting the activity of various enzymes and causing oxidative damage . 5. Conclusions In the present study, the mixture inclusion of CPC and CAP successfully decreased dietary fishmeal from 200 g/kg to 100 g/kg without adverse effects on the growth performance, nutrient utilization, serum biochemical, or intestinal and hepatopancreas histology of rainbow trout. Acknowledgments The authors thank Xia Lin, Menglu Li, Yuanyuan Wang, Hongyu Ke, Zhifen Xu, and Jinyu Zheng for their help in fish sampling. Author Contributions Conceptualization, methodology, date analysis and writing this paper, H.H. and X.L. (Xiaoqin Li); software, formal analysis, K.C.; guidance, writing--review and editing, X.L. (Xiangjun Leng). All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All the procedures for handling animals involved in this experiment are in accordance with the regulations of the Experimental Animal Ethics Committee and the Institutional Animal Care Committee of Shanghai Ocean University (Approval code: SFI 2020-23 of 20 May 2020). Informed Consent Statement Not applicable. Data Availability Statement All dates are contained within the article. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Effect of replacing fishmeal with cottonseed protein concentrate (CPC) and Clostridium autoethanogenum protein (CAP) mixture on foregut morphology of rainbow trout (10x). (A): CON; (B): FM-15; (C): FM-10; (D): FM-5; (E): FM-0; VH: villus height; VW: villus width; MT: muscle thickness. Scale bar = 200 mm. Figure 2 Effect of replacing fishmeal with CPC and CAP mixture on hepatopancreas morphology of rainbow trout (40x). (A): CON; (B): FM-15; (C): FM-10; (D): FM-5; (E): FM-0; LD: lipid droplet. Scale bar = 50mm. animals-13-00817-t001_Table 1 Table 1 The diet formulation and proximate composition (air-dry basis, g/kg). Ingredients 1 CON FM-15 FM-10 FM-5 FM-0 Fishmeal 200.0 150.0 100.0 50.0 0.0 Clostridium autoethanogenum protein (CAP) 0.0 24.0 47.0 71.0 94.0 Cottonseed protein concentrate (CPC) 0.0 24.0 47.0 71.0 94.0 Soy bean meal 70.0 70.0 70.0 70.0 70.0 Soy protein concentrate 120.0 120.0 120.0 120.0 120.0 Wheat gluten 30.0 30.0 30.0 30.0 30.0 Wheat flour 270.0 268.0 268.0 266.0 266.0 Corn gluten meal 80.0 80.0 80.0 80.0 80.0 Meat meal 80.0 80.0 80.0 80.0 80.0 Beer yeast 50.0 50.0 50.0 50.0 50.0 Fish oil 20.0 24.0 28.0 32.0 36.0 Soybean oil 20.0 20.0 20.0 20.0 20.0 Soybean lecithin 20.0 20.0 20.0 20.0 20.0 Ca(H2PO4)2 20.0 20.0 20.0 20.0 20.0 Vitamin premix 2 10.0 10.0 10.0 10.0 10.0 Mineral premix 3 10.0 10.0 10.0 10.0 10.0 Total 1000.0 1000.0 1000.0 1000.0 1000.0 Proximate composition (g/kg) Moisture 63.0 61.1 59.5 71.7 73.7 Crude protein 438.0 439.5 435.7 429.0 430.1 Crude lipid 77.9 74.9 77.1 82.7 83.7 Crude ash 111.6 105.0 98.0 95.5 90.4 1 The protein contents of soy bean meal, soy protein concentrate, wheat flour, corn gluten meal, and meat meal were 426.7 g/kg, 648.9 g/kg, 145.5 g/kg, 588.6 g/kg, and 650 g/kg, respectively. 2 Vitamin premix (mg or IU/kg diet): vitamin A, 1000 IU; vitamin D3, 3000 IU; vitamin E, 150 IU; vitamin K3, 12.17 mg; vitamin B1, 20 mg; vitamin B2, 20 mg; vitamin B3, 100 mg; vitamin B6, 22 mg; vitamin B12, 0.15 mg; vitamin C, 300 mg; biotin, 0.6 mg; inositol, 400 mg; and folic acid, 8 mg. 3 Mineral premix (mg/kg diet): I, 1.5 mg; Mn, 11.45 mg; Co, 0.6 mg; Cu, 3 mg; Zn, 89 mg; Se, 0.24 mg; Mg, 180 mg; and Fe, 63 mg. animals-13-00817-t002_Table 2 Table 2 The proximate composition and amino acid profile of the four ingredients (g/kg). Items CAP CPC CPC + CAP (1:1) FM Proximate composition (air-dry basis) Crude protein 842.1 615.1 728.6 682.1 Crude lipid 1.9 23.6 12.8 90.0 Crude ash 32.7 62.0 47.4 149.5 Moisture 71.4 52.5 62.0 68.0 Essential amino acids (dry-matter basis) Lysine 87.0 24.7 55.9 52.1 Methionine 22.9 8.5 15.7 20.3 Arginine 34.0 78.9 56.5 40.9 Histidine 16.8 18.0 17.4 20.7 Isoleucine 52.8 18.9 35.9 27.5 Leucine 63.8 34.4 49.1 52.6 Phenylalanine 33.0 35.3 34.2 35.9 Threonine 40.2 19.0 29.6 28.7 Tryptophan 6.2 8.1 7.2 7.0 Valine 54.4 26.6 40.5 33.7 Non-essential amino acids (dry-matter basis) Aspartic acid 95.4 56.6 76.0 61.0 Serine 32.1 26.5 29.3 26.1 Glutamic acid 97.8 123.7 110.8 87.5 Glycine 38.7 25.0 31.9 41.3 Alanine 46.3 23.6 35.0 44.2 Cysteine 7.1 9.5 8.3 4.1 Proline 24.0 21.7 22.9 28.4 Tyrosine 31.4 20.2 25.8 22.9 Total amino acids 783.9 579.2 681.6 634.9 animals-13-00817-t003_Table 3 Table 3 Effect of replacing fishmeal with cottonseed protein concentrate (CPC) and Clostridium autoethanogenum protein CAP mixture on growth performance of rainbow trout. Items CON FM-15 FM-10 FM-5 FM-0 IBW (g) 35.05 +- 0.05 34.97 +- 0.06 35.00 +- 0.05 34.95 +- 0.05 35.02 +- 0.03 FBW (g) 125.73 +- 0.46 a 125.47 +- 2.16 a 122.47 +- 1.77 ab 119.93 +- 0.88 b 117.8 +- 1.41 b WG (%) 258.72 +- 1.42 a 258.82 +- 5.75 a 249.90 +- 5.03 ab 242.89 +- 2.18 bc 236.57 +- 4.04 c FCR 1.19 +- 0.01 c 1.20 +- 0.03 c 1.24 +- 0.02 bc 1.28 +- 0.01 ab 1.31 +- 0.02 a FI (g/fish/day) 1.93 1.93 1.93 1.93 1.93 SGR (% BW/day) 2.28 +- 0.01 a 2.28 +- 0.03 a 2.24 +- 0.03 ab 2.2 +- 0.01 bc 2.17 +- 0.02 c Survival (%) 100 100 100 100 100 K (g/cm3) 1.63 +- 0.01 1.55 +- 0.1 1.61 +- 0.08 1.64 +- 0.12 1.55 +- 0.01 VSI (%) 11.00 +- 0.50 10.13 +- 0.75 10.89 +- 0.99 10.42 +- 0.59 9.87 +- 0.7 HSI (%) 1.26 +- 0.09 1.23 +- 0.13 1.37 +- 0.13 1.28 +- 0.06 1.31 +- 0.02 In the same row, values with different small letter superscripts mean significant difference (p < 0.05). IBW: initial body weight; FBW: final body weight; WG: weight gain; FCR: feed conversion ratio; SGR: specific growth rate; K: condition factor; VSI: viscerosomatic index; HSI: hepatosomatic index. animals-13-00817-t004_Table 4 Table 4 Effect of replacing fishmeal with CPC and CAP mixture on whole-body composition of rainbow trout (fresh weight, g/kg). Items CON FM-15 FM-10 FM-5 FM-0 Moisture 708.5 +- 3.7 713.4 +- 15.7 714.8 +- 20.1 706.4 +- 15.7 700.2 +- 19.2 Crude ash 22.4 +- 1.6 22.7 +- 0.2 20.7 +- 0.2 21.0 +- 0.4 22.0 +- 1.7 Crude lipid 64.9 +- 2.7 b 63.2 +- 2.5 b 61.2 +- 5.0 b 64.0 +- 3.9 b 71.1 +- 1.0 a Crude protein 180.4 +- 1.6 181.5 +- 2.1 179.5 +- 6.4 178.4 +- 3.2 181.0 +- 1.8 In the same row, values with different small letter superscripts mean significant difference (p < 0.05). The same as below. animals-13-00817-t005_Table 5 Table 5 Effect of replacing fishmeal with CPC and CAP mixture on nutrient utilization and intestinal digestive enzymes of rainbow trout. Items CON FM-15 FM-10 FM-5 FM-0 Protease (U/mg prot) 16.10 +- 0.75 a 14.26 +- 4.90 ab 10.32 +- 0.67 bc 8.16 +- 0.71 cd 5.12 +- 0.21 d Amylase (U/mg prot) 0.70 +- 0.02 a 0.60 +- 0.01 ab 0.62 +- 0.03 ab 0.44 +- 0.08 bc 0.34 +- 0.02 c PER 1.91 +- 0.01 a 1.90 +- 0.04 ab 1.85 +- 0.04 abc 1.83 +- 0.01 bc 1.78 +- 0.02 c PR (%) 41.02 +- 0.18 a 41.08 +- 0.81 a 39.76 +- 0.67 ab 39.12 +- 0.24 b 38.99 +- 0.45 b LR (%) 80.42 +- 4.02 a 81.00 +- 4.49 a 77.35 +- 2.46 a 68.43 +- 2.31 b 77.59 +- 1.59 a In the same row, values with different small letter superscripts mean significant difference (p < 0.05). PER: protein efficiency ratio; PR: protein retention; LR: lipid retention. animals-13-00817-t006_Table 6 Table 6 Effect of replacing fishmeal with CPC and CAP mixture on serum biochemical indices of rainbow trout. Items CON FM-15 FM-10 FM-5 FM-0 AST (U/mL) 1.13 +- 0.07 1.09 +- 0.16 1.12 +- 0.19 1.17 +- 0.11 1.39 +- 0.26 ALT (U/mL) 6.78 +- 0.54 6.16 +- 0.73 6.40 +- 0.54 7.11 +- 0.09 7.40 +- 0.66 TG (mmol/L) 2.55 +- 0.07 2.56 +- 0.07 2.42 +- 0.12 2.68 +- 0.06 2.44 +- 0.05 GLU (mmol/L) 4.67 +- 0.34 b 4.84 +- 0.28 ab 4.58 +- 0.39 b 5.09 +- 0.17 ab 5.47 +- 0.23 a TCHO (mmol/L) 7.11 +- 0.48 b 6.70 +- 0.25 b 7.01 +- 0.11 b 7.31 +- 0.33 b 8.32 +- 0.22 a TP (g/L) 27.41 +- 2.11 28.48 +- 1.26 27.41 +- 2.41 26.56 +- 2.00 29.35 +- 0.84 MDA (nmol/mL) 14.03 +- 0.29 b 14.68 +- 0.88 ab 14.46 +- 1.15 ab 16.45 +- 0.91 a 16.29 +- 1.19 a CAT (U/mL) 4.24 +- 0.92 3.73 +- 0.50 3.72 +- 0.37 3.17 +- 0.61 2.76 +- 0.64 SOD (U/mL) 49.93 +- 1.38 a 50.78 +- 5.18 a 50.57 +- 2.55 a 40.49 +- 1.04 b 42.16 +- 1.46 b T-AOC (mmol/L) 0.79 +- 0.04 a 0.78 +- 0.05 a 0.80 +- 0.05 a 0.67 +- 0.03 b 0.68 +- 0.01 b In the same row, values with different small letter superscripts mean significant difference (p < 0.05). AST: aspartate aminotransferase; ALT: alanine aminotransferase; TG: triglyceride; GLU: glucose; TCHO: total cholesterol; TP: total protein; MDA: malondialdehyde; CAT: catalase; SOD: superoxide dismutase; T-AOC: total antioxidant capacity. animals-13-00817-t007_Table 7 Table 7 Effect of replacing fishmeal with the CPC and CAP mixture on the foregut morphology of rainbow trout. Items CON FM-15 FM-10 FM-5 FM-0 Villus height (mm) 734.9 +- 27.0 a 737.2 +- 39.2 a 764.4 +- 37.8 a 670.4 +- 32.8 b 603.1 +- 58.4 c Villus width (mm) 161.9 +- 17.6 a 165.6 +- 4.9 a 168.4 +- 11.7 a 167.0 +- 23.5 a 127.6 +- 9.0 b Muscle thickness (mm) 140.6 +- 5.8 a 141.1 +- 6.4 a 139.2 +- 20.1 a 120.0 +- 9.3 b 119.4 +- 6.0 b In the same row, values with different small letter superscripts mean significant difference (p < 0.05). 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PMC10000055

Cryptosporidium parvum is an important zoonotic protozoon that threatens the health of humans and animals, but the interaction mechanisms between C. parvum and hosts are poorly understood. Our previous study indicated that the expression levels of C3a and C3aR were up-regulated in mice during C. parvum infection, but the mechanisms of C3a/C3aR signaling during C. parvum infection have not been elucidated. In the present study, an optimized BALB/c suckling mouse model infected with C. parvum was used to explore the function of C3a/C3aR signaling during C. parvum infection. The expression levels of C3aR in the ileum tissues of mice infected with C. parvum were analyzed using real-time PCR, Western blot and immunohistochemistry. The mRNA levels of the Cryptosporidium 18S rRNA gene, tight junction proteins (zo-1, claudin 3, and occludin), intestinal stem cell marker lgr5, cell proliferation marker ki67, Th1 cell-related cytokine ifn-g, and Treg cell-related cytokine tgf-b in mouse ileum tissues were analyzed by real-time PCR. The pathological injury of ileal mucosa was examined by histopathology analysis. The mRNA expression levels of Cryptosporidium 18S rRNA gene were significantly up-regulated in the ileum tissues of C3aR-inhibited mice during C. parvum infection. Meanwhile, histopathology analysis of ileal mucosa in mice showed that inhibition of C3aR significantly aggravated the changes in villus length, villus diameter, mucosal thickness and the ratio of villus length to crypt depth during C. parvum infection. Further studies found inhibition of C3aR aggravated the down-regulation of occludin at most time points during C. parvum infection. The mRNA levels of ki67 and lgr5 in the ileum tissues of mice infected with C. parvum were significantly down-regulated. Inhibition of C3aR significantly down-regulated the mRNA expression levels of lgr5 at most time points, but significantly up-regulated the mRNA expression levels of ki67 at most time points. The mRNA expression levels of ifn-g and tgf-b were significantly up-regulated and down-regulated in the ileum tissues of mice infected with C. parvum, respectively. However, inhibition of C3aR significantly increased the mRNA expression levels of ifn-g and tgf-b in the ileum tissues of mice infected with C. parvum. Taken together, C3a/C3aR signaling could possibly affect the propagation of C. parvum in mouse ileum tissues by regulating the gut barrier, cell proliferation and CD4+ T cell main effectors, which would contribute to our understanding of the interaction between Cryptosporidium and hosts. Cryptosporidium parvum C3a/C3aR signaling propagation intestinal barrier function cell proliferation CD4+ T cell-related cytokines National Natural Science Foundation of China32072890 31572509 32202838 This research was funded by grants from the National Natural Science Foundation of China (32072890; 31572509; 32202838). pmc1. Introduction Cryptosporidium spp. are important diarrheal pathogens threatening the health of humans and hundreds of animals . Cryptosporidium spp. are primary diarrheal pathogens in dairy cattle, with over 20% of young animals infected with the pathogens . Meanwhile, children and immunosuppressed populations are highly susceptible to Cryptosporidium . Cryptosporidium spp. have been recognized as one of five major diarrheal pathogens in children under two years of age in developing countries , and have led to hundreds of outbreaks of diarrhea in industrialized nations . Nitazoxanide is the only FDA-approved drug in the treatment of human cryptosporidiosis, but it is almost useless in children and immunosuppressed populations . Therefore, hitherto, there are still no effective strategies for the treatment of cryptosporidiosis in humans and animals. Knowledge on the interaction between Cryptosporidium and hosts can shed the light on the discovery of novel targets for the development of vaccines and drugs, providing resources for the prevention and control of cryptosporidiosis in humans and animals. Previous studies have found that both innate and acquired immune responses played important roles in the early defense and late elimination of Cryptosporidium . During the early stages of acute infection, innate immune response participates in the host defense against Cryptosporidium invasion by monitoring immunocytes, cytokines, chemokines and complement molecules . More importantly, activated innate immune response can trigger and regulate acquired immune responses . Acquired immune responses are necessary to eliminate Cryptosporidium, especially for the cellular immune response dominated by CD4+ T cells . As the main components of innate immune response, complement molecules can play roles in dissolving pathogens, regulating phagocytosis, and mediating inflammation, immune adhesion and cytotoxicity in infected areas through the classical pathway, alternative pathway and lectin pathway . Moreover, activated complement components (e.g., C3d, complement receptor CD21/35 and C5aR) can be involved in the regulation of inflammation and acquired immune response dominated by CD4+ T cells, and linked with the innate immunity with acquired immunity . Complement C3 hydrolyzes to form anaphylaxis toxin C3a after the activation of the complement system, and C3a can bind to its specific receptor C3aR on the surface of cells; it then functions in inflammation, intestinal injury repairment, and CD4+ T cell differentiation and development . Our previous studies indicated the up-regulation of C3a and C3aR in mice during C. parvum infection, but the role of C3a/C3aR signaling during Cryptosporidium infection was still unknown . Therefore, the present study applied an optimized BALB/c mouse model infected with C. parvum to explore the function of C3a/C3aR signaling during C. parvum infection, which would contribute to our understanding of the interaction between C. parvum and hosts. 2. Materials and Methods 2.1. Parasites The C. parvum IIdA19G1 strain was isolated from one pre-weaned dairy calf with diarrhea in the Shaanxi province of China, and identified based on sequence analysis of the 18S rRNA and the gp60 gene loci . The strain was isolated in the lab of parasitology of Northwest A&F University and passaged in pre-weaned dairy calves in specific pathogen-free conditions. Cryptosporidium parvum oocysts in the faeces were purified using the Sheather's sugar flotation technique and cesium chloride density gradient centrifugation, then stored in PBS with 100 U/mL penicillin, 0.1 mg/mL streptomycin and 0.25 mg/mL amphotericin B solutions for less than 3 months, as in the previous study . 2.2. In Vivo Infection Model An established BALB/c suckling mouse model infected with C. parvum in our group was optimized in the present study by increasing the concentration of sodium taurocholate from 0.1% to 1%, and the excystation time from 30 min to 1 h. Briefly, each suckling mouse of the infection group and control group was orally administrated with 1 x 107 oocysts and equal volume of PBS, respectively. Suckling mice in the infection group and the control group were kept in separate cages to avoid contamination. At 1 day post infection (dpi), 2 dpi, 4 dpi, 6 dpi, 8 dpi, 9 dpi, 10 dpi, 29 dpi, 30 dpi, 35 dpi and 36 dpi, three animals were randomly selected for the isolation of ileum tissues from both control and infected groups at each time point, respectively. The above-mentioned time points and the range were selected since they represented specific milestones in the infection development. The ileum tissues collected from three animals for each group at each time point were washed in PBS and stored at -80 degC for RNA and protein isolation. Meanwhile, the ileum tissues at the peak of infection (6 dpi), based on RT-qPCR of 18S rRNA gene in mouse ileum tissues, were also stored in 4% paraformaldehyde fix solution for histopathology analysis, and the contents of large intestine at the peak of infection were kept at 4 degC for acid-fast stain analysis. All the experiments were performed thrice. The contents of the large intestine at the peak of infection from both the infected and control groups were analyzed using acid-fast stain to identify C. parvum oocysts, as previously reported . Histopathology analyses of mouse ileum tissues at the peak of infection were conducted as previously reported . Five slides of each mouse ileum tissue sample were randomly selected for calculating villus length, villus diameter, mucosal thickness and the ratio of villus length to crypt depth. For each slide, five complete intestinal villi with the highest length were analyzed. Furthermore, a C3aR-inhibited BALB/c suckling mouse model infected with C. parvum was established by intraperitoneally injecting animals with C3aR antagonist SB290157 (MedChemExpress, Princeton, NJ, USA) at a dose of 10 mg/kg before C. parvum infection . Collection of ileum tissue, RNA isolation and cDNA synthesis, expression level analysis of Cryptosporidium 18S rRNA gene, and histopathology analyses were conducted to explore the effect of C3a/C3aR signaling on the propagation of C. parvum in the ileum of mice. 2.3. Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR) Extraction of total RNA from mouse ilea and subsequent synthesis of cDNA were the same as previous study . The relative expression levels of Cryptosporidium 18S rRNA gene, C3aR, zo-1, claudin 3, occludin, lgr5, ki67, ifn-g and tgf-b in mouse ileum tissues were evaluated by qPCR using SYBR Green Fast RT-qPCR Mix (ABclonal, Wuhan, China) according to the manufacturer's recommendations and with the primers listed in Table 1. The gapdh gene was also included for data normalization (Table 1). Three independent experiments for tissues collected from three animals were performed, with data calculated by applying the 2-DDCt method to assess mRNA expression. 2.4. Western Blot Analysis The protein expression levels of C3aR in mouse ileum tissues at 2 dpi and 6 dpi were analyzed by Western blotting. Briefly, the mouse ileum tissues were homogenized by grinding, and treated with 1 mL RIPA (ComWin, Beijing, China) containing 1% PMSF (Beyotime, Shanghai, China) for the isolation of proteins. Proteins were separated by SDS-PAGE analysis, and transferred to nitrocellulose membranes pre-activated with methanol. Subsequently, the membranes were blocked with TBST containing 5% non-fat milk for 2 h, and incubated with anti-C3aR antibodies (Sanying, Wuhan, China) with 1:1,000 dilution at 4 degC overnight. The membranes were washed in TBST for 5 min thrice. HRP-conjugated goat-anti-rabbit antibodies (Sangon, Shanghai, China) with 1:5,000 dilution were applied as the secondary antibodies to incubate the membranes for 1 h at room temperature. Then, the membranes were washed in TBST for 5 min thrice and treated with Super ECL Plus (Applygen, Beijing, China) for photography under an automatic gel imaging analysis system (Sage, Beijing, China). 2.5. Immunohistochemistry Analysis The mouse ileum tissues at the peak of infection were analyzed by immunohistochemistry using SP-0023 HistostainTM-Plus Kits (MBL, Beijing, China). Briefly, paraffin sections were made from the ileum tissues at the peak of infection and subsequently incubated with anti-C3aR antibodies (Sanying, Wuhan, China) with 1:200 dilution at 4 degC overnight and HRP-conjugated goat-anti-rabbit antibodies (Sangon, Shanghai, China) for 15 min at room temperature. These were then photographed under a microscope (Olympus, Hataya, Japan). Image-Pro Plus was used to assess the distribution of C3aR on the surface of mouse ileum tissues by calculating the mean optical density value. Accumulative optical density divided by the area of cell distribution equals the mean optical density value. 2.6. Data Analysis Statistical difference analysis was conducted by GraphPad Prism V.8.0 accessed on 7 July 2022) using Student's t test and ANOVA . Significant difference was identified if the p value was less than 0.05. 3. Results 3.1. Optimization of a Mouse Model Infected with C. parvum Optimization of the excystation scheme could significantly increase the excystation rate from 40% to over 85%, as shown in Figure 1A (p < 0.05). Compared with the control group, the relative mRNA expression levels of Cryptosporidium 18S rRNA gene in the mouse ilea in C. parvum-infected group increased gradually and reached a peak at 6 dpi, then gradually decreased. The duration period of C. parvum infection in the mouse model reached 29 days . Acid-fast stain analysis of the large intestine contents at 6 dpi recognized rosy oocysts in the infected group . Histopathology analyses of mouse ileum tissues at 6 dpi found the existence of C. parvum and the infiltration of the inflammatory cells in the ileum tissues of the infected group ; subsequently statistical analysis indicated C. parvum infection significantly deceased the length of villi and the ratio of villus height to crypt depth, and significantly increased the diameter of villi . 3.2. Expression of C3aR in Mouse Ileum Tissues during C. parvum Infection Compared with control group, the mRNA expression levels of C3aR in mouse ileum tissues of the infected group were significantly down-regulated from 1 to 8 dpi, and then up-regulated until 36 dpi . Western blot analysis also indicated the down-regulation of protein levels of C3aR in mouse ileum tissues of the infected group at both 2 dpi and 6 dpi . Further immunohistochemistry analysis indicated C3aR was majorly distributed on the surface of ileal epithelial cells at 6 dpi , and subsequently statistical analysis also showed significantly decreased distribution of C3aR in mouse ileum tissues of the infected group . 3.3. Effect of C3a/C3aR Signaling on the Propagation of C. parvum in the Ilea of Mice As shown in Figure 3A, the protein level of C3aR in mouse ileum tissues was significantly down-regulated three hours after intraperitoneal injection with SB290157 . Further studies indicated that inhibition of mouse C3aR significantly up-regulated the relative mRNA expression levels of the Cryptosporidium 18S rRNA gene in mouse ileum tissues during C. parvum infection (p < 0.05). Meanwhile, the infection duration period increased from 29 days to 35 days in the C3aR-inhibited infected group . Significant differences were found between the relative transcriptional expression of Cryptosporidium 18S rRNA among the three groups (PSB, Oocyst, and Oocyst + SB290157) at 1 dpi (F = 359.612, p < 0.001), 2 dpi (F = 58.443, p < 0.001), 4 dpi (F = 44.112, p < 0.001), 6 dpi (F = 215.622, p < 0.001), 8 dpi (F = 393.826, p < 0.001), 9 dpi (F = 114.706, p < 0.001), 10 dpi (F = 427.847, p < 0.001), 29 dpi (F = 41.950, p < 0.001), 30 dpi (F = 55.628, p < 0.001), 35 dpi (F = 25.905, p < 0.001) and 36 dpi (F = 13.106, p = 0.006). Histopathology analyses of mouse ileum tissues at 6 dpi found the existence of C. parvum in the ileum tissues in the infected group and the C3aR-inhibited group . Subsequent statistical analysis indicated that C. parvum infection in C3aR-inhibited group further decreased the length of villi and the ratio of villus height to crypt depth, and increased the diameter of villi and mucosal thickness compared with the normal mouse in the infected group , suggesting that C3aR likely alleviated the intestinal injury caused by C. parvum. 3.4. Transcriptional Expression Level Analysis of Tight Junction Proteins in the Ileum Tissues of Mice during C. parvum Infection Significant differences were found between the relative transcriptional expression levels of the zo-1 gene among three groups (PSB, Oocyst, and Oocyst + SB290157) at 4 dpi (F = 253.975, p < 0.001), 6 dpi (F = 434.805, p < 0.001), 8 dpi (F = 21.897, p = 0.002), 9 dpi (F = 164.639, p < 0.001), 10 dpi (F = 18.990, p = 0.003), 29 dpi (F = 9.539, p = 0.014), 30 dpi (F = 14.252, p = 0.005), 35 dpi (F = 7.212, p = 0.025) and 36 dpi (F = 14.015, p = 0.005), while differences at 1 dpi (F = 3.316, p = 0.107) and 2 dpi (F = 0.146, p = 0.867) were not significant. Cryptosporidium parvum infection significantly up-regulated the mRNA expression levels of the zo-1 at 4 dpi and down-regulated at 6 dpi, 8 dpi, 9 dpi, 30 dpi and 35 dpi. Inhibition of C3aR led to dynamic changes in the zo-1 gene during C. parvum infection, reflected by the down-regulation at 8 dpi, 9 dpi, 10 dpi and 29 dpi, and up-regulation at 4 dpi, 6 dpi, 30 dpi, 35 dpi and 36 dpi . Significant differences were found between the relative transcriptional expression of the claudin 3 gene among three groups (PSB, Oocyst, and Oocyst + SB290157) at 2 dpi (F = 29.079, p = 0.001), 4 dpi (F = 89.677, p < 0.001), 6 dpi (F = 10.677, p = 0.011), 8 dpi (F = 37.588, p < 0.001), 9 dpi (F = 119.767, p < 0.001), 10 dpi (F = 13.474, p = 0.006), 29 dpi (F = 210.694, p < 0.001), 30 dpi (F = 380.076, p < 0.001), 35 dpi (F = 52.630, p < 0.001) and 36 dpi (F = 60.535, p < 0.001), while the difference at 1 dpi (F = 3.593, p = 0.094) was not significant. Compared with the control group, the mRNA expression levels of the claudin 3 gene of mouse ileum tissues in C. parvum-infected group were significantly down-regulated at 10 dpi, 29 dpi, 30 dpi, 35 dpi and 36 dpi, and up-regulated at 4 dpi, 8 dpi and 9 dpi. Inhibition of C3aR led to dynamic changes in the claudin 3 gene during C. parvum infection, reflected by the down-regulation at 6 dpi, 8 dpi, 9 dpi, 29 dpi and 30 dpi, and up-regulation at 2 dpi, 10 dpi, 35 dpi and 36 dpi . Significant differences were found between the relative transcriptional expression of the occludin gene among three groups (PSB, Oocyst, and Oocyst + SB290157) at 1 dpi (F = 15.306, p = 0.004), 4 dpi (F = 13.151, p = 0.006), 6 dpi (F = 257.643, p < 0.001), 8 dpi (F = 33.276, p = 0.001), 9 dpi (F = 29.416, p = 0.001), 10 dpi (F = 62.741, p < 0.001), 29 dpi (F = 219.563, p < 0.001), 30 dpi (F = 28.790, p = 0.001), 35 dpi (F = 67.671, p < 0.001) and 36 dpi (F = 13.095, p = 0.006), while the difference at 2 dpi (F = 4.718, p = 0.059) was not significant. Cryptosporidium parvum infection significantly up-regulated the mRNA expression levels of the occludin gene at 9 dpi, and down-regulated at 1 dpi, 6 dpi, 8 dpi, 10 dpi, 29 dpi, 30 dpi, 35 dpi and 36 dpi. Inhibition of C3aR led to significant up-regulation of the occludin gene at 1 dpi, 35 dpi and 36 dpi, but down-regulation from 4 dpi, 6 dpi, 8 dpi, 9 dpi, 10 dpi and 29 dpi . 3.5. Transcriptional Expression Level Analysis of the lgr5 and ki67 in the Ileum Tissues of Mice during C. parvum Infection Significant differences were found between the relative transcriptional expression levels of the lgr5 gene among three groups (PSB, Oocyst, and Oocyst + SB290157) at 1 dpi (F = 115.620, p < 0.001), 2 dpi (F = 8.921, p = 0.016), 4 dpi (F = 20.221, p = 0.002), 6 dpi (F = 41.482, p < 0.001), 8 dpi (F = 15.293, p = 0.004), 9 dpi (F = 6.992, p = 0.027), 10 dpi (F = 16.161, p = 0.004), 29 dpi (F = 23.714, p = 0.001), 30 dpi (F = 35.947, p < 0.001), 35 dpi (F = 11.568, p = 0.009) and 36 dpi (F = 25.923, p = 0.001). Compared with the control group, the mRNA expression levels of the lgr5 gene of mouse ileum tissues in the C. parvum-infected group were significantly down-regulated at 1 dpi, 2 dpi, 6 dpi, 8 dpi and 10 dpi, and up-regulated from 30 dpi, 35 dpi and 36 dpi. Inhibition of C3aR led to significant up-regulation of the lgr5 gene at 1 dpi, 2 dpi and 4 dpi, but down-regulation at 6 dpi, 8 dpi, 29 dpi, 30 dpi and 35 dpi . Significant differences were found between the relative transcriptional expression levels of the ki67 gene among three groups (PSB, Oocyst, and Oocyst + SB290157) at 1 dpi (F = 136.699, p < 0.001), 2 dpi (F = 621.666, p < 0.001), 4 dpi (F = 1282.733, p < 0.001), 6 dpi (F = 15.791, p = 0.004), 8 dpi (F = 19.258, p = 0.002), 9 dpi (F = 10.979, p = 0.010), 29 dpi (F = 12.055, p = 0.008), 30 dpi (F = 12.545, p = 0.007), 35 dpi (F = 119.732, p < 0.001) and 36 dpi (F = 69.487, p < 0.001), while the difference at 10 dpi (F = 1.225, p = 0.358) was not significant. Cryptosporidium parvum infection significantly up-regulated the mRNA expression levels of the ki67 gene at 9 dpi, 30 dpi, 35 dpi and 36 dpi, but down-regulated at 1 dpi, 2 dpi, 4 dpi, 8 dpi and 29 dpi. Inhibition of C3aR led to the significant up-regulation of the ki67 at 1 dpi, 2 dpi, 4 dpi, 6 dpi, 29 dpi and 36 dpi, but down-regulation at 9 dpi and 35 dpi . 3.6. Transcriptional Expression Level Analysis of CD4+ T cell-Related Cytokines in the Ileum Tissues of Mice during C. parvum Infection Significant differences were found between the relative transcriptional expression levels of the ifn-g gene among the three groups (PSB, Oocyst, and Oocyst + SB290157) at 1 dpi (F = 69.841, p < 0.001), 2 dpi (F = 300.925, p < 0.001), 4 dpi (F = 58.746, p < 0.001), 6 dpi (F = 72.761, p < 0.001), 8 dpi (F = 97.252, p < 0.001), 9 dpi (F = 38.113, p < 0.001), 10 dpi (F = 12.446, p = 0.007), 29 dpi (F = 48.334, p < 0.001), 30 dpi (F = 19.592, p = 0.002), 35 dpi (F = 41.574, p < 0.001) and 36 dpi (F = 45.236, p < 0.001). Compared with the control group, the mRNA expression levels of the ifn-g gene of mouse ileum tissues in C. parvum-infected group were significantly down-regulated at 1 dpi, but up-regulated at most time points. Inhibition of C3aR led to the significant up-regulation of ifn-g at 1 dpi, 2 dpi, 8 dpi, 9 dpi and 29 dpi, but down-regulation at 6 dpi, 30 dpi and 35 dpi . Significant differences were found between the relative transcriptional expression levels of the tgf-b gene among three groups (PSB, Oocyst, and Oocyst + SB290157) at 1 dpi (F = 1721.105, p < 0.001), 2 dpi (F = 35.969, p < 0.001), 6 dpi (F = 30.388, p = 0.001), 8 dpi (F = 51.836, p < 0.001), 9 dpi (F = 94.612, p < 0.001), 10 dpi (F = 8.058, p = 0.020), 29 dpi (F = 87.382, p < 0.001), 30 dpi (F = 19.085, p = 0.003), 35 dpi (F = 205.922, p < 0.001) and 36 dpi (F = 8.847, p = 0.016), while the difference at 4 dpi (F = 4.687, p = 0.059) was not significant. Cryptosporidium parvum infection significantly down-regulated the mRNA expression level of the tgf-b at most time points. Inhibition of C3aR led to the significant up-regulation of tgf-b at all time points post infection . 4. Discussion Cryptosporidiosis is an important zoonotic disease caused by Cryptosporidium spp., which greatly threatens the health of humans and animals, especially young and immunocompromised hosts . However, there are still no effective drugs and vaccines for the prevention and control of cryptosporidiosis. One of the main reasons for this is a lack of knowledge on the interaction between Cryptosporidium and hosts. Previous studies indicated C3a/C3aR signaling played some important roles in the recovery of intestinal injury . Our previous study showed that C. parvum infection could lead to the up-regulation of C3a and C3aR in mouse ileum tissues , but the role of C3a/C3aR signaling during C. parvum infection is still unknown. The present study applied an optimized mouse model with C. parvum infection to explore the function of C3a/C3aR signaling during C. parvum infection, enriching our knowledge on the interaction between Cryptosporidium and hosts. The present study optimized an established suckling mouse model infected with C. parvum in our group by increasing the concentration of sodium taurocholate and the excystation time, and the results indicated that the optimized model significantly increased the excystation of C. parvum. Similar to the previous model, the optimized model also found pathological damage and an inflammatory reaction in the intestines in mice during C. parvum infection . Compared with other infection models, the optimized mouse model in the present study simulated the natural infection process of animals and provided a candidate model for studies on the interaction between Cryptosporidium and hosts without the use of dexamethasone. In the present study, the optimized mouse model found that the up-regulation of C3aR in the ileum of mice during C. parvum infection and inhibition of C3aR significantly aggravated intestinal damage caused by C. parvum infection. Although opposite results were found for the C3aR mRNA expression at 1 dpi and 2 dpi between the present study and our previous work, the mRNA levels of this gene were increased at later stages of infection for both studies . The differences at the early stage of infection may be due to the distinct immunity of animal models. The present study used suckling mice for parasite infection, while older mice aged 3 weeks were used in the previous study. Tight junctions are an important structural basis of the intestinal mucosal mechanical barrier, which plays an important role in maintaining the integrity of the intestinal morphology and resisting the invasion of intestinal pathogens . To explore the mechanisms of C3a/C3aR signaling, the mRNA expression levels of three tight junction proteins, namely zo-1, claudin 3 and occludin, were detected by using qPCR. The results found that C. parvum infection down-regulated the mRNA expression levels of the zo-1, claudin 3 and occludin genes at most time points, indicating that C. parvum likely destroyed the integrity of intestinal barrier by down-regulating tight junctions, which was similar to the C57BL/6 mouse model infected with C. parvum . Meanwhile, Cryptosporidium andersoni infection could also destroy the expression of the ZO-1 in the epithelial cells of humans and cattle . Interestingly, inhibition of C3aR significantly down-regulated the mRNA expression of the occludin gene at half of the time points during C. parvum infection, suggesting that C3a/C3aR signaling likely maintained the integrity of the intestinal barrier by upregulating the expression of the tight junction-related gene occludin, thus reducing the infection and propagation of C. parvum in mouse ilea. Meanwhile, the proliferation and differentiation of intestinal stem cells and the normal renewal of intestinal epithelial cells also play some important roles in maintaining the structure and function of intestinal mucosa . Furthermore, the mRNA expression levels of intestinal stem cell marker LGR5 and cell proliferation marker KI67 of ileum tissues during C. parvum infection were detected by qPCR. The results indicated that C. parvum infection down-regulated the mRNA expression levels of the lgr5 and ki67 genes in mouse ileum tissues at half of the time points, reflecting that C. parvum infection possibly retarded the proliferation and renewal of intestinal epithelial cells to prolong the survival time of the parasites in intestinal cells, and then aggravated Cryptosporidium infection in intestine, in accordance with previous reports . Inhibition of C3aR led to the down-regulation of the lgr5 gene at most time points, while leading to up-regulation of ki67 at most time points, suggesting that C3a/C3aR signaling likely promoted the regeneration of intestinal epithelial cells and inhibited the proliferation of intestinal epithelial cells to inhibit the propagation of C. parvum in the ilea of mice. Early moderate IFN-g can not only initiate intestinal immunity, but can also promote intestinal cell proliferation and mucosal damage repair. However, abnormal over-activation of the immune system will release a large amount of IFN-g, and excessive IFN-g can not only kill pathogenic microorganisms, but also activate the JAK/STAT1 pathway of intestinal epithelial cells, leading to programmed necrosis of intestinal epithelial cells and impairment of intestinal barrier function . Unlike IFN-g, TGF-b showed a protective function on the intestinal barrier . The aforementioned functions of IFN-g and TGF-b have also been confirmed in intestinal epithelial cells infected with Cryptosporidium . Meanwhile, C3a/C3aR signaling can function in resisting pathogen invasion by promoting/inhibiting the expression of IFN-g and TGF-b . To explore the link between C3a/C3aR signaling and the expression of IFN-g and TGF-b, we used qPCR to detect the mRNA expression levels of the ifn-g and tgf-b genes in mouse ilea during Cryptosporidium infection. The results revealed that C. parvum infection significantly up-regulated and down-regulated the mRNA levels of the ifn-g and tgf-b genes, respectively, indicating that C. parvum infection may induce a Th1-type immune response, and inhibit the differentiation and function of Treg cells. Therefore, mice infected with C. parvum were likely in a state of continuous inflammation. However, inhibition of C3aR led to the significant up-regulation of mRNA levels of both ifn-g and tgf-b in the ileum tissues of infected mice, which was possibly related to the dual anti-inflammatory and pro-inflammatory actions of C3a/C3aR signaling , suggesting that C3a/C3aR signaling likely participated in the anti-Cryptosporidium effect by down-regulating the expression of Th1 and Treg cells' main effect factors; however, these mechanisms need to be explored in future studies. 5. Conclusions The present study firstly explored the preliminary function of host C3a/C3aR signaling during C. parvum infection, and revealed C3a/C3aR signaling likely inhibited the propagation of C. parvum in the ilea of mice by regulating the gut barrier, cell proliferation, and CD4+ T cell main effectors of hosts, which could contribute to our knowledge on the interaction between Cryptosporidium and hosts. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Original Western blot picture for the protein expression level of C3aR in the mouse ileum tissues of PBS and Oocyst groups at 2 dpi; Figure S2: Original Western blot picture for the protein expression level of C3aR in the mouse ileum tissues of PBS and Oocyst groups at 6 dpi; Figure S3: Original Western blot picture for the protein expression level of C3aR in the mouse ileum tissues of PBS and SB290157 groups at three hours after intraperitoneal injection with SB290157. Click here for additional data file. Author Contributions Conceptualization, G.Z.; methodology, X.Y. and X.W.; software, S.H., Q.Y. and X.C.; investigation, J.S. and Y.F.; writing--original draft preparation, X.Y.; writing--review and editing, X.Y. and G.Z.; visualization, X.Y. and X.W.; supervision, G.Z. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study was conducted under the approval and instructions of the ethics committee of Northwest A&F University (DY2019042). Informed Consent Statement Not applicable. Data Availability Statement Data are contained within the article. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Optimization of mouse model infected with C. parvum. (A) Excystation results of C. parvum oocysts. Unexcysted and excysted oocysts appeared as light refractile spheres (red arrow) and dark black spheres (blue arrow), respectively. Bars = 20 mm. (B) qRT-PCR amplification results of Cryptosporidium 18S rRNA gene. ** means p < 0.01. (C) Acid-fast staining results for contents in large intestines of mice infected with C. parvum at 6 dpi. Oocysts appeared as rosy spheres (red arrow). Bars = 20 mm. (D) HE staining results in ileum tissues of mice in the control and infected groups at 6 dpi. C. parvum in the brush border of ileum villi in infected group (red arrow) and lymphocytes (green arrow) and neutrophils (blue triangle) infiltration in the lamina propria were indicated. Bars = 20 mm. (E) Analysis of pathological lesions in ileum tissues of mice infected with C. parvum at 6 dpi. Error bars mean standard deviation with each group, ** means p < 0.01, and ns means p > 0.05. Figure 2 Expression of C3aR in the ilea of mice infected with C. parvum. (A) Analysis of relative expression levels of C3aR in ileum tissues of mice infected with C. parvum; (B) C3aR protein expression levels in ileum tissues of mice. a and c: Western blotting analysis at 2 and 6 dpi; b and d: statistical analysis at 2 and 6 dpi; (C) Immunohistochemical staining results of C3aR protein in ileum tissues (6 dpi) of mice. a and e: no primary antibody (control group); b and f: with primary antibody (control group); c and g: no primary antibody (infected group); d and h: with primary antibody (infected group); (D) Statistic analysis of immunohistochemical staining results of C3aR protein. * means p < 0.05, ** means p < 0.01. Figure 3 Effect of C3a/C3aR signaling on the propagation of C. parvum in mouse ilea. (A) C3aR protein expression levels in ileum tissues of mice detected using Western blotting analysis. a: Western blotting results; b: statistical analysis of Western blotting results. ** means p < 0.01; (B) Analysis of relative transcriptional expression levels of Cryptosporidium 18S rRNA gene in the ileum tissues of mice. The relative expression levels of Cryptosporidium 18S rRNA between two groups at each time point were compared, and significant differences were found between the relative expression levels of Cryptosporidium 18S rRNA between two groups if lowercase letters above the bars were different (p < 0.05); (C) HE staining results in ileum tissues of mice infected with C. parvum at 6 dpi. a/b/c indicate ilea in the control group/infected group/C3aR-inhibited group, respectively; (D) Analysis of pathological lesions in ileum tissues of mice infected with C. parvum at 6 dpi. ** means p < 0.01, ns means p > 0.05. Figure 4 Effect of C3aR inhibition on the relative transcriptional expression of intestinal barrier-related genes in the ilea of mice infected with C. parvum. Analysis of the relative expression levels of the zo-1 gene (A), claudin 3 gene (B) and occludin gene (C) in the ileum tissues of mice. Different lowercase letters above the bars indicated significant differences (p < 0.05). Figure 5 Effect of C3aR inhibition on the relative transcriptional expression levels of the lgr5 gene (A) and ki67 gene (B) in the ileum tissues of mice infected with C. parvum. Different lowercase letters above the bars indicated significant differences (p < 0.05). Figure 6 Effect of C3aR inhibition on the relative transcriptional expression of the CD4+ T cell-related cytokines in the ilea of mice infected with C. parvum. Analysis of the relative expression levels of the ifn-g (A) and tgf-b (B) genes in the ileum tissues of mice. Different lowercase letters above the bars indicated significant differences (p < 0.05). animals-13-00837-t001_Table 1 Table 1 Primers for real-time PCR amplification in the present study. Target Gene Sequence (5'-3') Annealing Temperature (degC) 18S rRNA F: CTCCACCAACTAAGAACGGCC R: TAGAGATTGGAGGTTGTTCCT 55 gapdh F: GGTGAAGGTCGGTGTGAACG 55 R: CTCGCTCCTGGAAGATGGTG C3aR F: CTATTGGGACTGCTAGGCAA R: TGTCCTTGGAGAATCAGGTG 54 zo-1 F: GCCGCTAAGAGCACAGCAA R: GCCCTCCTTTTAACACATCAGA 54 claudin 3 F: ACCAACTGCGTACAAGACGAG R: CGGGCACCAACGGGTTATAG 55 occludin F: TGAAAGTCCACCTCCTTACAGA R: CCGGATAAAAAGAGTACGCTGG 54 lgr5 F: GGCAGCACTTTTCAGCA R: GGACGACAGGAGATTGGA 53 ki67 F: TCTGTGCTGACCCTGATG R: CCCTGATGAGTCTTGGCTA 51 ifn-g F: ATGAACGCTACACACTGCATC R: CCATCCTTTTGCCAGTTCCTC 55 tgf-b F: CCGCAACAACGCCATCTAT R: CCAAGGTAACGCCAGGAATT 55 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000056

We aimed to investigate the effects of dietary alpha-lipoic acid (a-LA) on the growth performance, serum biochemical indexes, liver morphology, antioxidant capacity, and transcriptome of juvenile hybrid groupers (Epinephelus fuscoguttatus x Epinephelus polyphekadion). Four experimental diets supplemented with 0 (SL0), 0.4 (L1), 0.6 (L2), and 1.2 (L3) g/kg a-LA were formulated and fed to three replicates of juvenile hybrid grouper (24.06 +- 0.15 g) for 56 d. The results indicated that dietary 0.4 and 0.6 g/kg a-LA significantly decreased the weight gain rate in juvenile hybrid groupers. Compared with SL0, the content of total protein in the serum of L1, L2, and L3 increased significantly, and alanine aminotransferase decreased significantly. The content of albumin in the serum of L3 increased significantly, and triglyceride, total cholesterol, and aspartate aminotransferase decreased significantly. In addition, the hepatocyte morphology in L1, L2, and L3 all showed varying degrees of improvement, and the activities of glutathione peroxidase and superoxide dismutase in the liver of L2 and L3 were significantly increased. A total of 42 differentially expressed genes were screened in the transcriptome data. KEGG showed that a total of 12 pathways were significantly enriched, including the pathway related to immune function and glucose homeostasis. The expression of genes (ifnk, prl4a1, prl3b1, and ctsl) related to immune were significantly up-regulated, and the expressions of gapdh and eno1 genes related to glucose homeostasis were significantly down-regulated and up-regulated, respectively. In summary, dietary supplementation of 0.4 and 0.6 g/kg a-LA inhibited the growth performance of juvenile hybrid groupers. A total of 1.2 g/kg a-LA could reduce the blood lipid level, improve hepatocyte damage, and increase the hepatic antioxidant enzyme activity. Dietary a-LA significantly affected the pathway related to immune function and glucose homeostasis. juvenile hybrid grouper a-lipoic acid growth performance serum biochemical indexes antioxidant capacity transcriptome National Key R&D Program Blue Granary Science and Technology Innovation Key Special Project2020YFD0900204 National Key R&D Program of China2020YFD0900200 This work was financially supported by National Key R&D Program Blue Granary Science and Technology Innovation Key Special Project (2020YFD0900204) and the National Key R&D Program of China (2020YFD0900200). pmc1. Introduction As global demand for animal protein increases, the entire animal production system is gradually moving towards intensification . The aquaculture industry is growing rapidly, but it is vulnerable to the interactions between the animals themselves, diseases, and the environment. Therefore, a new model of aquaculture management strategy has emerged as a result of a growing understanding of animal nutrition and feed. The core objectives of this model are to minimize the effects of stressors by neutralizing free radicals, repairing oxidative damage to biological macromolecules and membrane systems, enhancing immunity, and maintaining normal physiological homeostasis. The key points of this model are antioxidant supplementation and increasing endogenous cellular antioxidants . Supplementation of diets with antioxidants can improve the resistance of fish to environmental stresses and is an essential measure to reduce losses in the aquaculture industry . Alpha-lipoic acid (a-LA), also known as 1,2-dithiolane-3-valeric acid, with the molecular formula C8H14O2S2, was first isolated from pig liver by Lester J. Reed in 1951 . a-LA is a naturally occurring compound found in microorganisms, plants, and animals, and is considered to be an "ideal antioxidant" or "universal antioxidant" because of its strong antioxidant capacity . Studies have shown that a-LA can improve the survival rate, growth performance, and immunity of fish, and also improve the nutritional value of fish, which makes a-LA suitable for application in aquaculture . For example, dietary supplementation with an appropriate amount of a-LA could promote growth, fatty acid b-oxidation, and lipolysis of grass carp (Ctenopharyngodon idellus; Cuvier et Valenciennes, 1844), increase protein deposition, enhance immunity and antioxidant capacity, alleviate the inflammatory response, and reduce lipid oxidative damage. It also could promote the expression of peripheral anorexia factor mRNA and reduce the expression of peripheral appetite factor mRNA, thus, reducing the intake and body weight of grass carp . The enhancement of growth performance has also been found in other aquatic organisms with moderate amounts of a-LA in their diets, such as Nile tilapia (Oreochromis niloticus; Linnaeus, 1758) , African catfish (Clarias gariepinus; Burchell, 1822) , giant gourami (Osphronemus goramy; Lacepede, 1801) , Chinese mitten crab (Eriocheir sinensis; H. Milne Edwards, 1853) , and northern snakehead (Channa argus; Cantor, 1842) . In addition, dietary supplementation with moderate amounts of a-LA could promote the expression of gluconeogenesis-related genes induced by a high-fat diet in fish, reduce lipid accumulation under high-fat conditions , and enhance starch utilization in carp (Cyprinus carpio; Linnaeus, 1758) . The hybrid grouper (Epinephelus fuscoguttatus; Forsskal, 1775 x Epinephelus polyphekadion; Bleeker, 1849) is an important mariculture fish in southern China, with the characteristics of rapid growth and strong stress resistance, and it has a high economic value in China . Although a-LA has been studied for over 70 years and there have been numerous studies on its addition as an antioxidant of aquatic animal diets, there have been few studies of a-LA in terms of the supplementation of marine fish diets. There are no reports of a-LA being added to the diet of groupers. Therefore, the purpose of this experiment was to research the effects of diet supplementation with a-LA on the growth performance, serum biochemical indexes, hepatic morphology, antioxidant capacity, and transcriptome of juvenile hybrid grouper fish, and to expand theoretical knowledge for the application of antioxidants in the hybrid grouper diet. 2. Material and Methods 2.1. Preparation of Diets and Testing of Nutritional Levels Four isonitrogenous diets (SL0, L1, L2, and L3) were prepared with 0, 0.4, 0.6, and 1.2 g/kg of a-LA (99% purity, Yingbo biotechnology Co., Ltd.), respectively. The a-LA content was referenced from previous studies . Referring to the study on experimental diet formulation and nutrient levels of hybrid groupers by Xie et al. . The experimental diet formulations, as well as nutrient levels, are shown in Table 1. All feed raw materials were crushed and sieved through a 60-mesh sieve. We weighed the ingredients accurately according to the feed formula and mixed them well, then added the fish oil and soybean lecithin, rubbed the powdered ingredients and oil together manually, then added the right amount of water to knead all the ingredients into a dough. Finally, the raw material was processed into pellets with a particle size of 2.5 mm using a twin-screw extruder, air-dried, and stored in a -20 degC refrigerator. The nutritional levels of the diets were tested according to the AOAC standard method , and specific detection methods refer to An et al. . 2.2. Experiment Design The experiments were conducted in the Zhanjiang Marine Hi-tech Park of Guangdong Ocean University (Zhanjiang, China). The water used for the culture experiments was natural seawater treated by sand filtration and sedimentation with uninterrupted aeration. The water temperature was kept at 28.5 +- 2.0 degC, pH was maintained at 7.6-8.2, dissolved oxygen was kept above 6 mg/L, total nitrite and ammonia content was kept below 0.04 mg/L, and the photoperiod adopted a natural day-night cycle (12 h of light, 12 h of darkness). The experimental juvenile hybrid groupers were purchased from a grouper hatchery at the southeast quay of Zhanjiang City, Guangdong Province, China, and were temporarily reared in an indoor cement pond (2.0 m x 4.0 m x 2.0 m) for 14 days after being transported back to the base. At the end of temporary rearing, the fish were starved for 24 h. A total of 360 fish (24.06 +- 0.15 g) with similar sizes, intact scales, and normal diet were randomly allotted to 12 fiberglass tanks (0.5 m3). Twelve fiberglass tanks were divided into four groups (SL0, L1, L2, and L3) with three replicates per group. The experiment was carried out in an indoor flowing water aquaculture system for 56 days. During the experiment, the corresponding feed was fed at 8: 30 and 17: 00 every day at 5-8% of their body weight. The water was changed as necessary to maintain superior water quality. 2.3. Sample Collection After the end of the experiment, all the experimental fish were fished out and weighed after 24 h starvation treatment. Six fish were randomly fished out from each fiberglass tanks and anesthetized with eugenol (each 100 mg of eugenol is dissolved in 1 L of seawater, Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China). The body length and body weight were measured to calculate morphological indices. Subsequently, blood was collected from the tail vein of the fish using a 1 mL needle tube, and the needle tube was washed with heparin sodium before blood collection. After standing for 12 h at 4 degC, the blood was centrifuged using a refrigerated high-speed centrifuge (4 degC, 3500 rpm,10 min). The supernatant (serum) was collected and placed in a 2 mL centrifuge tube and stored at -80 degC for biochemical testing. The abdomen was dissected using sterilized scissors and tweezers, the visceral mass and liver were separated and weighed, and the liver was then washed with saline to remove other impurities. A liver tissue (5 mm x 5 mm x 5 mm) was cut from the center of the liver and placed in a 2 mL centrifuge tube containing 4% paraformaldehyde. After 24 h of fixation, the liver tissue was washed with 70% ethanol solution and stored in 70% ethanol solution for HE sections. About 1.0 g of liver tissue was cut from the remaining liver tissue and placed in a 2 mL cryopreservation tube, frozen in liquid nitrogen, and transferred to a -80 degC refrigerator for antioxidant capacity testing and transcriptome sequencing. 2.4. Growth Performance Index Measurement In this study, the calculation methods of the growth performance indexes of juvenile hybrid groupers are as follows:(1) Weight gain rate (WGR,%)=[final body weight (g) - initial body weight (g)] / initial body weight (g)x100%; (2) Survival rate (SR, %)= The number of final fish / The number of initial fishx100; (3) Feed conversion ratio (FCR)= feed intake (g) / [final body weight (g) - initial body weight (g)]; (4) Specific growth rate (SGR,%/d)={Ln[final body weight (g)] - Ln[initial body weight (g)]} / Number of days (d)x100%; (5) Condition factor (CF, g/cm3)=[final body weight (g) / Final body length (cm)3]x100%; (6) Feed intake (FI,% )=total feed weight (g){[initial body weight (g)+final body weight (g)]/2}xNumber of days (d)x100 (7) Hepatosomatic index (HSI, %)=[liver weight (g)/ weight of this fish (g)]x100%; (8) Viscerosomatic index (VSI, %)=[viscera weight (g)/ weight of this fish (g)]x100% 2.5. Determination of Serum Biochemical Indexes and Liver Antioxidant Parameters The serum biochemical indexes included triglyceride (TG, GPO-PAP enzymatic method), total cholesterol (TCHO, COD-PAP method), total protein (TP, BCA microplate method), albumin (ALB, bromocresol green method), low-density lipoprotein cholesterol (LDL-C, dual reagent direct method), high-density lipoprotein cholesterol (HDL-C, dual reagent direct method), aspartate aminotransferase (AST, Lai's method), and alanine aminotransferase (ALT, Lai's method). Antioxidant parameters of the liver included glutathione peroxidase (GSH-Px, colorimetric method), catalase (CAT, ammonium molybdate method), superoxide dismutase (SOD, WST-1 method), and malondialdehyde (MDA, TBA method). The above indicators were determined using kits produced by Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Experiments were conducted in strict accordance with the instructions, and all instructions can be found and downloaded at (accessed on 15 January 2023). 2.6. Preparation and Observation of Hematoxylin-Eosin Stained Liver Sections The fixed liver tissue was taken out and repaired with a scalpel and placed in a dehydration box. The liver tissue was dehydrated by gradient alcohol using a dehydrator (Donatello, DIAPATH) and then embedded in paraffin. The paraffinized liver tissue was embedded in an embedding machine (JB-P5, Wuhan Junjie Electronics Co., Ltd., Wuhan, China) to form a tissue block. After cooling, 4 mm thick sections were cut out in a paraffin sectioning machine (RM2016, Shanghai Leica Instrument Co., Ltd., Shanghai, China). After deparaffinization, the sections were stained with hematoxylin and eosin, and finally dehydrated and fixed in a glass slide. The morphology of liver cells was observed using an upright fluorescence microscope (Nikon ECLIPSE Ni-E, Tokyo, Japan). 2.7. Transcriptome Sequencing and Analysis 2.7.1. RNA Extraction and Detection, Library Construction and High-Throughput Sequencing Total RNA from SL0 and L3 hepatic tissues was extracted using a Trizol kit (Invitrogen, Carlsbad, CA, USA). The total RNA quality and integrity were examined using an Agilent 2100 biological analyzer (Agilent Technologies, Palo Alto, CA, USA) and RNase-free agarose gel electrophoresis, respectively. The library construction and high-throughput sequencing were completed by Genedenovo Biotechnology Co., Ltd. (Guangzhou, China), and high-throughput sequencing was performed using an Illumina NovaSeq 6000. 2.7.2. Data Quality Control, De Novo Assembly and Unigene Basic Annotation The raw data were quality-controlled using the quality control software fastp (version 0.18.0) to filter low-quality raw sequencing data. De novo assembly was performed using the short reads assembling the program, Trinity. The unigene sequence was compared with the SWISS-PROT protein database, NCBI non-redundant protein (Nr) database, Kyoto Encyclopedia of Genes and Genome (KEGG) database, and COG/KOG database using the BLASTx program, and then the protein function annotation was obtained according to the best alignment results. 2.7.3. Differentially Expressed Genes Analysis The analysis was performed using DESeq2 software. First, we normalized the read counts, then calculated the probability of hypothesis testing (p-value) based on the model, and, finally, we performed multiple hypotheses testing corrections to obtain the FDR value (false discovery rate). Based on the results of differential analysis, the genes of FDR < 0.05 and |log2(Fold Change)| > 1 were screened as differentially expressed genes (DEGs). Volcano plot analysis, KEGG pathway enrichment analysis, and GO functional enrichment analysis were performed according to the DEGs. 2.7.4. Real-Time Quantitative PCR (RT-qPCR) Validation Five genes, namely phosphatase inhibitor-1 (i-1), alpha-enolase (eno1), thioredoxin-interacting protein (txnip), parvalbumin (pvalb), and dual specificity phosphatase 1 (dusp1), were randomly selected from DEGs for RT-qPCR to verify the reliability of RNA-Seq data. Total RNA extraction, cDNA synthesis, and RT-qPCR detection of SL0 and L3 liver tissues were performed using TransGen Biotech kits (Beijing, China). The specific primers (Table 2) were designed by Primer Premier 5.0 software and was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). According to the RT-qPCR kit instructions, qualified cDNA and primers were tested using a LightCycler 96 real-time fluorescent quantitative PCR instrument (Roche, Basel, Switzerland) with four replicates for each sample. The reaction procedures were as follows: 94 degC for 30 s (1 cycle); 95 degC for 5 s, 60 degC for 15 s and 72 degC for 10 s (40 cycles); 95 degC for 10 s, 60 degC for 60 s and 95 degC for 1 s (1 cycle); 37 degC for 30 s (1 cycle). According to the measured Ct value, the relative expression of each gene was calculated using the 2-DDCt method . 2.8. Data Statistical Analysis One-way ANOVA was performed on the experimental data using SSPS 21.0. For WGR, SR, SGR, VSI, and HSI, data transformation was required to remove the % before performing ANOVA, and, after completing ANOVA, data transformation was performed and the % was added. Tukey's test was used for multiple comparisons if there were significant differences between groups. The experimental data were expressed as mean +- standard deviation (mean +- SD). Here, p < 0.05 represents a significant difference between groups. 3. Results 3.1. Growth Performance The effects of dietary a-LA supplementation on the growth performance, morphology, and feed utilization of juvenile hybrid groupers are shown in Table 3. The WGR was significantly lower in the L1 and L2 groups than in SL0 and L3, and the FCR was significantly higher in the L2 group than in the other groups (p < 0.05). There was no significant difference in other indicators. 3.2. Serum Biochemical Indexes The effects of dietary a-LA supplementation on the serum biochemical indexes of juvenile hybrid groupers are shown in Table 4. The TG level of L3 was significantly lower than that of SL0 and L1, and the TCHO level of L3 was significantly lower than that of SL0 (p < 0.05). The TP content of SL0 was significantly lower than the other three groups (p < 0.05). The ALB level of L3 was significantly higher than that of SL0 and L1 (p < 0.05). The LDL-C level of L2 was significantly higher than the other three groups, and the LDL-C level of L3 was also significantly higher than SL0 and L1 (p < 0.05). The AST level of L3 was significantly lower than the other three groups, while the ALT level of SL0 was significantly higher than the other three groups (p < 0.05). 3.3. Hepatic Morphology The effect of dietary a-LA supplementation on the hepatic morphology of juvenile hybrid groupers is shown in Figure 1. It was observed that the hepatic cells of the SL0 (control) group showed serious cell vacuolation, swelling, disordered arrangement, and nuclear migration. Compared with SL0, L1, L2, and L3 liver cells were slightly vacuolated, the phenomenon of nuclear migration was reduced, and cell morphology was more regular. 3.4. Antioxidant Capacity of Liver The effect of dietary a-LA supplementation on the hepatic antioxidant capacity of juvenile hybrid groupers is shown in Table 5. The GSH-Px activity of L3 was significantly higher than that of the other three groups, and the GSH-Px activity of L1 and L2 was also significantly higher than that of SL0 (p < 0.05). The activity of SOD in L2 and L3 was significantly higher than that in SL0 and L1 (p < 0.05). There was no significant difference in CAT activity and MDA content (p > 0.05). 3.5. Liver Transcriptome 3.5.1. Transcriptome Sequencing Results The transcriptome data are shown in Table 6. A total of 37760784900 bp of RawData was obtained. After data quality control and filtering low-quality data, a total of 37,229,629,218 bp of CleanData was obtained. Base quality and composition analysis showed that the GC content range in each liver tissue sample was 49.58-50.10%, the percentage of Q20 bases was higher than 98.13%, and the percentage of Q30 bases was higher than 94.62%. 3.5.2. DEGs Analysis A total of 42 DEGs were identified using DEseq2 software under FDR < 0.05 and |log2(Fold Change)| > 1. Compared with the SL0, 31 DEGs were up-regulated and 11 DEGs were down-regulated in L3 liver tissue . A part of the DEGs is shown in Table 7. 3.5.3. GO Function Analysis of DEGs GO functional enrichment analysis was performed on DEGs. Based on sequence homology, all DEGs were classified into the following three major branches of GO: molecular function, biological process, and cellular component, including 40 functional subcategories, involving 7 molecular functions, 12 cellular components, and 21 biological processes . Among them, the biological process is mainly composed of the single organism process, metabolic process, and cellular process. Cellular components were mainly the membrane, organelle, cell part, and cell. The main molecular functions were molecular function regulation, catalytic activity, and binding. 3.5.4. KEGG Pathway Enrichment Analysis of DEGs In the KEGG pathway database, biological metabolic pathways are divided into six categories, namely human diseases, organismal systems, cellular processes, environmental information processing, genetic information processing, and metabolism. In this experiment, a total of 17 DEGs were annotated into these six categories. DEGs were mostly enriched in the two KEGG main classes of biological systems and human diseases; they were also enriched in the overall and overview maps, signal transduction, endocrine system, immune system, cardiovascular disease, and infectious disease KEGG subclasses . When performing KEGG pathway enrichment analysis on DEGs, the top 20 pathways with the smallest p-value were used to make KEGG enrichment bubble maps, and the results were shown in Figure 5. DEGs are significantly enriched in the JAK/STAT signaling pathway, glycolysis/gluconeogenesis, amino acid biosynthesis pathways, cytokine-cytokine receptor interaction, lysosomes, and so on. However, each KEGG significantly enriched pathway contained no more than three DEGs. 3.5.5. Validation of RNA Sequencing Data To verify the accuracy of RNA-Seq results, five DEGs (two down-regulated and three up-regulated genes) were randomly selected, and their expression levels were detected by RT-qPCR. The results are shown in Figure 6. The results of the gene expression obtained were consistent with the trend of the results obtained from RNA-Seq, indicating that the RNA-Seq data had a certain feasibility. 4. Discussion a-LA is a multifunctional antioxidant that can promote growth performance as a feed additive for poultry animals . However, a-LA could also inhibit AMPKa in the hypothalamuses of chickens (Gallus; Linnaeus, 1758) to reduce food intake , and could activate AMPKa in the liver to inhibit the synthesis of glycogen synthase in the liver, resulting in a decrease in glycogen synthesis, thereby changing energy homeostasis and delaying the growth of chicken weight . Therefore, the growth-promoting effect of a-LA needs to be analyzed specifically in combination with the amount of a-LA added. In the study of a-LA as a diet supplement for aquatic animals, more studies have shown that with the increase in a-LA dose, the growth performance of aquatic animals exhibited a trend of increasing first and then decreasing, and high doses of a-LA were still able to improve the growth performance, such as in catfish , giant gourami , northern snakehead , and Chinese mitten crab . However, some studies have suggested that high doses of a-LA had an inhibitory effect on the growth of aquatic animals, such as in Nile tilapia and oriental river prawn (Macrobrachium nipponense; de Haan, 1849) . The recommended addition amounts were 439-528 mg/kg and 1354.8 mg/kg, respectively, but their growth performance was inhibited at 2400 mg/kg and 4000 mg/kg, respectively. In the present study, dietary supplementation with low doses of a-LA (0.4 and 0.6 g/kg) significantly reduced the WGR of juvenile hybrid groupers. Similar to the experimental results of Zhang et al. , the addition of lower a-LA to the diet reduced the WGR of abalone (Haliotis discus hannai; Ino, 1952), which may be the result of a-LA increasing energy consumption in juvenile hybrid groupers . However, the addition of 1.2 g/kg a-LA had no significant effect on the WGR of juvenile hybrid groupers, which may be the result of a high dose of a-LA promoting lipolysis to consume energy by activating the AMPKa-ATGL pathway without causing weight loss . In addition, Ding et al. found that a-LA in diets could reduce the growth rate of oriental river prawn fed with low carbohydrate diet but had no significant effect on the growth rate of oriental river prawn fed with high carbohydrate diet. This indicates that the composition of the diet may affect the mechanism of a-LA. Huang et al. discovered that dietary supplementation of 1.2 g/kg a-LA could inhibit the growth performance of grass carp. In this experiment, dietary supplementation of 1.2 g/kg a-LA had no significant effect on the WGR of juvenile hybrid groupers, indicating that different species had different sensitivities to a-LA. At present, the optimal a-LA addition amount for juvenile hybrid groupers with WGR as a reference still needs further study, and 1.2 g/kg a-LA has a certain reference value. Serum biochemical indexes can reflect the overall health status, physiological stress response, and nutritional status of fish . TG and TCHO are the main components of blood lipids . The contents of TG and TCHO in serum are important indicators to measure lipid metabolism in fish . Studies have shown that a-LA has the effect of lowering blood lipids and could reduce the content of TG and TCHO in mice and rats . Samuki et al. reported that dietary supplementation of 0.3, 0.6, and 0.9 g/kg a-LA reduced the content of TG in the serum of giant gourami; similarly, Siagian et al. also reported that 1.0 and 1.5 g/kg a-LA reduced the content of TG in the serum of African catfish. In this experiment, a-LA not only reduced the content of TG in L3 serum but also reduced the content of TCHO. Butler et al. suggested that a-LA could reduce the TG content in blood and the liver by inhibiting the expression of liver lipogenic genes, reducing hepatic TG secretion, and stimulating the clearance of TG-rich lipoproteins. Zulkhairi et al. believed that a-LA may reduce the TCHO content in the blood by cholesterol metabolism or lipoprotein lipase activity in the liver. TP and ALB are important indicators of protein synthesis and metabolism and immune function . Shi et al. discovered that a-LA regulates the AMPKa-CPT-1a pathway to reduce protein consumption in grass carp to increase protein deposition. In addition, Liu et al. found that a-LA could enhance the immune function of the grass carp skin, spleen, and head kidney. In this experiment, the contents of TP and ALB in the serum of L3 were significantly increased, but the growth performance of L3 did not change significantly. Therefore, the increase in TP and ALB may be the result of a-LA enhancing the immune function of juvenile hybrid groupers. ALT and AST are low in serum and are mainly distributed in liver cells. When liver cells are damaged, they can release ALT and AST to increase their activity in serum, which is consistent with the extent of hepatic cell damage . In this experiment, the levels of ALT and AST in the serum of L3 decreased significantly, which was similar to the experimental results of adding a-LA in grass carp and African catfish . This indicated that the degree of hepatic damage of L3 was lower than that of SL0, i.e., dietary supplementation of a-LA could improve the damage of liver cells in juvenile hybrid groupers. At the same time, by observing the morphology of hepatic tissue cells, it was found that compared with SL0, the morphology of hepatic tissue cells of juvenile hybrid groupers fed with a-LA was improved to varying degrees. This further confirmed that dietary supplementation of a-LA can improve hepatic cell damage. The antioxidant system can protect fish from oxidative stress and is essential for fish health . Antioxidant enzymes (GSH-Px, CAT, and SOD) can scavenge free radicals to reduce oxidative stress, and their activities can reflect the health status of aquatic animals . GSH-Px can remove hydrogen peroxide and lipid peroxide in the body . SOD is a common antioxidant enzyme in the body and can remove superoxide anions . a-LA is considered to be an "ideal antioxidant" or "general antioxidant", which can reduce oxidative damage and enhance the antioxidant defense systems of fish by scavenging excessive ROS and regenerating other antioxidants . In this experiment, the activity of GSH-Px in the livers of juvenile hybrid groupers fed with a-LA was increased to varying degrees, and the activity of SOD in L2 and L3 was significantly increased. At present, many studies have reported similar results, and a-LA could improve the antioxidant capacity of aquatic animals. Xu et al. found that 0.3 g/kg a-LA significantly increased the activities of SOD and GSH-Px in the liver of Nile tilapia. Li et al. discovered that 600, 900, and 1200 mg/kg a-LA significantly increased the activities of SOD and GSH-Px in the liver, head kidney, and spleen of northern snakehead. Zhang et al. found that 800 mg/kg a-LA significantly increased the activities of SOD and GSH-Px in abalone. In summary, the results of this experiment showed that an appropriate amount of a-LA could increase the activity of antioxidant enzymes in the livers of juvenile hybrid groupers, thereby enhancing antioxidant capacity. The transcriptome includes RNA transcripts expressed in a specific cell or tissue types under environmental conditions or specific developmental conditions . In recent years, transcriptome analysis has been widely used in aquaculture, and can be used for effective identification and expression analysis of candidate genes, such as growth, development, reproduction, disease, immunity, stress, and toxicology genes . In this experiment, according to serum biochemical indicators and liver antioxidant capacity, liver samples of SL0 and L3 were selected for transcriptome sequencing analysis. Functional analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that a total of 10,810 unigenes were annotated into 48 KEGG pathways, of which 12 pathways were significantly enriched, including the JAK/STAT signaling pathway, prolactin signaling pathway, antigen processing and presentation, glycolysis/gluconeogenesis, and so on. The JAK/STAT signaling pathway is a common pathway for signal transduction of many cytokines, which is closely related to apoptosis, cell proliferation, inflammatory response, and differentiation. It is very important for coordinating adaptive immune mechanisms, initiating innate immunity, and inhibiting inflammatory responses . In this experiment, the JAK/STAT signaling pathway included three genes: ifnk, prl4a1, and prl3b1. In addition, the prolactin signaling pathway also includes prl4a1 and prl3b1 genes. The ifnk gene is a new type I interferon subclass . Interferons are proteins that are crucial to the human immune system. They are formed in various cells of fish, mammals, reptiles, and amphibians. IFN type I and IFN type II are found in ray-finned fish (Actinopterygii), and IFN type III is also found in phylogenetically older cartilaginous fishes. IFN type I in ray-finned fish (Actinopterygii) can activate the JAK/STAT signaling pathway and induce the expression of IFN-stimulated genes containing IFN-stimulated response elements complexes and, thus, possessing antiviral activity. In addition, in Perciformes, IFN I has been shown to exert antibacterial effects through macrophage phagocytosis . The grouper belongs to Osteichthyes, Actinopterygii, and Perciformes. Both prl4a1 and prl3b1 are members of the prolactin family, and prolactin is a multifunctional polypeptide hormone with immunomodulatory and protective effects . Studies have shown that prolactin can induce the expression of genes encoding major phagocytic NADPH oxidase components and ROS production in fish macrophages through the JAK2/Stat/IRF-1 signaling pathway . Antigen processing and presentation is the mechanism by which the entire antigen is degraded and loaded onto MHC molecules (class I and II) to display on the cell surface of T cells . Zhang and Chen found that a novel CC chemokine may be involved in the adaptive immune response by regulating MHC class I antigen processing and presentation in large yellow croaker (Pseudosciaena crocea; Richardson, 1846). In this experiment, only the expression of the ctsl gene was significantly up-regulated in antigen processing and presentation. Cathepsin L (ctsl) is a member of the papain family of cysteine proteases which plays an important role in the biological activities of fish, including antigen processing , antigen presentation , protein degradation , and anti-microbial invasion . Recently, the key role of ctsl in the innate immune system of many fish species has been further revealed . In summary, combined with the significant increase in TP and ALB in the serum of L3 and the significant up-regulation in ifnk, prl4a1, prl3b1, and ctsl in the JAK/STAT signaling pathway, prolactin signaling pathway, and antigen processing and presentation in liver, it is speculated that dietary supplementation of a-LA can enhance the immune function of juvenile hybrid groupers by regulating the JAK/STAT signaling pathway, prolactin signaling pathway, and antigen processing and presentation. Glycolysis/gluconeogenesis is an opposing metabolic pathway involved in carbohydrate degradation and synthesis and plays an important role in maintaining glucose homeostasis . In this experiment, the glycolysis/gluconeogenesis pathway included two genes, gapdh and eno1. Glyceraldehyde-3-phosphate dehydrogenase (gapdh) plays a key role in the glycolytic pathway. It can catalyze the formation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, which produces NADH. NADH can synthesize ATP through the electron transport chain in mitochondria . a-enolase (eno1) plays a functional role in glycolysis/gluconeogenesis. It can catalyze the conversion of 2-phosphate-D-glycerate to phosphoenolpyruvic acid during glycolysis and phosphoenolpyruvic acid to 2-phosphate-D-glycerate during glycogen synthesis . Huang et al. showed that the addition of a-LA to the diet could enhance the expression of glycolysis, gluconeogenesis, and glucose transport-related genes in zebrafish (Danio rerio; Hamilton, 1822) livers. In this experiment, the expression of gapdh gene was significantly down-regulated, and the expression of eno1 gene was significantly up-regulated. Therefore, it is speculated that a-LA can maintain the glucose homeostasis of juvenile hybrid groupers by regulating the expression of gapdh and eno1 genes in the glycolysis/gluconeogenesis pathway. Therefore, the optimal addition of a-LA in the diet of hybrid groupers needs further study. However, this experiment showed that without affecting the growth of hybrid groupers, it could reduce the blood lipid level of hybrid groupers, improve the damage of liver cells, and increase the activity of antioxidant enzymes in the liver. This indicates that an appropriate amount of a-LA can be used as an additive to improve fish health in actual production. In addition, the transcriptome results provide some theoretical knowledge for the further study of a-LA in immune and glucose homeostasis. 5. Conclusions In summary, in this experiment, 0.4 and 0.6 g/kg a-LA inhibited the growth performance of juvenile hybrid groupers. Although 1.2 g/kg a-LA had no significant effect on the growth performance, it could reduce the blood lipid level of juvenile hybrid groupers, improve hepatocyte damage, and increase the antioxidant enzyme activity of the liver. Transcriptome analysis showed that dietary a-LA supplementation significantly affected the pathway related to immune function (JAK/STAT signaling pathway, prolactin signaling pathway, and antigen processing and presentation), and significantly up-regulated the expression of genes related to immunity (ifnk, prl4a1, prl3b1, and ctsl). In addition, a-LA also changed the pathway related to glucose homeostasis (glycolysis/gluconeogenesis), significantly down-regulated the expression of the gapdh gene, and up-regulated the expression of the eno1 gene. Acknowledgments We acknowledge National Key R&D Program of China (2020YFD0900200) and National Key R&D Program Blue Granary Science and Technology Innovation Key Special Project (2020YFD0900204), and Chen Gang and other teachers in the laboratory for their patience and support during this research work. Author Contributions G.O. was responsible for designing the experiments, breeding experiments, data processing, and writing articles; R.X. provided experimental guidance; J.H. (Jianpeng Huang), Z.W., Y.L. and X.J. helped with sample collection and handling of the experimental samples. Q.M. and J.H. (Jiansheng Huang) revised the original articles and reviewed them. G.C. revised the experiment design, reviewed the article, and provided funding. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study was reviewed and approved by the Guangdong Ocean University Research Council for the care and use of laboratory animals on 25 November 2021 (approval number: GDOU-LAE-2021-021). Informed Consent Statement Not applicable. Data Availability Statement Upon reasonable request, the data supporting this study are accessible from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Effect of diet supplementation with a-LA on the hepatic histomorphology of juvenile hybrid groupers. The labels in the upper right corner represent the different experimental diets. (A) SL0; (B) L1; (C) L2; (D) L3. The red arrow represents cell vacuolation, the blue arrow represents cell nuclear migration, and the green arrow represents cell swelling. Scale bars = 50 mm; 400x magnification. Figure 2 Volcano map of DEGs (SL0 vs L3). Red dots represent up-regulated genes, blue dots represent down-regulated genes, and grey dots represent genes with no significantly difference. Figure 3 GO enrichment analysis of DEGs in the livers of juvenile hybrid groupers (SL0 vs L3). The abscissa is the enriched gene ontology (GO) term, and the ordinate is the number of differentially expressed genes in the term. The red columns represent up-regulated DEGs, and the green columns represent down-regulated DEGs. Figure 4 KEGG pathway enrichment analysis of DEGs in the livers of juvenile hybrid groupers (SL0 vs L3). The ordinate is the enriched KEGG subclass, and the abscissa is the number of genes enriched in this subclass. The number on each KEGG subclass item represents the number of differentially expressed genes on that item. Different colors represent different categories. Figure 5 Top 20 KEGG pathways with significant enrichment of DEGs in the livers of juvenile hybrid groupers (SL0 vs L3). Ordinate represents the name of the pathway. The abscissa represents the enrichment factor, and the circle's color represents Q. The deeper the red color, the more reliable the significant enrichment, and the larger the circle, the greater the number of enriched genes. Figure 6 RT-qPCR verification of DEGs in the livers of juvenile hybrid groupers (SL0 vs L3). animals-13-00887-t001_Table 1 Table 1 Composition and nutritional levels of the experimental diets (g/kg). Ingredients Diet Groups SL0 L1 L2 L3 Fish meal 420 420 420 420 Shrimp med 50 50 50 50 Spray-dried blood meal 20 20 20 20 Soybean meal 160 160 160 160 Wheat gluten 50 50 50 50 Cottonseed meal 50 50 50 50 Wheat flour 150 149.6 149.4 148.8 Soybean lecithin 30 30 30 30 Fish oil 40 40 40 40 Alpha lipoic acid 0.0 0.4 0.6 1.2 Calcium monophosphate 10 10 10 10 Vitamin and mineral premix 1 20 20 20 20 Total 1000.0 1000.0 1000.0 1000.0 Nutrient levels Moisture 9.2 9.5 10.0 9.1 Crude protein 49.6 49.3 49.4 49.7 Crude lipid 10.4 10.3 10.3 10.3 Ash 11.0 10.9 10.8 11.0 1 Vitamin and mineral premix (g/kg of mixture): vitamin A, 5.0 g; vitamin B1, 10.0 g; vitamin B2, 10.25 g; vitamin B6, 22.5 g; vitamin B12, 0.05 g; vitamin D, 20.0 g; vitamin E, 45.0 g; vitamin K3, 2.275 g; Ca(H2PO4)2*H2O, 25.0 g; CoCl2*6H2O, 2.175 g; CuSO4*5H2O, 9.425 g; KIO3, 0.015 g; KCl, 8.87 g; Na2SeO3, 1.0 g; MgSO4*H2O, 6.095 g; MnSO4*7H2O, 0.055 g; ZnSO4*7H2O, 14.56 g; biotin, 1.0 g; cellulose, 239.485 g; D-calcium pantothenate, 30.335 g; folic acid, 3.085 g; ferric citrate, 7.175 g; inositol, 70.02 g; nicotinic acid, 41.0 g; zeolite powder, 425.63 g. animals-13-00887-t002_Table 2 Table 2 Primer sequences for RT-qPCR verification. Gene Name Primer Sequence Accession Number Product Length b-actin F: GCAGGAGTACGATGAGTCCG KU200949.2 102 R: GCTGAAGTTGTTGGGCGTTT i-1 F: TCCCCAACCATAACGCTGAAC Unigene0003248 115 R: CAGACCACTGTTTTCTCATCTCCGT eno1 F: CATCAACGGCGGCTCACAT Unigene0018779 185 R: CGAAACCTCCCTCGTCTCCTACA txnip F: CGGCAGGTCCTTCTACAGCA Unigene0047552 146 R: GGCTCTTGAGTTTCTTCCCACC pvalb F: TGGTTTCATTGAGGAGGAAGAGC Unigene0012447 128 R: CAATCTTGCCATCACCGTCAG dusp1 F: CGTCCGCTTCAGCACCATA Unigene0043927 155 R: TCGCCTGGCTCAAGTCAAAG animals-13-00887-t003_Table 3 Table 3 Effect of diet supplementation with a-LA on the growth performance, morphology, and feed utilization of juvenile hybrid groupers. Parameter Diet Groups SL0 L1 L2 L3 IBW (g) 23.88 +- 0.30 23.98 +- 0.23 24.13 +- 0.06 24.10 +- 0.00 FBW (g) 103.42 +- 6.13 98.87 +- 8.30 95.65 +- 4.38 105.60 +- 4.72 WGR (%) 333.26 +- 30.76 b 312.25 +- 3.37 a 296.33 +- 17.75 a 338.19 +- 19.59 b SR (%) 100.00 +- 0.00 100.00 +- 0.00 100.00 +- 0.00 96.67 +- 2.89 FCR 0.82 +- 0.06 a 0.83 +- 0.05 a 0.88 +- 0.06 b 0.81 +- 0.04 a SGR (%/d) 2.62 +- 0.13 2.53 +- 0.01 2.46 +- 0.08 2.64 +- 0.08 CF (g/cm3) 6.56 +- 0.69 6.52 +- 0.82 7.07 +- 0.46 6.96 +- 0.71 FI (%) 1.59 +- 0.07 1.60 +- 0.11 1.67 +- 0.08 1.58 +- 0.04 VSI (%) 12.33 +- 1.12 13.0 +- 1.41 12.44 +- 2.07 12.11 +- 1.27 HSI (%) 3.30 +- 0.70 3.38 +- 0.79 3.54 +- 0.82 3.46 +- 0.75 Notes: data are expressed as mean +- SD (n = 3), and values with different superscript letters in the same row are statistically significant (p < 0.05). IBW, initial body weight; FBW, final body weight; WGR, weight gain rate; SR, survival rate; FCR, feed conversion ratio; SGR, specific growth rate; CF, condition factor; FI, feed intake; VSI, viscerosomatic index; HIS, hepatosomatic index. animals-13-00887-t004_Table 4 Table 4 Effect of diet supplementation with a-LA on the serum biochemical indexes of juvenile hybrid groupers. Parameter Diet Groups SL0 L1 L2 L3 TG (mmol/L) 1.19 +- 0.22 b 1.05 +- 0.11 b 1.01 +- 0.05 ab 0.80 +- 0.06 a TCHO (mmol/L) 1.60 +- 0.14 b 1.40 +- 0.07 ab 1.40 +- 0.06 ab 1.21 +- 0.15 a TP (g/L) 19.32 +- 0.58 a 24.42 +- 1.57 b 28.48 +- 2.85 bc 29.44 +- 3.52 c ALB (g/L) 4.45 +- 0.32 ab 4.03 +- 0.51 a 5.26 +- 0.70 bc 5.99 +- 0.35 c LDL-C (mmol/L) 0.34 +- 0.05 a 0.29 +- 0.01 a 0.80 +- 0.02 c 0.42 +- 0.06 b HDL-C (mmol/L) 1.10 +- 0.12 1.25 +- 0.06 1.11 +- 0.18 1.37 +- 0.26 AST (U/L) 41.89 +- 3.77 b 43.78 +- 4.38 b 44.23 +- 2.42 b 33.40 +- 2.12 a ALT (U/L) 581.11 +- 18.34 b 400.42 +- 45.54 a 382.46 +- 30.51 a 390.53 +- 19.07 a Notes: data are expressed as mean +- SD (n = 3), and values with different superscript letters in the same row are statistically significant (p < 0.05). TG, triglyceride; TCHO, total cholesterol; TP, total protein; ALB, albumin; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; AST, aspartate aminotransferase; ALT, alanine aminotransferase. animals-13-00887-t005_Table 5 Table 5 Effect of diet supplementation with a-LA on the antioxidant capacity of the livers of juvenile hybrid groupers. Parameter Diet Groups SL0 L1 L2 L3 GSH-Px (U/mgprot) 312.35 +- 12.26 a 346.87 +- 6.48 b 350.37 +- 6.88 b 371.72 +- 7.18 c CAT (U/mgprot) 10.41 +- 0.36 11.28 +- 0.78 12.59 +- 1.40 10.47 +- 0.42 SOD (U/mgprot) 51.64 +- 1.61 a 44.02 +- 3.86 a 62.01 +- 4.33 b 61.03 +- 5.33 b MDA (nmol/mgprot) 1.60 +- 0.43 1.24 +- 0.15 1.30 +- 0.06 1.22 +- 0.03 Notes: data are expressed as mean +- SD (n = 3), and values with different superscript letters in the same row are statistically significant (p < 0.05). GSH-Px, glutathione peroxidase; CAT, catalase; SOD, superoxide dismutase; MDA, malondialdehyde. animals-13-00887-t006_Table 6 Table 6 Information of the transcriptomic read of each sample. Sample RawData (bp) CleanData (bp) Q20 (%) Q30 (%) GC (%) SL0-1 7,981,551,300 7,908,021,279 98.09% 94.62% 49.83% SL0-2 5,738,949,600 5,643,980,098 98.15% 94.73% 50.02% SL0-3 5,812,150,800 5,713,500,475 98.19% 94.82% 49.65% L3-1 6,519,141,900 6,430,009,609 98.13% 94.69% 50.10% L3-2 5,679,499,500 5,590,980,794 98.22% 94.86% 50.08% L3-3 6,029,491,800 5,943,136,963 98.13% 94.66% 49.58% animals-13-00887-t007_Table 7 Table 7 Part of the DEGs of SL0 vs L3. Genes ID Genes Description log2(FC) p-Value FDR Unigene0038423 ifnk Interferon kappa precursor +10.632 0.000 0.039 Unigene0049822 prl3b1 Prolactin-3B1 precursor +12.004 0.000 0.000 Unigene0036965 prl4a1 Prolactin-4A1 precursor +11.728 0.000 0.000 Unigene0046207 ctsl Cathepsin R precursor +7.349 0.000 0.000 Unigene0018198 gapdh Glyceraldehyde-3-phosphate dehydrogenase -6.806 0.000 0.000 Unigene0018779 eno1 enolase +1.640 0.000 0.010 Unigene0003248 i-1 Beta-galactoside-binding lectin-like isoform X1 +2.216 0.000 0.001 Unigene0012447 pvalb Parvalbumin beta-like -7.204 0.000 0.000 Unigene0043927 dusp1 MAP kinase phosphatase 1 -1.880 0.000 0.015 Unigene0047552 txnip Thioredoxin-interacting protein-like +1.577 0.000 0.034 Notes: "+" and "-" represent up-regulation and down-regulation of the expression of DEDs, respectively. 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PMC10000057

The feeding habits of organisms are important elements in their ecological role and are affected by several factors. The present study provides for the first time information on the diet and feeding strategy of Dentex maroccanus (Valenciennes, 1830) and examines the effects of various factors on the species' feeding activity. Various indices (vacuity index, numerical and weight proportion, frequency of occurrence, alimentary coefficient, index of relative importance, diet breadth and overlap, Shannon-Wiener index, and trophic level) were estimated. The diet of the species consisted of 18 different prey taxa. The most important prey taxon was Decapoda. The study of the feeding strategy showed the narrow width of the species. Body size was found to significantly affect the species' feeding habits. Polychaeta and Stomatopoda were found only in individuals with size >=165 mm, Bivalvia mainly in sizes <=120 mm, and Decapoda in the intermediate sizes. The largest individuals showed the lowest overlap with all other size groups. The trophic level increased from 3.7 in young individuals to 4.0 in larger sizes, indicating the carnivorous character of the species. The results of the present work contribute to a better knowledge of the species' feeding ecology. Dentex maroccanus diet ecology feeding strategy size effect trophic level Aegean Sea Mediterranean Sea This research received no external funding. pmc1. Introduction Stomach content analysis constitutes an essential component for understanding, exploring, and contrasting trophic relationships between organisms, as well as population and community dynamics . These studies are essential and fundamental in Marine Ecology and Fisheries Dynamics, providing useful information for fisheries management and ecosystem status assessment . Several factors may affect the diet of the species, such as season, sex, and body size . The Morocco dentex, Dentex maroccanus Valenciennes, 1830, is a ray-finned fish species of the family Sparidae. It is distributed in the Eastern Atlantic, from the Bay of Biscay to the Gulf of Guinea, sometimes even further north, and in the Southern and Eastern Mediterranean; it is not recorded in the Adriatic and Black Sea . It is a demersal species and may be found in a range of bottom types, mostly gravels or conglomerates . It is mainly an inshore species, found in the Mediterranean at depths of 20 to 250 m with the highest abundance between 50-70 m in depth . However, recent studies have revealed that the species' bathymetric distribution extends to 316 m in depth . The distribution of the species was found to be related to environmental factors such as depth, temperature, and season . It is a by-catch species of low economic value mostly fished by trawl and artisanal fisheries . Studies on various biological aspects of D. maroccanus such as age, growth, length-weight relationship, reproduction, fecundity, and morphometry have been conducted in the Mediterranean to date . Only one study on the diet of the species in the Atlantic Ocean has been published . Two relevant studies in the Mediterranean are also known , while no information is available for the species in the Greek waters. The objective of the present study is to investigate D. maroccanus' diet and feeding strategy, and the effect of various factors (body size, season, sex) on its feeding activity. Diet overlap, breadth, and trophic level were also studied. The case study was conducted in the South Aegean Sea. The aim was to provide new information on the species' diet in the study area and improve existing knowledge of the species' feeding ecology. 2. Materials and Methods Sampling was carried out with a commercial bottom trawl in the South Aegean Sea (E. Mediterranean) in September 2014 and May 2015, during the daytime and within the framework of the national project EPILEXIS. The sampling scheme included 84 hauls in a wider geographical area; however, a few specimens of the species occurred in most of them. Therefore, the samples for this study were taken from the hauls where the species was more abundant. A total of 416 specimens were collected at depths between 68 and 255 m. Fish were frozen promptly after capture and dissected in the laboratory, where the total weight (TW) (to the closest 0.001 g), the total length (TL) to the closest mm, the sex, and the maturity stage were recorded. TL ranged from 92 to 233 mm. Stomachs were extracted and sectioned, and their content was weighted. A stereomicroscope was used to examine the content of the stomachs in order to identify prey items, which were counted and weighted. The intestinal contents were also extracted and examined qualitatively, but they were not taken into account in further analyses. Prey items were identified to the lowest feasible taxonomic level, depending on their digestion condition. However, most of the prey items in the stomachs were particles of the entire organism (e.g., otoliths, eyes or flesh of Cephalopoda, Decapoda appendages, parts of Gastropoda, and Bivalvia shells) or digested to the point that did not permit the identification to the species or genus level. When the organism was intact or a specific characteristic of an organism was found, the identification of the species level was successful. The vacuity index (VI) was estimated as the proportion of empty stomachs. Stomach fullness was determined using two methods: (i) the repletion index (%RI) = stomach content weight x 100/body weight and (ii) a fullness empirical scale (with 0 representing empty stomach and 5 representing full stomach). The study of the diet composition of the species was based on the following indices proposed by Hyslop : numerical proportion (%N), frequency of occurrence (%F), and weight proportion (%W). Two additional indices were used to determine the importance of each prey in the diet of D. maroccanus: (i) the alimentary coefficient Q (Q = %F x %W) ; prey was divided into three groups based on the Q coefficient : principal for Q > 200, secondary for 20 < Q < 200, and accidental for Q < 20 and (ii) the index of relative importance (IRI) of Pinkas et al. , modified by Hacunda : IRI = (%N + %W) x %F. This index was calculated as %IRI = (IRI/S IRI) x 100, where S IRI was the sum of all IRI values. The feeding strategy was determined using the method of Amundsen et al. , which plots prey-specifying abundance (pi) against the frequency of occurrence (%F). Expressed as a percentage, prey-specific abundance is a given prey taxon proportion in relation to all prey items observed in only those predators' stomachs that contained the given prey taxon: pi = 100 x Si x Sti-1, where Si is the sum of the stomachs comprising prey i, Sti is the sum of all prey items found in only those predator stomachs that contained prey i. In order to identify the effects of various factors on the feeding habits of D. maroccanus, permutational multivariate analysis of variance (PERMANOVA) was performed based on the Bray-Curtis similarity resemblance matrix for the prey abundance (N) using three factors: sex (male/female), season (summer/autumn), body size (juvenile/adult), and their interactions. The discrimination of the size groups was based on Mohdeb and Kara findings that estimated the species' first maturity at 144 mm TL. Distance-based redundancy analysis (db-RDA) was also used to demonstrate the resemblance of the prey items in the different identified groups. Based on the results db-RDA, the largest individuals showed differentiation in their diet compared to the other individuals. Therefore, ontogenetic changes in the diet of D. maroccanus were further examined for the following 4 size groups (<=120 mm, 121-142 mm, 143-164 mm, >=165 mm). Thus, 2 groups of juveniles and 2 groups of adults were created. In our samples, there were many prey taxa with zero values or values equal to 1. For this reason, the mean abundance and biomass of each prey taxon were considered for each one of the above-mentioned size groups. Based on these data, a cluster analysis was performed based on the Bray-Curtis similarity resemblance matrix of the average prey taxon abundance. Shade plots were produced to show the relationship between the cluster of size groups and prey taxa abundance. SIMPER analysis was used to identify the contribution of each prey taxon in differentiating the four examined size groups. The diversity in prey items of the four size groups was also examined using the Shannon-Wiener index (H'). All the above-mentioned analyses were implemented using the Primer v7 software package . Diet overlap between the defined size groups of D. maroccanus was determined using Schoener's formula: Cxy = 1 - 0.5 ( |pxi - pyi|). Cxy is the dietary overlap, pxi is the proportion of prey taxon i (i terms of the relative abundance N) in the diet of group x, and pyi is the proportion of prey taxon i in the diet of group y. The values of this index range from 0 to 1, with 1 indicating complete overlap and 0 indicating no overlap. A value of index >=0.6 was considered to indicate high overlap. Diet breadth B for the size group i was calculated in this work for D. maroccanus using Levins index : B = (n - 1)-1 [(j p2ij)-1] - 1, where pij is the proportion of the diet of size group i on prey j and n is the number of prey taxa. The index values vary from 0 (the species consumes a simple item) to 1 (the species consumes different prey in equal proportion). Values of B >= 0.6 are considered high, between 0.4-0.6 moderate and below 0.4 low . The trophic level (TrL), which expresses the position of organisms within the food webs, was calculated for each size group according to Pauly and Christensen formula:TrLi=1+j=1G (DCijxTrLj) where TrLj is the fractional trophic level of prey j, DCij represents the proportion (%W) of prey j in the diet of specimen i, and G is the total number of prey species. Trophic levels of most fishes take values between 2 and 5 . The trophic level values of each specimen were calculated using TrophLab routine . 3. Results Out of 416 D. maroccanus examined, 349 stomachs were empty (VI = 83.89%). Only 67 stomachs contained food remains. The size of the latter individuals ranged from 99 to 186 mm TL. There was no indication of regurgitation. The fullness index of non-empty stomachs based on RI was 0.74% (+-0.1). From the stomachs with food, the empirical fullness scale of the stomachs showed a clear dominance of categories 1 and 2 . Stomach content analysis of the 67 individuals revealed a total of 125 prey items, from 18 taxa (Table 1), weighing a total of 22.98 g. The examination of the intestinal content showed the following additional prey taxa: Algae, Foraminifera, Nematode, Echinodermata (Echinoidea), Gastropoda (Calliostomatidae, Cerithiidae, Drilliidae, Homalopoma sanguineum, Trochidae), Bivalvia (Myidae), and Crustacea (Amphipoda, Galatheidae, Nephrops norvegicus, Thalassinidae). Microplastics were also found in the intestine of some individuals. The quantitative diet analysis of stomachs revealed that Decapoda unidentified was the most abundant prey identified in the stomachs of D. maroccanus (%N = 34.4), followed by Gastropoda (%N = 20.0). The heaviest prey taxon was also Decapoda unidentified (%W = 35.0), followed by Cephalopoda (%W = 18.9). In terms of frequency, Decapoda unidentified predominated followed by Gastropoda. The alimentary coefficient Q index showed that the primary prey in the diet of D. maroccanus was Decapoda unidentified, while benthic Decapoda, Gastropoda, Cephalopoda, Dendrobranchiata, Teleostei, and Bivalvia were identified as secondary prey. All the other taxa were classified as accidental prey. Similar results were also derived by the analysis of %IRI (Table 1). The feeding strategy showed that most prey taxa were located on the left side of the diagram with low prey-specific abundances (<20%) and relatively low frequency of occurrence (<40%), indicating that most prey taxa were rare in the diet of the species. Benthic Decapoda, Decapoda unidentified, and Gastropoda, which were more frequent, were located below the prey importance axis (50%), showing a kind of generalized feeding behavior towards these prey taxa. PERMANOVA analysis showed that among the examined factors of body size, sex, and season, the only statistically significant factor affecting the D. maroccanus diet was body size (Table 2). The results of db-RDA analysis, although not very clear for the juveniles and adults of D. maroccanus, showed that the largest of the individuals of the size group of adults determined by Teleostei, Rissoides desmaresti, and Polychaeta were grouped separately than the other examined individuals . Due to the results of the db-RDA analysis, the following analyses were performed for four size groups: group 1--smaller juveniles (<=120 mm), group 2--larger juveniles (121-142 mm), group 3--smaller adults (143-164 mm), and group 4--larger adults (>=165 mm). The results of the cluster analysis for the similarity of the mean abundance of prey items in the four size groups are presented in Figure 5. It is obvious that size group 4 is clearly distinguished from all other size groups, while size group 1 is more discrete than the two intermediate-sized groups (2 and 3). The shade plot for the combined information on the mean abundance of the prey taxa and the four cluster size groups shows their feeding preferences in Figure 6. Polychaeta and R. desmaresti are exclusively presented in the diet of size group 4. Size group 1 fed primarily on Bivalvia, followed by Decapoda unidentified. Size groups 2 and 3, presenting the highest similarity, showed a more mixed diet (group 2: mainly Gastropoda, Decapoda unidentified, benthic Decapoda, and Bivalvia; group 3: mainly Decapoda unidentified, Gastropoda, and Bivalvia). SIMPER analysis showed that size group 4 presented the highest values of dissimilarity (48.0-56.8%) with all other groups (Table 3). The lowest dissimilarity (16.6%) was found between size groups 2 and 3. The prey taxon with the highest contribution in the dissimilarity between the size groups was Bivalvia. Bivalvia and Dendrobranchiata contributed equally to the dissimilarity between groups 2 and 4, while Teleostei was between the intermediate size groups. The diet overlap indicated the lowest values for size group 4. High overlap was defined between the two intermediate-size groups (2 and 3) and between the size groups 1 and 3 (Table 3). The mean value of the diversity index H' was relatively low for all size groups (Table 4) with lower values for size groups 1 and 4. No statistically significant differences were defined between the four groups (ANOVA, F-Ratio = 1.66, Df = 66, p-value = 0.18). Diet breadth was increased from group 1 to 4 (Table 4) indicating that the diet of the smallest individuals was characterized by the high dominance of one prey taxon, while the largest individuals fed equally on different prey taxa. The trophic level also increased from the smaller-sized to the larger-sized groups (Table 4). 4. Discussion Knowledge of the feeding biology and ontogenetic changes of a species is important in population and community dynamics, which in turn is crucial for evaluating the status of the ecosystem where the species is encountered . The present study investigated for the first time the diet of D. maroccanus in the South Aegean Sea. Additionally, the effect of body size on the feeding activity of this species was studied, an issue that has presented limited published information from other areas to date . Although an important number of samples were examined in the current work, the number of specimens with empty stomachs was very high, a fact that may produce limits in our analyses. Indeed, although our samples included specimens of up to 233 mm TL, the larger specimen with food in the stomach was 186 mm TL. The high number of empty stomachs in the present study may be related to the sampling period, which included summer and early autumn. Bayhan et al. also found a higher percentage of empty stomachs in summer, which is the species' spawning period. It is generally known that fish reduce their feeding intensity during the reproductive period . D. maroccanus feeding habits showed a tendency towards a more generalized feeding on Decapoda and Gastropoda, although a kind of specialization for Polychaeta and the stomatopod R. desmaresti was also observed. However, the majority of prey items were occasionally found in the stomach contents, reflecting the narrow feeding width of the species. This was in accordance with the results of the Shannon-Wiener diversity index, which indicated relatively low values. In the present work, among the three examined factors of season, sex, and body size, only the latter was found to significantly affect the diet of D. maroccanus. Simper analysis and the shade plot showed that small individuals prey mainly on Bivalvia, while the intermediate-sized individuals (<165 mm) fed mainly on various categories of Decapoda, small Gastropoda, and small Bivalvia. The larger preys of R. desmarsti and Polychaeta, which require larger mouth dimensions to prey, were found only in the largest of the adults (>=165 mm). Mohdeb et al. found that adult individuals target mainly Crustacea and Teleostei, as opposed to young individuals for which Crustacea were the preferred prey. However, they did not detect statistically significant differences in the diet of the species for the factors of season, sex, and body size. In contrast, Bayhan et al. found that the species presented a different diet in summer compared to the other seasons. These differences may be related to the different prey availability and frequency in each study area. The results of the diet breadth and shade plot showed that the largest size group fed equally on various prey taxa, while smaller individuals were characterized by the high dominance of a few prey categories. Similarly, Simper analysis and the estimation of the diet overlap confirmed the above-mentioned results by indicating higher dissimilarity and lower overlap, respectively, between the largest size group and the smaller ones. The estimation of the trophic level indicated higher values for the adults (>143 mm) than juveniles. Similar results for the trophic level of D. maroccanus have been mentioned by Mohdeb et al. . The values of the trophic level confirm the carnivorous character of the species with only the larger individuals classified close to the top predators while the smaller ones presented the lowest trophic level values of this category . The results of the present study showed that D. maroccanus is a predator that forages on benthic and demersal organisms and thereby serves as a vector of energy between benthic and pelagic ecosystems. The feeding pattern of this demersal species seems to be related to body size. Therefore, diet studies for this species should take into consideration the effect of this factor. Furthermore, the high vacuity index values have implications for a large number of samples and an extended spatiotemporal sampling design. The outcomes of the present work are useful in further understanding the species' biology and ecology and may support the development of ecosystem-based fisheries management. 5. Conclusions This study has updated our knowledge of D. maroccanus feeding habits and has presented for the first time information on this issue in the context of the South Aegean Sea. In this study area, D. maroccanus showed a narrow-width feeding strategy with a preference for Decapoda. The diversity of the prey was also relatively low. This species' feeding habits are affected by the body size of the individuals. Therefore, large-sized prey such as Polychaeta and Stomatopoda were found only in the diet of the largest individuals, while small-sized prey like Bivalvia was mainly included in the stomach content of the smallest D. maroccanus. The trophic level confirmed the carnivorous character of the species with the larger individuals classified close to the top predators while the smaller presenting the lowest values of this category. Acknowledgments The authors would like to acknowledge Dimitra Stromplou and Georgia Pappou for their help in the laboratory processing of the samples, John Dokos for the map design, the HCMR personnel and the crew of the commercial vessel "Takis-Mimis" for the collection of the data sampled within the frame of the project ELIPEXIS (EPAL 2007-2013, Measure 3.5, Pilotic Planning, Code Action 185365). Author Contributions Conceptualization, A.M., C.M. and A.A.; data curation, A.M., A.K. and A.R.; formal analysis, A.M., C.M., A.K., A.R. and A.A.; methodology, A.S.; resources, C.M. and A.A.; software, A.S.; supervision, C.M. and A.A.; validation, C.M. and A.A.; writing--original draft, A.M.; writing--review & editing, C.M. and A.A. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethical review and approval was not required for the animal study because no ethics committee was available when the present study was performed. In general, the followed procedure in the samples collection and elaboration was the common procedure used in fisheries and diet studies. Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Sampling sites (as black spots) of Dentex maroccanus in the South Aegean Sea. Figure 2 Percentage of the empirical fullness scale categories of non-empty stomachs (fullness scale with increasing order from 1 to 5). Figure 3 Feeding strategy plot of Dentex maroccanus. Prey types (as squares) are: 1. Polychaeta; 2. Rissoides desmaresti; 3. Teleostei; 4. Cephalopoda; 5. Dendrobranchiata; 6. Bivalvia; 7. Benthic Decapoda; 8. Gastropoda; 9. Decapoda unidentified. Figure 4 Distance based Redundancy Analysis (db-RDA plot). Size 1 = juvenile size group, Size 2 = adult size group. Figure 5 Dendrogram of four clusters of body sizes based on the Bray-Curtis distance. Size group 1: smaller juveniles (<=120 mm), group 2: larger juveniles (121-142 mm), group 3: smaller adults (143-164 mm), and group 4: larger adults (>=165 mm). Figure 6 Shade plot for the mean abundance of the prey taxa and the four defined size groups of D. maroccanus. Shading intensity shows the relative presence of each prey in each size group. Size group 1: smaller juveniles (<=120 mm), group 2: larger juveniles (121-142 mm), group 3: smaller adults (143-164 mm), and group 4: larger adults (>=165 mm). animals-13-00939-t001_Table 1 Table 1 Quantitative dietary composition of D. maroccanus. Prey Taxa %N %F %W Q %IRI ANNELIDA Polychaeta 0.8 1.06 0.30 0.24 0.04 MOLLUSCA Gastropoda 20.00 19.15 5.34 106.80 8.18 Gastropoda unident. 12.80 12.77 4.27 54.64 7.69 Chilodontaidae 0.80 1.06 0.21 0.17 0.04 Fasciolariidae 2.40 1.06 0.12 0.29 0.09 Naticidae 1.60 2.13 0.35 0.56 0.15 Pyramidellidae 2.40 2.13 0.39 0.94 0.21 Bivalvia 12.80 11.70 2.82 36.10 2.66 Bivalvia unident. 4.80 5.32 1.35 6.50 1.15 Nuculanidae 0.80 1.06 0.91 0.73 0.06 Pectinidae 7.20 5.32 0.55 3.95 1.45 Cephalopoda 4.80 6.38 18.89 90.67 5.34 ARTHROPODA-Crustacea Decapoda unident. 34.40 28.72 34.98 1203.30 70.32 Benthic Decapoda 15.20 18.09 10.08 153.22 6.54 Brachyura 8.80 10.64 4.13 36.34 4.85 Alpheidae 0.80 1.06 1.37 1.10 0.08 Munididae 0.80 1.06 1.22 0.97 0.08 Paguridae 4.80 5.32 3.36 16.12 1.53 Dendrobranchiata 6.40 7.45 6.65 42.58 3.43 (pelagic decapoda) Stomatopoda Rissoides desmaresti 1.6 2.13 11.70 18.72 1.00 CHORDATA-Teleostei 4.00 5.32 9.23 36.93 2.48 Length range (mm) 99-186 N of individuals 67 %N = relative abundance, %F = frequency of prey occurrence, %W = percentage weight, Q = alimentary coefficient, %IRI = index of relative importance. Length range and number of examined individuals are also presented. animals-13-00939-t002_Table 2 Table 2 Results of PERMANOVA analysis with the factors affecting the abundance of prey items in D. maroccanus diet (statistically significant values in bold). PERMANOVA Abundance p-Value Size 0.012 Sex 0.470 Season 0.146 Sex x Size 0.511 Sex x Season 0.474 Size x Season 0.140 Sex x Size x Season 0.516 animals-13-00939-t003_Table 3 Table 3 SIMPER analysis of the prey taxa contributing 90% to the diet of the four size groups of D. maroccanus. Contrib. % = contribution in abundance of the most important prey for each size group. Diet overlap values are also presented. Size group 1: smaller juveniles (<=120 mm), group 2: larger juveniles (121-142 mm), group 3: smaller adults (143-164 mm), and group 4: larger adults (>=165 mm). Size Groups Average Dissimilarity (%) Prey Taxa Contrib. (%) Diet Overlap 1-2 40.55 Bivalvia 28.51 0.55 1-3 29.67 Bivalvia 28.36 0.65 2-3 16.58 Teleostei 34.91 0.65 1-4 54.72 Bivalvia 41.38 0.34 2-4 56.80 Bivalvia and Dendrobranchiata 15.85 0.26 3-4 48.00 Bivalvia 21.66 0.32 animals-13-00939-t004_Table 4 Table 4 Results of Shannon-Wiener index (H'), diet breadth (B), and trophic level (TrL) for D. maroccanus by size group. Size group 1: smaller juveniles (<=120 mm), group 2: larger juveniles (121-142 mm), group 3: smaller adults (143-164 mm), and group 4: larger adults (>=165 mm). Size Group H' B TrL 1 0.10 +- 0.09 0.35 3.70 +- 0.47 2 0.32 +- 0.08 0.46 3.70 +- 0.43 3 0.31 +- 0.07 0.50 4.10 +- 0.54 4 0.12 +- 0.14 0.90 4.00 +- 0.62 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000058

In this study, we assessed the PPR disease status, its economic cost, the financial viability of vaccination, and the perspectives of field veterinarians on the PPR vaccination programme implemented in Karnataka state, India. In addition to secondary data, cross-sectional surveys undertaken during 2016-17 (survey I) and 2018-19 (survey II) from 673 sheep and goat flocks and data collected from 62 veterinarians were analysed. The economic costs and perceptions of veterinarians were analysed using deterministic models and the Likert scale, respectively, and the financial viability of vaccination programmes under the best (15%), base (20%), and worst-case (25%) PPR incidence scenarios, considering two different vaccination plans (plan I and plan II), was assessed. The disease incidence in sheep and goats was found to be 9.8% and 4.8% in survey I and survey II, respectively. In consonance with the increased vaccination coverage, the number of reported PPR outbreaks in the state declined significantly. The estimated farm-level loss of PPR varied between the surveyed years. Even under the best-incidence scenario, under vaccination plan-I and plan-II, the estimated benefit-cost ratio (18.4:1; 19.7:1), the net present value (USD 932 million; USD 936 million) and the internal rate of return (412%) implied that the vaccination programmes were financially viable and the benefits outweighed the cost. Though the majority of veterinarians perceived that the control programme was well planned and rolled out in the state, a few of them disagreed or were neutral towards the plan per se, towards the coordination between functionaries, the availability of funding, and the programme acceptance by farmers. Despite many years of vaccination, PPR still persists in the Karnataka state for various reasons and in order to eradicate the disease, a review of the existing control programme with strong facilitation from the federal government is needed. PPR incidence eradication financial viability of vaccination Karnataka India The Indian Council of Agricultural Research (ICAR), Agricultural Extension DivisionA. Extn.26/5/2106-AE-I/13 Department of Animal Husbandry and Dairying (DAHD), Government of IndiaK-11053(5314)/11/2018-LH The Indian Council of Agricultural Research (ICAR), Agricultural Extension Division, for funding the project through the Extramural project (A. Extn.26/5/2106-AE-I/13) and the Department of Animal Husbandry and Dairying (DAHD), Government of India, for funding the project (K-11053(5314)/11/2018-LH). pmc1. Introduction Peste des Petits Ruminants (PPR) is an acute febrile viral disease of sheep and goats and is generally referred to as 'sheep and goat plague'. The disease is characterised by mucopurulent nasal and ocular discharges, necrotising and erosive stomatitis, enteritis, diarrhoea, and bronchopneumonia . The causative agent, PPR virus (PPRV), belongs to the genus Morbillivirus under the family Paramyxoviridae. It is closely related to the Rinderpest virus that causes cattle plague, which was eradicated globally by 2011. The disease was first reported in sheep and goats in Cote d'Ivoire, West Africa in 1942 and later spread to different countries in Asia and Africa. The global annual loss due to PPR is estimated to range from USD 1.45 billion to USD 2.1 billion . Small ruminants are an important asset of landless, marginal and small farmers and disease outbreaks in flocks undermines the food and livelihood security of these poor farmers. Hence, considering the importance of PPR for small holder's, the Food and Agriculture Organisation (FAO) and the World Organisation of Animal Health (WOAH) launched a global plan to eradicate PPR by 2030 . In India, the disease was first reported in 1987 in a small sheep flock in the village of Arasur in Tamil Nadu state, which was caused by a lineage III virus . Subsequently, large outbreaks were reported in 1994-1995, which was caused by a lineage IV virus. Later, the disease spread to the entire country, and now PPR is enzootic in India and outbreaks are reported regularly among small ruminant population . Very high rates of morbidity and mortality (> 50 percent) due to high fever, pneumonia, diarrhoea, and dehydration have been reported in the affected flocks . Epidemics have enormous consequences on livestock productivity and affect the livelihoods of farmers and associated stakeholders in the sheep and goat value chain. Outbreaks have been reported regularly in the majority of the states of India since 2002. The losses due to PPR, as estimated by various researchers in India during different time periods, were INR 16116 million (USD 230.2 million) during 2015 , INR 88,951 million (USD 1270.7 million) based on reported outbreaks during 2008 to 2012 , and INR 45,710 to 46,830 million (USD 653-669 million) during 2016 . Considering the devastating nature of the disease, an effective Vero-cell-line-based live attenuated indigenous freeze-dried vaccine (Sungri 96) with a shelf life of more than one year at 4 degC, which provides immunity for three to six years, was developed by the ICAR-Indian Veterinary Research Institute . In India, since 2002, focused vaccinations of sheep and goats have been undertaken, and since 2010-2011, in southern states, the PPR-Control Programme (PPR-CP), with mass vaccination drives, has been implemented. This programme was implemented even before the global framework to control and eradicate PPR was developed. The Department of Animal Husbandry, Dairying and Fisheries (DADF), Government of India, sponsors the PPR-Control Programme (PPR-CP) in India and the participating states adopted a plan to carry out 100% vaccination of the risk population of small ruminants in the first year, followed by 30% bi-annual vaccination in the subsequent years to cover the naive population . Among the southern states, Karnataka and its contiguous state, undivided Andhra Pradesh, have implemented focused vaccination since 2002 and carried out mass vaccinations during 2004 and 2007-2008, respectively. Thereafter, they conducted annual vaccination programmes until 2010. After the first phase of the implementation of PPR-CP in these states during 2010-11, a significant reduction in the number of outbreaks in Karnataka state and a 99% reduction in PPR burden, with a flock immunity level of 81% to 95.6%, was observed in undivided Andhra Pradesh . This was achieved due to the adoption of a systematic vaccination programme under PPR-CP. In central India, Chhattisgarh state has carried out annual mass vaccination campaigns (MVCs) since 2010 on the 'pulse polio vaccination mode' for a designated period of 11-12 days in a year, resulting in no PPR outbreaks having been reported in the state since 2013-14 . In the second phase of PPR-CP, since 2015, although the rest of India's states and Union Territories (UTs) have implemented PPR-CP , the continuation of the programme is largely elusive in these states. Karnataka accounts for 17.21 million sheep and goats.PPR is endemic in the state and is the single largest cause of mortality in small ruminants. Hence, to control the disease, 'focused vaccination' has been undertaken since 2003-2004, and the PPR-CP strategy, including 'mass vaccination', was implemented in 2010-2011 . The main objectives of PPR-CP are 100% vaccination coverage, with the aim of bringing the outbreaks and epizootics to the level of zero and to make the state a PPR-free zone. The number of outbreaks has significantly decrease in the state due to the implementation of the vaccination programme. Now the state has planned to eradicate the disease by 2025-2026, implementing a further planned mass vaccination programme in consonance with the government of India's recent PPR eradication plan. In general, the economic costs and financial effectiveness of PPR vaccination programmes are limited in PPR-endemic countries, including India. Hence, the present study was undertaken to understand the disease status after implementing the vaccination programme, vaccination coverage vis-a-vis outbreaks, the economic cost of the disease, the financial viability of the vaccination programme, and the perceptions of veterinarians in the PPR-endemic state of Karnataka, India. 2. Materials and Methods 2.1. Study Area Karnataka is the 6th largest state, comprising 30 districts, which cover an area of 191,791 square kilometers or 5.83% of the total geographical area of India. Karnataka is the only southern state to have land borders with all of the other six southern Indian sister states. It is bordered by the Arabian Sea to the west, Goa to the northwest, Maharashtra to the north, Telangana to the northeast, Andhra Pradesh to the east, Tamil Nadu to the southeast, and Kerala to the south. As per the 20th livestock census, 2019, the total livestock population in Karnataka state was 29 million, of which 59.35% (17.21 million) comprised sheep and goat, of which, sheep constitute 64.21% (11.05 million). To understand the PPR incidence and the disease cost, the primary survey was undertaken during 2016-2017 and 2018-2019 and the geographical locations of the survey districts are presented in Figure 1. 2.2. Sampling Procedure Cross-sectional surveys were undertaken in six districts of Karnataka state among sheep- and goat-rearing households during 2016-2017 (survey I) and 2018-2019 (survey II). Initially, the districts in the state were grouped into three risk groups (high, medium, and low) based on the cumulative number of outbreaks that occurred in the districts in the three years preceding 2016-2017, the total number of PPR attacks per 1000 heads of sheep and goats, and the frequency of outbreaks and livestock density per 100 km2, and one district was selected in each of the risk group for the primary survey. During survey I, three districts, Kolar (high PPR risk), Bangalore rural (medium PPR risk), and Bagalkote (Low PPR risk), and in survey II, three districts, Chikkaballapura (high PPR risk), Kalaburgi (medium PPR risk) and Bidar (low PPR risk), were surveyed. A multistage random sampling procedure was followed to conduct survey I and survey II. In the first stage, one district from each of the risk groups was selected randomly and in the second stage 2-4 blocks (a block is a smaller administrative unit in a district and each district may comprise 2 to 5 or more blocks, depending on the population of the district) were randomly selected in each of the selected districts. In the third stage, in each selected block, one veterinary dispensary/hospital was selected randomly. In the fourth stage, the sheep- and goat-rearing villages under the jurisdiction of the dispensary/hospital were enlisted, of which 5-10 villages were selected randomly. In the final stage, in each of the identified villages, the individual farmers/households were selected randomly for the primary survey. The number of flocks to be surveyed in the identified villages was determined based on the proportion of sheep- and goat-rearing households in the village. 2.3. Sample Size The sample size for the primary survey was estimated as below , (1) SS=z2p1-pe2 where SS is the required sample size; Z is the z-value at a 95% confidence interval (1.96); p is the proportion of sheep- and goat-rearing households to the total number of households rearing livestock (0.224), and e is the acceptable sampling error (5%). The estimated total sample size for the survey was 267 sheep- and goat-rearing flocks. However, the primary data was collected from 350 flocks during 2016-2017 (survey I) and 323 flocks during 2018-2019 (survey II) using pre-tested schedules. 2.4. Data Collection and Identification of PPR-Affected Flocks The primary data on socio-economic parameters, sheep and goat inventories, production parameters, morbidity and mortality, PPR vaccination and treatment cost, etc. were collected from the selected sheep and goat farmers in the surveyed villages as per the sampling plan. Among the sampled farmers, photographs of various clinical signs of PPR were shown to the farmers and based on their observations of clinical signs, the flocks were diagnosed and grouped into PPR-affected and non-affected groups. Furthermore, the PPR incidence reported by the farmers during the survey was triangulated with the data provided by jurisdictional veterinary doctors to confirm the occurrence of PPR in the surveyed flocks, as these veterinarians are the 'gatekeepers' for the animal diseases occurring in their jurisdiction. 2.5. Estimation of the Flock-Level Economic Cost of PPR Sheep and goat inventory, clinically diagnosed PPR cases, and deaths were considered in order to estimate the primary metrics of morbidity and mortality levels. The mortality loss was estimated based on the number of animals that died in each age and sex group multiplied by the market value of the apparently healthy animals and subtracting the recovered value of the animal, if any. To calculate the weight loss after PPR outbreaks, measurements of the pre- and post- disease weights of the animals across the breed, age, and sex were required. As this was a cross-sectional survey-based study, on the day of the survey, it was possible that diseased flocks may not have been encountered. Hence, to calculate the weight loss due to PPR, the actual weight of the animals of various breeds, ages, and sexes from the apparently healthy flocks were documented by physically weighing the animals during the survey. A conservative 10% reduction (although a 15% weight reduction for sheep and goat pox was reported earlier ) in the recorded weights for the healthy flocks was considered as the weight loss in the PPR-affected flocks, and accordingly, the weight loss was calculated across breed, age, and sex for the flocks reported to be affected by PPR. The distress sale loss was calculated based on the number of animals sold under distress and the difference in the actual price obtained during a healthy state and the distress sale value of the animals. The treatment cost was calculated based on the expenditure made for drugs/medicines and veterinarian charges during the PPR outbreak in the flock and converted to the per-animal cost. Similarly, the opportunity cost of labour engaged for nursing the sick animals was calculated based on the incremental labour hours spent by the farm family or hired members multiplied by the prevailing wage rate in the PPR-affected villages. After calculating the loss per flock, appropriate weights were considered based on the number of animals that died and that were infected and recovered among the sheep and goats reared by the sample farmers to calculate the per-animal loss. The market value/prices of animals of various age and sex groups and labour wage rate prevailing in the surveyed villages during the survey periods (2016-2017 and 2018-2019) were considered in order to estimate the various losses. The estimated disease cost per animal during survey I (2016-2017) was converted to the 2018-2019 constant price, based on the prevailing consumer price index during the respective years . 2.6. Estimation of the Financial Viability of Vaccination 2.6.1. Vaccination Period The cash-flows were estimated in order to assess the benefit versus the cost of the vaccination programme from the initial year of PPR vaccination in Karnataka (2003-2004) up to the year selected for the potential control and eradication of PPR (2025-2026) as envisaged in the draft plan for PPR eradication in India. The vaccination period was divided into two stages--vaccination before PPR-CP implementation in the state (from 2005-2006 to 2010-2011) and vaccination after PPR-CP implementation (2011-2012 to 2025-2026). 2.6.2. Vaccination Coverage and Plans The data on the number of sheep and goats vaccinated in Karnataka state were collected from secondary sources from 2003-2004 to 2020-2021 in order to calculate the vaccine coverage (%). The benefits and costs of vaccination were estimated under two methods of vaccination plans. The first plan involved actual vaccination coverage from 2003-2004 to 2020-2021, covering 100% of the risk population from 2021-2022 to 2023-2024, followed by the need-based vaccination of 10% for the next two years (2024-2025 and 2025-2026) and the second plan was as per the PPR-CP plan, i.e., the actual vaccination coverage from 2003-2004 to 2020-2021 and the vaccination of 100% of the at-risk population during 2021-2022, followed by 30% bi-annual coverage for the two subsequent years (2022-2023 to 2023-2024) and 10% need-based vaccination coverage for next two years (2024-2025 and 2025-2026). 2.6.3. Population Projection The livestock population census in Karnataka was available only for quinquennial periods (i.e., 2003, 2007, 2012, and 2019) and hence to calculate the sheep and goat population in Karnataka for in-between years, the interpolation method was employed, using the compound annual growth rate (CAGR) of the sheep and goat population between respective livestock censuses. Furthermore, to predict the population for the period from 2020-2021 to 2025-2026, the average CAGR of the sheep and goat population between the 2003 and 2019 livestock censuses was considered. 2.6.4. Incidence Interpolation The PPR incidence levels with and without vaccination intervention were crucial for our financial evaluations. Due to various surveillance bottlenecks in developing countries, the reported disease incidence differs from the field-level incidence. For Karnataka state, the PPR incidence in the field based on surveys was available only for two years (2016-2017; 2018-2019). Hence, the 8% incidence level reported in the literature during 2008-2009 for Madhya Pradesh state was considered as the initial incidence level in the study state (Karnataka) for the first year of financial assessment (i.e., 2003-2004). Furthermore, in determining the PPR incidence under a scenario with a vaccination intervention, we assumed that this 8% incidence level increased gradually and reached 12% during 2010-2011, as the progress of vaccination in the initial years was very slow due to the adoption of the 'focussed vaccination' policy in the state. Thereafter, mainly after PPR-CP implementation (since 2010-2011), the incidence level gradually decreased due to the adoption of the 'mass vaccination' drive. In the primary surveys undertaken in this study, we estimated an incidence of 9.8% and 4.8% in Karnataka during 2016-2017 and 2018-2019, respectively. Considering the initial incidence (2003-2004), the incidence under focussed vaccination (2010-2011), and the estimated incidence after PPR-CP implementation (2016-2017 and 2018-2019), the possible incidence levels in in-between years under the vaccination scenario were derived via interpolation through the linear method. At the end of 2023-24, as per the draft national strategic plan, 100% of the sheep and goat population will be vaccinated, and as a result, the disease incidence will be very low during 2023-2025 to 2025-2026. Hence, for the financial analysis, a PPR incidence of zero was assumed during 2023-2025 to 2025-2026 (In India, the financial year lasts from April until March of the following year (for example, the year 2010-2011 year represents the period from April 2010 to March 2011). Similarly, the stream of incidences in different years between 2003-2004 and 2025-2026 under the scenario without vaccination intervention was derived via interpolation through the linear method. The three possible incidence scenarios (Supplementary Table S1) were as follows: an 8% incidence level during 2003-2004 that increased to 15% by 2010-2011 and remained at the same level until 2025-2026 (low incidence); an 8% incidence level during 2003-2004 that increased to 20% by 2025-2026 (medium incidence); and an 8% incidence level during 2003-2004 which increased to 25% by 2025-2026 (high incidence). These differences between incidence levels under scenarios with and without vaccination in different years were considered in order to estimate the avoided costs/benefits of morbidity and mortality reductions that occurred due to PPR vaccination. The details of interpolated incidence levels in scenarios with and without vaccination (with three possible incidences under without vaccination) are presented in Supplementary Table S1. 2.6.5. Benefit Stream The benefits of vaccination in different years were calculated based on the difference in the PPR incidence (the disease avoidance level) under scenarios with and without vaccination, the projected risk population in the respective year, the per-animal disease cost, the morbidity and mortality levels, and the effectiveness of vaccination (80%). The disease cost included mortality, body weight reductions, distress sales, treatment costs, and the opportunity cost of labour per animal due to PPR. The disease cost, incidence, morbidity, mortality, and distress sale proportions were calculated based on the data collected in the primary surveys undertaken in Karnataka state during 2016-17 and 2018-19. An annual 3% deflation rate (from 2003-2004 to 2017-2018) and inflation rate (from 2019-2020 to 2025-2026) were applied to calculate disease cost per animal at current prices. The total benefit of vaccination against PPR was calculated as follows. (2) Bv=i=1n(DIixPi) MpixLmi+WpixLwi+DpixDli+Tci+OcixVEi where Bv = total benefits of vaccination/PPR avoidance cost (USD); (DI)i = difference in PPR incidence under scenarios with and without vaccination in the ith year (%), Pi = projected population in the ith year (No.), Mpi = proportion of mortality due to PPR in the ith year (%), Lmi = average mortality loss per animal in the ith year (USD), Wpi = proportion of morbidity due to PPR in the ith year (%), Lwi = average body weight reduction loss per animal in a PPR-recovered animal in the ith year (USD), Dpi = distress sale proportion due to PPR in the ith year (%), Dli = average distress sale loss per animal in the ith year (USD), Tci = average treatment cost per animal in the ith year (USD), Oci = average opportunity cost of labour per animal in the ith year (USD), VEi = vaccination effectiveness in the ith year (80%), and n = number of years (i = 1, 2, 3, ...n). 2.6.6. Vaccination Cost Stream The total vaccination cost per annum was calculated based on the proportion of sheep and goats vaccinated, multiplied by the vaccination cost (vaccine cost per dose and the vaccination logistics and accessories cost per dose/animal). The vaccine cost and vaccination logistics and accessories cost varied depending on the vaccination strategy adopted, viz., focussed/routine vaccination or using the PPR-CP model. In Karnataka, the focussed/routine vaccination strategy was adopted from 2003-2004 to 2010-2011 and, thereafter, the PPR-CP vaccination strategy was implemented up to 2025-26. Hence, for the focussed/routine vaccination period (from 2003-2004 to 2009-2010), the vaccine cost per dose was assumed to be twice the vaccine cost for the control programme vaccination period (2010-2011 to 2025-2026) the vaccine cost was INR 1.8/dose and the vaccination logistics and accessories cost was INR18.2/dose . The vaccination logistics and accessories cost included expenditure for accessories (needles, syringes, etc.); payment for hired vaccinators, vaccine storage, and transportation; the cost of sensitisation and technical workshops, extension awareness materials, programme dissemination, and publication; expenditure directed towards the strengthening of district diagnostic laboratories, staff salaries, etc. . The total vaccination cost for the respective year was calculated as follows. (3) Cv=i=1nVpix Vci where Cv = total cost of vaccination (USD million), VPi = the proportion of sheep and goats vaccinated in the ith year (%); Vci = cost of vaccination (including the vaccine cost and the vaccination logistics and accessories cost per dose) per animal in the ith year (USD million), and n = number of years (i = 1, 2, ..., n). All the benefit and cost streams were estimated in INR and converted to millions of USD (One USD = INR 75). 2.6.7. Financial Assessment of PPR Vaccination The evaluation of the financial benefits of PPR vaccination was based on the benefit:cost ratio (BCR), net present value (NPV), and the internal rate of returns (IRR) as per standard procedure under two vaccination plans, designated as plans I and II. Plan I refers to the actual vaccination coverage from 2003-2004 to 2020-2021 and vaccinating 100% risk population from 2021-2022 to 2023-2024 (for three years), followed by 10% need-based vaccination coverage from 2024-2025 to 2025-2026 (for two years). Plan II refers to the actual vaccination coverage from 2003-2004 to 2020-2021 and vaccinating 100% of the naive population during the year 2021-2022, followed by 30% bi-annual coverage from 2022-2023 to 2023-2024 (the two subsequent years) and 10% need based vaccination coverage from 2024-2025 to 2025-2026 (the next two years), as per the PPR-CP plan. 2.7. Perspectives of Veterinary Officers on PPR-CP A survey was conducted among field veterinarians, who are the custodians of implementing the PPR vaccination programme for the sheep and goats in their jurisdictional veterinary hospitals/dispensaries through the support of the Animal Disease Surveillance Scheme (ADSS), Department of Animal Husbandry and Veterinary Services, Government of Karnataka, using the pre-tested schedules. In addition to implementing PPR-CP, field veterinarians also implement other disease prevention programmes and animal treatment and extension activities. The schedules were mailed to the 176 veterinarians working in veterinary hospitals/dispensaries in the state via e-mail and 62 responded to the survey. The survey schedule comprised statements about the planning and rollout of the PPR control programme in the state, the performance of the functional components of the programme, and the respondent's opinions regarding its improved implementation. The statements were assessed through a two-point assessment (Yes/No) and a three-point Likert scale (Disagree (SD), Neutral (N), and Agree (A)) with positive and negative statements. The frequencies were calculated based on the responses of the field veterinarians to various statements about PPR control and eradication in Karnataka state. 2.8. Statistical Analysis Descriptive statistics and a two-sample z-test for proportions were used to test the significant differences in terms of diagnosed and dead cases in sheep and goats in the sample farms between survey I (2016-2017) and survey II (2018-2019). These were carried out using SPSS version 22.0 (SPSS Inc., Chicago, IL, USA). 3. Results The results obtained from the secondary data on the reported PPR outbreaks and vaccination coverage in Karnataka state and the results of the cross-sectional primary surveys undertaken in the sampled districts are presented below. 3.1. Reported Outbreaks and PPR Vaccination Coverage Karnataka practiced focussed vaccination from 2003-2004 to 2010-2011. The vaccination coverage increased from 6% during 2003-2004 to 51% during 2009-2010, whereas after the implementation of PPR-CP in 2010-2011, the vaccination coverage increased consistently and reached 100% in the period from 2014-2015 to 2020-2021, except during 2017-2018 . In consonance with this increased vaccination coverage, the reported PPR outbreaks in the state declined significantly . 3.2. Socio-Economic Characteristics of Sheep- and Goat-Rearing Farmers The socio-economic characteristics of flocks surveyed in survey I and survey II are presented in Table 1. The median ages of farmers rearing sheep and goats during the two surveys were 49 and 46, respectively. The majority of the farmers were illiterate, and the majority of them were small farmers having less than 2.0 ha in land holdings. A considerable proportion of surveyed farmers were landless and they depended entirely on sheep and goats for their livelihoods. The observed average numbers of sheep and goats reared/flock sizes were 40 and 55 during survey I and survey II, respectively (Table 1). The majority (40%) of the sample farmers' nominal incomes were less than USD 715 during 2016-2017, whereas the majority (52%) of the sample farmers' nominal incomes were between USD 715 and USD 1429 during 2018-2019. The other socio-economic details of the surveyed flocks in different surveyed districts in Karnataka state during survey I and survey II are presented in Table 1. 3.3. PPR Incidence in the Surveyed Flocks The PPR-affected flocks and the disease incidence in sheep and goats among the sample flocks during surveys I and II are presented in Figure 3. The proportion of PPR-affected flocks among the surveyed sheep and goat flocks was 19.7% and 16.1%, respectively , whereas the PPR incidence at the animal level was 9.8% and 4.8% in survey I and survey II, respectively . 3.4. Distribution of Incidence, Mortality and Case Fatality Rate (CFR) The age- and sex-wise distributions of PPR incidence, mortality, and CFR in sheep and goats in survey I and survey II are presented in Table 2. The PPR incidence in sheep and goats was 9.8% in survey I, whereas it was 4.8% in survey II. In sheep, age-wise and sex-wise comparisons of incidence showed a significant difference (p < 0.01) between the two surveys, whereas, in goats, we observed a significant difference in all the age groups (< 6 months (z = 5.65, p < 0.01), 6-12 months (z = 2.52, p < 0.1) and > 1 year (z = 5.43, p < 0.01)), whereas a non-significant difference was observed in the sex-wise comparison between the two surveys. In sheep, age-wise and sex-wise comparisons of mortality levels showed a significant difference between two surveys, whereas, in goats, a significant difference was observed in the group < 6 months old (z = 6.75, p < 0.01) and the 6-12 month old (z = 2.62, p < 0.01) group. Furthermore, the CFR was lower in survey I, compared to survey II (Table 2). 3.5. Estimated Loss of PPR The various components of the farm-level loss per animal in surveys I and II are presented in Table 3. The estimated mortality loss ranged between USD 30.1 and USD 128.6 per animal, depending on the severity of the disease in the respective years and the distress sale loss ranged between USD 19.6 and USD 101.8. The body-weight reduction, treatment cost, and opportunity cost of labour ranged between USD 1.7 and USD 7.4, USD 0.3 and USD 7.3, and USD 0.2 and USD 8.3 during the two surveys, respectively. The estimated loss components in sheep and goats independently during the two surveys are presented in Table 3. 3.6. Financial Viability of PPR Vaccination The results of the financial viability assessment of PPR vaccination in Karnataka under the best-, base-, and worst-case PPR incidence scenarios of 15%, 20%, and 25%, respectively, under two methods of vaccination plans are presented in Supplementary Tables S2 and S3. The estimated BCR under the best-, base-, and worst-case incidence scenarios were 18.36:1, 23.45:1, and 28.55:1, whereas the NPV was USD 931.97 million, USD 1205.75 million, and USD 1479.52 million, respectively, under vaccination plan I (Supplementary Table S2). Under vaccination plan II, the estimated BCR was 19.65:1, 25.11:1, and 30.57:1 and the NPV was USD 935.51 million, USD 1209.29 million, and USD 1483.07 million in the best-, base-, and worst-case incidence scenarios, respectively (Supplementary Table S3). Remarkably, an IRR of 412% was observed in all three incidence level scenarios with the two methods of vaccination plans (Supplementary Tables S2 and S3). 3.7. Perspective of Field Veterinarians on PPR-CP 3.7.1. Planning and Rollout of PPR-CP The perceptions of veterinarians with regard to the planning and rollout of PPR-CP in Karnataka state are presented in Table 4. The majority (93.5%) of the surveyed field veterinarians perceived that the PPR-CP had been planned well in the state. For the statement regarding the 'proper coordination of various programme functionaries', although the majority agreed on the existence of co-ordination, around 19% remained neutral. Regarding the negative statement concerning the weak support from local Panchayat authorities, the 42% level of agreement indicates that there is still scope for fostering support from the local authorities. Regarding the statement that a 'delay in the release of funds affected the PPR-CP implementation', 26% agreed and 42% remained neutral, indicating that there were delays in the release of funds, which in turn affected the implementation of PPR-CP. 3.7.2. Performance of Functional Components of PPR-CP The majority (82%) of veterinarians perceived that the required quantity of PPR vaccine doses was supplied on time and 72% were able to cover the targeted population in the stipulated time. Regarding the statement on the 'availability of cold-chain facilities', 86% of the veterinarians perceived that good facilities were available (Table 5). 3.7.3. Opinions of the Veterinarians on Improving the Implementation of PPR-CP The majority (84%) of the veterinarians opined on the need for training regarding vaccine efficacy and effectiveness and 77% opined that provision of funds, mainly for mobility and contingency expenditure, were necessary for the success of the PPR-CP implementation in the state. The majority (92%) of the field veterinarians opined that intensive awareness meetings for farmers before the implementation of vaccination would benefit the programme considerably (Table 6). 4. Discussion The socio-economic profile of the sampled farmers revealed that the majority of them were illiterate and small farmers (with landholdings < 2.0 ha) and their annual income was <1500 USD. An outbreak of disease in flocks affects the animal asset pattern, as well as financial and social security, and pushes these farmers to transitional poverty. Hence, to ensure the livelihoods of these farmers, it is important to control and prevent the incidence of PPR in their flocks. Karnataka state reported 150 to 200 outbreaks per year during 2004 to 2006 . Due to the adoption of PPR vaccination in the state, the numbers of reported outbreaks have declined significantly since 2006-2007. The maximum vaccination coverage during the focussed vaccination period (2003-2004 to 2009-2010) was only 52%, whereas after the implementation of PPR-CP (2010-2011), a significant increase was observed. In consonance with the increased vaccination coverage, the number of reported PPR outbreaks in the state has declined considerably over the years. A similar pattern of a decline in outbreaks due to mass vaccination was observed in the undivided Andhra Pradesh area, which is a contiguous state of Karnataka. Though there were fewer reported outbreaks in Karnataka in recent years, the two primary surveys conducted here, covering 673 flocks, revealed incidences of 9.8% (survey I) and 4.8% (survey II), respectively. Similar results showing a higher incidence (17.5%) before mass vaccination (Singh et al., 2014) and a lower incidence (0.8%) after a mass vaccination campaign (MVC) were reported in Chhattisgarh state, India . Age-wise and sex-wise comparisons of incidence and CFR revealed a significant difference between the two surveys. The observed PPR incidences during surveys I and II implied that a significant number of animals became infected with PPR across species, age, and sex groups and a considerable burden was being inflicted upon various stakeholders associated with small-ruminant rearing despite the state being a vaccine-adopted state since 2003-2004. This also implies that vaccination coverage in this particular state alone will not be sufficient to eradicate the disease and the implementation of parallel programmes in the neighboring and contiguous states are also equally important as the regular movement of sheep and goats between these states occurs for trade, transit, and for grazing. Though the number of reported outbreaks significantly decreased in Karnataka state after the implementation of the vaccination programme, in order to obtain a further definitive decline and disease eradication by 2025-2026 through 'mass vaccination' in the state, in consonance with the government of India's PPR eradication plan, an appropriate strategy covering vaccination in all the contiguous regions or epi-systems--mainly in the regions where the maximum level of animal movement is observed between the states--is warranted. The observed variations in the estimated flock-level losses due to PPR in the surveyed years could be due to differences in the risk population, the number and severity of outbreaks, the age and sex compositions of the diseased flocks, the vaccination coverage in the preceding years, and the time and season of outbreaks. These findings are also in agreement with those of earlier reports . Furthermore, the sensitivity analysis for the various disease incidence scenarios under the two vaccination strategies indicated high gains even under the low-incidence scenario (BCR 18.36 and 19.65; NPV 931.97 and 935.51; and IRR 412% under vaccination plan I and plan II, respectively). A previous study conducted in Chhattisgarh state, India, reported BCRs of 4.9:1, 12.4:1, and 13.5:1 under low, medium, and high PPR incidence levels, respectively, for a 100% yearly vaccination strategy and BCRs of 13.7:1, 34.7:1, and 37.8:1 for a five-year vaccination cycle (100% vaccination in the first year, followed by 30% coverage for three years and need-based coverage in the fifth year) . A study on the economic feasibility of the PPR control programme using the PPR vaccine at the national level, which used an economic surplus model , revealed a significant NPV, IRR, and BCR, with values of USD 6988 million (at 1 USD= INR 70), 119%, and 123:1, respectively. Furthermore, a study using a dynamic herd model estimated a BCR of 12.0 for adopting a 5-year period vaccination cycle in Niger . These results imply that PPR vaccination is economically viable and generates more outflows (benefits) than inflows (costs). However, if the Karnataka state intends to eradicate PPR in consonance with the government of India's plan by 2025-2026, in addition to vaccination, other bio-security measures also need to be promoted in order to reduce the PPR incidence to zero. The perception of the veterinarians regarding the PPR-CP's rollout revealed that the majority of the veterinarians concurred with the various activities implemented in Karnataka state, viz., the control programme was planned well, the vaccination programme was accepted by the farmers, proper coordination existed between various functionaries of PPR-CP, and they agreed upon the effectiveness of extension services. Furthermore, regarding the performance of the functional components of the control programme, the majority of the veterinarians perceived that vaccines were available on time in sufficient quantities, they could vaccinate the targeted animals on time, storage and cold-chain facilities were available, and they could manage the outbreaks on time. Though the majority of veterinarians expressed positive opinions on the existing PPR control plan, including its rollout and the performance of the various functional components implemented in the state, a few of the veterinarians disagreed and some remained neutral towards the plan per se, as well as towards the level of coordination between the functionaries, the availability of funding, and the programme's acceptance by farmers. The veterinarians' opinions regarding the improvement of the existing control programme included imparting training on vaccine efficacy and effectiveness, increasing the provision of funds for various activities, and increased coordination with various authorities. This implies that there is scope for improvement in the existing programme. Furthermore, since India is planning to eradicate PPR by 2025-2026, all facets of the programme need to be revisited, revised, implemented, and monitored on a regular basis and, more importantly, the perspectives of the important 'gatekeepers' in the PPR eradication programme, such as field veterinarians, need to be included in the current programme. Although the availability of effective vaccines, diagnostic tests, veterinary infrastructure, and technical manpower were what prompted the government to initiate PPR control and eradication efforts in small ruminants in India , it may be difficult to achieve the eradication target unless shortfalls in the existing programme are addressed. Previous studies also reported the need for strengthening of the veterinary infrastructure, vaccine production, disease diagnoses, and surveillance measures, as well as faster notification in order to achieve PPR disease eradication in Nigeria and in Karnataka . Furthermore, a study on the eradication and control of animal diseases revealed that a combination of measures may be employed to avoid the spread of disease from infected animals to clean animals and their success is dependent on a variety of factors, including the strength and capacity of veterinary services, cross-border efforts for disease vaccination and disease surveillance, political will and support, diagnostic facilities, and financial support. Furthermore, another obstacle that will limit the success of the eradication efforts in India is the unabated movement of animals between and within states for trade and transit, as well as for migration in search of fodder. Vaccination is an important palliative method to prevent infectious diseases . However, voluntary efforts by farmers to vaccinate their animals on a regular basis are limited in developing countries such as India and hence all the stakeholders in the livestock sector, including farmers, need to be involved for the success of the eradication programme that is planned in India. The results of this study provide evidences and necessary directions for the up-scaling of this strategy in similar socio-economic environments with the aim of eradicating PPR from India and the world by 2030 . At a broader level, the study provides evidence on the operational and financial feasibility of PPR-CP, as implemented in Karnataka state, India. However, the results of the present study need to be visualised with certain limitations, for instance, PPR was confirmed based on the clinical signs observed by farmers and triangulated with those of field veterinarians in the respective jurisdictions and was not based on laboratory confirmation; we also did not consider concomitant diseases; only major farm-level costs were considered in the financial assessments; and only 62 field veterinarians who responded to the survey were considered in the study. 5. Conclusions Though Karnataka state followed a 'focussed vaccination' strategy after 2003-2004 and a 'mass vaccination' strategy after 2010-2011 under the PPR-CP plan, the disease continues to persist in the state. If the state and the country plan to eradicate PPR, the revisiting of the existing programme is warranted. Furthermore, strong facilitation measures are needed from the federal government for the effective implementation of the eradication programme in these states and to ensure effective coordination between the states. Despite long years of vaccination against PPR, the benefits outweigh the costs, mainly because of the low cost of the vaccine (USD 2.4/100 doses). However, developing countries such as India cannot afford to extend the current control strategies and continue to observe incidences of PPR. Hence, a strong mass vaccination programme that does not exclude a single susceptible animal from vaccination for a considerable period is necessary. Furthermore, the migration routes, trade routes, state borders, and high-risk regions within the states need to be covered during vaccination drives to eradicate the disease. Furthermore, the syndromic surveillance and attending the outbreaks on time, as well as stamping out policy, if needed with compensation to farmers need to be implemented in the final stage of the eradication process. Acknowledgments The contribution of project and other staff of ICAR-NIVEDI is acknowledged. The authors appreciate the efforts of Karnataka state animal husbandry department officials, field veterinarians, para-veterinarians, and participating sheep- and goat-rearing farmers for their active participation in the surveys. Supplementary Materials The following supporting information can be downloaded at: Table S1: Different interpolated PPR incidence levels under scenarios with vaccination and without vaccination--(low (15%), medium (20%), and high (25%)) from 2003-2004 to 2025-26 in Karnataka. Table S2: Financial viability of the PPR vaccination in Karnataka under vaccination plan I (actual vaccination coverage (from 2003-2004 to 2020-2021) with the vaccination of 100% of the at-risk population for three years (from 2021-2022 to 2023-2024), followed by 10% need-based vaccination coverage for the next two years (from 2024-2025 to 2025-2026)). Table S3: Financial viability of the PPR vaccination in Karnataka under vaccination plan II (actual vaccination coverage (from 2003-2004 to 2020-2021) with the vaccination of 100% of the naive population during the year 2021-2022, followed by 30% bi-annual coverage during the two subsequent years (from 2022-2023 to 2023-2024) and 10% need-based vaccination coverage for the next two years (from 2024-2025 to 2025-2026) as per the PPR-CP plan). Click here for additional data file. Author Contributions Conceptualisation, G.N.G.; methodology, G.N.G.; validation, G.N.G.; formal analysis, G.S.N. and G.N.G.; investigation, G.N.G., V.B., G.B.M.R., R.Y. and S.P.; resources, P.R.; data curation, G.S.N., K.V.K., H.R.C. and A.Z.B.; writing--original draft preparation, G.N.G.; writing- review and extensive editing, S.P., F.N., P.R. and B.R.S.; visualisation, G.N.G.; supervision, G.N.G. and T.S.R.; project administration, P.R. and B.R.S.; funding acquisition, G.N.G., V.B. and P.R. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Informed verbal/oral consent was obtained from the survey participants by explaining the pertinent information. Data Availability Statement The data presented in this study are available in the manuscript and in the Supplementary Files. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study: the collection, analyses, or interpretation of data; the writing of the manuscript; nor in the decision to publish the results. Figure 1 Districts with a high risk (Kolar (1), Chikkaballapur (2)), medium risk (Bangalore rural (3), Kalaburgi (4)), and low risk (Bidar (5), Bagalkote (6)) surveyed in Karnataka during survey I (2016-2017) and survey II (2018-2019). Figure 2 Reported PPR outbreaks and vaccination coverage in Karnataka between 2003-2004 and 2020-2021. Figure 3 Number of flocks surveyed and proportion of flocks infected with PPR (A) and animals at risk and animals infected with PPR (B) in survey I and survey II in Karnataka. animals-13-00778-t001_Table 1 Table 1 General characteristics of sample farms in Karnataka. Particulars Unit Survey I (2016-2017) Survey II (2018-2019) Districts Pooled Districts Pooled Kolar Bangalore Rural Bagalkote Chikkaballapur Kalaburagi Bidar No. of farms/flocks surveyed No. 200 90 60 350 205 44 74 323 Age of the farmers (Average) Years 50 51 44 49 48 45 43 46 Family size (Median) No. 6 5 8 6 5 6 5 5 Education Illiterate No. 101 (51.0) 29 (32.2) 39 (65.0) 205 (58.6) 141 (38.1) 37 (84.1) 60 (81.1) 238 (73.7) Primary No. 40 (19.8) 9 (10.0) 7 (11.7) 39 (11.1) 22 (29.3) 4 (9.1) 5 (6.8) 31 (9.6) High school No. 40 (19.8) 37 (41.1) 12 (20.0) 80 (22.9) 32 (32.7) 2 (4.6) 5 (6.8) 39 (12.1) College and above No. 19 (9.4) 15 (16.7) 2 (3.3) 26 (7.4) 10 (0) 1 (2.3) 4 (5.4) 15 (4.6) Land holdings Landless No. 51 (25.5) 14 (15.5) 18 (30.0) 81 (23.1) 32 (15.6) 27 (61.4) 45 (60.8) 104 (32.2) Small (< 5 Acre) No. 140 (70.0) 68 (75.6) 21 (35.0) 229 (65.4) 124 (60.5) 13 (29.6) 24 (32.4) 161 (49.9) Medium (5 to 10 Acre) No. 8 (4.0) 6 (6.7) 15 (25.0) 30 (8.6) 41 (20) 3 (6.8) 4 (5.4) 48 (14.9) Large (> 10 Acre) No. 1 (0.5) 2 (2.2) 6 (10.0) 10 (2.9) 8 (3.9) 1 (2.3) 1 (1.4) 10 (3.1) Income levels (USD) < 715 No. 78 (39.0) 34 (37.8) 10 (16.7) 122 (34.9) 51 (24.9) 0 (0) 17 (22.9) 68 (21.1) 715-1429 No. 73 (36.5) 36 (40.0) 34 (56.7) 141 (40.3) 135 (65.9) 9 (20.5) 26 (35.1) 170 (52.6) 1429-2858 No. 33 (16.5) 15 (16.7) 16 (26.7) 64 (18.3) 18 (8.8) 15 (34.1) 15 (20.3) 48 (14.9) > 2958 No. 18 (9.0) 5 (5.6) 0 (0.0) 23 (6.6) 1 (0.5) 20 (45.5) 16 (21.6) 37 (11.5) Total No. of sheep and goats in surveyed farms/flocks No. 4070 1970 6297 12,337 7765 2649 4897 15,311 Average No. of sheep and goats per farm/flock No. 27 22 108 40 38 60 66 55 Total No. of sheep and goats sold per year No. 1706 599 1843 4148 1825 1033 1371 4229 Average No. of sheep and goats sold per year per farm/flock No. 9 7 31 12 9 23 19 17 Note: Figures in the parenthesis indicate percentages of the total. One USD = INR.70. animals-13-00778-t002_Table 2 Table 2 Distribution of morbidity and mortality in sheep and goats due to PPR in Karnataka. Groups Category No. of Surveyed Animals/Animals at Risk No. of Affected Animals/ Incidence (Morbidity) No. of Death Cases (Mortality) CFR (%) 2016-2017 2018-2019 2016-2017 2018-2019 z-Value |z| 2016-2017 2018-2019 z-Value |z| 2016-2017 2018-2019 Animal/species Type Sheep 10,006 9794 1036 (10.4) 279 (2.8) 21.20 *** 455 (4.5) 207 (2.1) 9.52 *** 43.9 74.2 Goats 2331 5517 169 (7.3) 460 (8.3) 1.62 NS 81 (3.5) 388 (7) 6.07 *** 47.9 84.3 Total 12,337 15,311 1205(9.8) 739 (4.8) 15.97 *** 536 (4.3) 595 (3.9) 1.91 * 44.5 80.5 Sheep Age-wise <6 months 2112 2812 336 (15.9) 137 (4.9) 13.01 *** 204 (9.7) 113 (4) 7.98 *** 60.7 82.5 6-12 months 947 3426 108 (11.4) 102 (3) 10.73 *** 30 (3.2) 69 (2) 2.11 * 27.8 67.6 >1 year 6947 3556 592 (8.5) 40 (1.1) 15.08 *** 221 (3.2) 25 (0.7) 7.94 *** 37.3 62.5 Sex-wise Male 2029 3939 328 (16.2) 134 (3.4) 17.47 *** 153 (7.5) 99 (2.5) 9.14 *** 46.6 73.9 Female 7977 5855 708 (8.9) 145 (2.5) 15.45 *** 302 (3.8) 108 (1.8) 6.65 *** 42.7 74.5 Goats Age-wise <6 months 633 1476 43 (6.8) 234 (15.9) 5.65 *** 28 (4.4) 217 (14.7) 6.75 *** 65.1 92.7 6-12 months 267 1830 9 (3.4) 139 (7.6) 2.52 * 5 (1.9) 104 (5.7) 2.62 *** 55.6 74.8 >1 year 1431 2211 117 (8.2) 87 (3.9) 5.43 *** 48 (3.4) 67 (3) 0.54 NS 41 77 Sex-wise Male 631 2182 61 (9.7) 227 (10.4) 0.53 NS 30 (4.8) 192 (8.8) 3.32 *** 49.2 84.6 Female 1700 3335 108 (6.4) 233 (7) 0.45 NS 51 (3) 196 (5.9) 4.47 *** 47.2 84.1 Sheep + Goats Age-wise < 6 months 2745 4288 379 (13.8) 371 (8.7) 6.83 *** 232 (8.5) 330 (7.7) 1.14 NS 61.2 88.9 6-12 months 1214 5256 117 (9.6) 241 (4.6) 6.94 *** 35 (2.9) 173 (3.3) 0.73 NS 29.9 71.8 > 1 year 8378 5767 709 (8.5) 127 (2.2) 15.51 *** 269 (3.2) 92 (1.6) 5.99 *** 37.9 72.4 Sex-wise Male 2660 6121 389 (14.6) 361 (5.9) 13.44 *** 183 (6.9) 291 (4.8) 4.05 *** 47 80.6 Female 9677 9190 816 (8.4) 378 (4.1) 13.26 *** 353 (3.6) 304 (3.3) 1.27 NS 43.3 80.4 Figures in parentheses represent percentages. CFR = case fatality rate, *** indicates significance at 1%; * indicates significance at 10%, NS = non-significant. animals-13-00778-t003_Table 3 Table 3 Components of estimated loss (per animal) on the effects of PPR on sheep and goats in Karnataka. Parameters Sheep Goats Pooled (Sheep + Goats) 2016-2017 2018-2019 2016-2017 2018-2019 2016-2017 2018-2019 Loss due to reduction in body weight (USD) 4.2 (0.4, 1.7-12.5) 4.3 (0.5, 2.9-7.3) 3.9 (0.4, 3.9-7.6) 4.0 (0.4, 2.0-7.4) 4 (0.4, 1.7-12.5) 4.2 (0.4, 2.0-7.4) Mortality loss (USD) 49.1 (2.4, 25.8-91.9) 56.4 (2.6, 34.3-128.6) 51.2 (2.5, 34.7-99.6) 56.5 (2.5, 42.9-120.0) 50.2 (2.5, 30.1-99.6) 56.5 (2.6, 34.3-128.6) Distress sale loss (USD) 43.3 (1.9, 26.1-76.5) 0 (0, 0-0) 38.3 (1.5, 15.3-68.9) 84.9 (3.7, 35.7-101.8) 40.8 (1.7, 19.6-76.5) 84.9 (13.7, 35.7-101.8) Treatment cost (USD) 1.6 (0, 0.3-9.2) 2.6 (0.07, 0.9-7.3) 1.6 (0, 0.3-9.2) 2.6 (0.07, 0.9-7.3) 1.6 (0, 0.3-9.2) 2.6 (0.07, 0.9-7.3) Opportunity cost of labour (USD) 1.8 (0.1, 0.2-4) 2.4 (0.05, 0.4-8.3) 1.8 (0.1, 0.2-4) 2.4 (0.05, 0.4-8.3) 1.8 (0.1, 0.2-4) 2.4 (0.05, 0.4-8.3) Figures in parentheses represent standard error and range values. animals-13-00778-t004_Table 4 Table 4 Survey results on the perceptions of veterinarians regarding the planning and rollout of the PPR control programme (PPR-CP) in Karnataka (N = 62). Statements Disagree Neutral/Undecided Agree The PPR vaccination programme has been well planned in your jurisdiction. 4 (6.5, 0.3-12.6) 0 (0, 0-0) 58 (93.5, 87.4-99.7) Proper coordination exists between various functionaries. 1 (1.6, -1.5-4.7) 12 (19.4, 9.5-29.2) 49 (79, 68.9-89.2) Funding delays hindered the implementation of PPR-CP. 20 (32.3, 20.6-43.9) 26 (41.9, 29.7-54.2) 16 (25.8, 14.9-36.7) Weak support from local authority such as the Panchayat was experienced. 20 (32.3, 20.6-43.9) 16 (25.8, 14.9-36.7) 26 (41.9, 29.7-54.2) The vaccination programme was acceptable for farmers 1 (1.6, -1.5-4.7) 10 (16.1, 7-25.3) 51 (82.3, 72.7-91.8) Creating awareness about PPR and vaccination for farmers and other functionaries through mass media is effective in its implementation. 1 (1.6, -1.5-4.7) 4 (6.5, 0.3-12.6) 57 (91.9, 85.2-98.7) Figures in parentheses indicate percentages and 95% confidence intervals. animals-13-00778-t005_Table 5 Table 5 Perceptions of veterinarians regarding the performance of the functional components of PPR-CP in Karnataka (n = 62). Statements Disagree Neutral/Undecided Agree Timely availability of vaccines 4 (6.5, 0.3-12.6) 7 (11.3, 3.4-19.2) 51 (82.3, 72.7-91.8) Supply of sufficient quantity of vaccines and quality of vaccination materials (syringes, gloves, etc.) 6 (9.7, 2.3-17) 5 (8.1, 1.3-14.8) 51 (82.3, 72.7-91.8) Able to cover the targeted population within the stipulated time 9 (14.5, 5.7-23.3) 8 (12.9, 4.6-21.2) 45 (72.6, 61.5-83.7) Appropriate training was imparted to the vaccination team to vaccinate animals at the farmer's doorstep 10 (16.1, 7-25.3) 9 (14.5, 5.7-23.3) 43 (69.4, 57.9-80.8) Storage and cold-chain facilities were good 6 (9.7, 2.3-17) 3 (4.8, -0.5-10.2) 53 (85.5, 76.7-94.3) Veterinarians were able to manage the reported outbreaks in time 1 (1.6, -1.5-4.7) 8 (12.9, 4.6-21.2) 53 (85.5, 76.7-94.3) Figures in parentheses indicate percentages and 95% confidence intervals. animals-13-00778-t006_Table 6 Table 6 Opinions of veterinarians regarding statements for the improved implementation of PPR-CP in Karnataka (n = 62). Statements Yes No Training should be supplied to veterinarians and para-veterinary workers on the vaccine's efficacy and effectiveness 52(83.9, 74.7-93.0) 10(16.1, 6.9-25.3) Provision of funds for mobility, contingencies, etc., is key for success 48(77.4, 67.0-87.8) 14(22.6, 12.2-32.9) Involving various local authorities in vaccination will strengthen the programme 45(72.6, 61.5-83.7) 17(27.4, 16.3-38.5) Organising farmer-awareness meetings is one way to make the programme better 57(91.9, 85.2-98.7) 5(8.1, 1.3-14.8) Figures in parentheses indicate percentages and 95% confidence intervals. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000059

This study aimed to provide information on the presence and frequency of viral and parasitic agents in wildlife presented to a Veterinary Teaching Hospital in 2020-2021. Serum and faecal samples were collected from 50 rescued animals (roe deer, fallow deer, foxes, badgers, pine martens, and porcupines) and examined by serological, molecular, and parasitological techniques. Transtracheal wash (TTW) was also collected post-mortem from roe deer. Overall, the results of the different techniques showed infections with the following viral and parasitic agents: Bovine Viral Diarrhea Virus, Small Ruminant Lentiviruses, Kobuvirus, Astrovirus, Canine Adenovirus 1, Bopivirus, gastrointestinal strongyles, Capillaria, Ancylostomatidae, Toxocara canis, Trichuris vulpis, Hymenolepis, Strongyloides, Eimeria, Isospora, Dictyocaulus, Angiostrongylus vasorum, Crenosoma, Dirofilaria immitis, Neospora caninum, Giardia duodenalis, and Cryptosporidium. Sequencing (Tpi locus) identified G. duodenalis sub-assemblages AI and BIV in one roe deer and one porcupine, respectively. Adult lungworms collected from the TTW were identified as Dictyocaulus capreolus (COX1 gene). This is the first molecular identification of G. duodenalis sub-assemblage AI and D. capreolus in roe deer in Italy. These results show a wide presence of pathogens in wild populations and provide an overview of environmental health surveillance. wildlife central Italy viruses helminths protozoa interspecies transmission zoonotic pathogen Fondi di Ateneo (Univesity of Pisa)Maurizio MazzeiMicaela SgorbiniRoberto Amerigo PapiniThis research was funded by Fondi di Ateneo (Univesity of Pisa) and Maurizio Mazzei. Micaela Sgorbini, and Roberto Amerigo Papini. pmc1. Introduction In recent decades the incidence of introduced and emerging infectious diseases in humans and animals has increased worldwide . Wildlife plays an important role in the epidemiology of infectious and parasitic diseases, resulting in a source of various pathogens with significant implications on human health, wild and domestic animal health, biodiversity, and economy, as it harbours most of the emerging and re-emerging pathogens and could play a significant role in their spread and persistence in ecosystems . Numerous studies on wildlife have shown that wild animals act as hosts for some known and unknown pathogens that are transmissible to several other species, including domestic animals and humans. Therefore, in recent years, research on wildlife diseases has become an increasing international interest as a crucial aspect of wildlife conservation projects, such as reintroduction and translocation programmes, especially for species of high conservation value, and programs of disease surveillance for domestic animals and humans . Unfortunately, the elusive and unique nature of these animals makes it difficult to sample and conduct health surveillance actions in these species and to understand their real role in the epidemiology and ecology of diseases . Wildlife rescue centres can play an important sentinel role and represent an underutilised source of information on pathogens circulating in ecosystems, particularly at the wildlife-domestic animal interface . Due to the increasing proximity of wild animals to urban areas, often in search of food, and the growing public awareness of biodiversity conservation, the number of wild animals admitted to rescue centres is steadily increasing. Each year, rescue centres recover hundreds of animals belonging to many different species, some of which were impossible to sample in the wild. In addition, wildlife rescue centres represent a system in which many injured, diseased, and stressed animals interact and where the risk of introducing, amplifying, and spreading pathogens is considerable. Similarly, the release of rescued animals into the wild habitats can lead to the spread of pathogens in the area, posing a health risk to the local population. It is, therefore, important to gather information on the pathogens that are or could be circulating among hospitalised animals. Bovine viral diarrhoea virus (BVDV), family Flaviviridae, infects a variety of ungulate species and is associated with severe economic losses in livestock production worldwide. Several wild and domestic ruminant species cohabit during the grazing period, and BVDV circulation has been demonstrated in both domestic and wild ruminants and interspecific transmission has already been reported . In Italy, a serosurvey was carried out in four large alpine ungulates in the High Valley of Susa, and antibodies were detected in chamois, wild boar, and red deer, but all roe deer were negative . A similar result was obtained a few years later by Fernandez-Sirera and colleagues. Small Ruminant Lentivirus (SRLV), genus Lentivirus, family Retroviridae, are widespread throughout the world and have a detrimental economic impact on the small ruminant industry due to increased mortality and reduced animal performance . Bopivirus is a novel genus of the family Picornaviridae recently discovered in faecal samples from wild and domestic ruminants. To date, genus Bopivirus contains the species Bopivirus A, detected in cattle, the novel Bopivirus B, detected in sheep and goats in Hungary; and the newest, tentatively designed Bopivirus C, detected for the first time in faeces of Australian fallow and red deer . Astroviruses (AstV) are small, round, non-enveloped RNA viruses. First discovered in children with diarrhoea, they have been discovered in a variety of domestic and wild animals, both with and without gastroenteritis . The wide host range and genetic diversity within the Astroviridae family have made classification difficult, and they are currently divided into two genera, Mamastrovirus (with 19 species) and Avastrovirus (with 3 species), infecting mammalian and avian hosts, respectively. However, numerous unclassified AstVs have yet to be accepted as species . Kobuvirus is a genus belonging to the Picornaviridae family and consists of three species, Aichivirus A (formerly Aichi virus), Aichivirus B (formerly Bovine kobuvirus) and Aichivirus C (porcine kobuvirus). First recognised in humans with gastroenteritis, kobuviruses have recently been detected in a variety of animals in several countries with an ever-increasing number of hosts. In particular, they have been found in domestic and wild carnivores, ruminants, and suids . Canine adenovirus 1 (CAdV-1), family Adenoviridae, genus Mastadenovirus, is the causative agent of infectious canine hepatitis in dogs and is characterised by high pathogenicity due to differential tissue tropism. In addition to the dog, CAdV-1 infection has been documented in several free-ranging wild mammalian carnivores, foxes, wolves, brown bears, striped skunks, and Eurasian otters, causing from subclinical to fatal diseases and sporadic epizootics . As far as parasitic agents are concerned, coccidia are protozoa with a direct life cycle that infect the epithelial cells of the intestinal tract of their hosts and that usually have high host-specificity. The following Eimeria species are found in roe deer: Eimeria capreoli; Eimeria catubrina; Eimeria panda; Eimeria patavina; Eimeria ponderosa; Eimeria rotunda; and Eimeria superba . Instead, Isospora canis, Isospora canivelocis, Isospora ohioensis, Isospora pavlodoratica, Isospora vulpina, and Isospora vulpis may occur in red foxes, though it must be considered that coccidia of canids is an exceptionally confused group of coccidia . Eimeria melis and Isospora melis have been reported in badgers . Gastrointestinal strongyles (GSI) are very common among wild ruminants and always occur in mixed infections with different nematode species whose eggs are morphologically indistinguishable from one another. Infections with helminths belonging to the genera Bunostomum, Chabertia, Haemonchus, Nematodirus, Oesophagostomum, Ostertagia, and Thychostrongylus have been reported in the cervids . Hosts get infected by direct ingestion of third-stage larvae whilst grazing. Capillaria bovis and Capillaria bilobata have been reported in roe deer . This intestinal nematode of ruminants has a direct life cycle and is considered to be of low pathogenic significance. Dictyocaulus capreoli has previously been reported as a parasitic agent of bronchopulmonary infection in roe deer . In particular, this lungworm is a specific parasite for C. capreolus, as the name suggests, and Alces alces (moose) . The life cycle of the Dictyocaulus species is direct. Adult lungworms live in the bronchi and bronchioles of their hosts. Females are ovoviviparous. First-stage larvae hatch in the respiratory or intestinal tract and are expulsed with the faeces into the environment. Hosts get infected by ingesting L3 larvae whilst grazing. Intestinal worms (Toxocara canis, Trichuris vulpis, Ancylostomatidae), lungworms (Capillaria spp.), and heartworms (Dirofilaria immitis, Angiostrongylus vasorum) can be found in red foxes. Their life cycle can be direct (T. vulpis, Capillaria spp.), indirect (D. immitis, A. vasorum), or both (T. canis, Ancylostomatidae), depending on the species. Within the family Ancylostomatidae, both Ancyclostoma caninum and Uncinaria stenocephala have been reported in V. vulpes . Among Capillaria species, both Capillaria aerophila (syn. Eucoleus aerophilus) and Capillaria boehmi (syn. Eucoleus boehmi) are commonly found in the respiratory system of fox populations . Overall, within the family Ancylostomatidae, four species belonging to the Uncinaria or Tetragomphius genera and unidentified species belonging to the Ancylostoma genus have been reported in badgers. Uncinaria criniformis is the species widely reported in European badger populations . Lungworm species reported in Meles include Crenosoma melesi and Crenosoma vulpis , as well as C. aerophile . Crenosoma spp. has an indirect life cycle with terrestrial gastropods as intermediate hosts, while C. aerophila has a direct life cycle. Strongyloides is a genus of small parasitic nematodes infecting the small intestine of mammals, characterised by an unusual life cycle involving one or more generations of free-living adult nematodes. Strongyloides sp. infections have been reported in European badgers . Results of phylogenetic analysis in different carnivorous hosts suggested that badgers can be infected with a distinct species of Strongyloides . The European badger may also be a definitive host for three species of heartworms. These include D. immitis as well as A. vasorum and Angiostrongylus daskalovi, which was considered a relatively rare species . Angiostrongylus spp. has snails and slugs as intermediate hosts, while D. immitis is transmitted by mosquito bites. Reported prevalence values in badgers are 0.87% for D. immitis, 6.4 to 24% for A. vasorum, and 72% for A. daskalovi . Neospora caninum, an apicomplexan protozoan, is among the main causes of abortion in cattle and has both horizontal and vertical transmission patterns. The existence of a sylvatic life cycle of N. caninum has been supported by some authors . Giardia duodenalis is a flagellate protozoan with a direct orofecal life cycle. Molecular investigations have revealed that G. duodenalis is a complex species, including eight morphologically indistinguishable genot ypes or assemblages with different host ranges classified as A to H. Genotypes A and B infect humans but can also be found in animals with their respective sub-assemblages . The Veterinary Teaching Hospital (VTH) of the Department of Veterinary Sciences of the University of Pisa has been providing a 24-h emergency service for wild mammals rescued in the Pisa area since 2010. The aim of the present study was to retrospectively investigate the presence and frequency of different virological and parasitic agents in a cohort of 50 injured wild mammals admitted to the VTH from September 2020 to August 2021. In addition, the study area was divided into three zones based on the different levels of urbanisation, population density, and land use and differences in the prevalence of viral pathogens concerning the rescue area were also investigated. 2. Materials and Methods 2.1. Study Area Tuscany is the region of the Italian peninsula with the highest number of wild ungulates, with about 400,000 animals in constant increase, and it is estimated that 40% of roe deer, 45% of fallow deer, and 30% of wild boar of the entire country live there . The province of Pisa, composed of 39 municipalities, covers an area of 2450 km2; 25% of the territory is made up of plains, with most of the inhabited centres, and the rest of the territory consists of hills and mountains suitable for the development of numerous and diverse wildlife populations . Despite this, the data on land use shows that almost the entire territory of the province is used for industrial or agricultural purposes and that unused part is very limited . Therefore, the uncontaminated and uninhabited areas are very limited, and wildlife habitats are fragmented and interrupted by an extensive network of roads and urban areas with many opportunities for contact and interaction between wildlife, domestic animals, and humans. Moreover, the territory of the province of Pisa is very diverse due to its considerable size, with the main urban settlements located in the northern area, where, in addition to the city of Pisa, some of the most important inhabited centres are located. For this study, the province was divided into three zones of similar size, characterised by different demographic densities, road networks, and land use . The zones have been named Zone A, B, and C . Data on zone extension, number of inhabitants, population density, and forest cover, expressed as its relationship with the total area surface, are shown in Table 1 . 2.2. Enrolled Animals In this study, a cohort of 50 wild animals, injured and hospitalised during a one-year period (between September 2020 and August 2021), was retrospectively assessed for the presence and frequency of different virological and parasitic agents. The enrolled animals were older than 12 months (based on general conformation and teeth eruption), were of both sexes, and belonged to the following species: 23 roe deer (Capreolus capreolus); 4 fallow deer (Dama dama); 12 red foxes (Vulpes vulpes); 6 badgers (Meles meles); 4 porcupines (Hystrix cristata); and 1 pine marten (Martes martes). For each animal, information on the place and the cause of the rescue, as well as on age, sex, and species, was collected from the person who brought the animals (volunteers, citizens, local authorities) and during the first clinical examination and recorded in the hospital database. Data on the species and sex of the enrolled cohort are presented in Table 2. All the animals enrolled were hospitalised because they were injured. Overall, 34/50 (68%) of animals were injured in car accidents, 3/50 (6%) by predators, and 2/50 (4%) by fighting with conspecifics. In 11/50 (22%) animals, the cause for injuries could not be readily determined. 2.3. Sample Collection Upon admission to the VTH, during the first clinical examination, a blood sample was usually collected from the jugular or cephalic vein of each animal under sedation or general anaesthesia to reduce the stress and handling time . The blood sample was immediately used for routine laboratory analyses in order to perform a rapid diagnosis, while an aliquot was collected separately in a serum tube and centrifuged within 30 min after collection at 1000 rpm for 10 min. The serum sample obtained was stored at -20 degC to be used for future analyses. A faecal sample was also usually collected from each animal after natural voiding; an aliquot of the faecal sample was immediately stored at +5 degC and examined within 12 h for routine parasitological examinations (passive flotation, Baermann technique, coproantigen detection) while an aliquot was stored at -20 degC pending further analyses. Therefore, all the serological and molecular analyses on serum and faecal samples were performed after the period of hospitalisation and on stored frozen samples. 2.4. Viral Pathogens A species-specific serological and molecular analysis panel was designed in agreement with previously reported data in the available literature on the most commonly detected viral agents in the enrolled species . Molecular analyses were performed on faecal samples. 2.4.1. Serological Analysis Roe deer (n = 23) and fallow deer (n = 4) sera were tested for the detection of antibodies against Bovine Viral Diarrhea Virus (BVDV) and Small Ruminant Lentivirus (SRLV), and all animals' (n = 50) sera were tested for antibodies detection against Hepatitis E virus (HEV). BVDV and SRLV antibodies were detected using the BVDV p80 Ab (IDEXX, Westbrook, ME, USA) and by Eradikit(r) SRLV Genotyping Kit (IN3 diagnostic, Grugliasco, Italy), respectively, after adaptation. The SRLV kit is suitable for the detection and genotyping of SRLV antibodies as it can discriminate whether a positive sample belongs to genotype A (Maedi Visna-like), B (CAEV-like), or E (Roccaverano strain). HEV antibodies were tested in all enrolled animals using the HEV Ab version ULTRA kit (DIA.PRO, Sesto San Giovanni, Italy). Optical density was measured using a plate reader (Multiscan FC; Thermo Scientific, Waltham, MA, USA) at a wavelength of 450 nm. The procedures and interpretation analysis of the analyses were carried out according to the manufacturer's instructions. 2.4.2. Molecular Analysis Species-specific panels for molecular analysis are summarised in the Supplementary Material (Table S1). For species with little data in the literature about previous virological studies, such as porcupine and pine marten, large panels were developed that included viruses identified in related species. Due to the multiple species tested in this study and the paucity of data on virus circulation in wildlife, broad-spectrum PCR protocols with consensus primers covering as many viral species as possible were used. Molecular analyses were performed on DNA and RNA extracted from 200 mg of faecal samples using the AllPrep PowerFecal DNA/RNA Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA and RNA were eluted in 30 mL of Buffer AE and RNase-free water, respectively, and stored at -80 degC until analysis. PCR for the detection of Canine bocavirus-2 (CboV-2), Feline/canine parvovirus (FPV/CPV), Torque teno virus 1 and 2 (TTV-1, TTV-2), and Canine adenovirus (CadV), and both rounds of nested PCR for CboV-1 and 3 were performed on DNA using the HotStarTaq(r) Plus Master Mix Kit (Qiagen, Germany). RT-PCR for the detection of Canine distemper virus (CDV), Bovine viral diarrhea virus (BVDV), Bopivirus (BoPV), and Kobuvirus (KoV), and first rounds of the nested RT-PCR for Coronavirus (CoV) and Astrovirus (AstV) were performed on RNA using the OneStep RT-PCR Kit (Qiagen, Germany). Secondary rounds were performed using the HotStarTaq(r) Plus Master Mix Kit (Qiagen, Germany). The protocol used for CadV detection is reported to discriminate between CadV 1 and CadV 2, resulting in distinct amplicons of 508 bp and 1030 bp, respectively . Samples positive at molecular analysis were subjected to sequence analysis (BMR genomics, Padova, Italy) to confirm PCR results and obtain phylogenetic information. The primer sets used in the PCR and rt-PCR were included as Supplementary Data (Table S2) . PCR was performed according to the manufacturer's instruction. 2.5. Parasitological Investigations 2.5.1. Faecal Examinations A passive flotation technique was carried out on the refrigerated aliquots to detect nematode eggs and coccidian oocysts, using a commercial saturated solution of sodium chloride (specific gravity = 1.20) as flotation fluid (Coprosol(r), Candioli Farmaceutici, Beinasco, TO, Italy). The parasite burden was estimated by determining the number of eggs per gram (EPG) and oocysts per gram (OPG) of faeces by the modified McMaster method, with the lowest detection limit of 50 EPG/OPG per gram. In addition, faecal samples were examined via the Baermann technique to detect bronchopulmonary and cardiopulmonary nematode larvae. The number of larvae per gram (LPG) of faeces was estimated as above. The mean and range of OPG, EPG and LPG were calculated. 2.5.2. Commercial Assays A panel of endoparasite infections was investigated by rapid in-clinic tests in a different range of species. In particular, a rapid diagnostic test to detect both Cryptosporidium and Giardia coproantigens (Fastest(r) Crypto-Giardia Strip, Megacor Diagnostik, Austria) was performed on all 47 faecal samples examined for parasitological infections. Moreover, sera from foxes (n = 10) and badgers (n = 5) were screened for the presence of antibodies against Leishmania infantum (Snap Leishmania, Idexx, USA), Angiostrongylus vasorum (Angio Detect Test, Idexx, Westbrook, ME, USA), and Dirofilaria immitis (Snap Heartworm Antigen Test, Idexx, Westbrook, ME, USA). Screening of seropositivity for Neospora caninum (Fastest(r) Neospora caninum test kit, Megacor Diagnostick, Gemeinde Horbranz, Austria) was carried out on foxes, badgers, roe deer (n = 23), and fallow deer (n = 4). All the rapid in-clinic tests were performed according to their respective manufacturer's instructions. Finally, serum samples from all animals were screened for Toxoplasma gondii infection by a commercial ELISA kit (ID Screen(r) Toxoplasmosis Indirect Multi-species, IDVET, Montpellier, France) according to the manufacturer's protocol. 2.5.3. Transtracheal Wash Technique In 4/13 (30.8%) roe deer that tested positive for lungworm larvae via the Baermann technique (see above), a transtracheal wash (TTW) was performed immediately after humane euthanasia due to severe injuries. Briefly, a polyurethane catheter (14G) (Terumo, Roma, Italy) was introduced 5 cm distal to the larynx at 45deg to the skin between two tracheal rings identified by palpation. Once the catheter entered the tracheal lumen, the needle was retracted, and the stylet advanced. Then a catheter (1.3 mm x 50 cm) (Buster dog catheter, Kruuse, Denmark) was passed through the stylet, down the trachea, and towards the lungs, until resistance was found. A bolus of 30 Ml of sterile saline was injected using a 60 Ml syringe and the fluid was immediately aspirated to recover at least 10 Ml of fluid . When the aspiration of fluid was complete, the catheter and stylet were withdrawn simultaneously. The TTW fluid was split into two 4 Ml EDTA tubes for parasitological examination. When lungworm specimens were detected in the amount of fluid recovered, they were stored for 24-48 h in physiological saline at 4 degC before the morphological examination and then stored in 70% alcohol pending molecular procedures for identification to species level. 2.5.4. Molecular Analysis for Giardia duodenalis Genotyping and Cryptosporidium Species Identification Molecular investigation of G. duodenalis and Cryptosporidium was performed on faecal samples testing positive for coproantigens test (Fastest(r) Crypto-Giardia Strip, Megacor Diagnostik, Gemeinde Horbranz, Austria). Genomic DNA extraction was performed by QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Giardia genotyping was determined through the amplification of tpi and b-giardin genes. In detail, a 530 bp fragment of the tpi gene was obtained via a two-step nested PCR protocol using AL3543/AL3546 primers for primary PCR and AL3544/AL3545 for secondary PCR . A 384 bp fragment of the b-giardin gene was amplified with a forward primer G7, a forward inner primer G376, and a reverse primer G759 . Cryptosporidium genotyping was determined through the amplification of 800-850 bp of the GP60 gene. In a two-step nested PCR protocol, AL3531/AL3535 primers were utilized for primary PCR and AL3532/AL3534 for the secondary PCR . All amplicons obtained were purified using the mi-PCR Purification Kit (Metabion International AG, Planegg, Germania), and sequencing was performed in the laboratory of Bio-Fab Research (Rome, Italy). In order to check the quality of the sequences, the resulting chromatograms were manually analyzed using Finch TYV1.4 software (Geospiza, Inc, Seattle, WA, USA). Consensus sequences were then compared with those available in the GenBank database by using the Standard Nucleotide BLAST search and aligned by Clustal Omega implemented in MEGA X with representative sequences of tpi, bg, and GP60 loci and used as references. 2.5.5. Molecular Identification of Adult Lungworms Adult lungworms collected by TTW were identified to species level by PCR technique. Molecular analyses were performed by BMR Genomics (Padua, Italy, accessed on 7 January 2022). Genomic DNA was extracted from the stored adult lungworms using the commercial PCRBIO Rapid Extract Lysis Kit (PCR Biosystems) following the manufacturer's instructions. A fragment (about 700 base pairs in length) of the mitochondrial gene for cytochrome C oxidase subunit 1 (COX1) was used as a DNA barcoding system and amplified. The PCR amplification was carried out in a final mixture containing 12.5 mL of AmpliTaq GoldTM 360 Master Mix (Thermo Fisher), 2 mL of gDNA extracted, 1 mL (10 mM) of LCO1490 primer (5'-GGTCAACAAATCATAAAGATATTGG-3'), 1 mL (10 mM) of HCO2198 primer (5'-TAAACTTCAGGGTGACCAAAAAATCA-3'), and deionized water, reaching a total reaction volume of 25 mL. The PCR reactions were subjected to the following conditions in a thermal cycler (Mastercycler(r), Eppendorf): 95 degC x 2 min, then 5 cycles (95 degC x 40 s, 45 degC for 90 s, and 72 degC for 90 s), followed by 35 cycles (95 degC for 40 s, 50 degC for 90 s, and 72 degC for 60 s), and finally, 72 degC for 10 min. The amplification products were visualized after electrophoresis on 1.5% agarose gel. PCR products were purified with ExoSAP-ITTM (Thermo Fisher Scientific, Massachusetts, MA, USA), sequenced, and aligned via BLAST analysis to detect their identity by retrieving similar sequences deposited in NCBI's GenBank database . 2.6. Statistical Analysis The prevalence of virological and parasitic agents was determined as the number of positive animals/numbers of examined animals X 100. The Fisher test was used to determine statistical differences between rescue areas on positivity/negativity for Astrovirus, Kobuvirus, Bopivirus, and Adenovirus. 3. Results 3.1. Viral Pathogens 3.1.1. Serological Analysis One out of 23 roe deer (4.3%) scored positive for antibodies against BVDV and one for SRLV, genotype A Maedi Visna-like, while all the fallow deer were serologically negative for both viruses. The positive roe deer was a male rescued in zone B. All the animals (50/50, 100%) scored negative for HEV antibodies. 3.1.2. Molecular Examination All animals tested negative for CBoV, FPV, CPV, TTV, BVD, CDV, and CoV. Animal distribution in the three-rescue area and results of virological molecular analysis presented by viruses, species and rescue area are reported in Table 3 and Table 4. Six out of 50 animals (12%) tested positive for KoV: 1/23 roe deer; 2/12 foxes; 2/4 porcupines; and 1/6 badgers, respectively. All positive roe deer, badger, foxes, and one porcupine were male; the other porcupine was female. All positive animals belonged to zone A; thus, no statistical comparison among zones was performed. Eight out of 50 (16%) animals tested positive for AstV: 3/23 roe deer (1 male and 2 female) and 5/12 foxes (4 male and 1 female). Four out of 8 (50%) animals belonged to zone A (3 foxes and 1 roe deer), 3 (37.5%) animals belonged to zone B (2 foxes and 1 roe deer) and 1 (12.5%) roe deer belonged to zone C. No differences were found between zone A vs. B (p = 0.626), zone A vs. C (p = 0.536), or zone B vs. C (p = 0.779). Foxes (n = 12), badgers (n = 6), and pine martens (n = 1) were tested for CAdV, and 5 out of 19 (26%) animals tested positive for CAdV type 1. Three out of 12 foxes and 2/6 badgers were positive; all the positive foxes were male, 2 from zone B and 1 from zona A. Both positive badgers were male, 1 from zone A and 1 from B. No animals tested for CAdV have been rescued in zone C. No statistical differences were found in CAdV type 1 prevalence between zones A vs. B (p = 0.161). Roe deer (n = 23) and fallow deer (n = 4) were tested for BoPV, and 1 out of 27 (3%) tested positive for BoPV. This was a female fallow deer from zone A; thus, no statistical comparison among zones was performed. Table S3 shows the accession numbers of the sequences entered in the GenBank database, along with the results of their analysis with Blast software. For each sequence in this study, the analysis identified a reference sequence and determined the per cent of nucleotide identity and E value obtained from their comparison. 3.2. Parasitological Investigations Overall, parasitological analyses were performed in a total of 47/50 (94%) animals, including 10 foxes, 5 badgers, 4 porcupines, 23 roe deer, 4 fallow deer, and 1 marten, as faecal and serum samples from 2 foxes and 1 badger were not available. Results of faecal examinations by flotation and Baermann techniques are shown in Table 5. 3.2.1. Faecal Examinations Roe deer. Overall, 19/23 (82.6%) roe deer were found to be coprologically positive by flotation and/or Baerman techniques. Ten out of 23, 8/23, and 1/23 roe deer showed two, one, or three parasitological infections, respectively. Eggs of gastrointestinal strongyles (mean = 7, range <= 50-100 EPG), Eimeria oocysts (mean = 250, range <= 50->1000 OPG), and Capillaria spp. eggs (mean <=50 EPG), were found in 14/23, 5/23, and 2/23 roe deer, respectively. Lungworm larvae (mean = 23, range <= 50-150 LPG) consistent with first-stage larvae of Dictyocaulus sp. were detected in 13/23 (56.2%) roe deer. Red foxes. All foxes (10/10, 100%) were found to be parasitised. Two, three, four, and even six parasitic co-infections were detected in 5/10, 2/10, 1/10, and 1/10 foxes, respectively. Therefore, only one fox out of 10 showed a single parasitic infection with coccidia oocysts. Parasite prevalence was as follows: Ancylostomatidae eggs (mean = 344, range <= 50->1000 EPG) were found in 8/10 foxes; Toxocara canis eggs (mean <=50 EPG) in 5/10; Capillaria spp. eggs (mean = 75, range <= 50-300 EPG) in 4/10; Trichuris vulpis eggs (mean <=50 EPG) in 3/10; and coccidia oocysts (mean = 600, range = 200->1000 OPG) in 2/10. In addition, the spurious passage of Hymemolepis eggs (<50 EPG) was detected in 1/10 foxes. Larvae of A. vasorum (mean = 275, range < 50->1000 LPG) were detected in stool samples from 4/10 foxes. Badgers. All badgers (5/5, 100%) harboured parasites. Infections with two and four parasitic agents or only a single infection were detected in 3/5, 1/5, and 1/5 of them, respectively. Ancylostomatidae eggs (mean = 80, range <= 50-300 EPG) and larvae of Crenosoma sp. (mean = 137, range <= 50-400 LPG) were found in stool samples from 5/5 and 4/5 badgers, respectively, while Strongyloides eggs (<50 EPG), Capillaria spp. eggs (<50 EPG), and coccidia oocysts (<50 OPG) were detected in 1/5 each. All the fallow deer, all the porcupines, and the marten were found negative for both the flotation and Baermann techniques. 3.2.2. Commercial Assays Results of the rapid in-clinic tests and the ELISA kit are summarized in Table 5. Briefly, eleven, six, and two samples from different host species showed positive results for N. caninum, G. duodenalis, and C. parvum, respectively. Six and four samples from foxes tested positive for A. vasorum and D. immitis, respectively, and one sample from badgers tested positive for D. immitis. No samples tested positive, either for L. infantum or T. gondii. 3.2.3. TTW Results Four adult lungworms were detected in the TTW fluid from one of the four (25%) roe deer found positive for Dictyocaulus sp. larvae via the Baermann technique. The specimens measured on average about 5 cm in length and 0.4 mm in width at the mid-body and had an elongated oral opening, dorso-ventrally flattened. Based on the length of the body and the shape of the oral opening, the lungworms were presumptively identified as Dictyocaulus sp. 3.2.4. Molecular Identification of Giardia duodenalis and Cryptosporidium spp. A total of six samples for G. duodenalis and two for Cryptosporidium spp., carried out positive by the coproantigens test, were tested by the two-step nested PCR protocols. By sequencing at the tpi locus, 1/6 (16.7%) sample from a roe deer and 1/6 (16.7%) from a porcupine were assigned to Assemblage A-AI and Assemblage B-BIV, respectively. Sequences obtained in this study were deposited in the GenBank database and are available under the accession numbers OP946512 and OP946513, respectively. 3.2.5. Molecular Identification of Adult Lungworms The BLAST search results showed that COX1 of adult lungworms collected from a euthanized roe deer had 99-100% DNA homology with sequences of Dictyocaulus capreolus available in the GenBank database. The sequence obtained in this study was deposited in the GenBank database and is available under the accession number: OQ209836. To the best of our knowledge, this is the first molecular identification of D. capreolus in roe deer in Italy. 4. Discussion Serological surveys in wildlife are a diagnostic tool to demonstrate the presence of antibodies against specific pathogens and to provide epidemiological data on pathogens; however, serological sampling in wild animals is not always feasible and can be very stressful for the animals themselves. In the present study, the hospitalisation of wild animals allowed blood sampling during clinical practice, and we decided to investigate by serological analysis a panel of pathogens for which direct virological diagnosis on faecal samples is not applicable. The detection of BVDV antibodies indicates the presence and circulation of the virus in wild animals; moreover, the absence of vaccination plans for wildlife removes doubts about possible vaccine seropositivity. In our survey, one roe deer out of 21 was serologically positive. This result is in agreement with data from European studies, which reported the absence of seropositive animals (Spain and England) up to 12% of seroprevalence (Norway) . In Italy, a serosurvey was carried out in four large alpine ungulates in the High Valley of Susa, and antibodies were detected in chamois, wild boar, and red deer, but all roe deer were negative . A similar result was obtained a few years later when Fernandez-Sirera and colleagues tested domestic ruminants, cattle, sheep, wild ruminants, chamois, and roe deer in the same research area for the presence of antibodies against BVD. High prevalence was found in both domestic ruminants, 25% in cattle, 17% in sheep, and 42 % in chamois, but none of the 213 roe deer tested positive . The results of the Italian studies and the low seroprevalence found throughout Europe, even in areas with regular contact with domestic animals with high prevalence, suggest that roe deer play a marginal role in the epidemiology of the virus and that they are mostly an occasional guest and does not represent a major risk to domestic ruminants. The low prevalence of the virus among the European roe deer populations compared to other wild deer, especially the chamois, is explained by the different habits of the roe deer. The roe deer is mainly a solitary animal that prefers bushy feeding areas to open pastures where it would be easy to encounter domestic ruminants . In addition, it is possible that this species has low susceptibility to common pestiviruses. Indeed, Fischer and colleagues have discovered a new pestivirus subtype in roe deer , and this may influence the serological results. However, as the presence of BVDV infection in roe deer is rarely detected, it would be advisable to conduct further surveys in the study area to increase the number of samples and obtain a more reliable estimate of prevalence. In addition, 1 roe deer tested positive for SRLV, genotype A Maedi Visna-like. Although SRLV were originally thought to be species-specific pathogens, several lines of evidence showed that lentiviruses can efficiently cross the species barrier and adapt to new hosts . Several studies reported the detection of SRLV antibodies in wild ruminants: Rocky Mountain goats; red deer; roe deer; and mouflon . Therefore, it is possible that contact between wild and domestic small ruminants could result in cross-transmission of SRLV. Regarding viral molecular analysis, positivities in foxes (CAdV-1, KoV, AstV), roe deer (KoV, AstV,) fallow deer (BoPV), badgers (CAdV-1, KoV), and porcupine (KoV) have been detected. A recent investigation in Italy revealed the presence of bopiviruses in sheep, roe deer, chamois, and Alpine ibex, with sequences clustering with bopivirus strains previously detected in goats and sheep from Hungarian farms and in red deer with sequences closely related to Bopivirus C sequences identified in fallow and red deer in Australia . In our study, one fallow deer sample resulted positive for Bopivirus, whereas all samples from roe deer were negative. According to BLAST analysis, the sequence shared 94-92% nucleotide (nt) identity with deer Bopivirus strain (Bopivirus C), 88-85% with bovine Bopivirus strain (Bopivirus A), and 72-70% with ovine-goat Bopivirus strain (Bopivirus B). The sample, therefore, showed the highest nucleotide identity to Bopivirus C previously identified in Australian fallow deer and Italian red deer. Although numerous viruses of the Picornavidae family are responsible for diseases with relevant clinical forms in humans and animals, the pathogenicity of Bopivirus in deer remains undetermined. The population In our study, as in previous studies, was apparently healthy or not affected by symptoms attributable to an infectious disease. Astroviruses were detected in foxes and roe deer representing, to our knowledge, the first report of astrovirus in these two species in Italy. Astroviruses have already been identified in foxes in the Netherlands and Australia using metagenomic techniques. In the first study, the sequences were mainly identified as two novel astroviruses called Fox Astrovirus F5 and F4, in the second, sequences clustered with feline astrovirus with high nt identity. Little research has been reported on AstVs in deer, but astroviruses have been identified in European roe deer in Denmark in 2010 from animals with gastrointestinal disease, and in Slovenia from healthy hunted animals . The following sequence analysis identified a new species of Mamastrovirus, the Capreolus capreolus Astrovirus (CcAstV strain 1 and 2), related to other bovine, porcine, yak, porcupine, and dromedary AstVs strains, possibly forming a group of currently unassigned sequences that are distantly related to mAstV genogroups I and II. Our sequences, both from foxes and roe deer, were obtained using broad-range primers amplifying a portion of RdRp without phylogenetic information; however, BLAST analysis of the amplicons confirms that this virus clustered with other mammalian-associated viruses within the mamastrovirus genera. In general, AstV infection is associated with mild to severe gastrointestinal tract disease, typically with diarrhoea and vomiting , but has also been associated with other diseases, such as shaking syndrome in mink, neurological disease in cattle and sheep, and encephalitis in humans ; infection may be asymptomatic . There is no information on the symptoms in foxes, although it is likely that CcAstV can cause gastroenteritis in roe deer. In our study, the sampled animals did not have gastrointestinal disease or other disorders attributed to astroviruses. In Italy, kobuviruses have been detected in dogs, foxes, wolves, roe deer, goats, swine, cats, and cattle . In this study, kobuviruses were detected in two foxes, one roe deer, one badger, and two porcupines, and none of the positive animals showed symptoms suggestive of enteritis. In foxes, Canine kobuvirus (CaKoV) has been detected in free-ranging foxes in Italy with a prevalence of 14.7%. To date, the known host range of CaKoV includes several species of Canidae (domestic dog, red fox, wolf, golden jackal, and side-striped jackal), and the virus has been detected in both diarrhoeic and asymptomatic animals, so the pathogenic potential of kobuviruses in carnivores remains to be elucidated. Our sequence of RdRp partial region from fox shared the highest identity with the CaKoV strains from Italian foxes with an nt sequence identity of 89-94% showing E-value ranging from 2e-30 to 9e-39. CaKoV is often found in co-infections as a potential secondary infection after primary infections by mainly viral agents responsible for immunosuppression, such as canine distemper virus or parvovirus . In the studies of Di Martino and colleagues, both a fox and some dogs were co-infected by Kobuvirus and cCoV . In our study, all foxes were negative to molecular analysis for CPV, CDV, and cCoV, but two foxes positive for kobuvirus were also PCR-positive for astrovirus, and one also for adenovirus; both were responsible for clinical gastroenteritis. Coinfection with CaKoV and cAstV and with CaKoV and cAdV has been described in diarrhoeic dogs in China and Korea, respectively . In roe deer, kobuvirus RNA was found in 6.6% of rectal swabs collected from healthy roe deer in northern Italy between 2012 and 2014; most of these strains showed high homology with bovine kobuvirus, and others with caprine kobuvirus . In our study, kobuvirus has been detected in 1/23 roe deer with a prevalence in line with previous studies. BLAST analysis of the partial 3D gene sequence showed 75% and 71% of nucleotide identity, respectively, with bovine and caprine kobuvirus detected in Italian roe deer, while the highest identities were shared with Italian fox kobuvirus. This result contrasts with what resulted in the previous study and it cannot be excluded that the roe deer may have ingested food contaminated with faecal contents of other species. However, it is important to consider that this is the second study to report Kobuvirus in wild ruminants and that very little is still known about which strains circulate among the populations of these animals and about the possibility of transmission of strains between different species. Kobuvirus has only been identified in one member of Mustelidae, a domestic ferret in the Netherlands, using a metagenomic approach, and the almost complete kobuvirus genome clustered with bovine and ovine kobuvirus (Aichivirus B) possibly indicates a past cross species transmission events . No data is currently present about the circulation of kobuvirus in free-ranging mustelids that share the ecosystem with other wild animals susceptible to infection. In the present study, kobuvirus has been identified in 1/6 badger representing its first detection in this species. The badger resulted also positive for adenovirus. Our sequence clustered with sequences of CaKoV detected in wild and domestic carnivores with an nt sequence identity of 85-87% and an e value of 3 e-80-1 e-65. Finally, in our study, kobuvirus was also identified in two porcupines representing its first detection in this species. To date, kobuvirus has been detected in other rodents, such as rabbits, grey squirrels, and some species of mice (Peromyscus crinitus, Apodemus agrarius, Rattus norvegicus, Rattus losea, and Rattus argentiventer, Mus musculus, Rattus losea, Rattus tanezumi, and Rattus norvegicus) . The BLAST analysis of our sequence showed a high homology both with both CaKoV (90-95% of nt identity, e value of 9 e-98-2 e-42), and murine kobuvirus (85-90% of nt identity, e value of 6 e-81-1 e-64), both belonging to the same genetic cluster within Aichivirus A, and more in-depth phylogenetic studies would be needed to better classify the virus. Interestingly, a high percentage of nt identity resulted between all our sequences (87-95%). This evidence, together with the common origin of all positive animals from zone A, suggests the possibility that a similar kobuvirus could infect different animal species sharing the same habitat. Moreover, three foxes and two badgers tested positive for cAdV-1. In foxes, cAdV was first identified in 1925 as the causative agent of severe neurologic disease, the epizootic fox encephalitis . Subsequently, evidence of cAdV circulation in foxes has been reported in several geographical areas, with serological prevalence in the European population ranging from 3% in Germany to 64.4% in the UK . In Italy, and more specifically in the Pisan area, the virological prevalence reported in previous studies is around 28% , which is in line with the value of 25% obtained in our study. All the positive animals were traumatised, and none showed clinical signs attributable to infectious diseases. Our data, together with the high prevalence values reported worldwide and the identification of the virus in healthy animals reported in previous studies , suggest that red foxes may play a role in maintaining cAdV-1 in the territory and may be a source of viral spread in wild ecosystems. In addition, although cAdV-1 can be controlled in domestic dogs by vaccination, cases of infectious canine hepatitis in domestic dogs have been diagnosed in Italy in recent years , and the presence of infected foxes may also be a source of infection for domestic dogs, especially foxes living in peri-urban areas . Adenovirus has previously been detected in Mustelidae, in a young striped skunk with hepatitis, in a seriously diseased Eurasian otter in the Seoul Grand Park Zoo, and in several pine martens and Eurasian otters in the UK, with prevalences of 33% and 88%, respectively . However, our evidence is the first detection of adenovirus in badgers. Both fox and badger viral sequences clustered with Canine adenovirus 1 with a high percentage of nt identity (94-98%). The presence of cAdV-1 in badgers was expected as specific adenoviruses have also been identified to date in otters and pine martens (marten adenovirus and lutrine adenovirus) and it is already known that mustelids are susceptible to CAdV-1 infection . Moreover, the home ranges of mustelids and other predators such as red foxes (Vulpes vulpes) may overlap and there is potential for cross-infection with pathogens through indirect contact with urine, faeces, and other infected fomites. Moving on to discuss the parasitological findings, although coccidiosis was presumably harmless for the animals examined in this study, coccidia may pose a major threat to some species of wildlife; for instance, juvenile coccidiosis may be associated with impaired growth and increased mortality in badger cubs . In this study, oocysts of Eimeria were detected in fecal samples from roe deer, red foxes, and badgers, but identification to species level could not be reached. Normally GSI are of little clinical significance in wild ruminants, but if combined, they can cause profuse and watery diarrhea, anaemia of varying degrees, edema under the lower jaw extending along the abdomen, progressive weight loss, rough hair coat, anorexia, hypoproteinemia, reduced growth, and even death in severe cases . Since a much higher prevalence of C. bovis (26.6%) than C. bilobata (6%) has been detected in C. capreolus , it is likely that C. bovis was the Capillaria species involved in roe deer of this study. Adults of D. capreoli were detected in the transtracheal wash fluid from a roe deer, as shown by the results of the molecular analysis. D. capreoli has been reported to cause anorexia, poor haircoat quality, delayed haircoat change, diarrhea, small apical atelectatic areas, peri-bronchial inflammation, and total bronchopneumopatia in roe deer . In agreement with previous studies carried out in Italy , combining results of coprological analyses and commercial assays, intestinal worms, lungworms, and heartworms were found in red foxes. It has been reported that the infection with intestinal helminths, especially T. canis, and the number of helminth species per individual may negatively affect the host body condition in red foxes . All parasites of foxes are shared with dogs , and some species can be zoonotic agents . In particular, foxes may be considered an important source of infections with cardiopulmonary nematodes, which may be responsible for overt clinical disease in dogs . The eggs of A. caninum and U stenocephala cannot be reliably distinguished microscopically. Likewise, eggs of C. aerophila and C bohemi are microscopically indistinguishable from one species to another because all Capillaridae eggs are morphologically characterized by a similar barrel shape with polar plugs . Therefore, it was not possible to determine whether the foxes examined in this study were infected with A. caninum, U. stenocephala, or both and with C. aerophila, C. boehmi, or both. In addition, the spurious expulsion of Capillaria hepatica (syn. Calodium hepaticum) eggs, after preying or scavenging on infected hosts, cannot be excluded. Indeed, foxes are known to scavenge on carcasses and eat a wide range of rodents, including mice and rats, which commonly show a very high prevalence of infection with C. hepatica . In this study, the spurious expulsion of typical eggs of Hymenolepis spp. was detected. Hymenolepis is a genus of zoonotic cestodes that commonly infect rodents, mostly mice and rats, with usually a high prevalence . This suggests that at least some of the foxes examined were feeding on rodents at the time of sampling and that foxes may act as carriers for the dispersal of the eggs of Hymenolepis species into the environment. Conversely, though Capillaria plica may be highly prevalent in foxes in central Italy , shedding of C. plica eggs in faeces was excluded because adult parasites of C. plica live in the bladder of definitive hosts, and eggs are expulsed with urine. Within the family, Ancylostomatidae U. criniformis is the species widely reported in European badger populations . Therefore, it may be suggested that U. criniformis was the species infecting badgers examined in this study. Lungworm infections in M. meles can be identified based on the typical morphology of L1 larvae (Crenosoma spp.) and eggs (C. aerophila). However, the limited information available on the morphological differences between L1 larvae of C. melesi and C. vulpis did not allow differentiation from one species to another. Therefore, it is likely that C. aerophila was the Capillaria species involved in the badgers of this study, but it was not possible to determine whether C. melesi, C. vulpis, or both were infecting the badgers examined. Similarly, to foxes, the shedding of spurious C. hepatica eggs, merely passing through the intestinal tract, cannot be completely excluded. Indeed, C. aerophila and C. hepatica eggs are hardly distinguishable by microscopy, while badgers are known to be scavenger animals and to eat small mammals, including mice and rats (often heavily infected by C. hepatica, as aforementioned). Oval, thin-shelled eggs with a fully developed coiled larva inside (typical of rhabditiform nematode parasites) were found in a badger faecal sample and were identified as eggs of Strongyloides sp. To our knowledge, the identification of Strongyloides species infecting the European badger is still unknown since Strongyloides specimens recovered in M. meles have not been identified to the species level . Strongyloides specimens isolated from the Japanese badger (Meles anakuma) are closely related to Strongyloides procyonis on a phylogenetic tree and are morphologically similar to S. procyonis and Strongyloides martis . However, the results of molecular phylogenetic analyses using mitochondrial genome sequencing as a marker suggested that they belonged to a species genetically separate from S. procyonis Concerning Leishmania and Toxoplasma, it cannot be excluded that the negative results found in this study may be due to limitations (uncertain sensitivity) attributable to the screening tests. A rapid immunochromatographic test (marketed for dogs) has been previously used to determine the seroprevalence of anti-Leishmania antibodies in wild canids, such as red foxes and coyotes . Likewise, a commercial indirect multi-species ELISA (marketed for ruminants, swine, dogs, and cats) has been previously used to determine the seroprevalence of anti-Toxoplasma antibodies in free-ranging and captive large African carnivores . Therefore, at present, it can be assumed that these two commercial assays may be suitable for wildlife species, but they have not yet been validated for the diagnosis of Leishmania and Toxoplasma infections in the species examined in this study. Neospora caninum-antibodies were detected in 3/4 fallow deer, 7/23 roe deer, and 1/5 badgers. These findings agree with those of other studies where the reported seroprevalence values for Neospora were 11% in fallow deer and 28% in roe deer , while 10.9% of badgers were found positive for Neospora DNA in brain, liver, or neck muscles . Red foxes are considered natural intermediate hosts for N. caninum, with reported prevalences of 10.7% by the PCR to 69.8% by the agglutination test . However, no foxes tested positive for Neospora in this survey. The possible existence of a sylvatic life cycle of N. caninum has been postulated . Therefore, the present results suggest that fallow deer, roe deer, and badgers may contribute to the sylvatic life cycle of N. caninum, if any, in some areas of central Italy. Further investigations are needed to elucidate the role they may play in maintaining the circulation of the parasite in nature. For the detection of A. vasorum infection in red foxes and badgers, the Angio Detect test was used in combination with the typical recovery of cardiopulmonary larvae by the Baermann technique. Angio Detect is commercialized as an in-clinic assay to detect A. vasorum circulating antigen in dogs but it has also been successfully used in other host species, such as red foxes and badgers . Likewise, commercially available antigen detection tests, commercialized for use in dogs, have been successfully used to identify D. immitis infection in other host species, such as wolves and red foxes, coyotes, and island foxes . In this study, the use of the Angio Detect rapid test enhanced the global sensitivity of the diagnostic procedure in foxes as it allowed the detection of two additional positive subjects concerning the traditional Baermann apparatus. It is likely that these two A. vasorum-antigen positive/Baermann negative foxes were in the prepatent period that characterises the early phase of any parasitic infection. The European badger may be a definitive host for the cardiopulmonary nematodes D. immitis, A. vasorum, and A. daskalovi . In this study, D. immitis circulating antigen was detected in 1/5 badgers by the in-clinic test, but no one was found positive either for circulating antigen or larvae of Angiostrongylus spp. In this study, the molecular analysis of G. duodenalis was successfully amplified at the tpi locus in one roe deer and in one porcupine. Among isolates of G. duodenalis from C. capreolus, assemblage A was previously identified in the Netherlands and Poland, while assemblage B was detected in Poland . Identification to sub-assemblage level was not carried out in those investigations. G. duodenalis sub-assemblage AII was reported in roe deer in Spain . In the present study, sub-assemblage AI was identified. To the best of our knowledge, this is the first report of G. Duodenalis sub-assemblage AI in C. capreolus in Italy. This sub-assemblage has previously been isolated in other wild species in the same geographical area . Likewise, the finding of G. duodenalis sub-assemblage BIV in a porcupine is consistent with data previously reported for the same host species in the same geographical area . G. duodenalis sub-assemblages AI and BIV are mainly found in animals and, to a lesser extent, in humans. Therefore, they are considered potentially zoonotic . 5. Conclusions The results highlight the widespread presence of pathogens in the population studied, confirming the role of wildlife in the epidemiology of several viruses and parasites. Although the impact on wild species' health is not yet known for all the pathogens identified, they can pose a threat to wildlife and can potentially have important effects on the population dynamics of their hosts, especially when they are experiencing the cumulative effects of other pathogens and stressors. Moreover, some of the parasitic agents found can be transmitted to domestic animals, and some of them are zoonotic. Therefore, they can affect the health status of livestock or pets and can have public health implications, especially in a highly urbanised area such as the study area. The study highlights the key role that structures admitting wild animals can play as focal points for collecting information on the health of wildlife populations and monitoring the health of the environments in which they live. Supplementary Materials The following supporting information can be downloaded at: Table S1: Summary of the specie-specific panels for virological molecular investigations; Table S2: The primer sets used in the study; Table S3: Accession number of the sequences entered on the GenBank database and results of their analysis with the Blast software. Click here for additional data file. Author Contributions Conceptualization, M.I.P., M.M., M.S. and R.A.P.; methodology, M.I.P., M.M., M.S. and R.A.P.; software, M.I.P. and M.S.; formal analysis, M.I.P., M.M., M.S., R.D. and R.A.P.; investigation, M.I.P., R.D. and R.A.P.; resources, M.M., M.S., R.D. and R.A.P.; data curation, M.I.P. and R.A.P.; writing--original draft preparation, M.I.P., M.M., M.S., R.D. and R.A.P.; writing--review and editing, M.I.P., M.M., M.S., R.D. and R.A.P.; supervision, M.S.; project administration, M.M., M.S. and R.A.P.; funding acquisition, M.M, M.S. and R.A.P. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Our study was carried out on injured animals admitted to the Veterinary Teaching Hospital (VTH). Since July 2010, the Fauna Defense Service of Tuscany has entrusted the VTH to provide a 24-h emergency service for wild animals found injured within the province of Pisa, and the ethical approval of clinical procedures was implicit in this agreement. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Map of the province of Pisa divided into the 3 zones. Figure 2 Development of the road network in the province of Pisa. Figure 3 Municipalities in the province of Pisa classified according to population density. Figure 4 Municipalities in the province of Pisa classified according to the forest/total surface ratio. animals-13-00931-t001_Table 1 Table 1 Data relating to area extension, number of inhabitants, population density, and forest extension (expressed as its relationship with the total area surface) for each zone. Surface (kmq) Population Population Density Forest/Total Surface Ratio ZONE A 824.4 328,873 398 0.3 ZONE B 588.3 63,313 107 0.2 ZONE C 939.6 25,763 27 0.5 animals-13-00931-t002_Table 2 Table 2 Number of enrolled animals presented by species and sex. Species Sex Total Males Females Roe deer 17/23 (74%) 6/23 (26%) 23 Fallow deer 3/4 (75%) 1/4 (25%) 4 Foxes 9/12 (75%) 3/12 (25%) 12 Badgers 4/6 (67%) 2/6 (33%) 6 Pine martens - 1/1 (100%) 1 Porcupines 2/4 (50%) 2/4 (50%) 4 Total 35/50 (70%) 15/50 (30%) 50 animals-13-00931-t003_Table 3 Table 3 Animal distribution in the three-rescue zones. Species Zone Total Zone A Zone B Zone C Roe deer 9/23 (39%) 11/23 (48%) 3/23 (13%) 23 Fallow deer 4/4 (100%) - - 4 Foxes 9/12 (75%) 3/12 (25%) - 12 Badgers 5/6 (83%) 1/6 (17%) - 6 Pine martens - 1/1 (100%) - 1 Porcupines 3/4 (75%) - 1/4 (25%) 4 Total 30/50 (60%) 16/50 (32%) 4/50 (8%) 50 animals-13-00931-t004_Table 4 Table 4 Results of virological molecular analyses presented by viruses, species and rescue zones. A, B, and C refer to rescue zones. CAdV 1 KoV AstV BoPV Roe deer -- 1 3 0 A B C A B C 1/9 0/11 0/3 1/9 1/11 1/3 Fallow deer -- 0 0 1 A B C 1/4 0/0 0/0 Foxes 3 2 5 -- A B C A B C A B C 1/9 2/3 0/0 2/9 0/3 0/0 3/9 2/3 0/0 Badger 2 1 0 -- A B C A B C 1/5 1/1 0/0 1/5 0/1 0/0 Pine Marten 0 0 0 -- Porcupine -- 2 0 -- A B C 1/3 0/0 0/1 Total 5 6 8 1 A B C A B C A B C A B C 2/14 3/5 0/0 6/30 0/16 0/4 4/30 3/16 1/4 1/13 0/11 0/3 animals-13-00931-t005_Table 5 Table 5 Parasite infections found by faecal flotation, the Baermann technique, and different commercial assays in the cohort of 47 wildlife animals examined. * GIS = Gastrointestinal strongyles; ^ ND = Not Determined. Host Species Faecal Flotation Baermann Technique Commercial Assays Giardia Cryptosporidium Toxoplasma Neospora Leishmania Dirofilaria Angiostrongylus Roe deer (n = 23) * GIS (10) Eimeria (5) Capillaria (2) Dictyocaulus (13) 1 0 0 7 ND ND ND Fallow deer (n = 4) Negative Negative 0 0 0 3 ND ND ND Red foxes (n = 10) Ancylostomatidae (8) T canis (5) Capillaria (4) T vulpis (3) Coccidia (2) Hymenolepis (1) Angiostrongylus (4) 1 1 0 0 0 4 6 Badgers (n = 5) Ancylostomatidae (5) Capillaria (1) Coccidia (1) Strongyloides (1) Crenosoma (4) 2 0 0 1 0 1 0 Porcupines (n = 4) Negative Negative 1 1 0 ND ^ ND ND ND Marten (n = 1) Negative Negative 1 0 0 ND ND ND ND Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000060

Cystic echinococcosis (hydatidosis) is a world-wide zoonotic disease of mainly humans, livestock and dogs, caused by Echinococcus granulosus. The disease can negatively impact food production and animal welfare and causes socio-economic hardship. Here, we aimed to identify the local bovine hydatid cyst fluid (BHCF) antigen for developing a sero-diagnostic assay to be used for the pre-slaughter screening of food animals. In total, 264 bovines approved for slaughter in Pakistan were subjected to serum collection and post-mortem screening for hydatid cysts. These cysts were assessed microscopically to assess fertility and viability, and by PCR for molecular confirmation of species. A BHCF antigen was identified from positive sera via SDS-PAGE, confirmed by Western blot, and quantified via a bicinchoninic acid (BCA) assay. The quantified crude BHCF antigen (iEg67 kDa) was then used in ELISA screening to test all sera collected from known positive and negative animals based on hydatid cyst presence/absence. Of the 264 bovines examined, 38 (14.4%) showed hydatid cysts during post-mortem examination. All of these individuals, plus an additional 14 (total: 52; 19.6%) tested positive based on less time-consuming ELISA examination. Based on ELISA, occurrence in females (18.8%) was significantly higher than in males (9.2%) and was higher in cattle (19.5%) compared to buffalo (9.5%). The infection rate increased with age in both host species: cumulatively, 3.6% in animals aged 2-3 years, 14.6% in 4-5-year-olds and 25.6% in 6-7-year-olds. The occurrence of cysts in cattle was significantly higher in the lungs (14.1%) compared to their livers (5.5%), whereas the opposite was true in buffalo (6.6% livers, 2.9% lungs). For both host species, most cysts in the lungs were fertile (65%), while the majority in the liver were sterile (71.4%). We conclude that the identified iEg67 kDa antigen is a strong candidate for the development of a sero-diagnostic screening assay for the pre-slaughter diagnosis of hydatidosis. zoonoses One Health neglected tropical disease Echinococcus granulosus hydatid cyst sero-diagnosis International Research Support Initiative Program1-8/HEC/HRD/2022/12612/IRSIP HEC-National Research Program for UniversitiesNRPU-7018 HEC-Grand Challenge FundGCF-273 Pakistan Agriculture Research BoardPARB-18-476 Punjab Higher Education CommissionPHEC/ARA/PIRCA/2020-6/8 S.K. received funding from Higher Education Commission (HEC) under the International Research Support Initiative Program (1-8/HEC/HRD/2022/12612/IRSIP 51 Agri 20); M.Y. received funding from HEC-National Research Program for Universities (NRPU-7018); H.A. and M.I.R. received funding from HEC-Grand Challenge Fund (GCF-273), Pakistan Agriculture Research Board (PARB-18-476) and the Punjab Higher Education Commission (PHEC/ARA/PIRCA/2020-6/8). pmc1. Introduction Pakistan has a large proportion of farmers entirely dependent on livestock for their livelihood. Livestock has a major role in the economy of Pakistan, contributing >60% to agriculture, and showing an annual growth rate of 3.06% per year and 11.5% for gross domestic product (GDP) (Pakistan economic survey 2020-2021). The livestock are affected by a range of infectious diseases that adversely reduce growth rates and overall production, particularly affecting food animals. Cystic echinococcosis (hydatidosis) is one such zoonotic disease, caused by the cestode Echinococcus granulosus that infects both humans and animals and has a cosmopolitan distribution . The disease occurs in South America, Eastern Europe, Russia, East Africa, Central Asia, Iran, Iraq, Syria, Jordan, Saudi Arabia and Pakistan . According to the Global Burden of Disease (GBD), 9186 symptomatic cystic echinococcosis patients were reported in Pakistan in 2016, and new cases reported in the same year totalled 2932 per 100,000 population (Global Burden of Disease Results Tool 2021). In many parts of the world, e.g., Mediterranean regions, the Americas, Asia, Australia, Africa, and Europe , echinococcosis is categorised as a neglected tropical disease . Two types of hosts are responsible for the transmission of Echinococcus spp.: ruminants and canines. Transmission to intermediate hosts (sheep, cattle, buffalo and goats) occurs through the ingestion of cestode eggs expelled by the definitive host . Dogs and other canines serve as definite hosts when they ingest offal contaminated with Echinococcus granulosus larvae . Humans are accidental hosts for E. granulosus, acquiring the infection through close contact with dogs or by ingesting water or food contaminated with parasitic eggs . As accidental or dead-end hosts, humans cannot transmit the infection. In the intermediate hosts, the parasite develops in a fluid filled hydatid cyst in soft organs, often the liver and lungs, and rarely in the brain, kidney and bone marrow, resulting in morbidity and mortality . In humans, the infection causes bile duct obstruction and pleural fistula disorders . To prevent and control cystic echinococcosis, methods applied to break the life cycle between the definitive and intermediate hosts include the deworming of dogs, meat inspection, not feeding infected offal to dogs and public awareness of the risks to humans . Despite this, cystic echinococcosis is highly prevalent in ruminants within herd-keeping areas of the world , ranging from 53.9% in China and 22% in Ethiopia to 13.9% in Iran and 12% in India . The prevalence of echinococcosis in Pakistan ranges from 2.4 to 35% in different host species , and here the estimated economic loss caused by cystic echinococcosis is USD 1.65 per organ . To control cystic echinococcosis, early diagnosis is essential . The diagnosis of Echinococcus typically relies on post-mortem extraction of the parasites, followed by microscopy and PCR. Echinococcosis cannot be confirmed through clinical signs, and in many countries, there are limited or no microscopy and molecular facilities available for ante-mortem examination. Additionally, radiography and ultrasonography are ineffective in identifying newly formed cysts. Serological assays, on the other hand, could provide a powerful pre-slaughter diagnostic screening tool for cystic echinococcosis. This could reduce the spread of E. granulosus on farms and in the environment, important for the prevention of human infections. Bovine and sheep hydatid cyst fluid (8, 16, 24 and 38 kDa) and goat hydatid cyst fluid antigens (60 and 245 kDa) have been previously studied for their potential usage in sero-diagnostic assays . In Pakistan, hydatid cyst fluid antigens of goat, sheep, cattle and buffalo have been characterised , but so far have not been tested for use in sero-diagnostic assays. Hence, here we isolate the unique antigen/s from bovine hydatid cysts to assess their usefulness in developing an ELISA for the pre-slaughter sero-diagnosis of cystic echinococcosis in bovines. 2. Materials and Methods 2.1. Study Site and Selection of Animals for Sera and Cyst Collection Sampling was conducted at a slaughterhouse in the district of Sheikhupura (Pakistan) in December 2021, where 960 bovines (cattle and buffalo, mostly dairy animals) were slaughtered during that month. Animals with the lowest body condition score were targeted in this study. A total of 264 cattle and buffalo individuals were tagged for collection of sera and for post-slaughter examination for hydatid cysts. Blood samples were collected from the jugular vein using sterilised 10 mL syringes (22G needle; Shifa) and stored in serum vacutainers. The tubes were transported on ice to the Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan, for further processing the next day. The serum was separated by centrifuging the samples at 3000x g for 20 min. The supernatant (serum) was collected and stored at -20 degC until further use. 2.2. Hydatid Cyst Collection Following the slaughter of tagged animals, the carcasses were examined for hydatid cysts in the liver, lungs and kidneys. Cysts removed from the liver or lungs were placed individually in sterilised containers for transport to the laboratory and subsequent microscopic examination. No cysts were detected in the kidneys. The meat was not tested for the presence of Echinococcus granulosus. 2.3. Microscopy of Hydatid Cysts Cysts with viable protoscoleces were classified as fertile and those with no protoscoleces were classified as sterile. To check fertility, fluid from each hydatid cyst was gently aspirated through a 1 mL sterilised syringe, and 0.2 mL was dropped onto a microscopic slide with a cover slip . To assess the viability of protoscoleces, the fluid (0.1 mL) from each hydatid cyst was mixed with 0.1% eosin stain for microscopic examination at 400x, with only viable protoscoleces taking up the stain . All slides were examined with an Olympus CX21FS1 microscope at 400x magnification. 2.4. Molecular Identification of Echinococcus Granulosus DNA was extracted from the germinal layer and fluid of the hydatid cyst using the WizPrepTM gDNA Tissue Kit (Wizbiosolutions, South Korea, W71060-300). Echinococcus granulosus mtDNA ND1 primers were used: Eg1F81 5' GTT TTT GGC TGC CGC CAGAAC 3' and Eg1R83 5' AAT TAA TGG AAA TAA TAACAA ACT TAA TCA ACA AT3', which generate a 226 bp amplicon . The PCR was performed in a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA) as described previously . Briefly, a total reaction volume of 20 mL included 2 mL of genomic cyst DNA (11.5 ng/mL), 10 mL master mix 2X AmpMasterTM Taq (GeneAll(r), ExgeneTM, catalogue number 541-001), 6 mL of UltrapureTM DEPC water (Invitrogen, 750023) and 1 mL of each forward and reverse primer solution (50 mM). PCR conditions were as follows: initial denaturation step at 94 degC for 3 min, followed by 28 cycles of denaturation at 94 degC for 30 s, annealing at 59.8 degC for 30 s and extension for 1 min at 72 degC, and a final extension step for 5 min at 72 degC. PCR products underwent electrophoresis in 2% agarose gels (1 h at 120 V, current 400 mA) using an SYBR safe DNA gel stain (catalogue no. 2291850; Invitrogen, Waltham, MA, USA alongside 7 mL of DNA ladder (100 bp DNA ladder Gene-direx, Catalogue # DM001-R500), and were observed under a GelDoc 100 imaging system (BioRad, Hercules, CA, USA). 2.5. Preparation of Bovine Hydatid Cyst Fluid (BHCF) Antigen Fluid was aseptically aspirated using a sterilised syringe (Shifa) from a hydatid cyst present in the liver of a buffalo, transferred to a 50 mL falcon tube, according to , and centrifuged at 7000x g for 30 min. Acetate buffer (0.005 M; pH 5) was used to dialyse the supernatant, and then the falcon tube was incubated overnight at 4 degC before centrifuging at 20,000x g for 30 min at 4 degC (ultra-high-speed centrifuge, Sigma 3K30). The precipitates were dissolved in 10 mL of phosphate buffer (0.2 M, pH 8) saturated with 40% ammonium sulphate and were centrifuged at 3000x g for 25 min. The supernatant was then incubated for 10 min at 60 degC and centrifuged again at 20,000x g for 1 h. The supernatant containing the soluble antigens of HCF was filtered with a 0.2 mm filter and stored at -20 degC . 2.6. SDS-PAGE and Western Blotting The native antigens separated from the bovine hydatid cyst fluid (BHCF) were analysed via SDS-PAGE and Western blotting according to . Briefly, each lane of a 12% w/v polyacrylamide gel was loaded with 30 mL native antigen (11.65 m g/mL), protein-loading (Laemmli) buffer and protein marker (PageRulerTm Cat# 26614, thermo scientific), separated by 12% w/v polyacrylamide gel and transferred to the nitrocellulose membrane (NCM, 0.22 mm, Trans-Blot(r) TurboTM Midi nitrocellulose transfer packs # 1704159, Bio-Rad, USA) using the Trans-Blot(r) TurboTM transfer system (Bio-Rad, USA). The blots were cut into strips, labelled and blocked with (5% w/v) skimmed milk in tris buffer saline (20 mM Tris-HCL, pH 7.2, 150 mM NaCl). The positive serum collected from infected tagged animals (three buffalo, each with 2-3 hydatid cysts in the liver, confirmed after post-mortem) was used as the primary antibody. The strips were charged with the anti-bovine antibody (Anti-bovine IgG-alkaline phosphatase conjugate Sigma Cat # A0705-25ML) as the secondary antibody (1:20,000) after three washings. A chromogenic substrate (NBT/BCIP bio-WORLD, Dublin, OH, USA) was used as a colour marker. 2.7. Quantification of Native Antigen The bovine hydatid cyst fluid protein was quantified using bicinchoninic acid (BCA) assay according to the manufacturer's instructions (Cat. 786-570, G-Biosciences, St. Louis, MO, USA). OD values were recorded at 650 nm using a multiskan sky microplate (1530-800580) spectrophotometer. 2.8. ELISA ELISA was performed according to with minor modifications. Hydatid cyst fluid antigen (100 mL/well in 50 mM Na2CO3 coating buffer at the concentration of 5 mg/mL) was coated on to a 96-well flat-bottom polystyrene plate (JET BioFil, Hong Kong, China, Code # TCP011096) and incubated at 37 degC overnight. The plate was washed 5 times with 250 mL/well 0.001 M PBS/0.05% Tween-20, then blocked with 4% BSA in 0.01 M PBS (200 mL/well), incubated for 2 h at 37 degC and washed 5 times with washing buffers. The primary antibody (serum) was added to the plate at a dilution of 1:99. Two wells were coated with positive sera (confirmed after slaughtering and PCR) and two wells were coated with negative sera. The plate was incubated at 37 degC for 2 h and re-washed 5 times. The plate was then coated with anti-bovine antibody 100 mL/well (Anti-bovine IgG-alkaline phosphatase conjugate Sigma Cat # A0705-25ML) as the secondary antibody (1:5000), incubated for 2 h at 37 degC and then re-washed 5 times. The substrate p-nitrophenyl phosphate (pNPP) (Thermo Scientific, Waltham, MA, USA, Ref # 34045) was added with 1 mg/mL of DAE substrate buffer (ThermoFisher, Waltham, MA, USA, Cat # 34064) at 100 mL/well. After 15 min, the stop solution (1 M NaOH at 100 mL/well) was added to stop the reaction. The microplate ELISA reader (ELX-800, Tennessee, BioTek, Winooski, VT, USA) recorded OD values at 405 nm. 2.9. ELISA Sensitivity, Specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV), Likelihood Ratio Positive (LR+), Likelihood Ratio Negative (LR-) and Diagnostic Odds Ratio (DOR) Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), likelihood ratio for a positive test result (LR+), likelihood ratio for a negative test result (LR-) and diagnostic odds ratio (DOR) were calculated according to the formulae in Table 1. 2.10. Statistical Analyses All statistical analyses were performed using R-Studio version 1.0.143 (R Development Core Team, 2015). We conducted a generalised linear model (GLM) with a Gaussian error family with loglink function with the dependent variable being the percentage of ELISA-positive animals within the following categories (independent variables): species, sex and age. A separate GLM for each species, both with binomial error families and logit functions, was then used to test whether the percentage of ELISA-positive animals varied significantly in terms of age, sex, location of cyst and cyst viability. Chi-square tests determined the association of the disease with species, sex, location of cyst and age groups. The Medcalc software (version 11.4.4.0) was used for the ROC curve line and interactive dot diagram. The ROC curve shows the sensitivity, specificity and criterion value (cut-off value) on the basis of which samples are declared positive or negative. The dot diagram displays (i) the separation of samples into positive (indicated as 1) and negative (indicated as 0) based on the gold standard (here, post-mortem examination); (ii) the cut-off value (indicated by a horizontal line) of ELISA separating ELISA-positive (above the line) and ELISA-negative (below the line) . For comparing data in Medcalc software, we used "1" to indicate post-mortem positive samples (n = 38) and "0" to indicate post-mortem negative samples (n = 226). On the basis of two negative control samples (negative in post-mortem), we calculated the cut-off value to differentiate between sero-negative and sero-positive samples . The mean OD value (0.136) of two negative controls was multiplied by 2.5 according to to obtain the cut-off value of 0.340. In parallel, the OD values of all 264 samples were put into the Medcalc software while telling the software either positive and negative based on post-mortem examination. Based on this, the software also determined the cut-off value as being 0.341. 3. Results Out of 264 bovines (128 cattle and 136 buffalo) examined at post-mortem, we found that 38 out of 264 (14.4%) contained hydatid cysts. All of these animals were subsequently confirmed as positive via serological examination using an ELISA kit based on the crude antigen (67 kDa) of BHCF. This ELISA approach also identified an additional 14 bovines, increasing the cystic echinococcosis infection rate to 52 out of 264 (19.6%). 3.1. Molecular Confirmation, Identification of Echinococcus-Specific Immunogen and BHCF ELISA For the antigen characterisation of BHCF, the hydatid cyst fluid collected from buffalo was confirmed to be Echinococcus granulosus based on species-specific primers targeting the mtDNA ND1 gene. The BHCF recovered from animals positive for echinococcosis, analysed via SDS-PAGE and stained with Coomassie blue, revealed an infected E. granulosus 67 kDa (IEg67 kDa) band and this was confirmed via WB analysis . There was also a 130 kDa band present in the SDS-PAGE, potentially representing a dimer that was not explored further. The sensitivity and specificity of ELISA based on the crude antigen was 100% and 93.8%, respectively . For ELISA, the positive predictive value (PPV), negative predictive value (NPV), LR+, LR- and DOR are given in Table 2. 3.2. Echinococcus Occurrence Based on ELISA, cattle had significantly higher infection levels than buffalo (GLM: std.err = 0.26, t = 2.5, p = 0.03). For both species, the number of infected individuals was positively associated with age (GLM: std.err = 0.09, t = 3.3, p = 0.009), and fewer males were infected than females (GLM: std.err = 0.25, t = -2.2, p = 0.05). The occurrence of cysts in cattle was significantly higher in the lungs (14.1%) compared to the liver (5.5%), whereas the reverse was true for buffalo (6.6% in liver and 2.9% in the lungs; kh2 = 10.69, p = 0.005). In each host species, neither location of cyst nor cyst viability were significantly related to host age nor gender (GLMs). From post-mortem examination, the occurrence of cysts was higher in the lungs of cattle (72%) compared to the liver (28%), whereas the reverse was true for buffalo (30.7% in lungs and 69.2% in the livers; kh2 = 10.69, p = 0.005). Fertile cysts were more prevalent in lungs (64.5%) than in livers (28.6%), whereas sterile cysts were more frequently observed in the liver (71.4%) than in lungs (35.5%) (kh2 = 285.028 and p < 0.001). The percentage of fertile cysts was higher in buffalo (84.6%) than in cattle (80%), and there were significantly more sterile cysts in cattle (20%) than in buffalo (15.4%; kh2 = 285.028 and p = 0.06). The distribution of Echinococcus-positive animal sera is shown in Table 3. 4. Discussion Cystic echinococcosis is a major disease in livestock and humans . Its diagnosis in intermediate hosts is mainly based on necropsy procedures and in most cases the disease is asymptomatic in the early stages , so there is an urgency to develop alternative diagnostic methods , such as antibody detection through ELISA. Here, we identified and successfully deployed a potential candidate antigen, the iEg67 kDa crude antigen (from cystic fluid collected from buffalo liver) in a sero-diagnostic assay (Ab-detection ELISA) for the diagnosis of cystic echinococcosis in cattle and buffalo with much improved sensitivity and specificity. This antigen showed highly promising results as a candidate for ELISA kit development, and similar to previous studies (e.g., ) was more accurate than post-mortem examination, with very high sensitivity (100%) and specificity (93.8%) (Table 4). We also observed a 130 kDa band, possibly a dimer of iEg67 kDa, consistent with the previously reported dimerisation of Echinococcus antigens . Previous studies have characterised the antigenic profile of hydatid cysts from cows, buffalo, sheep, goats and camels, and of humans infected with cystic echinococcosis, in different geographical areas to identify the potential diagnostic or vaccine candidate proteins . The sensitivity and specificity of diagnostic tests used to include or exclude a disease (i.e., 'rule in' or 'rule out', respectively), for previous isolates of the HCF crude antigen, partially purified the crude antigen and recombinant antigens are compared in Table 5. Factors influencing the sensitivity and specificity of ELISA include the source and quality of cyst fluid, the antigen purification process, laboratory techniques used for diagnosis and the target species/strains. The low sensitivity detected by Ibrahem et al. might be related to the smaller size of their recombinant antigen potentially having fewer antigenic epitopes compared to the native one. This notion is strengthened by Ibrahem et al. who found that the sensitivity of recombinant-protein-based ELISA (25%) was lower compared to crude/native antigens from camel and sheep (see Table 5). Lower specificity might also arise due to the presence of other infections such as Taenia hydatigena, which can be mistaken as hydatid cysts, but would not have been detectable in an HCF-antigen-based ELISA . Gatti et al. used three forms of antigens (LHT, S2B and B) in their enzyme immunoassay (EIA) and recorded the sensitivity and specificity of each (see Table 4), concluding that either LHT or S2B should be used for the sero-diagnosis of cystic echinococcosis. Kandil et al. used two types of antigens: protoscolex antigen and the germinal layer antigen of the hydatid cyst. They observed higher sensitivity (83%) and specificity (70.3%) in the protoscolex antigen than the germinal layer antigen (Table 5). We used total hydatid cyst fluid for our crude HCF antigen preparation and obtained higher sensitivity and specificity values than in previous work; the reasons for this could be numerous. The occurrence of hydatid cysts in Pakistan was determined in the current study to be 14.4% through post-mortem examination, and 19.7% when screened through the ELISA, similar to the 21% prevalence reported previously in cattle and buffalo from Punjab, India . The increase in the detection rate of ELISA relative to the post-mortem between the current study and Yakubu et al. can be explained by two complementary factors. Firstly, a known limitation of post-mortem lies in the lower detection probability for small developing cysts, possibly occurring more frequently when overall infection rate is higher in a population, as in . Secondly, our ELISA approach (IgG Detection ELISA) did not allow us to differentiate between present and past infections, likely leading to the detection of recovered animals that would have remained undetected in PMs. ELISA can be further developed by targeting IgM for acute infections and by the detection of the IgG titer in paired sampling to have a clearer picture about active and past infections. The gap between the detection rate of ELISA and PM may increase when aged animals (with a higher expected proportion of recovered animals) are sacrificed and examined for the presence of cysts. As previously reported by , infection prevalence was higher in female (18.75% post-mortem and 25% ELISA) than in male (9.2 and 13.3%) hosts. Pregnancy, parturition and lactation seem to enhance the risk of female infections . Risk of infection also increased with age , presumably related to the increased exposure of older individuals. The current study also suggests that cattle are more prone to hydatidosis than buffalo (similar to , but in contrast to ). Apart from geographical location and temperature variation, the infection rate observed by was influenced by the higher number of cattle amongst the slaughtered animals. In the current study, cattle had a higher occurrence of cysts in the lungs rather than the liver, with the reverse found in buffalo (as reported by , but in contrast to . This difference may be due to host specificity, with one organ being more permissive to the development of cysts than the other, but this finding could potentially also be influenced by different strains of the parasite. With regard to hydatid cyst fertility and sterility, most studies on livestock populations in Punjab, Pakistan, have reported higher numbers of fertile than sterile cysts . Variation in fertility may be due to differences in Echinococcus strain, time post-infection, development rate of the cysts and host species. 5. Conclusions Cystic echinococcosis in food animals reduces their suitability for human consumption globally. Here, we show that an iEg67 kDa antigen identified from bovine hydatid cyst fluid is a suitable candidate for developing sero-diagnostic assays for cystic echinococcosis. An ELISA based on the use of the iEg67kDa antigen improved diagnosis accuracy for cystic echinococcosis in bovines compared to post-mortem examination. Our study showed 100% sensitivity and 93.8% specificity, an improvement compared with all previous studies. This approach can be used for large-scale sero-epidemiological studies of cystic echinococcosis in food animals (i.e., sheep, goat and camels) from diverse climatic conditions and agro-ecological zones, as well as for the screening of humans. We anticipate that this will provide increased information on the distribution and prevalence of cystic echinococcosis in animals and humans, informing on suitable control strategies and contributing towards the formulation of a One Health approach. Acknowledgments We thank Shafqat Shabir and Sarfraz Ur Rahman, PhD scholars at the Department of Parasitology, UVAS, Lahore, for technical help, and Numair Masud for statistical advice. Author Contributions Conceptualisation, H.A., M.I.R. and M.Y.; methodology, S.K., H.A. and J.C.; validation, J.C. and H.A.; data analysis, S.K. and J.C.; investigation, S.K.; resources, H.A.; data curation, S.K.; writing--original draft preparation, S.K., H.A. and J.C.; writing--review and editing, H.A., J.C. and F.H.; supervision, H.A.; project administration, H.A.; funding acquisition, H.A. and M.I.R. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All of the experimental procedures used were approved by the Institutional Guidelines of the Ethical Review Committee of UVAS, Lahore, vide letter no. 939-1, dated 05-09-2019. Informed Consent Statement Not applicable. Data Availability Statement The data will be available from the corresponding author upon request. Conflicts of Interest The authors have declared no conflict of interest. Figure 1 (A) Coomassie-blue-stained SDS-PAGE showing protein separation and identification of Echinococcus granulosus native antigen. Lane MW = protein marker, lanes 1-3 = samples, lane 4 = empty well. (B) Western blot of hydatid cyst fluids from slaughtered buffalo using sera from naturally infected buffalo and developed by an anti-bovine antibody conjugated with alkaline phosphatase. Lane MW = protein marker (run on the same gel as the test samples, but image cropped to remove samples unrelated to the current study), lanes 1, 2 and 3 = samples and lane 4 is an empty well. Figure 2 (A) Sensitivity, specificity and cut-off value of crude-antigen-based detection of Echinococcus-specific IgG antibodies in bovine sera. (B) Interactive dot plot for negative and positive sera. animals-13-00866-t001_Table 1 Table 1 Definition and formulas to calculate sensitivity, specificity, PPV, NPV, LR+, LR- and DOR of ELISA, based on . Name Definition Formula Sensitivity "The capability of a test to correctly identify the patients with disease" a/(a + c) x 100 Specificity "The potential of a screening test to identify the patients without the disease" d/(b + d) x 100 Positive predictive value (PPV) "Prediction of positive patients before test (identifying true positive)" a/a + b Negative predictive value (NPV) "Prediction of negative samples before test (identifying true negative)" d/c + d Likelihood ratio positive (LR+) "Ratio of probability that a positive test outcome would be expected in a patient with a disease to the probability that a positive test outcome would be expected in a patient without a disease" (a/a + c)/(b/b + d) Likelihood ratio negative (LR-) "The ratio of probability of a patient testing negative who has a disease to the probability of a patient testing negative who does not have a disease" (c/a + c)/(d/b + d) Diagnostic odd ratio (DOR) "Odds of a positive test in those with disease relative to the odds of a positive test in those without disease" LR+/LR- a = true positive, b = false positive, c = false negative and d = true negative. animals-13-00866-t002_Table 2 Table 2 Sensitivity, specificity, PPV, NPV, LR+, LR- and DOR values of iEgELISA. Sensitivity Specificity PPV NPV LR+ LR- DOR 100% 93.8% 73.07 99.96 16.33 0.012 1360.83 animals-13-00866-t003_Table 3 Table 3 Distribution of Echinococcus-positive sera categorised by host species, sex and age group. Age Class Buffalo Cattle Female Male Female Male 2-3 years 2/21 (9.5%) 0/23 (0%) 0/11 (0%) 4/28 (14.2%) 4-5 years 2/31 (6.5%) 3/19 (15.7%) 12/31 (38.7%) 4/22 (18.1%) 6-7 years 9/28 (32.1%) 1/4 (25%) 11/22 (50%) 4/14 (28.5%) animals-13-00866-t004_Table 4 Table 4 Comparison of post-mortem and serological examination results among studies. Host Species Sample Size Post-Mortem (%) ELISA (%) Antigen Used in ELISA References Cattle 128 19.5 27.3 Ammonium sulphate ((NH4)2SO4) precipitated HCF Current study Buffalo 136 9.5 12.5 Ammonium sulphate ((NH4)2SO4) precipitated HCF Current study Cattle 256 1.6 35.5 Freeze-dried precipitated HCF Camel 304 14.14 52.6 Freeze-dried precipitated HCF Camel 200 6 8 Phosphotungstic acid and 2M magnesium chloride used for HCF precipitation HCF = hydatid cyst fluid. animals-13-00866-t005_Table 5 Table 5 Comparison of sensitivity and specificity of iEgELISA based on crude antigen (this study) with results from previous work. Arrows denote the direction of change compared with values from the present study. Sensitivity (%) Specificity (%) Host Origin Antigen Size (kDa) Type of Antigen References 100 93.8 Buffalo 67 Crude antigen Current study 82.8 | 62.5 | Camel Not mentioned Crude antigen 79.3 | 75 | Camel Not mentioned Partially purified crude antigen 36 | 93 | Sheep 8-24 Crude antigen 25 | 99 | Sheep 6 Recombinant antigen 96% | 71% | Camel Not mentioned Crude antigen 99% | 90% | Camel Not mentioned Camel antigen B 89.2 | 89.5 | Sheep 8-12 Total hydatid liquid (LHT) 80.0 | 93.9 | Sheep 16 Purified portion of total hydatid liquid (S2B) 86.4 | 92.8 | Sheep 20-24 Purified antigen (B) 83 | 70.3 | Camel Not mentioned Protoscolex antigen 46.5 | 41.7 | Camel Not mentioned Germinal layer antigen LHT = total hydatid liquid, S2B = purified portion of total hydatid liquid and B = purified antigen. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000061

Pre-emptive capture or translocation of wildlife during oil spills and prior to pest eradication poison applications are very specific conservation goals within the field of conservation translocation/reintroduction. Protection of wildlife from contamination events occurs during either planned operations such as pest eradication poison applications, or unplanned events such as pollution or oil spills. The aim in both incidences is to protect at-risk wildlife species, ensuring the survival of a threatened regional population or entire species, by excluding wildlife from entering affected areas and therefore preventing impacts on the protected wildlife. If pre-emptive capture does not occur, wildlife may unintentionally be affected and could either die or will need capture, cleaning, and/or medical care and rehabilitation before being released back into a cleared environment. This paper reviews information from pre-emptive captures and translocations of threatened wildlife undertaken during past oil spills and island pest eradications, to assess criteria for species captured, techniques used, outcomes of responses, and lessons learned. From these case studies, the considerations and planning needs for pre-emptive capture are described and recommendations made to allow better use and preparedness for pre-emptive capture as a preventative wildlife conservation tool. pre-emptive capture translocation conservation island eradication oil spill wildlife This research received no external funding. pmc1. Introduction As a wildlife management tool, the use of pre-emptive capture and translocations has risen rapidly in the past two decades . In 2020, a review of 145 studies on wildlife capture and translocations indicated that 77% had been carried out for conservation purposes, predominantly to reintroduce or increase species' presence within their indigenous range . Here, we review pre-emptive capture and hold or translocation as techniques for preventing wildlife from entering contaminated areas or removing wildlife from areas before they are oiled or before the use of poisons for pest eradication purposes. The pre-emptive capture, holding, and translocation process undertaken for wildlife during oil spills and pest eradication poison applications is a specific conservation goal within the field of conservation translocation and reintroduction techniques . The aim is to protect a significant proportion of a range-restricted species or significant regional population to reintroduce individuals back to their original range after an impact (oils spill or poison) has been removed to repopulate the area. The first priority of wildlife protection during contamination events is to minimise the impact of the contaminant on wildlife through prevention. If a contamination event occurs, either planned (poisoning events for pest eradication) or unplanned (oil spills), the protection of wildlife can occur by: (1) stopping the contaminant from reaching and affecting non-target wildlife by containing poison in equipment that does not allow wildlife access or not spreading poison in critical wildlife areas, or containing the oil spill at or close to the source or stopping oil entering the wildlife habitat; (2) stopping wildlife being affected through the removal of wildlife from an affected or about to be affected area, or preventing wildlife from entering affected areas through hazing, deterrence, or pre-emptive capture. If these preventative measures are not undertaken, wildlife may die or need capture, cleaning, and/or medical treatment and rehabilitation before being released back into a clean environment. Avoiding wildlife from being impacted is always the highest priority as it prevents the duress, injury, and possible death of wildlife. Additionally, while it does have its own risks, it will significantly lessen the cost of a wildlife response if wildlife does become affected, and reduces the negative public and media reactions to reports and visual images of impacted wildlife . This review concentrates on the priority of preventing wildlife from entering impacted areas or removing wildlife from areas before the area is impacted, and specifically focuses on pre-emptive capture either to translocate wildlife (move to another location) or to hold wildlife in captivity until release into a clean environment can occur. The first step needed in all protective and preventative processes is developing a plan, based on analysis of areas at risk of impact (either oiling or where poison will be spread), the vulnerability of species (to both the contaminant and any proposed action), and potential response options for species at risk . Each protective and preventative technique is species- and area-specific, and is usually initially based on a species population size and distribution, with species that have a high threat classification (i.e., listed as endangered), are range-restricted, and/or have high cultural importance/or public profile being most likely to be considered for pre-emptive capture. Threatened species usually have a small population size or restricted distribution or endemism, meaning an impact on their habitat could mean the extinction of that species or the local population. Other factors to consider are habitat use, therefore exposure risk, season, and biological factors, such as if the species is breeding at the time of impact . For example, for pest eradication/poisoning events, how species forage is important, i.e., nectar-feeding birds are unlikely to be impacted by an aerial application of cereal poison bait; however, herbivores or omnivores may be vulnerable to primary poisoning as they could eat the bait directly, or omnivores, carnivores, or scavengers that could get secondary poisoning from scavenging poisoned individuals. In oil spills, any species that contacts, digests, or inhales fumes from oil can be affected, and, like poison operations, carnivores or scavengers can get secondary poisoning or oiling from predating or scavenging on other oiled wildlife. Undertaking pre-emptive capture of some species may not be practical or viable, i.e., large animals such as whales cannot be pre-emptively captured; therefore, hazing or deterrence are better options to undertake. For all species, the different stages in life cycles, such as breeding or moulting, can prevent other techniques such as deterrence or hazing from working effectively, and pre-emptive capture may be the only technique that could be successful. This was the case for New Zealand dotterels (Charadrius obscurus) in 2011 during the MV Rena spill in New Zealand as the dotterels were breeding when the oil spill occurred, making individuals very territorial, and animals would not have left their nesting sites, eggs, or chicks regardless if disturbance techniques were used. This manuscript uses past oil spills and pest eradications using toxicants on islands as case studies of pre-emptive capture and holding, or translocation, to highlight lessons learned and considerations of species-specific response option restrictions, and outlines recommendations to allow better use, preparedness, and planning for pre-emptive capture as a conservation tool for threatened wildlife during contamination events. 2. Materials and Methods An online literature search was undertaken aligned with the PRISMA 2020 guidelines with the aim of creating a list of publicly available articles or reports on the use of pre-emptive capture during oil spill response or island eradication, from 1970 to 2022. Primary sources of information were sourced from scientific journal articles, conference proceedings, and any other grey literature through searches on Google, Google Scholar, or the Web of Science database (search terms were in English and included singular words or combinations of pre-emptive, pre-emptive, capture, wildlife, oil spill, oiled wildlife, and eradication). Additionally, searches were made through the Oil Spill conference websites for Interspill, IOSC, and translocation information from IUCN, including Proceedings of the International Conference on Eradication of Island Invasives. Experts in both fields were also contacted for any additional grey literature that was available but not yet published. 3. Results The most striking result from this research is how few of the undertakings of pre-emptive capture of wildlife for prevention from contamination have been written into publicly available reports, journal articles, conference proceedings, or grey literature (Table 1). There have been over 600 island eradications of invasive rodents, many of which were multi-species eradications , and 1000s of oil spills that have affected wildlife . There are multiple articles that highlight the need, advantages, and brief outlines on why pre-emptive capture should be undertaken but not many examples of when it has been undertaken or recommendations for what species should be considered, planning considerations needed, or factors to be taken into account before attempting pre-emptive capture . However, even from the articles that do mention pre-emptive capture or translocation being undertaken, most only mention that it occurred, and there are few reports on how wildlife was captured or held, with what methodology, what proportion of the population was captured, processes during captivity, or short- or long-term survival or reproduction results after their release. Outlined below and in Table 1 are summaries of the 11 documented case studies of pre-emptive capture of wildlife during oil spill responses or island pest eradications that were assessed. Locations of case studies are shown in Figure 1. 3.1. Case Studies--Oil Spills 3.1.1. Australia MV Iron Barron Oil Spill 1995 On 10 July 1995, the MV Iron Barron encountered bad weather coming into the port of Launceston in northern Tasmania, Australia, grounding on Low Head, Hebe Reef, leaking an estimated 325 tonnes of heavy bunker fuel oil . Little blue penguins (Eudyptula minor) were significantly impacted by the spill with an estimated 10,000 to 20,000 killed and 1894 oiled birds captured, cleaned, and rehabilitated in an improvised rehabilitation facility . The penguins were ready for release before their habitat had been cleaned, particularly as it was a large complex area with many islands over which the oil had spread. Rather than prolonging captivity, which increases the risk of disease and stress, and as breeding was imminent, a translocation strategy to release cleaned and rehabilitated penguins at different distances from the oiled site was trialled. This was undertaken to determine the optimal distance to release rehabilitated penguins so that they returned to their habitat after it had been cleaned. Twenty-five VHF-tagged penguins were translocated 360 km from the spill site on the east coast of Tasmania, and their movements were tracked from the air. Two birds returned to their original capture site within 3 days, not enough time to clean up the area, prompting a new release site 120 km further south (480 km in total). After the first trial, it was decided that the translocation site 480 km away was appropriate for the circumstances, and a further 863 penguins were translocated. At least 56% of the birds released further south returned to Low Head in four months, after their habitat had been cleaned. Monitoring found no differences in the survival rate of translocated and non-translocated birds. Lessons learned: While translocation was considered effective in this situation, it is recommended that translocation protocols should be trialled before being implemented . 3.1.2. South Africa MV Treasure Oil Spill 2000 The MV Treasure spilled approximately 400 tonnes of heavy fuel oil onto the coast of South Africa near Cape Town on 23 June 2000. The spill occurred near the two major breeding colonies, Robben and Dassen Islands, of the endangered African Penguins (Spheniscus demersus). A total of 19,000 oiled penguins were caught, cleaned, rehabilitated, and returned to a clean environment. Over 1660 birds died during captivity, most from the negative impacts of the oil . To prevent even more penguins from being oiled, a further 19,506 penguins were captured, relocated, and released at Cape Recife near Port Elizabeth, ~700 km to the east of Cape Town . These penguins, whether oiled or pre-emptively captured, represented over half of the known, endangered, declining population of African penguins at the time of the spill . Relocated birds returned quickly to their breeding islands, with the faster returning in 11 days and most returned within two to four weeks . This indicated that Cape Recife was an appropriate location for release because it was a suitable distance to allow time for the oil to be cleared before the birds returned, but close enough for birds to return within a month, thereby minimising any disruption to breeding and moulting. Of the 19,506 penguins translocated, 241 died between being captured and release at Cape Recife due to some being transported in closed trucks causing CO2 poisoning. Additionally, before transport, those kept on Dassen Island were kept fenced in an area on the island with limited access to drinking water and no areas to swim. Both factors contributed to the higher mortality of those pre-emptive captured birds . Additional to the adults, 3350 orphaned chicks were also pre-emptively captured and reared in captivity and released back into their clean environment when they had fledged. Of the 3350 chicks collected, approximately 2300 were fledged and released . Prior to the MV Treasure spill, South Africans' seabird oil spill rescue plans focused on catching and treating oiled birds as soon as possible, before releasing them back into the wild; preventing birds from becoming oiled was not part of any plan . This wildlife response is still the largest relocation response for oiled wildlife globally and, due to its success, the implementation of relocating birds before they became oiled has been implemented as a response option and documented to have been an effective conservation measure . One year after the MV Treasure spill, 84% of the evacuated birds had been re-sighted, compared with 55% of the captured, cleaned, and released birds. Lessons learned: The two overall lessons from the pre-emptive capture of African penguins were greater consideration of conditions prior to and during transport to translocation sites to prevent deaths, and consideration of distance transported so that the wildlife's return allowed enough time for the oiled areas to be cleaned, but the distance was not too far to cause individuals to get disorientated or lost, or to cause major disruption to breeding or moulting cycles. A second conclusion is that pre-emptive capture and raising of penguin chicks is a successful conservation practice that continues today for African Penguins, not only during oil spills, but also droughts, colony disturbances, and other human and natural impacts on this endangered species accessed on 15 February 2023). 3.1.3. USA Deepwater Horizons Oil Spill 2010 On 20 April 2010, the Deepwater Horizon well exploded 66 km off the coast of Louisiana, in the Gulf of Mexico, and before being capped, three months later, more than 780,000 tonnes of crude oil were spilled. There were numerous impacts on the environment and wildlife, and because of the length of time oil continued to be spilled, some wildlife that had been cleaned and rehabilitated were ready for release long before their environment was cleaned. Brown Pelicans (Pelecanus occidentalis) were one of the species impacted, with more than 700 rehabilitated in south-eastern Louisiana alone . To overcome the lack of a clean habitat for their release, 182 oil-rehabilitated pelicans were translocated from south-eastern Louisiana to Rabbit Island in south-western Louisiana, an island that was not impacted by the spill and had non-impacted pelicans breeding on it. The aims of this translocation were to enable monitoring of movements of translocated groups and determine if translocation would delay pelicans returning to their habitat, and therefore getting re-oiled, and to be able to monitor mortality, determine the integration of translocated pelicans with local pelican groups, and determine if supplemental feeding of translocated birds prolonged occupation on the island, therefore again reducing the likelihood of re-oiling . Daily surveys were undertaken at the island for six weeks from the date of translocations, with supplementary feeding occurring twice a day for four weeks. There was no mortality of rehabilitated birds recorded and it was observed that translocated pelicans mixed readily with local pelican flocks. Many of the local and translocated pelicans moved away from the island within 4 to 6 weeks, likely due to natural and human-induced factors. Lessons learned: The translocations and supplementary feeding program of the brown pelican were considered successful at reducing the movement of pelicans back into oiled areas. However, habituation to the feeding vessel and supplementary feeding were observed both from the rehabilitated and local pelicans. For future translocations, it is suggested shorter time periods of supplemental feedings should occur, using alternative feeding strategies such as blinds or remote feeders due to the easy habituation of pelicans to humans. For tracking of movement of rehabilitated birds, it is recommended a subset of individuals be radio/satellite tagged for documentation of movements and mortality. 3.1.4. New Zealand MV Rena Oil Spill 2011 On 5 October 2011, the container vessel MV Rena ran aground on Astrolabe Reef, Bay of Plenty, New Zealand, and within days spilled approximately 350 tonnes of heavy fuel oil. The endangered Northern New Zealand dotterels (Charadrius obscurus aquilonius--a small ~140 g shorebird) were pre-emptively captured as part of the oiled wildlife response to ensure the survival of a regional population. The pre-emptive capture occurred as it was considered that if these small birds became significantly oiled their chances of survival were minimal despite cleaning/rehabilitation . Sixty dotterels were caught, with over half the birds already having some level of oil contamination. This population of dotterels represented ~6% of the global population of this species and the majority of the local population within the area of the spill. Many pairs were already breeding and nesting at the time of the spill, so other deterrence or hazing activities would not have worked as the birds are territorial and would not have moved away from their nests. This was the first time wild adult New Zealand dotterels pairs had been held in captivity for a prolonged period. Birds were caught in their breeding pairs, with each pair held in individual enclosures, blocked from view of other pairs to prevent territorial and fighting behaviours which would have been normal during breeding. There was a 90% survival rate of the New Zealand dotterels held in captivity during the MV Rena oil spill response over a ~2-month period . Dotterels took 1-15 days (median 5 days) to convert to the captive diet. Sixty-one percent of birds obtained minor abrasions from contact with enclosure netting during captivity due to their flighty behaviour which did not affect survival; however, seven birds (11.7%) developed respiratory disease, with six of these dying from aspergillosis causing pneumonia-type deaths . Intensive captive husbandry was needed to convert the birds to a captive diet, minimise injuries, and manage pododermatitis/foot sores. Lessons learned: It was critical to have a dedicated captive management team for these birds. The challenges that come with managing wild adult shorebirds in captivity and converting to captive diets are well recognised within the wildlife rehabilitation community. Additionally, shorebirds are species considered to respond poorly to the stresses of capture and captivity . Therefore, although the pre-emptive capture and management of shorebirds during an oil spill to minimise the effects of oil spills carries significant costs and risks to the birds, it is considered essential in emergency management situations for high-priority/at-risk species. Additional to normal capture stressors, clinical signs of respiratory disease were not observed until the last half of the time the birds were in captivity. Therefore, a strong recommendation for the management of shorebirds that are pre-emptively captured is that the clean-up of their habitat is prioritised to enable the early return of birds to the wild. 3.2. Case Studies--Island Eradications 3.2.1. New Zealand--Mice and Rat Eradication/Poisoning, Kapiti Island 1996 After the eradication of cats (Felis catus), deer (Cervidae spp.), pigs (Sus spp.), goats (Capra spp.), and possums (Trichosurus vulpecula), by hunting and trapping, from the rugged 19.65 km2 Kapiti Island off the south-west coast of the North Island, New Zealand, the Department of Conservation of New Zealand also successfully eradicated Norway and Pacific rats (Rattus norvegicus and R. exulans) in 1996 using helicopter broadcast of brodifacoum cereal baits . Trials with non-toxic baits were carried out on North Island weka (Gallirallus australis grey) and little spotted kiwi (Apteryx owenii), both flightless birds found on the island, to help determine the risks of poisoning for these non-target species . North Island weka at the time were classified as endangered and were expected to be affected by the eradication activities both from primary and secondary poisoning, particularly as weka are omnivores and will scavenge and kill other species. From the non-toxic trials, measures to minimise the effects of the poison application on fauna at risk were put in place, which included the capture and holding in captivity or translocation to reserves on mainland New Zealand of 243 weka, and the transfer of 66 New Zealand robins (Petroica australis), which had previously been identified as being at risk to nearby Mana Island. Post-poisoning call rate monitoring indicated that weka call rates were significantly lower after poisoning; however, it could not be determined if that was caused by the removal of weka from the island (not yet returned or released at the time of the call counts) and/or the poisoning operation, because no call rate monitoring was undertaken in the period between the removal and the poisoning for comparison. However, the fact that weka calls were heard meant that some survived the poisoning operation, and together with the birds released after the operation, they are now distributed throughout Kapiti Island and breeding prolifically . Lessons learned: Species at risk should be identified through both non-toxic bait trials and knowledge from species at risk from previous operations. Monitoring between pre-emptive capture and poison applications should be undertaken to allow the determination of the impacts of both. 3.2.2. New Zealand--Rat Eradication/Poisoning, Whenua Hou Nature Reserve/Codfish Island 1998 Whenua Hou Nature Reserve/Codfish Island is located 3 km NW of Stewart Island, New Zealand, and is the protected island home to the largest population of the endangered Kakapo (Strigops habroptilus), a large flightless native parrot. Following the removal of possums and South Island weka (Gallirallus australis australia), eradication of the Pacific rat was undertaken on Codfish in August 1998, using a combination of aerial applications and bait station cereal pellets containing brodifacoum. In preparation for the eradication, a smaller island, Putauhinu (96 ha), was eradicated of Pacific rats the year before in 1997, so that a population of fernbirds (Bowdleria punctata wilsoni), endemic to Codfish Island, could be established . Additional to the transfer, a 37 ha block of the best fernbird habitat known on Codfish, containing the densest population of fernbirds, was poisoned using bait stations at 25 m intervals instead of using aerial baiting, which had been shown during field trials elsewhere to cause a high death rate in fernbirds. All Kakapo (except one that could not be found) were removed from the island prior to the poison application and temporarily held on a separate island. Short-tailed bats (Mystacina tuberculatus tuberculatus) were also managed, with 50 being captured and released onto Ulva Island, a predator-free island off Stewart Island; however, this was unsuccessful. Additionally, during the poison applications, four purpose-designed "batteries" were constructed on Codfish Island with 386 short-tailed bats held for nearly three months. There was no observable loss to the bat population linked with the bait application although individuals are likely to have been lost. Nine bats were lost up until the last week of the capture program, when 42 died in one event due to heat stress in one of the roost boxes. Despite this sad event, the operation was still considered a success given how difficult bat husbandry can be. The bat protection and monitoring was undertaken by a team of 5-7 people and this investment of single-task personnel is one of the main reasons for its success. The 21 fernbirds that were transferred to Putauhinu were confirmed to have bred, and follow-up checks on Putauhinu have shown that the fernbird population has continued to increase and expand its range on Putauhinu. It appeared most fernbirds were lost on Codfish due to the bait application, despite the management, with very few recorded for 2 years after. However, enough survived to rebuild and recover not only to the population's original range, but to also expand into a variety of habitats in the absence of rats . This meant the planned reintroduction from Putauhinu was not required. Lessons learned: Dedicated husbandry teams are needed for the pre-emptive capture of species during eradication projects. Although the bait stations in the areas of the fernbirds achieved the goal of reducing fernbird mortality, it was thought that as fernbirds outside the area affected by the aerial bait died, fernbirds within the bait station area expanded their range and therefore became more exposed to aerially laid bait. Therefore, it was thought that the impact may have been lessened by expanding the size of the core area in which only bait stations were laid, thus increasing the percentage of birds within the core area. This result also led to the conclusion that the additional cost of rat eradication and transfer of a security population to Putauhinu was warranted even though it proved to not be necessary. This eradication also proved that field trials are important for poison eradication, as fernbirds were thought to be insectivores mainly preying on spiders and hence at little risk from the baiting operation. However, field trials showed that fernbirds when presented with brodifacoum bait would eat it, and indicated that the species would be heavily impacted by aerial bait, therefore leading to the mitigation work of bait stations in the area where the fernbirds were in high abundance (Pete McClelland pers comm). 3.2.3. Seychelles--Cat, Rabbit, Rats and Mice Eradication 1996-2000 Between 1996 and 2000, attempts were made to eradicate five introduced mammal species, feral cat, rabbit (Oryctolagus cuniculus), ship rat (Rattus rattus), Norway rat, and house mouse (Mus domesticus), on four inhabited Seychelle islands. As there were no rat-free islands in close proximity for the transfer of species at risk, 590 individuals from three threatened native species, the Seychelles magpie-robins (Copsychus sechellarum, n = 39), Seychelles fodys (Foudia sechellarum, n = 330, 50% of the known population), and the Aldabran giant tortoises (Geochelone gigantea, n = 218), thought to be at risk from primary and/or secondary poisoning, or for public goodwill in the case of tortoises, were held in captivity for the three months of the eradication program . During the captivity of these species across the islands, the avicultural knowledge and capability of staff increased enormously. The captivity of these species during eradication was very successful, with magpie-robins breeding successfully during three months in captivity . All tortoises, Seychelles fodys, and magpie-robins were successfully released within 3 months after bait application. Lessons learned: Dedicated husbandry teams are essential for success and allow for increased knowledge and capability for the aviculture of species and in the region. This was one of the first major human-occupied island eradication programs and its success led to the planning of eradications on the likes of Galapagos and Lord Howe Islands (see below). It was an important conclusion at the time that land held privately, human habitation, or tourism activities need not be seen as barriers to eradication projects, as island-based tourism activities can provide the financial and human resources to restore and maintain threatened endemic biodiversity. 3.2.4. California, USA--Rat Eradication/Poisoning, Anacapa Islands 2001-2002 Eradication of black/ship rats from Anacapa Islands, US Channel Islands National Park, California, was undertaken in 2001 and 2002 . This was the first aerial application of a rodenticide in North America and the first attempt in the world to eradicate a rodent from islands while preserving a native endemic rodent on the same islands. There are three islands in this group and, to ensure the presence of the native deer mouse (Peromyscus maniculatus anacapae), the rodenticide application was staggered over two years so that a wild population was always present on one or more islands . Concurrently, mice populations from each island were held in captivity during poison applications. Additional to the mice, to avoid as much as possible birds being affected by the application, bait was made using colouring and sizing that deterred gulls and granivorous birds, resident raptors were captured and held or translocated, and a 15 ha no-drop zone was established on West Anacapa to create a refuge for granivorous birds, particularly the Santa Cruz Island rufous-crowned sparrow Aimophila ruficeps obscura. In the no-drop zone, rats were poisoned using bait stations that were inaccessible to granivorous birds. Prior to the first poisoning in 2001, 185 deer mice were live captured from East Anacapa and held for five months. Of these, 174 were released after poisoning. Prior to the second poisoning in 2002, 373 and 365 deer mice were captured from Middle and West Anacapa, respectively, and held in captivity, while concurrently 715 and 308 mice from Middle and West Anacapa were captured and translocated to rat-free East Anacapa. Five months after the second eradication, 358 and 360 captive mice were reintroduced to Middle and West Anacapa, respectively. Raptors were live captured prior to rodenticide applications (including eight peregrine falcons Falco peregrinus, nine red-tailed hawks Buteo jamaicensis, four barn owls Tyto alba, and six burrowing owls Athene cunicularia). Most were released onto suitable habitat on mainland California, except peregrine falcons, which were held and released back onto Anacapa 3 weeks after rodenticide applications. A total of 94 birds (16 species) were identified from carcass searches following rodenticide applications. Of the 63 birds tested for brodifacoum, 59 (94%) tested positive . Lessons learned: The successful recovery of the Anacapa deer mouse following the eradication demonstrates that it is feasible to eradicate invasive rodents from islands when native rodents or other susceptible native animals can be held in captivity and kept away from poison. Captive holding and translocation significantly reduced raptor mortality. However, captive holding or other mitigation measures (no-drop zones) may be necessary for sedentary granivorous passerines, as previously used for fernbirds during the eradication of rats from Codfish Island. 3.2.5. Galapagos--Rat Eradication/Poisoning, Pinzon Island 2012 In December 2012, brodifacoum bait was spread on Pinzon Island (1815 hectares), Galapagos, to eradicate black rats which had prevented the Pinzon giant tortoise (Chelonoidis duncanensis) from breeding successfully for nearly a century. Two years prior to the poisoning, 15 adult Pinzon tortoises were brought into captivity and housed on Santa Cruz Island for release after the eradication; all survived, and breeding has been recorded since the eradication. The two other species of concern were Pinzon lava lizards (Microlophus duncanensis) and Galapagos hawks (Buteo galapagoensis). Forty Pinzon lava lizards were taken into captivity prior to baiting and maintained in enclosures on Pinzon Island, and were released 10 days after the second bait application as it was determined that, due to bait degradation, the risk of poisoning would by then be minimal . Two lava lizards escaped captivity and five captive lizards died during captivity, resulting in a survival rate of 87%. Sixty Galapagos hawks were taken into captivity and held in purpose-built aviaries on Pinzon Island. All survived captivity and were released 12-14 days after the second aerial bait application. However, within 12 to 170 days after release, 22 mortalities of tracked Galapagos hawks were recorded . Unfortunately, reported to be due to the arid conditions of the island, residual poison persisted in lava lizards. The remaining Pinzon Island Galapagos hawk population (n = 10) was recaptured, returned to captivity, and treated with Vitamin K1, while the toxicological levels of Pinzon lava lizards were monitored . These captive Galapagos hawks represented 15% of the original population and were released when risk was considered acceptable, in July and August 2016. As of 2018, eight hawk nests had been observed on Pinzon with chicks and fledglings confirmed. Lessons learned: The rodenticide used in this eradication remained in the ecosystem much longer than in any previous rodent eradication project worldwide. This resulted in the secondary poisoning of predatory hawks long after expected; therefore, understanding the longevity of poisons in the local environment and possible pathways into at-risk species is essential to ensure captive wildlife are held for an appropriate length of time so as not to be impacted. Similar to the Whenua Hou fernbird experience, this eradication also highlighted the importance of field base trials, as laboratory trials do not always reflect the response of wildlife in the field. Lava lizards did not eat the rodent bait in the laboratory; however, they did in the field, leading to a greater impact on themselves and the hawks than expected. 3.2.6. Australia--Rat Eradication/Poisoning, Lord Howe Island 2019 Lord Howe is a permanently human-inhabited island group approximately 1455 hectares in size and having a diverse landscape, where rats have already been implicated in the extinction of five endemic bird species and at least 13 species of endemic invertebrates. After the successful eradication of cats, pigs, and feral goats from the Lord Howe Island group, ship rats and mice were then targeted. In 2019, brodifacoum baits were distributed across the island depending on habitat type and land use using aerial distribution in the uninhabited areas, and hand broadcast and locked bait stations in the inhabited areas. Following field observations on a range of species on the island in which they were presented with non-toxic bait, two species were thought to be at risk from the bait, Lord Howe woodhen (Gallirallus sylvestris) and Lord Howe pied currawong (Strepera graculina crissalis), and successful pre-emptive captive trials were undertaken for these species in 2013, prior to baiting. Twenty-two woodhens and ten currawongs were captured and held in captivity, with all individuals subsequently released successfully back into the wild. The woodhens were captured in family groups or pairs and held together in pens, and initial trials showed the need to be careful with the species' diet as they put on weight quickly on the captive diet . From this trial, despite the woodhens normally being very territorial, they were held in groups of 20-30 with great success. The idea to hold the woodhens together was undertaken from the experience with weka (a similar bird to the woodhen), on Kapiti Island, New Zealand (see above). For the main poison application, to minimise any potential impact, at least 85% of the woodhen population and 50% of the pied currawong population were placed into captivity. Birds were held for at least one month before baiting, and until risks of primary or secondary poisoning were considered no longer present. From ongoing surveys of the island, by the second autumn woodhen survey following the rodent eradication, 778 woodhens were recorded over a two-week period. This number nearly quadruples the population survey results prior to rodent eradication. Lessons learned: The Lord Howe Island eradication showed how important it was to build on the learning from previous operations based on similar species, i.e., woodhens vs. weka, not only to decide which species need managing but how they can be managed. The undertaking of pre-emptive capture trials before the poison application allowed a greater understanding of how animals would react to captivity, including understanding that they can put on weight easily with captive diets and can be held together in larger numbers than normal when needed, and assuring the local community of its success. This understanding allowed for better-conditioned individuals to be released back into their environment, with current surveys showing woodhen are thriving on the rodent-free island. 3.2.7. United Kingdom/South Atlantic--Mouse Eradication, Gough Island 2021 An attempt was made by the Royal Society for the Protection of Birds (RSPB) and Tristan da Cunha to eradicate mice from the rugged 6500 ha Gough Island between June and August 2021. Gough Island is part of a World Heritage Site in the southern Atlantic and is one of the world's most important seabird breeding areas, with 22 species of seabird species breeding on the island, many of which are globally threatened, as well as two endemic threatened land bird species. Invasive non-native mice have been responsible for demographically unsustainable levels of chick mortality in seabirds . However, it was the two endemic land birds, the Gough bunting (Rowettia goughensis) and Gough moorhen (Gallinula comeri), for which primary and secondary poisoning was of greatest concern during the eradication, as many of the seabirds would not be present on the island at the time of the poison application. Trials on the capture and holding of these two land bird species began early in the programme planning with 25 buntings and 30 moorhens captured and held for 6 weeks between April and September 2010. Over March to May 2021, 84 moorhens (pre-eradication population estimate of 3500-4250 pairs) and 100 buntings (from a population of 1041-1889 individuals; RSPB unpublished data) were captured and held in captivity during the mouse eradication poison application. Eighty moorhens and 103 buntings were subsequently released back into the wild after the completion of the bait application. Follow-up surveys had shown that there was still a significant wild population of buntings after the bait applications that was then joined by the safeguard bunting population. However, as expected, the wild moorhen population was significantly impacted, hence the importance of the aviculture operation. In late September 2021, once any sign of bait on the island and especially in the lowland moorhen habitat had disappeared (extensive searches were undertaken to validate this), the captive moorhen population was released into their preferred habitat. At the time of writing the status of the moorhen population is still not known: monitoring has proved difficult (e.g., few birds calling, larger potential habitat available, dense vegetation), and while moorhens remain on Gough, no breeding has as yet been recorded to show that the population is beginning to rebuild (A. Callender pers comm.). Lessons learned: It is highly recommended that the avicultural project be run as a separate parallel operation so as not to be overshadowed in its importance by the "high-profile" baiting operation. A dedicated husbandry team was essential for the capture, care, and survival of these two species. A comprehensive plan for all stages of the aviculture operation is required and it should be followed unless there is good justification to do otherwise. Adequate resourcing is crucial, especially considering sufficient capacity over the holding period to allow members of the team to have downtime, particularly on remote islands such as Gough. The documentation and recommendations from the pre-poisoning capture and holding of moorhens and buntings from Gough Island was an important tactic in the success of the survival of the species for release after the eradication attempt . Trials need to be carried out early and critically accessed, and final design undertaken by a combination of aviculturists and eradication specialists where appropriate so, if necessary, teams can work together. Trials should aim to hold the birds for as long as they are likely to be held for the operation when possible, as the initial Gough trials were not of sufficient length to test how issues such as pododermatitis might affect the birds. Unfortunately, the eradication attempt was not successful, possibly due to slug consumption of the aerial spread bait, which reduced the amount available for rodents and meant not all mice accessed a lethal dose . An independent review panel is currently assessing the Gough eradication attempt and will report its findings in 2023. 4. Discussion The conservation goal of pre-emptive capture/translocation of threatened wildlife during oil spills or eradication operations is to protect a biologically significant proportion of a range-restricted species or significant regional population to reintroduce individuals back to their original range to re-establish the population after an impact has been removed. There are examples both in oiled wildlife response and, particularly, eradications where wildlife was considered to be at risk, however, the at-risk population was not a biologically significant proportion of the species, range-restricted species, or significant regional population (i.e., could be reintroduced from other regions if impacted). Examples of these include eradications that impacted Giant Petrels (Procellariiformes spp.) and Skuas (Stercorariidae spp.) on Macquarie Island, Australian subantarctic , Antipodes Island, New Zealand subantarctic , and South Georgia, South Atlantic . It should also be noted that these species would also be difficult to hold in captivity, and particularly to hold sufficient numbers of individuals for the risk period to re-establish the population. The main result from this review is how few of the pre-emptive capture/translocations have occurred for oiled wildlife response and how few have been documented for island eradication operations considering the number of both that have occurred in the last three decades . Despite this, there are still valuable lessons to be learned from what has been documented. The most important lessons learned from both responses is the importance of planning and a specific, dedicated team for the capture and care of captive wildlife. In the case of eradications, planning can be very specific as the site and species present are known well in advance. Whereas planning for oiled spills is more likely to be generic because the specifics of the event, e.g., timing, location, season, etc., are uncertain. However, there will be known endangered or range-restricted species that can be identified within a region or country that can have plans developed for them in advance in the case of a spill in their area. In general, eradication operations have significantly more time and ability to learn from both laboratory and field-based trials, including non-toxic bait trials, to determine species likely to be at risk, and to be able to trial the capture and holding of species prior to poisoning event, as seen for Gough and Lord Howe . However, it is only recently that the capture and captive care of protected wildlife has been undertaken by specialist rehabilitators/zoological carers (Lord Howe, Taronga Zoo, and Gough, Royal Society for the Protection of Birds) for eradication operations, and that there has been greater documentation and reporting of the methodology of pre-emptive capture and care techniques, successes, and recommendations. Conversely, since the Exxon Valdez oil spill in 1989, oiled wildlife response has almost always been undertaken by professional and/or experienced wildlife veterinarians or rehabilitation centres, and has involved greater monitoring and documentation of events. Unfortunately for wildlife, due to the random, unexpected, and usually instantaneous nature of oil spills, and lack of planning, pre-emptive capture has not been undertaken frequently in oiled wildlife response. The speed at which the oil covers and impacts the environment and wildlife is often too fast to allow pre-emptive activities to occur; however, those that have occurred have been reasonably documented (i.e., ). Additionally, this review has highlighted that it is not only pre-emptive capture and translocation prior to wildlife being oiled that is a successful, useful management tool for oiled wildlife response. The translocation of cleaned rehabilitated birds outside the area of the oil, to lengthen the time before wildlife return to their habitat, has been shown not only to reduce their chance of being re-oiled, but also reduced the time spent in captivity, therefore reducing secondary problems that can occur in captivity such as pododermatitis . For both oil spills and eradications, an essential lesson is understanding and using the knowledge learned from previous operations to improve current operations. These learnings can be everything from understanding species likely to be impacted, to how to manage and care for species, and for how long to keep them in captivity. However, this type of information needs to be written up and made publicly available from past responses for these lessons to be learned and used in the future. All reports and articles on pre-emptive capture outline how logistically challenging it can be and, depending on preparation time prior to an oil spill or eradication poison application, pre-emptive capture can seem almost unfeasible. However, the case studies above outline how, with consideration and planning, particularly for planned eradication poison applications, pre-emptive capture or translocations can be successful and save significant proportions of populations or range-restricted species from potential extinction events. For an oil spill event, decisions for pre-emptive capture must be made in a time-critical window, meaning delays could result in wildlife being oiled or injured, or dying. Therefore, understanding the requirements as suggested below prior to a spill occurring, and therefore the activation of a predetermined wildlife plan and personnel immediately after an oil spill, is needed to ensure protective and preventive actions can be undertaken if the situation allows . Primary requirements for pre-emptive captures include:Before a spill or eradication--determine potential species at risk: consider the numbers and species of wildlife, their threat classification and geographic extent, the animals' behaviour (seasonal, feeding, breeding), response options available for each species, and whether it is practical for the species to be kept in captivity or if capture and translocation are more appropriate . For eradication activities, this includes developing an inventory of non-target species, including bait-competitors, and a simple food web model to try and understand all possible primary and secondary poison routes pathways (e.g., ). Both laboratory and field trials are recommended. Conduct applicable capture planning (techniques and personnel) to ensure animal welfare, i.e., conduct site assessment for capture and housing: consider site accessibility and the prioritisation of locations (accessibility, tide, weather), and have knowledge of species behaviour and the geographical area, and lists of experts and pertinent contacts. Plan for appropriate captive care arrangements (housing, husbandry, personnel expertise, etc.). Plan and possibly trial relocation solutions (release location, transport, site fidelity, predicted time to return, energetic costs of return, etc.). Ensure the plans for aviculture can logistically be undertaken given the species, scale of operation, and numbers of individuals or species that need to be held. Critically, gain approvals from relevant government agencies and first nations groups, where applicable, for the capture, handling, and holding and transfer/release of wildlife. 5. Conclusions The difficulty of capturing wildlife safely and providing for their health in a captive environment or during relocation must never be underestimated. The risk of impacts from oiling or primary or secondary poisoning must be weighed against the risks of injury, disease, or death of wildlife during the entire pre-emptive capture and holding process. To determine the effectiveness of a wildlife pre-emptive capture process, it is critical to monitor and quantify the short- and long-term success or failure of the project. Relative to the number of oil spills and eradication operations that have occurred, direct counts of mortality and pre- and post-event wildlife monitoring studies are still rare. These types of research are critical to fully understand the total and long-term benefits of pre-emptive capture operations of wildlife . One of the strongest recommendations from this review is that, once species are identified that are suitable and likely to require pre-emptive capture and holding or translocation, the development of prospective techniques for them should be undertaken by a dedicated and experienced team, and fully documented, with outcomes made publicly available to inform future conservation planning. Acknowledgments Thank you to Andrew Callender for his input into the section on Gough Island in this paper. Thank you also to Bridey White and the anonymous reviewers who contributed to the paper. The Gough Island Restoration Programme was undertaken by the Royal Society for the Protection of Birds and Tristan da Cunha. Thank you to Massey University. Author Contributions Conceptualization, B.L.C.; Methodology, B.L.C.; Formal Analysis, B.L.C.; Investigation, B.L.C. and P.J.M.; Resources, B.L.C. and P.J.M.; Writing--Original Draft Preparation, B.L.C.; Writing--Review and Editing, B.L.C. and P.J.M.; Visualization, B.L.C.; Supervision, B.L.C.; Project Administration, B.L.C.; Funding Acquisition, B.L.C. All authors have read and agreed to the publication version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement All data used in this review is already publicly available. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Locations of case studies. Oiled wildlife responses are shown as a black star. Eradication operations are shown as a grey star. animals-13-00833-t001_Table 1 Table 1 Case study summaries highlighting species protected, control measures, outcome and lessons. Case Study (Reference) Species Protected Control Outcome Lessons Oil Spill Response MV Iron Barron, Tasmaina, 1995 Little blue penguins (Eudyptula minor) Translocation 480 km from spill after wildlife cleaned and rehabilitated to allow time for area to be cleaned before wildlife returns 863 translocated, 56% reported returned within 4 months, no difference in survival rates recorded between translocated and non-translocated wildlife Translocation considered effective, recommend trialing distances before being implemented MV Treasure, South Africa, 2000 African Penguins (Spheniscus demersus) 19,506 penguins were pre-emptive captured and translocated ~700 km away to allow time for area to be cleaned before wildlife returns. 3350 orphaned chicks captured and hand reared One year after the spill, 84% of the translocated birds were re-sighted, compared with 55% of the captured, cleaned, and released birds. Of the 3350 chicks collected approximately 2300 were fledged and released Translocations considered effective however greater consideration of conditions prior to and during transport needed. Preemptive capture and hand rearing of chicks was a successful conversation practice which can be used for oil spills, droughts and other human and natural impacts. Deepwater Horizons, USA 2010 Brown Pelicans (Pelecanus occidentalis) Translocation and supplementary feeding away from spill area after wildlife cleaned and rehabilitated to allow time for area to be cleaned No morality of translocated birds reported, birds mixed with local flock and stayed for 4 to 6 weeks Translocations and supplementary feeding considered successful. Shorter time period of feeding suggested and tracking of translocated individuals MV Rena, New Zealand, 2011 Northern New Zealand dotterels (Charadrius obscurus aquilonius) 60 dotterels pre-emptively caught and held for 60 days 90% survival to release Critical to have a dedicated captive management team. Strong recommendation that if shorebirds are preemptively captured, that the clean-up of their habitat is prioritised to enable as early release as possible. Eradication Operation Kapiti Island, New Zealand 1996 North Island weka (Gallirallus australis grey) Capture and transfer of 243 weka to mainland NZ Some Weka not transferred survived the aerial poisoning and no reintroduction back to the island was made. Weka now breed prolifically on the island and are fully recovered Species at risk should be identified through both non-toxic bait trials and knowledge from species at risk from previous operations Whenua Hou/Codfish Island 1998 Fernbirds (Bowdleria punctata wilsoni), Short-tailed bats (Mystacina tuberculatus tuberculatus) Fernbirds--21 birds transferred to a nearby rat-free island and poison placed in bait stations in highest density fernbird habitat instead of aerial spread Bats--captured and translocated onto another island and 386 held in captivity on island for ~90 days Fernbirds--transferred birds successfully translocated, established, and bred and have not been transferred back. Most fernbirds on the island were thought to be killed. However enough survived or naturally reintroduced to recover and expanded their range without rats. Bats--capture and release unsuccessful, none know to survive. Capture and hold on the island was considered successful Dedicated husbandry teams are needed for the pre-emptive capture of species during eradication projects. The additional cost of an additional rat eradication and transfer of a security population to another island was considered warranted even though not needed in the end. Seychelles 2000 Seychelles magpie-robins (Copsychus sechellarum), Seychelles fodys (Foudia sechellarum), Aldabran giant tortoises (Geochelone gigantea) 590 individuals from the 3 species were held in captivity on the island for up to 90 days during eradication All individuals survived capture and were released. Magpie robins breed in captivity Dedicated husbandry teams are essential for success and allow for increased knowledge and capability for the aviculture of species Anacapa Islands, California 2001 and 2002 Anacapa deer mouse (Peromyscus maniculatus anacapae), Peregrine falcons (Falco peregrinus) Aerial poisoning was conducted over two years. Pior to each drop deer mice were live captured and held in captivity or before the second application mice (from the soon to be poisons island) were transferred into the wild on the now rat-free island Raptors were live captured prior to rodenticide applications (peregrine falcons, red-tailed hawks, barn owls, and burrowing owls). Most were released on the mainland in suitable habitat; peregrine falcons were held and released back onto Anacapa 3 weeks after rodenticide applications There were no signs of rats or wild deer mice on the islands after poison applications. Deer mice that had been captured were released back onto rat-free islands 5 months after applications. In both years, >90% of the deer mice taken into captivity were released. Captive holding and translocation significantly reduced raptor mortality. One granivorous bird species, rufous-crowned sparrow, Aimophila ruficeps Obscura ,showed an unexpected significant decline This was the first recorded rodent eradication that ensured a native endemic rodent, which showed to be equally susceptible to the bait as the rats, to survive. Eradication showed the importance of learning from previous operations, particularly based on species similar to raptors, as some granivorous birds may require captive-holding efforts or no-drop zones to minimize risk for non-target impacts as seen on Codfish Is, NZ. Demonstrates the need for well-designed data-driven mitigations. Galapagos 2012 Pinzon giant tortoise (Chelonoidis duncanensis), Pinzon lava lizards (Microlophus duncanensis), Galapagos hawks (Buteo galapagoensis) 15 tortoises captured and held on another island for 2 years 40 lizards held in captivity until 10 days after second bait spread 60 hawks were captured and held in captivity until 12-14 days after second bait spread All tortoises survived, were released, and have since bred 87% survival rate of lizards in captivity Unfortunately, 22 hawks died 12 to 170 days after the release of secondary poisoning therefore 10 were recapture treated with Vit K and not released until poison levels known to reduce Rodenticide lasted longer in the environment than expected. Lizards did not eat bait in laboratory trials, but did in the field, emphasising both laboratory and field trials should be undertaken to determine species at risk Lord Howe Island, Australia 2019 Lord Howe woodhen (Gallirallus sylvestris) and pied currawong (Strepera graculina crissalis) Trial preemptive capture of both species prior to poison spread. 85% of woodhen population and 50% of currawong population captured before eradication operation and held until one month after. All woodhen and currawong survived capitivity and woodhen population now quadruple pre-eradication population size Importance of pre-emptive capture trials to understand how to manage wildlife in captivity Eradication also showed the importance of learning from previous operations particularly based on similar species Gough Island, Tristan da Cunha, UK 2021 Gough bunting (Rowettia goughensis) and moorhen (Gallinula comeri) Bunting and moorhens were trial preemptively captured and held before poison spread 84 moorhens and 100 buntings captured and held during poisoning 80 moorhens and 103 bunting released Buntings continue to do well; however, the status of moorhens is unknown Unfortunately, the rodent eradication was not a success Recommended that the avicultural project be run separately but parallel to the eradication operation A dedicated husbandry team with a comprehensive plan was essential Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000062

In Corozalito beach, Costa Rica, Olive Ridley turtles (Lepidochelys olivacea) nest both solitarily and in arribadas. The predation of solitary nests was monitored from 2008 to 2021, recording date, time, sector of the beach, zone, status of nest (predated or partially predated) and predator when possible. We recorded 4450 predated nests in total (N = 30,148 nesting events); predation rates showed a fluctuating trend, with recent percentages reaching up to 30%, with four distinctive dips in 2010, 2014, 2016 and 2017. The spatial distribution of predated nests along the beach showed significant differences among the sectors regardless of the seasons (Friedman test, chi-squared = 14.778, df = 2, p-value = 0.000), with most predated nests (47.62%) occurring in the northern sectors of the beach. Predators were identified by their tracks and/or direct observations (N = 896, 24.08%). The most conspicuous predators identified were raccoons (55.69%) and black vultures (22.77%). As seen in Corozalito, predation rates have increased in recent years despite established conservation efforts. A comprehensive assessment of all threats towards the overall hatching success for clutches is needed, considering predation during mass nesting events, poaching and beach erosion, among other factors, to fully understand the nesting dynamics occurring in this beach. nest predation sea turtles nesting beach eastern pacific RIESTER FoundationThis project was funded by the RIESTER Foundation. pmc1. Introduction The Olive Ridley sea turtle (Lepidochelys olivacea Eschscholtz 1829) is widely distributed in circumtropical waters and reaches its greatest abundance along the eastern rim of the Pacific Ocean . It is known to be the most globally abundant sea turtle, being one of the two species in the genus Lepidochelys that nests in a unique massive synchronous fashion known as "arribada", with more than half a million female nesting turtles during a single season . Arribadas have been described in the Eastern Pacific, the Western Atlantic, and the Northern Indian Ocean , but the species also nests widely and abundantly throughout the beaches of the Central American coast in solitary events . Despite these large numbers and wide global distribution, the Olive Ridley sea turtle is currently listed as Vulnerable by the International Union for the Conservation of Nature's Red List of Threatened Species with a persistent decreasing population trend . In addition, the species is included in Appendix I of CITES, since it is considered an endangered species whose marketing control (in its entirety or for by-products) is quite strict . Simultaneously, Costa Rica protects Olive Ridley turtle populations under the Wildlife Conservation Law No 7317 , Protection and Conservation of Sea Turtles No 8325 and lists of national species (resolution No 092-2017) . The intense protection, either at a national or international level is due to the major cause of detriment to Olive Ridley turtle populations in the Eastern Pacific, which is thought to have been massive commercial overexploitation of their eggs, and to a lesser degree high coastal fishery induced mortality . The protection of sea turtle nesting beaches to enhance recruitment is a common strategy to assist in the recovery of their populations . Natural stressors, however, play an important role when resorting to this strategy, as sea turtle nests are subject to a series of natural threats such as habitat destruction caused by erosion resulting from high tides, sea level rise, or flash floods at river mouths . Nests and hatchlings are also highly vulnerable to predation, most frequently by ants, beetles, snakes, coatis, raccoons, pigs, foxes, coyotes, and domestic animals (e.g., dogs) . Secondary or opportunistic predation also occurs in previously raided nests by vultures, crabs, and maggots . The contribution of each predator to the estimated predation rate differs greatly between sites. Historic predation by raccoons (Procyon lotor, Linnaeus 1766) and armadillos (Dasypus novemcinctus, Linnaeus 1758) occurred upon 95% of Green (Chelonia mydas), Loggerhead (Caretta caretta) and Leatherback turtle (Dermochelys coriacea) nests laid in Hobe Sound, Florida in 2002 , whereas high Loggerhead turtle nest destruction (78%) resulted from nest infestation by beetle larvae (Lanelater sallei, LeConte 1853) in southeast Florida from 2002 to 2003 . Furthermore, 22 species of ants have been identified raiding Green, Loggerhead and Leatherback turtle nests in Palm Beach, Florida . According to Cornelius (1982), the most frequent and conspicuous mammal predators of Olive Ridley turtle nests and hatchlings along the Pacific coast of Costa Rica are the coatimundi (Nasua nasua), followed by raccoons (Procyon spp.) and coyotes (Canis latrans). Avian predators were also observed, with magnificent frigate birds (Fregata magnificens), black vultures (Coragyps atratus), caracaras (Caracara cheriway), and turkey vultures (Cathartes aura) creating the greatest impact. Additionally, 29 species of birds, mammals, reptiles, and crustaceans were also listed as either suspected or confirmed scavengers or predators of Olive Ridley eggs and/or hatchlings . Sea turtle conservation programs boomed throughout the Pacific coast of Costa Rica during the 90s at different Olive Ridley nesting beaches, to not only monitor nesting activity and estimate population trends , but also to protect nests with the intent of increasing hatchling production, thus preventing further population detriment . The Rescue Center for Endangered Marine Species (CREMA by its Spanish acronym) has been running a sea turtle nesting monitoring program for over 24 years along the Costa Rican Pacific . Although predation and other nesting events have been recorded over the years, these events have not been properly described nor analyzed until recently. Reavis et al. (2022) described the threats that predation and human interactions represent at two Olive Ridley nesting sites monitored by CREMA, with their findings showing similar tendencies and dynamics when compared to other nesting sites, where typically predation and poaching decrease considerably with time after conservation projects are initiated . Corozalito beach is a solitary and nascent arribada nesting beach in the Pacific of Costa Rica, which has been monitored uninterrupted from June to January since 2008 by CREMA. This location holds the highest number of solitary nesting events (over 2000 records per season) among the sites covered by CREMA's monitoring program , and more recently has had estimated numbers in arribadas ranging between 2000 and 21,000 egg-laying females in a single nesting event . These mass nesting events normally occur once per month, from August to December, overlapping with solitary nesting events . The aim of this study is to evaluate natural nest predation upon solitary Olive Ridley nesting events (quantity, percentage, distribution of predated and partially predated nests and most common predators). This evaluation intends to provide insights on the impact of predation upon Olive Ridley sea turtle nests at this important site and recommend effective conservation strategies. 2. Materials and Methods 2.1. Study Site Corozalito (9deg50'54.28'' N, 85deg22'43.86'' W), located in the Southern Nicoya Peninsula, Guanacaste, Costa Rica , is a beach flanked to the north and south by rocky outcrops, with an associated wetland and river mouth to the south . For monitoring purposes, only the available nesting area of the beach was divided transversally into 25 m sectors from north to south using numbered posts, which corresponded to 768 m of beach. Sector markers were placed in line with the vegetation, with numbers facing the water . These sectors were then grouped into three main areas: the northern area (sectors 1 to 10), characterized by dense shrubs and low-lying trees, the central area (sectors 11 to 20), characterized primarily by high standing palm trees and little to no ground cover (this area also contains multiple picnic tables and a shelter), and lastly the southern area (sectors 21 to 30), that is primarily ground cover with some mangrove trees encircling the small estuary . This beach in the Pacific of Costa Rica shows two seasons: a dry season, or "summer", from January through June, and a rainy season, or "winter", from July through December . In addition to the sectors, the beach was divided into three zones that lie parallel to the high tide line, determined by the proximity to either the vegetation and/or the average high tide line (Zone 1 = below the average high tide line; Zone 2 = between average high tide line and vegetation; Zone 3 = above the line of vegetation) . Overall, the beach shows the primordial characteristics of an Olive Ridley sea turtle nesting beach; it is a sandy beach, with a gentle inclination platform, easy access from the sea and an important river/estuary at one of its ends . Organic debris is abundant on this beach and includes logs and tree branches from the nearby river mouths. Palm trees are predominant, yet beach almond trees (Terminalia sp.), button wood mangrove (Conocarpus erectus), white mangrove (Laguncularia racemosa) and other small shrubs are also common . In addition, Corozalito not only hosts solitary nesting events for Olive Ridley, but also Green, Hawksbill and Leatherback turtles, and the mass nesting events (arribadas) previously mentioned . Nesting activity has showed an increase over the 13-year study, with an average of 2156 nesting events per year, with over 45% of nesting occurring on the northern sectors and approximately 60% in zone 2 (CREMA unpublished). Neither predation over arribada nests nor the impact of arribadas were considered on our analysis. 2.2. Data Collection and Analysis Data on sea turtle nest predation upon solitary nesters were compiled following the CREMA monitoring protocol . Field researchers checked the integrity of all nests laid during night patrols or morning surveys from 2008 to 2021 nesting seasons (running from June to January), recording date, time, sector, zone, status of nests (predated or partially predated) and predator (when possible) . A predation event was considered when broken eggshells were found around the nest, with evidence of non-human disruptions . Data were taken only on recently predated nests between 19:00 and 05:00 (times when night patrols and morning censuses were conducted) . The type of predator was determined by how the nest was entered (digging, poking holes, etc.) and tracks around the nests . The primary predator was determined by the fresher tracks in nests during night patrols, whereas secondary and/or third predators were confirmed during morning surveys on nests that have been previously recorded to be predated at least once . Sometimes, predators were observed searching around freshly laid nests and later on digging the nests to predate on them; when possible, photos were taken . Notes on predation were also taken when performing nests excavations . Movements or dynamics of predators were not analyzed. In contrast to other CREMA monitoring projects , all nests laid in Corozalito were left in situ due to the high number of sea turtles that come to nest, either in solitary fashion or in arribadas . Data from the 2020 season were collected only during the morning censuses due to the restrictions during the global COVID-19 health emergency. Descriptive and summary analyses were calculated, including means, standard deviations, minimum and maximum values, using Excel (r) 2007 spreadsheets and STATISTICA (r) v7 . Predation rates were calculated as the percentage of total nests that were destroyed and/or consumed by predators among the total of nesting events during the study . Due to the non-normal distribution of predated nests as shown by the Shapiro-Wilk test, non-parametric statistical analyses were performed using the Friedman test to detect any associations with their spatial distribution among sectors; statistical significance was set at a <= 0.05. This analysis was carried out in program R . 3. Results 3.1. Partial and Complete Predation of Nests We found a total of 4450 predated nests (partial and complete) from 2008 to 2021 nesting seasons (N = 30,148 nesting events recorded) (Table 1). Only 7.23% (n = 332) were considered partially predated, although predation was most certainly complete after several days. The follow up of partially predated nests, however, was not monitored. Predation rates showed a fluctuating trend during the sampling period, with increasing values over the years and four distinctive downfalls in 2010, 2014, 2016 and 2017 (Table 1). The lowest predation rate (4.711%) occurred in 2017 and the highest rate during the 2020 nesting season (28.35%). Alternatively, predation rates were the highest in August and September (3.55% and 3.36%, respectively), followed by a decreasing trend by the end of the season . 3.2. Distribution of Predation Events The distribution of predation events alongside the beach showed an heterogenous pattern, with 47.62% of the total predated nests in the northern area of the beach (sectors 1 to 10), followed by the central area (sectors 11 to 20) accounting for 28.67% of predated nests, and lastly the southern area (sectors 21 to 30), with 23.71% of predated nests (Friedman test, chi-squared = 14.778, df = 2, p-value = 0.000). Individually, sectors 4 and 5 showed the highest number of predated nests (Sector 4 = 27.86 +- 23.08 predated nests; Sector 5 = 23.36 +- 17.35 predated nests), while sectors 1 and 30 showed the lowest (Sector 1 = 1.93 +- 2.40 predated nests; Sector 30 = 1.00 +- 1.80 predated nests) . Indeed, the pairwise Wilcoxon signed rank test between these three areas revealed statistically significant differences in the number of predated nests between the northern and southern sectors (p-value = 0.006), a suggested difference between the center and northern sectors (p-value = 0.08) and no significant differences between center and southern sectors (p-value = 1.00). In addition, most predated nests were recorded in zone 2 (218.43 +- 170.69; 68.72%), while zone 1 showed the lowest number of predated nests (9.14 +- 13.67; 2.88%). 3.3. Nest Predators Nine species of mammals, birds, crustaceans and insects were identified and confirmed as nest predators (only eggs) in Corozalito (Table 2). In most of the cases (75.92%), nest predators remained unidentified as tracks were erased or were difficult to categorize due to rain, tides, debris, other turtle tracks, tourism, etc., which causes a bias in identifying predators . Predators were identified in 24.08% of the predated nests (n = 896 predation events). The most active and visible predators were raccoons (55.69%), followed by vultures (22.77%). One, two or even more predators at a time were recorded predating nests. In most cases (63.90%), two different species were recorded predating a nest as scavengers or secondary predators (mostly hermit crabs and vultures). These predators were observed during morning surveys predating over nests that had been partially predated during night patrols. 4. Discussion 4.1. Predation Rates In the past decade, nests from Corozalito have become an easy target for different predators. A noticeable increase in predation rates was detected, from 2008 with 7.95% to 2021 with close to 30% of nests predated. Predation rates in Corozalito showed four dips during the sampling period, with the lowest predation rate occurring in 2017 (Table 1), which is closely related to the total number of solitary nesting events (n = 3396) during that nesting season (CREMA, unpublished). The highest predation rate (28.35%) was recorded in 2020, when beaches and other public places were closed to researchers and the public due to the restrictions imposed by the Costa Rican government during the COVID-19 health emergency. Reavis et al. (2022) reported the predation dynamics of two solitary nesting sites, San Miguel and Costa de Oro, about 15 km south of Corozalito, finding an overall decreasing predation rate trend. They found that the differences regarding predation rates between both sites were primarily due to the length dissimilarity in conservation efforts on each site; with higher predation rates (25%) in the most recent project site (Costa de Oro, starting date 2012), in comparison to San Miguel (6%), with a longer trajectory on protecting sea turtle nests (starting date 1998) . Different authors have mentioned that, in the absence of monitoring efforts in nesting beaches, both the number of predators and frequency of predation increases significantly, resulting in changes for both predator and prey alike , a trend that has been observed in Corozalito during the 2020 COVID-19 lockdowns, and in San Miguel and Costa de Oro before the establishment of CREMA's conservation program. High nest predation has been observed in other nesting beaches worldwide with high nest densities , where nest predation rates ranging from 30% to 80% have been detected, before and/or during the incubation period . Corozalito is a high nest density beach, with up to 2800 solitary nests deposited per year (CREMA, unpublished), as well as several arribadas per year involving several thousands of nesting females over a period of 3 to 4 days . While arribada nesting may reduce the chance for predation due to predator satiation, it has been previously noted that it could also increase overall predation by attracting predators . In fact, the increased predation over time that we found in the present study agrees with the pattern exhibited by Corozalito's arribada, which displays a clear growing trend in annual frequency and days of duration (from 2008 to 2021) . Predation events were also highest in August and September, followed by a steady decrease in these percentages during the remaining months, which corresponded to the months with the lowest nesting activity in the area. Rojas-Canizales et al. (2022) suggested that Corozalito is in its early stages of development as an arribada beach, with arribadas occurring from August to December and the largest recording during the months of September and October . These events also coincide with the occurrence of the largest arribada events in the Eastern Tropical Pacific region. These conditions may suggest the hypothesis that arribada beaches attract more predators, which then may have an impact on nesters that choose the arribada beach to nest individually. Several authors have also suggested that predation might play a relatively small role in determining hatching success in arribada beaches, but it may be heavily detrimental in solitary nesting . In fact, in beaches where both reproductive strategies take place, other authors have reported that 51% of first-night solitary nests were predated in contrast to a low percentage of arribada nests (7.8%), supporting the hypothesis of predator satiation in the evolution of the arribada phenomena . Unfortunately, predation rates on arribada nests were not accounted for in this study, hence making it difficult to estimate the overall predation threats upon Olive Ridleys nests (both solitary and arribada) at Corozalito. 4.2. Spatial Distribution of Predation Events Several variables may determine the distribution of predation events alongside the beach. Kolbe and Janzen (2002) explained that there is a significant effect of distance from the edges (water or wooded edges) where the nests are located. In their study, the water edge represented an abrupt ecological edge, which demarcates where resources transition from completely unavailable to available to some degree . Similarly, the environmental characteristics in Corozalito suggests playing an important role in the dynamics of predation. For instance, a larger number of predated nests and, hence, a higher predation rate occurred at the northern sectors , where most nesting events (successful and unsuccessful) have been recorded ; this area shows ideal physical characteristics (such as grain size, slope and humidity, among others) for nesting, as described by several authors before . In addition, the presence of dense vegetation at the northern end of Corozalito might provide an ideal predator habitat and shelter . Subsequently, in the central area, the regular visits by community members, tourists, and researchers could have a barrier effect towards predators, excluding them from the nests laid in this area . Lastly, in the southern sectors are covered by rocks, which might represent a major factor that drives nest selection in Olive Ridleys, therefore resulting in lower predation rates . Additionally, beach profile results demonstrated that the highest incidence of predation occurred in zone 2 (between average high tide line and vegetation), which may be due to the significantly higher frequency of nesting events that take place in this zone as well . Several authors found similar results, with nests often located on the un-shaded center of the beach with a strong preference for nests to be located as far from the high tide line as possible . Even though most nests were placed far from the high tide line, some of them were flooded, damaged, or eroded by sea water (due to 'spring tides') and later found to be destroyed by predators. 4.3. Predator Identification According to several authors, some of the most common sea turtle nest predators are feral domestic animals and raccoons . Cortes-Briceno (2015) recorded nest predation in Mexico, where the complete predation of Green and Hawksbill turtle nests was often associated with feral animals (mainly dogs), while partial predation of nests was caused by other animals such as birds, ghost crabs, and ants (the latter being the most frequent) . Raccoons are the main raiders of nests in the United States, with up to 80% of total Loggerhead and Green turtle nests predated on certain southern beaches . Reavis et al., 2022, found that the main predators of Olive Ridley nests in San Miguel and Costa de Oro were domestic dogs (Canis familiaris), followed by raccoons, which accounted for 58% and 42% of depredated nests, respectively . The most common predators recorded in Corozalito were raccoons (55.69%), which were observed digging up undisturbed nests, leaving them vulnerable to secondary predators and/or scavengers such as vultures (22.77%) and hermit crabs (13.28%). Other predators, such as coatimundis (6.14%) and feral dogs (0.89%), were only recorded predating on well-developed clutches. The main predators and their impacts on the nests are different between Corozalito and the other two project sites (San Miguel and Costa de Oro), which could be due to the proximity of the population centers in these three localities; the town of Corozalito is 2.5 Km inland , whereas San Miguel and Costa de Oro communities are just a few steps from the beach , making it an easy access for domestic animals. Similar results according to predation richness were found in Playa Nancite (approximately 120 Km north of Corozalito) . Nelson and Mo (1996) indicated that nest predation was performed, in decreasing order, by coyotes (Canis latrans), raccoons (Proycon lotor), coatis (Nasua narica), humans (Homo sapiens sapiens), and caracaras (Polyborus plancus). Both Corozalito and Nancite's population centers are far inland from the nesting site, with the latter also located in the Santa Rosa National Park, known as a protected area . Other predators, such as caracaras (0.36%), skunks (0.45%), and tayras (0.12%), were considered rare encounters, perhaps opportunistic predators . Finally, maggots were only observed during excavations of old or non-developed nests . 4.4. Conservation Efforts Nest predation has recently become an issue in Corozalito, which has increased over the years and needs to be addressed with an appropriate approach . In fact, Corozalito is a public beach that was initially protected by a group of inhabitants from the nearby town, many of which have expressed that the increase in nesting sea turtles, and thus nests laid per season, was responsible for the increase of predators that find an endless source of food on this beach . Predator impacts on eggs and hatchlings of sea turtles are somewhat easy to document, a necessary task to plan an effective management program . Many conservation programs have addressed both nest predation and poaching (illegal taking) by implementing hatcheries, where nests are relocated for their protection, and monitoring programs to decrease and/or regulate the presence of people on the beach. These actions have helped in the conservation of several sea turtle populations worldwide . However, hatcheries have several limitations and may not always be the best approach . In high density nesting beaches such as Corozalito, preliminary assessments have concluded that less manipulative options are more practical and effective , and with the increase in frequency and number of arribadas the beach itself serves as an in-situ hatchery . The importance of Corozalito's sea turtle nesting populations and their nests are twofold; firstly, as mentioned before, this beach holds up approximately 2800 solitary nesting events of Olive Ridleys each nesting season, which could represent up to 80% of hatching success, that potentially would maintain part of the Eastern Pacific Olive Ridley turtle population . Secondly, Corozalito is now known as a nascent arribada beach, with higher hatching success of nests laid during arribadas, reaching up 50% , three times higher than already established arribada beaches such as Ostional and Nancite . This could also indicate that the hatching success is higher in solitary nesters regardless of high predation rates, although further research in this aspect is needed. As described by several authors in different nesting beaches, patrol-based monitoring programs have resulted in a successful reduction of several threats to both nesters and nests .We propose to continue CREMA's sea turtle nesting monitoring program, along with community-based programs (e.g., environmental education classes, incorporating locals in the monitoring program, among others), which will ultimately involve residents and visitors in the conservation of these reptiles . It is important to include predation rates from other sea turtle species' nests, as well as predation both during solitary and mass nesting events. Similarly, other threats, such as illegal egg extraction (poaching), have not been addressed since CREMA's nesting monitoring program started in 2008 . Viejobueno et al. (2012) estimated egg extraction rates in Corozalito from 2008 to 2011, observing a decreasing trend of poaching events from 30% to 6% during the three-year study . Nine years later, a study on illegal egg extraction in several localities both in the Caribbean and the Pacific coasts of Costa Rica tracked a poached turtle nest from Corozalito to a supermarket in the Central Valley of Costa Rica, 137 km away . Today, predation rates swivel around with similar values (5% in 2022, CREMA's unpublished data), nonetheless the general impact over nesters and nests has not been evaluated. Additionally, climate change must be assessed in order to give a complete report of all the threats that these species are facing in Corozalito and the impact on their populations . Long-term analysis of the nesting population on this beach would be beneficial in the future to elucidate the effects of all threats and conservation efforts . The design of a proper beach management plan is an imperative and valuable conservation strategy for the nesting sea turtles of Corozalito , which must include potential alternatives to manage predators' impact (e.g., translocation, altering predator foraging strategies , among others). These measures could help to reduce both human and natural loss of nests and improve measures to safeguard the Olive Ridleys and their nesting habitat . For lasting results, such alternatives must be supported and promoted by several entities such as the Environment and Energy Ministry, local communities, NGOs, other state institutions, and academia, among others. 5. Conclusions Our results suggest a clear increase in the predation of Olive Ridley turtle nests throughout the 13-year study period. This trend, although fluctuating over the years, could be also an indication of the increasing number of nesting events in Corozalito, and its full protection might be facilitated by the designation of Corozalito as a National Wildlife Refuge with a corresponding marine protected area, which together with the drive of the surrounding coastal communities and a continuation of long-term monitoring will better elucidate the possible effects on the nesting abundance of Olive Ridleys. Acknowledgments The authors would like to acknowledge all the research assistants, locals and volunteers who have helped us to collect data over the years in Corozalito. Thanks to Turtle Trax, S.A, for their collaboration and logistical support for this research during the sampling period. Special thanks to the RIESTER foundation, and its members Gary Kaasa, Mike Hopkins and John Lindsay. We would also like to thank the contribution of two valuable reviewers, who greatly helped to improve the quality of this paper. Author Contributions Conceptualization, N.E.-R.; methodology, N.E.-R., D.R.-C., C.M.-B. and I.N.; formal analysis, N.E.-R.; investigation, N.E.-R., C.M.-B. and D.R.-C.; resources, D.R.-C., I.N. and R.A.; data curation, N.E.-R. and D.R.-C.; writing--original draft preparation, N.E.-R.; writing--review and editing, N.E.-R., D.R.-C., C.M.-B., I.N. and R.A.; visualization, N.E.-R.; supervision, D.R.-C. and I.N.; project administration, D.R.-C. and I.N.; funding acquisition, D.R.-C., R.A. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This research was approved and authorized by MINAE, SINAC, and ACT under the research permits ACT-OR-DR-055-2020 and ACT-OR-DR-063-2021. Informed Consent Statement Not applicable. Data Availability Statement Restrictions apply to the availability of these data. Data were obtained from CREMA's dataset and are available from the authors with the permission of Isabel Naranjo (Director of CREMA). Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Figure 1 Geographical location of Corozalito and the surrounding Marine Protected Areas of the Camaronal and the Caletas-Ario National Wildlife Refuges, southern Nicoya Peninsula, Costa Rica. Bold red represents the monitored area. Figure 2 Division of the beach according to (a) sectors (markers indicated by arrows), and (b) zones in Corozalito (Photos: Espinoza-Rodriguez, 2019). Figure 3 Monthly predation rates (%) in Corozalito during the sampling period (2008-2021). Figure 4 Percentage of predated nests per sector in Corozalito, from 2008 to 2021; Northern Area (orange) = sectors 1-10, Central Area (blue) = sectors 11-20, Southern Area (green) = sectors 21-30. animals-13-00875-t001_Table 1 Table 1 Predation Rates (%) of Olive Ridley sea turtle nests in Corozalito, from 2008 to 2021. Percentage values are relative to the total number of nesting events. Total predation events include both partial and complete predated nests. Season Total Nesting Events Total Predation Events Annual Predation Rate (%) 2008 1119 89 7.95 2009 1782 162 9.09 2010 1763 98 5.56 2011 1512 150 9.92 2012 1588 275 17.32 2013 1772 453 25.56 2014 1918 107 5.58 2015 2923 445 15.22 2016 2403 157 6.53 2017 3396 160 4.71 2018 2313 489 21.14 2019 2656 500 18.83 2020 2303 653 28.35 2021 2700 712 26.37 Total 30,148 4450 14.80 animals-13-00875-t002_Table 2 Table 2 Olive Ridley sea turtle egg predators and scavengers identified at Corozalito during the sampling period. Class Scientific Name Common Name (% of Occurrence) Mammals Procyon spp. Nasua narica Conepatus semitriatus Eira barbara Didelphis marsupialis Canis lupus familiaris Raccoon (55.69%) Coati (6.14%) Stripped Skunk (0.45%) Tayra (0.12%) Common Opossum (0.05%) Dog (0.89%) Birds Caragyps atratus Caracara cheriway Black Vulture (22.77%) Caracara (0.36%) Crustaceans Coenobitidae compressus Ecuadorian Hermit Crab (13.28%) Insects Unidentified Maggots (0.12%) Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000063

Many of the micro- and macro-elements (MMEs) required by the body are found in environmental objects in concentrations different from their original concentration that can lead to dangerous animal diseases ("microelementoses"). The aim was to study the features of MME (accumulating in wild and exotic animals) in connection with particular diseases. The work using 67 mammal species from four Russian zoological institutions was completed in 2022. Studies of 820 cleaned and defatted samples (hair, fur, etc.) after "wet-acid-ashing" on an electric stove and in a muffle furnace were performed using a Kvant-2A atomic absorption spectrometer. The content of zinc, copper, iron, cadmium, lead, and arsenic was assessed. The level of MME accumulation in the animal body contributes not only to the MME status and the development of various concomitant diseases, but the condition itself can occur by intake of a number of micronutrients and/or drugs. Particular correlations between the accumulation of Zn and skin, oncological diseases, Cu--musculoskeletal, cardiovascular diseases, Fe--oncological diseases, Pb--metabolic, nervous, oncological diseases, and Cd--cardiovascular diseases were established. Therefore, monitoring of the MME status of the organism must be carried out regularly (optimally once every 6 months). animal health and diseases essential and toxic microelements classes of diseases general morbidity of animals atomic absorption spectrometry Ministry of Higher Education and Science of the Russian FederationFSMF-2022-0003 Research Laboratory of Ophthalmology, Oncology and Biochemistry of Animals, Federal State Budgetary Educational Institution of Higher EducationThe work was carried out within the framework of the topic: "Etiopathogenesis and development of methods for diagnosing, preventing and treating immune-mediated paraneoplastic ophthalmopathies in animals" (project code FSMF-2022-0003 of the Ministry of Higher Education and Science of the Russian Federation) of the Research Laboratory of Ophthalmology, Oncology and Biochemistry of Animals, Federal State Budgetary Educational Institution of Higher Education "Russian Biotechnological University (ROSBIOTECH)". (Moscow, Russia). pmc1. Introduction Many "micro- and macro-elements" (MMEs) are found at different concentrations in environmental objects (especially in areas with a high anthropogenic load) that can lead to animal diseases . The supply of microelements to all organisms should occur only in optimal amounts . This is well-known for all domestic and farm animals, as well as for humans . That is why here we would like to point out only a few recent reviews concerning the comparison of inorganic and chelate forms of MMEs for broilers' feeding and some requirements of MME diets for two commercial broiler strains . For example, the numerous data of the most important MMEs (such as iron, copper, zinc, manganese, selenium) indicated that the chelate forms of these MMEs (i.e., complexes of MME with organic ligands) provided better "protection" for broilers, for "the environment and also improve egg quality" . A substantial comparison of the 9th and 10th versions of nutrient requirements of poultry (NRC 1994 and 2017) with the particular recommendations of "the trace element requirements for commercial broiler strains" (Ross 308 and Cobb 500, since 2007 and 2008, respectively) was provided by Iran scientists . In the "Conclusion" part of this paper , they wrote "the iron requirements of broilers have been increased and the requirements of copper, manganese, zinc, selenium and iodine have decreased compared with the NRC (1994) recommendations". However, all data (in the paper ) presented the opposite tendencies. The iron requirements have been decreased from 80 mg/kg (NRC 1994 for broiler chickens) to 40 mg/kg (for broiler strains Ross 308, since 2007 and Cobb 500, since 2008 or 2013) or even to 20 mg/kg (for broiler strain Ross 308, since 2014). The requirements of copper, manganese, zinc, selenium, and iodine have been increased from 8, 60, 40, 0.15, and 0.35 mg/kg (NRC 1994 for broiler chickens) to 16, 120, 100-110, 0.30, and 1.25 mg/kg (for broiler strain Ross 308, since 2007-2014) or 15, 100, 100, 0.30-0.35, and 1.0 mg/kg (for broiler strain Cobb 500, since 2008-2013) . In general, less is known about MMEs in the tissues of the wild and exotic animals compared to those for the farm animals and birds. It is especially important now because of the high technological activity all over the world that results in the introduction of the increasing amounts of various "trace elements into biogeochemical cycles" . In recent decades, wild animals have been successfully and increasingly used as "bioindicators of environmental pollution" . Due to their wide distribution and often high trophic levels, birds and mammals are the most suitable indicators of pollution, and therefore the determination of trace element concentrations, including heavy metals, in various tissues of different bird and mammal species (for example, in blood, feathers, liver, kidneys, muscles, eggs, feces, etc.) is widely used in "biomonitoring studies" . However, the most prominent studies have been connected with the exposure of animals to pollution in heavily polluted areas or aquatic ecosystems, whereas the relatively moderately polluted urban and suburban environments have been less studied, despite their ecological significance . Most studies are aimed at elucidating the effect of one pollutant or elevated doses of several substances , but there are still no clear patterns concerning the effects of trace elements in animals. A number of Polish authors revealed significant differences in the level of accumulation of microelements in animals living in nature and kept in captivity (i.e., in urban facilities). Therefore, the study of the features of their accumulation in animal zoological institutions, taking into account the characteristics of their maintenance and nutrition, is significant . It is important to highlight that a deviation from the optimal level can lead to the animal diseases ("microelementoses") with various degrees of severity . 1.1. MMEs in Animal Tissues and Organs Animals need MMEs in certain concentrations for the implementation of optimal metabolism level in their tissues and organs, as well as in the whole body. The clinical picture of the pathology occurrence (taking into account the complex interaction between individual microelements, the physical and chemical properties of their compounds) leads to difficulties of data interpretation due to the presence of direct and indirect (not always explicit) effects . For these purposes, it is of great importance to choose an adequate ("correct") marker, i.e., "biosubstrate", and comprehensive monitoring of the MMEs level, which will allow their accurate quantitative analysis . Each MME has its own optimal level of content depending on the animal breed. If this level deviates towards an increase or decrease in the concentration of an element in the body, a biological effect occurs. Moreover, the degree of bioeffect development depends on the microelement dose. Acute poisoning develops when the body absorbs or accumulates relatively large doses of toxicant MME. Chronic poisoning develops with prolonged, cumulative, action of chemicals. In this case, the increase in symptoms and the clarity of the manifestation of the clinical picture occurs gradually. The contact of chemical compounds with the epithelium and skin leads to their entry into the blood and lymph. Absorption is accompanied by special "transformation" of compounds, distribution and accumulation of microelements by organs and tissues . Four major and minor zoos in Russia were selected for our research. 1.2. The Moscow Zoo The Moscow zoo is the first among the major zoos in Russia and one of the oldest zoos in Europe, which was opened on February 13 (January 31), 1864. Now, it has the largest zoological collection in Russia and is the leading methodological center in the system of zoos in our country. At the moment, this zoo includes: a center for the reproduction of rare species of animals (zoo nursery) of the Moscow State Zoological Park, a branch of the zoo in the Estate of Father Frost ("Veliky Ustyug"), and the main exposition parts in the center of Moscow city. In total, this zoo contains 1340 species of various breeds and exhibits 14,105 wild and exotic animals, birds, etc., in total . The research facility of this institution specializes in breeding rare species of predatory mammals, birds of prey and waterfowl, cranes, poisonous snakes, etc. . There are about 15% of mammals in the total number of animals in the institution collection and 24-26% of birds (depending on the studied period) . The change in the number of animals is associated mainly with the acquisition and exchange of species between zoological institutions and the reproduction of formed pairs. During the studied period, the total collection of mammals increased by 19% of species (from 174 to 207) and 8.7% in total head numbers (from 1462 to 1589 items) mammals; birds increased by 12.3% of species (from 292 to 328), but the total head numbers decreased by 0.2% (from 620 to 612 items) . 1.3. The Ivanovo Zoo Ivanovo zoo is a rather small institution located in the capitol of the textile regional center (officially opened in 1994). Today, the territory of the zoo is about 3.4 hectares. In total, this zoo contains 790 animals of 173 species. The zoological institution specializes in breeding birds of prey. The collection of the institution is represented mainly by birds (60.8-67.6%) and mammals (22.0-28.9%) . In this zoo, the species diversity of mammals decreased by 25.5% (from 51 to 38 species) and by 13.7% in numbers (from 256 to 221 items). The number of bird species and heads increased by 9.3% and 12% (from 107 to 117 species and from 475 to 532 items, respectively) . 1.4. The Yaroslavl Zoo Yaroslavl zoo is the first landscape-type zoo in Russia, where animals are kept in conditions as close to natural as possible. Today, the territory of the zoo is about 123 hectares (under the exposition--58 hectares). In total, 1791 animals of 441 species live in this zoo. The institution specializes in keeping and breeding animals of the central Russia population. The collection of the institution is represented by birds (18.3-20.8%) and by mammals (13.0-14.9%), depending on the year of the study . The species diversity of the mammalian exposition decreased by 11.5% species (from 104 to 92), but slightly increased by 0.5% in total head numbers (from 440 to 442). The number of bird species and heads decreased by 1.3% (from 160 to 158 and from 620 to 612 items, respectively). The change in the number of livestock is associated mainly with the physiological conditions of some old animals . 1.5. The Uglich Zoo Station The Uglich zoo station is one of the smallest in Russia and located in a historical building since 1936. During the last year, more than 5000 people from the region and city guests came to the station for excursions and public events. In total, this station contains 305 individuals belonging to 52 animal species. The institution specializes in keeping and exhibiting exotic and domestic animals (especially in the contact mode). The collection of the institution, depending on the year of the study, is represented by birds (18.2-28.8%) and by mammals (25.0-31.9%) . The species diversity of the mammal exposition remained at the level of 13 species, but total head numbers increased by 33.7% (from 89 to 119 items, due to exchange and birth) . The number of bird species and heads increased by 87.5% and 77.3%, respectively (from 8 to 15 species and from 22 to 39 heads) . The change in the number of livestock is associated with the organization of regular monitoring and veterinary care of animals . The aim of this study was to evaluate the features of the MME accumulation in the biological samples (hair, fur) of wild and exotic animals from these four zoos during some diseases of various etiologies. 2. Materials and Methods 2.1. Living Creatures as Objects The studies were carried out based on the Moscow, Ivanovo, Yaroslavl, and Uglich zoos. The objects were wild and domestic animals of different taxonomic groups. The studies were carried out during the whole year. The level of microelements, including heavy metals, in biological media was analyzed based on the results of our original research. The following species of wild, domestic, and exotic mammals kept in zoological institutions in territories with different anthropogenic pressure in the Central Federal District of Russia were selected for study: Moscow, Ivanovo, and Yaroslavl zoos. In particular: bristly armadillo--Chaetophractus (Euphractus) villosus (n = 15), globular armadillo--Tolypeutes matacus (n = 9), Egyptian flying dog--Rousettus aegyptiacus (n = 15), hare--Lepus timidus (n = 9), European hare--Lepus europaeus (n = 6), yellow pied--Eolagurus luteus (n = 15), Mongolian (clawed) gerbil--Meriones unguiculatus (n = 36), Bush-tailed gerbil--Sekeetamys calurus (n = 18), Eastern mole vole--Ellobius tancrei (n = 6), Cactus hamster--Peromyscus eremicus (n= 9), golden (Syrian) hamster--Mesocricetus auratus (n = 30), Djungarian hamster--Phodopus sungorus (n = 15), Baraba hamster (Chinese hamster)--Cricetulus barabensis griseus (n = 9), acacia rat--Thallomys loringi (n = 15), spiny mouse--Acomys cahirinus (n = 30), dwarf mouse--Mus minutoides (n = 9), gray rat--Rattus norvegicus (n = 18), house mouse--Mus musculus (n = 18), multi-mother mouse--Mastomys natalensis (n =9), chinchilla (home form)--Chinchilla laniger var. dom. (n = 18), degu--Octodon degus (n = 24), guinea pig--Cavia porcellus (n = 27), Indian porcupine--Hystrix indica (leucura) (n = 12), red fox--Vulpes vulpes (n = 18), Fennec fox--Vulpes (Fennecus) zerda (n = 6), Arctic fox--Alopex lagopus var. dom. (n = 12), Alaskan Malamute--Canis familiaris (n = 12), polar wolf--Canis lupus tundrorum (n = 6), wolf--Canis lupus (n = 39), raccoon dog--Nyctereutes procyonoides (n = 15), raccoon--Procyon lotor (n = 6), nosoha--Nasua nasua (n = 9), domestic ferret (furo, ferret)--Mustela putorius var. Dom. (n = 15), common genet--Genetta genetta (n = 9), brown bear--Ursus arctos (n = 12), Ussuri white-breasted (Himalayan) bear--Selenarctos (Ursus) thibetanus ussuricus (n = 6), lynx--Felis (Lynx) lynx (n = 15), puma--Puma (Felis) concolor (n = 9), snow leopard (Irbis)--Uncia (Panthera) uncial (n = 6), Far Eastern (Amur) leopard--Panthera pardus orientalis (n = 9), Amur tiger--Panthera tigris altaica (n = 12), white lion--Pantera leo var. alba (n = 6), lion--Panthera leo (n = 6), Bruce's hyrax--Heterohyrax brucei (n = 3), alpaca--Vicugna pacos (n = 12), bactrian camel--Camelus bactrianus (ferus) dom. (n = 21), camel--Camelus dromedarius (n = 6), reindeer--Rangifer tarandus (n = 28), spotted deer--Cervus nippon (n = 9), European fallow deer--Dama (Cervus) dama (n= 9), European elk--Alces alces (n = 18), musk ox--Ovibos moschatus (n = 6), domestic yak--Bos mutus (grunniens) var. dom. (n = 12), bison--Bison bonasus (n = 9), Dagestan tur--Capra cylindricornis (n = 9), Sichuan takin--Budorcas taxicolor tibetana (n = 6), blue sheep--Pseudois nayaur (n = 3), Cameroonian goat--Capra hircus hircus (n = 12), domestic horse--Equus caballus (n = 33), Grant's zebra--Equus burchelli boehmi (n = 6), Przewalski's horse--Equus przewalskii (n = 9), domestic donkey--Equus asinus dom. (n = 12), common marmoset (Wistity)--Callithrix jacchus (n = 6), ring-tailed lemur--Lemur catta (n = 6), mandrill--Mandrillus (Papio) sphinx (n = 6), rhesus monkey--Macaca mulatta (n = 6), and lapunder (pig-tailed macaque)--Macaca nemestrina (n = 9). 2.2. Methods The studies were carried out on a "Kvant-2A" atomic absorption spectrometer. The selection of biological samples (hair, fur, etc.) of all types was carried out from the whole body with a total sample weight of about 1-3 g. The samples were cleaned and degreased with acetone and bidistilled water for two days. Then, "wet-acid-ashing" was carried out on an electric stove, and then in a muffle furnace with a gradual increase in temperature from 250 to 450 degC with a half-hour exposure. The samples were assessed for the level of trace elements--zinc, copper, iron, cadmium, lead, and arsenic. The results obtained were processed statistically. Arithmetic mean values (M), mean errors (m), and standard deviation (d) were determined. To identify statistically significant differences in the compared groups and the contingency between the signs, the nature of the distribution of compatibility data, the nonparametric criterion (W criteria, Shapiro-Wilk test), Student's t-test, and Spearman's correlation coefficient were used. Databases were formed in the programs "Microsoft Office Excel" 2010 and "Statistica" version 10.0 (Windows XP). 3. Results and Discussion As a first part of this work, a study of the nosological profile of diseases of wild and exotic birds and mammals of zoos (the Yaroslavl, Moscow, Ivanovo, and Uglich zoos) was carried out. 3.1. The Moscow Zoo The annual mortality in the institution was about 0.3% of all animal specimens: birds--0.4-0.5%; mammals--0.2% . In the studied period, parasitic diseases were recorded. Most enclosures with predatory mammals are permanently unfavorable for ascariasis and toxocariasis. It is impossible to carry out a complete devastation of open enclosures, because a part of the development cycle of ascariasis and toxocariasis occurs in the soil, where eggs can be stored for months. For young and healthy animals, carriage is not dangerous and, as a rule, does not affect their condition. However, in aging or chronically ill animals, the presence of parasites can cause various clinical symptoms. A similar situation develops with helminths in ungulates--"strongyloidosis" and "ascariasis" were recorded. Animals are constantly dewormed and the degree of infection is maintained at a "safe level". Various types of nematodes were observed in almost all groups of birds living in outdoor enclosures, which is associated with the presence of synanthropic birds in enclosures. Some populations of parrots turned out to be a reservoir of megabacteriosis. There was a trend towards an increase in the number of gerontological diseases (chronic diseases of the musculoskeletal system, cardiovascular system, chronic kidney diseases, and tumors for aged animals), which is associated with the aging of the collection. It should be noted that one of the systemic problems is obesity, especially for mammals (diagnostic studies) . 3.2. The Ivanovo Zoo During the year, up to 9.5% of the animals from the total number of livestock were exposed to diseases. The survival rate of animals (after past diseases) was 94.7% in all cases. Of the total number of established diseases of animals, 50.7% were injuries, 17.3%--diseases of the respiratory system, and 16.0%--diseases of the digestive system. Then, other diseases were observed in 8.0% cases, metabolic disorders in 6.7% cases, and diseases of the reproductive organs in 13% cases . 3.3. The Yaroslavl Zoo About 12.8% of animals from the total number of livestock were exposed to diseases during the year . There is a regular increase in registered diseases by 34.2-76.3%, which was associated with an increase in the number of livestock and an improvement in the results of diagnostic tests due to the purchase of laboratory equipment. The effectiveness of the therapeutic measures taken was confirmed by the increase in the survival rate of animals after diseases from 55.4% of cases to 79.3% in recent years. Of the total number of established diseases of animals--31.2% were diseases of the digestive system, then we observed lesions of the musculoskeletal system--17.3% and the cardiovascular system--10.1%, diseases of the hearing organs and the nervous system are sporadically noted--1.1% and 1.3%, respectively . Cancer diseases were detected in 5.05% of individuals during the year . The main causes of diseases of non-contagious etiology in wild animals in zoos were the factors that limit their active movement and constant stress factors due to the specifics of the institution . 3.4. The Uglich Zoo Station During the year, up to 37.4% of animals from the total number of livestock were exposed to diseases. The percentage of survival of animals (after past diseases) was about 41.9% in all cases . Of the total number of established animal diseases, 45.3% occurred against the background of physiological conditions of some old animals (since most often individuals came to the institution from visitors), 24.4% were diseases of the digestive system, 11.08% were lesions of the musculoskeletal system, in 7.2% had diseases of the respiratory system . 3.5. A General Description of Animal Diseases It was found that up to 12.8% of animals from the total number of livestock were subjected to various diseases. Of the total number of established animal diseases, the main share was diseases of the digestive system, then lesions of the musculoskeletal system and the cardiovascular system. Moreover, diseases of the hearing organs and the nervous system were sporadically noted . During the research, an increase in the proportion of oncological diseases in all the studied institutions was established . Based on a retrospective analysis of records entered in the register of sick animals, 1208 heads of wild animals were treated, i.e., about 12.9% of animals from the total number of livestock. Clinical examination was carried out as follows: history taking, general clinical examination, laboratory tests. The information obtained was recorded in the medical history of each animal. Skin diseases (dermatitis, urticaria, fungal infections) accounted for 2.3-2.5% of the total number of non-communicable diseases. A significant increase in the number of animals with pathology of the hearing organs (otitis media, injuries) was observed, amounting to 2.5%, diseases of the musculoskeletal system in wild animals--21.6%. Most often during the study period, various traumatic injuries were noted that occurred in animals and birds as a result of intraspecific and interspecific aggression, a lot of injuries were observed in mixed species exposures. Some injuries that the animals inflicted on themselves on the structural elements of the enclosures were revealed. Respiratory system diseases were observed in 6.2% of cases, among them inflammatory pathologies were noted--acute and chronic pneumonia and bronchopneumonia (of unknown etiology), rhinitis, tracheitis. At the same time, diseases of the digestive system of non-contagious etiology were registered in 25.6% of the total number of non-contagious animal diseases. The main disorders of the gastrointestinal tract, as a rule, were interconnected with a violation of the functional state of the liver; hepatosis (fatty, granular, cholestatic) was established in 33.1% of cases of diseases, then enteritis (17.5% of diseases) and poisoning, 6.8%; intestinal obstruction was established in isolated cases only. The level of metabolic diseases during the research period was found between 4.8 and 4.9% of cases, including exhaustion and impaired calcium-phosphorus metabolism. During the research period, diseases of the cardiovascular system accounted for 10.2%, detected mainly at the autopsy of animals, and a high percentage of pathologies of the heart muscle was noted--25.8%. Diseases of this system were represented by atherosclerotic changes in the coronary arteries, myocardial infarction, hypertrophic cardiomyopathy, and myocardial dystrophy. Inflammatory diseases were represented by chronic lymphocytic pericarditis and chronic focal myocarditis (complicated by thrombosis of the right ventricle--in the fluffy-tailed gerbil), cardiomegaly myocardial dystrophy, as well as a case of hemopericardium of unknown etiology. The reasons may be inadequate feeding with a deficiency in the diet of carbohydrates and minerals, autointoxication from the liver and kidneys. Eye diseases were established in 8.0% of cases. Basically, conjunctivitis was observed, which is associated with a violation of the conditions of detention, for example, abundant contamination of the litter, strong dust formation due to too small a fraction of sawdust. The decrease in the level of morbidity was associated with the formation of new expositions, the operating time of suppliers, and the improvement of the skill level of service personnel. Among the diseases of the musculoskeletal system, the most common were arthritis, bursitis, discopathy, myositis, sprain, inflammation of the jaw bone plate, and various kinds of injuries. The incidence of the reproductive system was detected in 6.3% (dystocia, ovarian cyst complicated by secondary infection and abscess formation, follicular stasis). Often there was a pathology of labor and postnatal activity in ungulates, a delay in the placenta was observed. Predisposing factors for this may be multiple pregnancies (twins), long, difficult births, and lack of vitamins and minerals in the concentrated type of feeding. The increase in the number of diseases was associated with the formation of breeding pairs in the institution and the entry of young animals into the sexually mature stage. Insignificant fluctuations in the incidence of the nervous system during the study period from 1.4 to 1.6% were noted. The low level of diseases of the nervous system was explained by the presence of large open-air cages, the formation of species groups, mixed species exposures, and regular enrichment and diversity of the animal habitat. During the research period, diseases of the excretory system were detected in 9.5% of the total number of diseases (renal failure was due to various causes: chronic glomerulonephritis with outcome in nephrosclerosis, hydronephrosis, pyonephrosis, acute interstitial nephritis; unspecified nephropathy, ascites). The cause of kidney pathology may be an unbalanced diet, the presence of mycotoxins in feed. Oncological diseases accounted for 13.8% of the total number of diseases and 19.5% of the number of non-communicable diseases. The most common malignancies were carcinomas and adenocarcinomas. Based on the results obtained, a model of the nosological profile of non-communicable diseases (of exotic, wild, and ornamental animals kept in captivity) was obtained. Based on the nosological profile of non-communicable diseases, one can conclude that the most often there are the following diseases: of the digestive system--in 31.2% of cases, of the musculoskeletal system--17.3%, of the cardiovascular system--10.1%, of the nervous system--1.3%, and of the hearing organs--1.1% for exotic, wild, and decorative animals. To establish the relationship between the MME content in samples of biological media of animals with general morbidity during the study period, a correlation-regression analysis was carried out, the results of which are presented in Table 1. The diagnosis was established based on the study of individual animal maps, pathological anatomical acts, and registers of veterinary procedures that was proposed before . A significant relationship was established between the accumulation of Zn and the following diseases: skin, digestive, and vision systems, as well as oncological diseases, Cu--with diseases of the musculoskeletal and the cardiovascular system, oncological diseases, Fe--with diseases of the cardiovascular system, Pb--with metabolic diseases, nervous and excretory systems, oncological diseases, Cd--with diseases of the cardiovascular and nervous system, and As--with diseases of the excretory systems (Table 2), which corresponded to some other data . 3.6. Zinc Pairwise statistical analysis of the data (Table 2) revealed a significant (p < 0.05) decrease in the level of Zn in animals with diseases of the organs of vision, digestive system, and skin diseases. There was also a tendency of a decrease in the zinc concentration in the case of metabolic disorders and diseases of the reproductive system . Zinc is believed to be essential for optimal retinal cell metabolism, modification of photoreceptor plasma membranes, regulation of the light-rhodopsin response, and modulation of synaptic transmission. In mice, the ZnT3 and ZnT7 transporters present in different layers of the retina, along with MME, provide zinc homeostasis and are involved in the functions of the eye . Moreover, zinc is required to maintain the level of taurine in the retina by acting on the transporter responsible for the movement of taurine into tissues . In animals, the effect of low zinc levels on abnormalities in the functioning of photoreceptors, the onset of primary glaucoma, disorganization of the ultrastructure, and loss of color sensitivity has been shown . A decrease In the level of Zn in the digestive system may be associated with a decrease in food intake due to a decrease in animal weight or the development of various infections, diarrhea, which prevents MME's reabsorption . Skin diseases with a decrease in zinc levels may be associated with a violation of its homeostasis with a decrease in the zinc transporter present in epidermal skin cells, etc. . For example, ZIP4 and especially ZIP2 are highly expressed in keratinocytes and are involved in their proliferation . Moreover, ZIP10 is highly expressed in epidermal progenitor cells located in the outer root sheath of hair follicles and plays an important role in the regulation of zinc to maintain the skin epidermis . In vitro studies have shown that ZIP2 is activated by the induction of differentiation in cultured keratinocytes and that when this transporter is disabled, differentiation is inhibited . The effect of Zn on the excretory system may be due to the fact that the metal mainly accumulates in the liver , while the biliary system is the main pathway zinc excretion and enterohepatic circulation . A number of authors note a decrease in the level of the microelement in pathological lesions associated with the presence of metallothionein , which is regulated by a decrease in the level of intake of food and water in the body with low body weight caused by liver disease, cytokines and inflammatory mediators, general inflammation, stress, and medications such as glucocorticoids . The importance of zinc for reproductive function in males has been documented . Zinc ions in the seminal fluid play both structural and regulatory roles in the activity of prostate-specific arginine esterase, which maintains normal prostate and sperm function . For females, data on the effect of Zn on reproductive function are practically absent, there are only indications of its significance in the formation and development of pregnancy and fetal size . There are observations of a significant increase in the concentration of Zn in both malignant and benign tumor skin tissues . This was due to two possible reasons: intensive metabolic processes in neoplastic cells and increased activity of intracellular enzymes that require intracellular zinc for proper functioning, or an increase in intracellular Zn, which inhibits tumor cell apoptosis . In contrast, zinc was significantly lower and copper significantly higher in neoplastic tissue with hepatocellular carcinoma and females with mammary tumors compared to healthy tissues. This can be explained by zinc chelation, which is especially important in this context, since copper is essential for angiogenesis, and by reducing tissue copper levels, zinc can limit tumor growth . In the study, there were no significant differences in the accumulation of MME in the case of some oncological diseases. 3.7. Copper During the statistical analysis of the data, a significant (p < 0.05) decrease in the Cu level for animals with a disease of the musculoskeletal system was found. In contrast, an increase in the Cu level by oncological disorders and diseases of the cardiovascular system was established. Although a deficiency of copper, which is involved in normal iron transport, may be accompanied by the development of iron deficiency anemia , its excess can lead to antagonism between the both metals due to the presence of common transport pathways, primarily the DMT-1 protein . In particular, an increase in the level of copper in the hair of patients with iron deficiency anemia has been noted . It has been established that copper plays an important role in cancer cell proliferation and metastasis , since cancer cells are characterized by a high demand for copper . The impact on the mobility of copper ions can be one of the tools of anticancer therapy . At the same time, an increased concentration of copper in the blood was found in patients with pancreatic cancer . There are indications that high levels of copper in hair are associated with the risk of developing prostate cancer , as well as neoplasms in children . At the same time, it is noted that under the conditions of the physiological level of copper intake into the body (0.6-3 mg/day), the existence of a relationship between copper in the body and oncology is unlikely . These results, in relation to Cu, can be explained by the fact that the deficiency of this MME leads to deficient collagen synthesis, accompanied by skeletal deformity, and changes in elastic fibers, which further leads to the occurrence of orthopedic diseases or Cu deficiency in the body is a consequence of such a disease . 3.8. Iron A significant decrease in the level of iron was found by development of cardiovascular diseases. Dysregulation of Fe homeostasis, increased uptake, and accumulation of MME in the reticuloendothelial system leads to the removal of the element from the blood into the cells of the reticuloendothelial system following a decrease in the availability of iron for erythroid progenitor cells and iron-limited erythropoiesis . The acute phase protein hepcidin plays a key role in the development of anemia due to its ability to inhibit Fe absorption in the intestine. In addition, at the same time, there is an increase in the uptake of iron by macrophages and blocking of the export of iron from macrophages, mainly to the bone marrow. As a result, serum iron concentration decreases (with normal total body iron), which slows down erythropoiesis and causes anemia . However, sometimes such a drop in serum iron concentration can be beneficial, as it makes iron less available to micro-organisms that inhibit their growth . Regulation of systemic iron metabolism, including organs and cell types involved in systemic iron balance are discussed in details . For example, cells (such as enterocytes) are absorbing "dietary iron" by divalent metal transporter 1 "located on the apical surface upon reduction of Fe3+ to Fe2+" by ferrireductases such as duodenal cytochrome B . These cells in addition to the macrophages ("spleenic reticuloendothelial") that are recycling iron from "senescent red blood cells" , finally "release iron via ferroportin with the aid of hephaestin, which oxidizes of Fe3+ to Fe2+" . Circulating plasma transferrin (which represents "the most dynamic body iron pool") is transferring this metal all around the animal body. Another conditions "that affect iron metabolism indirectly" are the following: "inflammation, ER stress, erythropoiesis, and hypoxia" . 3.9. Lead (Plumbum) A significant increase in the content of Pb in animals with lesions of the cardiovascular system and a tendency to an increase in the content of metals in lesions of the excretory system and reproductive organs were revealed. Lead has a negative impact on health , which is associated with a pronounced toxicity of the metal for a number of systems and organs and primarily for the nervous system . The influence of lead on the maternal organism and the development of congenital heart anomalies in newborns , as well as congenital neural tube defects , has been established, which is associated with the ability of MME to disrupt the regulatory mechanisms of DNA methylation . Scientists note the role of Pb in the development of oncological diseases, but these data were not confirmed in our study . It is well-known that Pb is able to influence metabolic processes. 3.10. Cadmium The sample found a significant increase in the content of cadmium in animals with diseases of the cardiovascular and circulatory systems. The hematopoietic system is one of the targets of the toxic action of Cd. It has been shown that an increase in the level of the xenobiotic in the blood is caused by a decrease in the concentration of hemoglobin and increases the likelihood of developing iron deficiency anemia . Experiments have shown that exposure to cadmium is accompanied by a shift in the blood formula towards myelopoiesis , as well as hemolysis and insufficient production of erythropoietin . 3.11. Arsenic A significant increase in the content of As was found in the presence of diseases of the excretory system. Arsenic induces the formation of oxidized lipids, which in turn generate several bioactive molecules (ROS, peroxides, and isoprostanes), the main end products of which are aldehydes. There is an indication of chronic and acute exposure to As in the etiology of cancer, cardiovascular disease (hypertension and atherosclerosis), neurological disorders, gastrointestinal disorders, liver and kidney disease, reproductive health effects, skin changes, and other health disorders. The role of antioxidant defense systems against arsenic toxicity is also discussed in detail . 4. Conclusions The level of MME accumulation in the body of animals can be the key reason to the occurrence of microelementoses, i.e., the development of various diseases. A study of the "nosological profile" of diseases was carried out and it was found that up to 12.9% of the animals from the total number of livestock (from four Russian zoological institutions) were exposed to diseases. The following tendency of disease frequency for all animals in our research was obtained: digestive system > musculoskeletal system >= cardiovascular system > hearing organs >= nervous system. During the research, an increase in the proportion of oncological diseases in all the studied institutions was established. A significant relationship was established between the accumulation of Zn and the following diseases: skin, digestive, and vision systems, as well as oncological diseases, Cu--with diseases of the musculoskeletal and the cardiovascular system, oncological diseases, Fe--with diseases of the cardiovascular system, Pb--with metabolic diseases, nervous and excretory systems, oncological diseases, Cd--with diseases of the cardiovascular and nervous system, and As--with diseases of the excretory systems. There was a tendency of a decrease in the concentration of MMEs in case of metabolic disorders and diseases of the reproductive system. Therefore, monitoring of the MME status of the organism of all animals must be carried out regularly (optimally--once every 6 months). Author Contributions Conceptualization, M.V.S.; methodology, S.Y.Z. and L.F.S.; validation, M.V.S. and L.F.S.; formal analysis, L.F.S. and S.Y.Z.; investigation, M.V.S.; resources, M.V.S.; data curation, S.Y.Z. and L.F.S.; writing--original draft preparation, M.V.S. and S.Y.Z.; writing--review and editing, M.V.S., S.Y.Z., and L.F.S.; supervision, S.Y.Z.; project administration, M.V.S.; funding acquisition, L.F.S. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by the Institutional Review Board of the Institute of Veterinary Medicine, Veterinary Sanitary Expertise and Agrosafety of the Federal State Budgetary Educational Institution of Higher Education "Russian Biotechnological University (ROSBIOTECH)" (protocol code 11_2022, Moscow, 125080, Russia and date of approval: 7 November 2022). Informed Consent Statement Not applicable. Data Availability Statement The research data obtained during this study are subject of special protection (particular privacy restrictions of ROSBIOTECH), i.e. not available to public. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Figure 1 Simplified scheme of zinc metabolism: absorption and excretion from the intestine: ZIPS, ZIP4, ZnT1, and ZnT2--transporters of enterocyte membranes and intracellular components (shown in blue and green, respectively); excretion routes are shown by dotted arrows; ER--endoplasmic reticulum; MT--metallothionein; some organs (heart, brain, kidney, skin, liver, pancreas) that exchange zinc with the plasma pool are presented (adapted from ). Figure 2 Copper transport. Ctr1: Copper specific transporter; DMT1: Divalent Metal Transporter 1; MT: Metallothionein; TGN: Trans Golgi Network; Cox17 and Atox1: Copper chaperone proteins; SOD: Superoxide dismutase; CP: Ceruloplasmin; CCS: Cytochrome c oxidase; ATP7B: Copper-transporting P-type ATPase (adapted from ). animals-13-00852-t001_Table 1 Table 1 Correlation between the MME content (mg/kg) in samples of derived integument with general morbidity of certain classes of diseases (with a proven role of environmental factors). Group of Nosologies MME, mg/kg Zn Cu Fe Pb Cd As Skin diseases 0.43 * 0.19 0.41 0.72 * 0.08 -0.04 Diseases of the hearing organs -0.14 0.24 0.34 -0.11 0.17 0.01 Diseases of the musculoskeletal system 0.43 0.24 * 0.51 0.41 0.12 0.05 Diseases of the respiratory system -0.26 0.14 0.11 0.01 0.34 0.29 Diseases of the digestive system 0.38 0.12 0.10 0.62 0.23 0.11 Metabolic diseases 0.03 -0.05 0.12 0.27 * 0.18 0.11 Diseases of the cardiovascular system -0.01 0.37 * 0.24 0.63 0.18 * 0.16 Diseases of the organs of vision 0.42 0.22 0.41 0.22 -0.21 0.08 Diseases of the nervous system 0.02 0.11 0.09 0.11 * -0.24 0.23 Diseases of the reproductive system 0.21 0.17 0.13 -0.08 -0.01 0.41 Diseases of the excretory system 0.11 0.04 0.04 0.34 * -0.01 0.01 Oncological diseases 0.28 * 0.19 0.11 * 0.03 0.17 0.15 * p < 0.05. animals-13-00852-t002_Table 2 Table 2 The MME level (mg/kg) in animal biological media, taking into account past disease. Group of Nosologies ME, mg/kg Zn Cu Fe Pb Cd As Skin diseases 56.9 +- 8.4 * | 15.9 +- 2.45 543 +- 113 6.99 +- 2.48 1.01 +- 0.48 0.110 +- 0.102 Diseases of the hearing organs 129 +- 11.2 11.7 +- 14.5 428 +- 48.6 8.01 +- 4.24 1.52 +- 0.93 0.211 +- 0.071 Diseases of the musculoskeletal system 143 +- 2.42 3.95 +- 4.57 * | 395 +- 38.5 7.11 +- 3.24 3.15 +- 1.01 0.342 +- 0.241 Diseases of the respiratory system 166 +- 3.84 13.8 +- 9.42 288 +- 69.0 6.99 +- 2.97 1.13 +- 0.24 0.533 +- 0.042 Diseases of the digestive system 62.1 +- 6.51 * | 12.5 +- 7.15 343 +- 57.5 6.97 +- 2.11 1.56 +- 0.64 0.241 +- 0.112 Metabolic diseases 74.5 +- 19.5 | 14.6 +- 4.52 354 +- 15.8 8.01 +- 3.54 1.85 +- 1.03 0.653 +- 0.431 Diseases of the cardiovascular system 142 +- 54.9 18.7 +- 3.54 | 128 +- 11.8 * | 6.88 +- 1.02 5.01 +- 0.68 * | 0.342 +- 0.123 Diseases of the organs of vision 49.7 +- 33.8 * | 14.7 +- 8.41 302 +- 65.8 7.55 +- 1.24 2.03 +- 1.51 0.429 +- 0.221 Diseases of the nervous system 169 +- 64.5 11.6 +- 5.64 342 +- 54.2 14.6 +- 2.94 * | 3.48 +- 0.86 * | 0.538 +- 0.341 Diseases of the reproductive system 98.8 +- 29.6 | 11.9 +- 8.31 298 +- 51.4 11.68 +- 1.06 | 1.56 +- 1.02 0.668 +- 0.339 Diseases of the excretory system 146 +- 25.4 11.7 +- 5.42 270 +- 185 12.97 +- 2.84 | 2.11 +- 0.63 0.951 +- 0.022 * | Oncological diseases 155 +- 49.8 21.9 +- 2.48 * | 298 +- 67.8 9.01 +- 2.48 2.46 +- 1.39 0.638 +- 0.109 Average level 130 +- 14.3 17.7 +- 4.89 388 +- 48.8 5.73 +- 0.93 1.25 +- 0.17 0.768 +- 0.181 * p < 0.05. 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PMC10000064

Canine stifle joint osteoarthritis (OA) is characterized by damage and degeneration of the articular cartilage and subchondral bone, bony hypertrophy at the margins, and synovial joint membrane changes. Non-invasive imaging modalities, such as digital radiography (DR), computed tomography (CT), and magnetic resonance imaging (MRI), can be used to describe these changes. However, the value of MRI in diagnosing spontaneous canine OA and the comparison of different imaging modalities have seldom been addressed. This study compared multiple noninvasive imaging modalities in canine spontaneous stifle OA cases. Four client-owned dogs with five spontaneously affected OA stifle joints were recruited and underwent DR, CT, and MRI. Information on osteophytes/enthesophytes, ligament/tendon lesions, synovial effusion and membrane thickening, subchondral bone lesions, and meniscal and cartilage lesions were scored and compared. The results showed that MRI provides the most comprehensive and superior lesion detection sensitivity for ligament, meniscus, cartilage, and synovial effusions. DR provides adequate bony structure information, while CT provides the most delicate images of bony structure lesions. These imaging findings may provide further understanding of the disease and help clinicians draft a more precise treatment plan. canine stifle joint computed tomography digital radiography magnetic resonance imaging osteoarthritis Southern Taiwan Science Park Bureau, Ministry of Science and Technology, Taiwan, ROC110CB-1-04 This publication was supported by the Southern Taiwan Science Park Bureau, Ministry of Science and Technology, Taiwan, ROC, under contract 110CB-1-04. pmc1. Introduction Canine osteoarthritis (OA) is a common clinical disorder that affects approximately 20% of skeletally mature dogs . OA is characterized by damage and degeneration of articular cartilage and subchondral bone, bony hypertrophy at the margins, and synovial joint membrane changes, which lead to structural and functional decline of the joint and result in pain and lameness . In clinical practice, noninvasive physical examination and imaging modalities, such as digital radiography (DR), computed tomography (CT), and magnetic resonance imaging (MRI), are commonly used to diagnose and evaluate the severity of OA. DR has the advantages of less space requirements, not necessitating specifically trained staff, less requirements of anesthesia, and lower cost, and hence is mostly applied in diagnosing OA. Common radiographic findings of OA include narrowing of the joint space, subchondral sclerosis, and osteophytosis . However, information on ligament injuries and cartilage defects is limited. CT allows the evaluation of periarticular and articular tissues without superimposition on joint radiography . These images can be constructed with the desired window levels or widths for specific joint anatomic components . The disadvantage of CT is that the similar attenuation of complex intra-articular structures of the synovial joint may blur the details. MRI has recently been defined as the ''gold standard'' in human medicine for synovial joint examination because of the superior soft tissue contrast of the periarticular muscles, tendons, ligaments, and articular cartilage . Signal changes within the bone marrow adjacent to osteoarthritic joints are common findings noted on MRI in humans and the subchondral lesions noted on MRI are considered crucial for determining treatment options for OA patients . Nevertheless, the use of MRI in canine OA has not been fully explored. In addition, the settings of MRI for OA are challenging in dogs because the optimal sequences are often geared towards human use, and commercial customized coils for dog limbs are limited. Previous studies have indicated that spontaneously occurring canine OA and human OA share a similar disease progression phenotype . However, the MRI features of canine OA were reported in surgically induced experimental OA models rather than in spontaneous cases . Moreover, a comparison of imaging findings from different imaging modalities in the same patients has also not been reported. Therefore, this study aimed to describe and compare the imaging features of DR, CT, and MRI in spontaneous canine stifle OA cases. 2. Materials and Methods 2.1. Cases Selection Adult client-owned dogs with spontaneous chronic (>6 months) stifle OA and associated pain were enrolled in this study. Physical examination, synovial joint fluid analysis, and radiographic findings were used to diagnose osteoarthritis (OA). Dogs with neoplastic joint disease, immune-mediated joint disease, or dogs with a high anesthesia risk (>grade 4 of the American Society of Anesthesiologists) were excluded from this study. 2.2. Clinical Lameness Scoring System Clinical impact of OA on the affected limbs was assessed using an objective clinical scoring system (CLSS, scores: 3-15) (Table 1), which was adopted from a previous study . The CLSS assessed lameness, pain on palpation, and weight-bearing status. 2.3. Imaging Exam and Analysis X-ray examinations were performed using an X-ray machine (MRAD-A50S, Toshiba Medical Systems Corporation, Nasu, Japan) with a digital receptor (CXDI-701c wireless digital radiography system, Canon, Japan). The digital radiographic (DR) images of each affected stifle included caudocranial (CdCr) and mediolateral views. The kVp and mA settings were adjusted according to the radiographic technique chart provided by the manufacturer. Non-contrast CT was performed using a 64-slice positron emission tomography (PET)/computed tomography (PET/CT) (Discovery 64 Slice PET/CT Scanner, GE Healthcare, New York, NY, USA), with bone plus and standard desired reconstruction algorithm, 150 mAs and 120 kVp, and helical acquisitions with a table pitch factor of 0.516. The CT images were reformatted to soft tissue (window width: 400, window level: 40) and bone (window width: 1500, window level: 450). The dogs were placed in dorsal recumbency on V-shaped foam with a bilateral hindlimb stifle joint positioned at a 90-degree angle. MRI was performed using a 1.5-T MRI system (Signa 1.5T; GE Healthcare, New York, NY, USA) with a flex coil (GE Signa 1.5 T Flex Coil, GE Healthcare, New York, NY, USA). The dogs were in a laterally recumbent position with the target stifle joint on the non-dependent side at an angle of 135deg-145deg. The contralateral hind limb was placed at a different angle from the target stifle joint. To reduce artifacts and noise signals, towels and large MRI foam sponges were used to maintain the position of the contralateral hindlimb while the drip lines and capnography sensor were kept away from the target stifle joint. The sequences performed included T1-weighted (T1W), T2-weighted (T2W), proton-density-weighted (PDW), proton-density-weighted fat saturation (PDW fs), and T2 mapping. Standard planes included the sagittal plane, which was oriented parallel to the medial surface of the medial femoral epicondyle; the transverse plane, which was oriented perpendicular to the patellar ligament and sagittal orientation line; and the dorsal plane, which was parallel to the patellar ligament. The scanning parameters are listed in Table 2. All imaging procedures were performed by residents of veterinary radiology under general anesthesia. Each dog underwent the DR, CT, and MRI examination on the same day. Digital Imaging and Communications in Medicine (DICOM) format was evaluated using diagnostic imaging software (Osirix, Version 9.5.2, Bernex, Switzerland). The original DR, CT, and MRI images were assessed and analyzed by an experienced veterinarian with radiology expertise (L.-S.L.) and a chief resident of veterinary radiology (Y.-J.T.). The final decision was made by consensus. 2.4. Articular Lesions Evaluation Articular lesions of osteophytes and enthesophytes, ligament lesions, synovial effusions, synovial membrane thickening, subchondral lesions, and meniscal lesions were evaluated by grading or presented as "present" or "absent". 2.4.1. Osteophytes/Enthesophytes The osteophytes/enthesophytes in 12 anatomical compartments of the medial femoral epicondyle (ME), lateral femoral epicondyle (LE), medial femoral condyle (MC), lateral femoral condyle (LC), medial proximal tibial plateau to edge (MT), lateral proximal tibial plateau to edge (LT), femoral trochlear ridges (FT), tibial tuberosity (TT), medial fabella (MF), lateral fabella (LF), distal patella (DP), and proximal patella (PP) were evaluated using DR, CT, and MRI . The sum of the maximum width and height of osteophytes/enthesophytes obtained from the images was divided by the length of the anatomic midlines (AM). The results were then scaled from 0 to 3 (0: none; 1: <25%; 2: 25-50%; 3: >50%). 2.4.2. Ligament/Tendon Lesions Ligament/tendon lesions were assessed in 6 regions: medial collateral ligament (MCL), lateral collateral ligament (LCL), long digital extensor tendon (LDET), patellar ligament (PL), cranial cruciate ligament (CCL), and caudal cruciate ligament (CdCL). Because the ligament/tendon structure and the lesion cannot be well identified using DR and CT, the severity and incidence of ligament/tendon lesions were only recorded on MRI images and scaled from 0 to 3 (0: normal; 1: partial rupture; 2: partial rupture with thickening or increased peripheral effusion; 3: complete rupture), which was modified from a previous study . 2.4.3. Synovial Effusion and Membrane Thickening The presence of synovial effusion in the infrapatellar region (IP) was recorded in the lateral view of the DR or sagittal planes of the CT and MRI images, and the maximum potential distention of the synovial cavity was scaled from 0 to 3 (0: normal; 1: <=33%; 2: 33-66%; 3: >=66%) . The presence of synovial effusion extensions at the MF, LF, medial femoral trochlear ridges to the epicondyle region (MFTr), lateral femoral trochlear ridges to the epicondyle region (LFTr), and femoral trochlear groove (FTG) was recorded on MRI only . In addition, synovial membrane thickening was observed on MRI. 2.4.4. Subchondral Bone Lesions Subchondral bone defects, cysts, and bone-marrow-edema-like lesions were defined as subchondral bone lesions and evaluated in five anatomical compartments: femoral trochlear (FT), femoral intercondylar fossa (ICF), femoral lateral condyle (LC), femoral medial condyle (MC), and tibial plateau (TP) . The presence of subchondral bone lesions was evaluated using DR, CT, and MRI. Severity was assessed using a modified scale (0: normal; 1: a hypersignal < 1/3 of the surface of the subregion; 2: a hypersignal < 2/3 of the surface of the subregion, 3: a hypersignal > 2/3 of the surface of the subregion) . 2.4.5. Meniscal and Cartilage Lesions Because meniscal and cartilage lesions cannot be well identified in DR and CT images, these lesions were only evaluated using MRI. Meniscal lesions were evaluated at the medial and lateral menisci and recognized as linear or oval hyperintensity on T2W and PDW images. The severity of meniscal lesions was scaled from 0 to 3 (0, normal; 1, linear or oval core lesion; 2, linear or oval lesion extending to the articular surface; 3, complete rupture) . Cartilage lesions were recognized as heterogeneous signals of cartilage, irregular cartilage contour, and cartilage defects/rupture. The presence of cartilage lesions was recorded in 13 anatomical compartments and numbered from C1-C13: two compartments of MC (C1, C2) and LC (C3, C4), and nine compartments of the tibial plateau (C5-C13) . T2 relaxation times for evaluating cartilage lesions at the medial femoral condyle, lateral femoral condyle, medial tibial plateau, and lateral tibial plateau were calculated by manually drawing the region of interest (ROI) on color-coded T2 mapping images with a cartilage thickness > 0.5 mm (>1 pixel). The results were recorded and compared with those of a previous study . 2.5. Statistical Analysis Data were statistically analyzed using commercially available software (GraphPad Prism, ver. 7.00, San Diego, CA, USA). Descriptive statistics (mean, median, and percentage) were applied for continuous variables (age, body weight, and T2 relaxation time) and ordinal variables (CLSS and scores for each grading articular lesion). Categorical variables (signalments and affect joints) are presented in tabular form. Comparisons of scales obtained from DR, CT, and MRI images were performed using the Kruskal-Wallis test followed by Dunn's multiple comparisons test. 3. Results 3.1. Animals Four dogs (three Beagles and one Labrador retriever) with five stifle joints (three left and two right) met the inclusion criteria and were recruited. Three dogs were spayed females and one dog was an intact male. The median age of the dogs was 15 years (range, 10-17 years). The median weight was 11.7 kg (range, 7.8-28.0 kg). Hindlimb lameness was noted in all dogs, and the median CLSS score was 9.0 (range, 8-10). 3.2. Articular Lesions 3.2.1. Osteophytes/Enthesophytes Representative images of the severity of osteophytes/enthesophytes in the stifle joint are shown in Figure 5. The incidences and scores of osteophytes and enthesophytes in each anatomical compartment are listed in Table 3. In the ME, LE, MC, LC, MT, and LT subregions, osteophytes/enthesophytes were identified in all joints by all modalities. In the FT subregion, osteophytes and enthesophytes could not be identified in only one joint on DR. The tibial tuberosity subregion was the most uncommon site for the osteophytes/enthesophytes, and they were only found in one joint (20%) by different modalities. In the medial and lateral fabella subregions, CT (100%) had a higher sensitivity than DR (60%) and MRI (60%) . In the distal patella and proximal patella subregions, MRI (100%) had higher sensitivity than DR (60%) and CT (40%) (DP, PP) . No significant differences in osteophytes/enthesophytes lesion scores at different anatomic subregions among DR, CT, and MRI were noted. 3.2.2. Ligament/Tendon Lesions Ligament/tendon lesions were mostly noted in the CCL (5/5 [100%]), CdCL (5/5 [100%]), and LCL (5/5 [100%]) regions, followed by the MCL (3/5 [60%]), LDET (1/5 [20%]), and PL (1/5 [20%]) on MRI. The median (range) ligamental/tendon scores were 1 (0-2) for MCL, 1 (1-2) for LCL, 0 (0-1) for LDET, 0 (0-1) for PL, 1 (1-3) for CCL, and 1 (1-3) for CdCL. Representative images of the ligament/tendon ligament lesions are shown in Figure 7. 3.2.3. Synovial Effusion and Membrane Thickening The presence of synovial effusion at the IP was recorded on DR (4/5 [80%]), CT (5/5 [100%]), and MRI (5/5 [100%]), with median (range) synovial effusion extension scores 2 (0-2), 1 (1-3), and 1 (1-2), respectively, which were not significantly different between the modalities (p = 0.8519). In contrast, an extension of synovial effusion was identified on MRI in MF (4/5 [80%]), LF (3/5 [60%]), MFTr (3/5 [60%]), LFTr (1/5 [20%]), and FTG (1/5 [20%]). The MF was the most remarkable extension site of the synovial effusion . Synovial membrane thickening was identified only in the IP region of all joints on MRI . 3.2.4. Subchondral Bone Lesions Subchondral bone lesions were noted in all modalities . The incidence and scores of subchondral bone lesions in the different compartments on DR, CT, and MRI are listed in Table 4. ICF was the most common site of subchondral bone lesions, which could be noted in most joints, except for one joint on the DR images. In contrast, lesions were less commonly seen in FT, and only two joints were identified on DR images. Although CT and MRI provided similar sensitivities in detecting lesions, CT exhibited higher sensitivity in TP. In addition, no significant differences in subchondral bone lesion scores in each compartment were observed among the different modalities. 3.2.5. Meniscal and Cartilage Lesions Medial meniscal lesions were noted in two joints (2/5 [40%]), with scores of 1 and 3, respectively. Except for linear or oval core lesions, a meniscal maceration lesion, which is a specific term used in MRI to describe chronic degenerative meniscal lesions causing meniscal wasting and becoming separated fragments, was noted in one joint . A lateral meniscal lesion was only seen in one joint, which was encountered together with the medial meniscal lesion, with a score of 2. Cartilage lesions were noted in almost all subregions of all joints except for two subregions (C5 and C8) in one stifle joint. The T2 relaxation time of the cartilage lesions was generally lower than that of previously published data (Table 5). 4. Discussion This study recruited canine patients with naturally occurring chronic stifle OA with moderate lameness and evaluated DR, CT, and MRI imaging findings. Although similar clinical performances were noted in these patients, the individual associated soft and bony structural changes may differ, and the results could be altered using different modalities. Currently, various treatment options are available for canine OA, including surgery, rehabilitation, regeneration therapy, and nutritional supplementation. Detailed knowledge from these imaging modalities is essential for developing customized treatment for OA. Our results indicated that osteophytes/enthesophytes could form in almost all anatomical compartments of the stifle joint in chronic OA, except for the tibial tuberosity. This is considered an acceptable result, since the tibial tuberosity is an extra-articular structure and may not be involved in the OA process. In addition, the osteophytes/enthesophytes at the femoral epicondyle (ME, LE), femoral condyle (MC, LC), and fabella (MT, LT) were the most evident and could be identified by all modalities. CT and MRI may be more sensitive than DR at femoral trochlear ridges (FT) because the superimposition of osteophytes, enthesophytes, and distal femur can be avoided. In the fabella subregions (MF and LF), it may be difficult to identify osteophytes and enthesophytes by using DR and MRI. Severe osteophytes in the distal femur may superimpose on the fabella in the CdCr view, and bilateral fabellae may superimpose on each other in the lateral view. Moreover, multiple connective tissues may superimpose on each other at this site, such as the femoropatellar ligament and the origin of the gastrocnemius on DR images. These connective tissues also present hypointense signals on MRI; thus, they can sometimes be confounded by osteophytes and enthesophytes. In contrast, CT has the following advantages: multiple plane images; imaging processing by three-dimensional CT rendering and reconstruction; and the superior ability in differentiating the soft and bony tissues, allowing better identification of the osteophytes and enthesophytes better than the other modalities at the fabella site. In the patellar region (DP, PP), MRI provided the highest sensitivity in detecting osteophytes and enthesophytes. The texture of osteophytes and enthesophytes at an early stage is similar to cartilage and appears radiolucent on DR or CT images, whereas the osteophytes and enthesophytes around the patella in the early stage may present hyperintense signals compared with cortical bone on MRI owing to incomplete mineralization . This enables early detection of osteophytes and enthesophytes on MRI. In addition, the superimposition of incompletely mineralized osteophytes on DR images may provide better identification than CT. It is challenging to evaluate ligament/tendon lesions using non-invasive imaging examinations, especially DR and CT. Nevertheless, MRI is useful for diagnosing ligamental/tendon lesions. In this study, various severities of ligament/tendon lesions were identified in the six ligaments and tendons around the stifle joints. CCL, CdCL, and LCL lesions were the major ligament lesions observed in all joints. CCL disease is the most common cause of hindlimb lameness in dogs, and progressive stifle OA will develop . Although the exact pathogenesis of canine CCL disease remains controversial, aging changes resulting in degeneration of the CCL may be associated with programmed cell death, inflammatory cell infiltration, and extracellular matrix degeneration, and may contribute to the event . Therefore, it is not surprising that CCL lesions were notable. In addition, the CdCL, which is close to the CCL in the same space, may share the same process of degeneration. This might explain the same incidence and severity of CCL and CdCL lesions here, even though injury of the CdCL was less commonly noted in dogs encountering traumatic processes. Interestingly, the LCL also revealed notable changes simultaneously with CCL and CdCL. Since the LCL can be injured when the tibia is pushed outward or excessively internally rotated, lesions of the LCL may result from excessive tension after CCL injury . In contrast, the LDET and PL were the most common sites of ligament lesions. Unlike ligaments, tendons are responsible for the extension or flexion of joints, rather than the maintenance of joint stability. As a result, the lesions in the LDEL and PL were not notable and may not be related to the OA process. Synovial effusion at the IP subregion, which was strongly correlated with chronic OA, was found in all modalities. Consistent results have also been reported in experimental canine OA models and working dogs . Although all three modalities provided a high detection rate, CT and MRI were more sensitive than DR for detecting synovial effusion in the IP region. The extension of synovial fluid was mostly noted around the fabella in our cases, similar to the results reported in dogs undergoing experimental surgery after 26 weeks . This may indicate that the fabella region, including the femorofabellar joint and joint capsule, is also important in OA processing. In contrast, synovial extension is least seen in the trochlear region (FTG), and it is thought that the presence of the patellar bone in the trochlear groove may limit the extension. In addition, thickening of the synovial joint capsule in the IP region was also identified in all joints on MRI. These results indicated that MRI exhibited high sensitivity for fluid signals and synovial membrane structures in stifle joints, which were not well identified on DR or CT. The subchondral lesion or bone marrow lesion may indicate inflammation, edema, and ischemic condition of the bone marrow , which presents as T2W and PDW hyperintense lesions on MRI. Nevertheless, DR and CT provide details of bony structures, such as subchondral bone defects or cysts. Our results revealed that subchondral lesions at the FT were only noted on DR images. Combining the CT and MRI findings of the same joint, these lesions resulted from the superimposition of regional osteophytes with trochlear ridges rather than "true" subchondral lesions . Therefore, the findings of DR imaging, used to detect subchondral lesions in the FT subregion, should be interpreted with caution. Although subchondral bone plays a crucial role in the initiation and progression of human OA , the clinical values still can not be identified here due to the chronic OA stage and single time point for assessment. However, a previous study demonstrated that the abnormal bone metabolism was detected before subchondral bone lesion formatted in human OA, and the sodium fluoride positron emission tomography/CT coregistered with MRI could serve as a feasible molecular imaging modality to assess early post-traumatic OA . Meniscal lesions were observed less commonly in this study. Previous studies revealed that the degenerative change and cranial cruciate ligament rupture could be associated with meniscal lesions . However, the underlying causes could not be identified due to limited information on illness history. Cartilage lesions are common and extensive in canine stifle OA. Although a previous study showed that MRI could evaluate the morphology of articular cartilage , only the integrity of the cartilage could be documented in our study. Although a higher magnetic field MRI may be required to evaluate the details of stifle joint cartilage in small-to-middle-breed dogs, severe cartilage defects could still be detected using 1.5T MRI. A recent study applied T2 relaxation time to determine changes in cartilage composition in normal beagles . T2 relaxation time reflects the water composition change in articular cartilage and provides early detection of articular cartilage injury and subsequent mineralization. The increase in T2 relaxation time could be used to detect cartilage damage in dogs, whereas the decrease may be related to severe OA or articular cartilage mineralization in humans . The T2 relaxation time in our study was generally lower than previous results in normal beagle stifle joints . The profound cartilage lesions with severe OA observed in our cases may explain these changes. Our study had several limitations. The primary limitation is the lack of comparison between imaging findings with arthroscopy and histological examination because all cases were client-owned clinical cases. Another limitation of our study is its cross-sectional design with a small sample size of clinical OA cases, making it difficult to investigate the etiology of OA lesions. Further longitudinal studies including more dogs and patients with different severities of OA are warranted. 5. Conclusions In conclusion, comparing the imaging modalities for canine stifle OA, DR provides adequate bony structure information, the shortest examination time, and the least budget consumption; however, most soft tissue and cartilage lesions could not be seen. In addition, the superimposition of abundant osteophytes and enthesophytes may have misled the results. CT provides the most delicate bony structure lesion information with a moderate examination time. However, articular soft tissue lesions cannot be obtained without invasive techniques (e.g., contrast arthrography), and a higher cost than DR is required. MRI provides images of the superior ligament, meniscus, cartilage, and synovial effusion with adequate bony structure information. Although the disadvantages of long anesthesia time during the examination and high cost are still of concern, MRI provides the most comprehensive joint images compared to other non-invasive imaging modalities used in this study, and these findings provide detailed information on stifle OA. Acknowledgments We would like to thank Chuan Chiang and UniCore Animal Hospital, Taipei City, Taiwan. Author Contributions Conceptualization, C.-S.C., Y.-J.T. and L.-S.L.; methodology, L.-S.L.; validation, C.-S.C.; formal analysis, Y.-J.T. and L.-S.L.; investigation, Y.-J.T.; resources, L.-S.L.; data curation, Y.-J.T.; writing--original draft preparation, C.-S.C. and Y.-J.T.; writing--review and editing, L.-S.L.; visualization, C.-S.C.; supervision, L.-S.L.; project administration, L.-S.L.; funding acquisition, L.-S.L. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study protocol was approved by the Institutional Animal Care and Use Committee of the National Pingtung University of Science and Technology (NPUST-108-059, 2019/10). Informed Consent Statement Informed consent was obtained from the owners of all subjects involved in the study. Data Availability Statement The data presented in this study are available upon request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Schematic representation of stifle joint anatomic compartment subregions for evaluation of osteophytes/enthesophytes of each subregion, dorsal plane (MRI and CT) or caudocranial view (DR) (A), and sagittal plane (MRI and CT) or lateral view (DR) of stifle joint (B). Red dash lines are schematically represented as imaginary anatomic midline (AM) of the stifle joint for each subregion. Stifle joints are divided into 12 subregions for evaluating osteophytes/enthesophytes. Abbreviations: DP: distal patella; FT: femoral trochlear ridges; LC: lateral femoral condyle; LE: lateral femoral epicondyle; LF: lateral fabella; LT: lateral proximal tibia plateau to edge; MC: medial femoral condyle; ME: medial femoral epicondyle; MF: medial fabella; MT: medial proximal tibia plateau to edge; PP: proximal patella; TT: tibial tuberosity. Figure 2 Schematic representation of stifle joint anatomic compartments for evaluating synovial effusion in the dorsal plane (CT or MRI)/caudocranial view (DR) (A), and sagittal plane (CT or MRI)/mediolateral view (DR) of stifle joint (B). Stifle joint is divided into 6 subregions to evaluate synovial effusion. Abbreviations: FTG, femoral trochlear groove; IP, infrapatellar region; LF, lateral fabella; LFTr, lateral femoral trochlear ridges to epicondyle region; MF, medial fabella; MFTr, medial femoral trochlear ridges to epicondyle region. Figure 3 Schematic representation of stifle joint anatomic compartments for evaluating subchondral lesion in the dorsal plane (CT or MRI)/caudocranial view (DR) (A), and sagittal plane (CT or MRI)/mediolateral view (DR) of stifle joint (B). Stifle joint is divided into 6 subregions to evaluate subchondral lesions. Abbreviations: FT, femoral trochlear; ICF, femoral intercondylar fossa; LC, femoral lateral condyle; MC, femoral medial condyle; TP, tibia plateau. Figure 4 Schematic representation of stifle joint anatomic compartment subregions to evaluate cartilage lesion on MRI images: dorsal plane of the distal femur (A) and proximal tibia (B). Stifle joint is divided into 13 anatomic subregions to evaluate cartilage lesions: lateral part (A, C1) and medial condyle medial (A, C2); lateral condyle medial (A, C3) and lateral part (A, C4); tibia plateau medial region cranial (B, C5), middle (B, C6), and caudal part (B, C7); tibia plateau middle region cranial (B, C8), middle (B, C9), and caudal part (B, C10); tibia plateau lateral region cranial (B, C11), middle (B, C12), and caudal part (B, C13). Figure 5 The severity of osteophytes/enthesophytes of stifle joint using different imaging modalities. Representative DR (A-D), CT (E-H), and MRI (H-K) images, with score = 0 (A,E,H), score = 1 (B,F,I), score = 2 (C,G,J), and score = 3 (D,H,K). White arrows indicated osteophytes/enthesophytes formation at lateral proximal tibia edges. The score = 0 images were acquired from a dachshund which was not included in this study. Figure 6 Comparison of osteophytes/enthesophytes at fabella using different imaging modalities in the same leg. Caudocranial view of DR (A), bone window of CT (transverse plane) (B), and T2W MRI (transverse plane) (C) images. Enthesophytes/osteophytes at lateral fabella (arrows) are superimposed with the distal femur on DR image (A), but can be easily identified on CT image (B), while exhibiting similar hypointense signal with the attached femoropatellar ligament on MRI image (C). Mediolateral view of DR (D), and sagittal plane of CT (E) and MRI (F) images in the same leg. A hyperintense projection of the distal patella (arrow), which can be seen on MRI (F) with a score = 2, is graded score = 1 and score = 0 on DR (D) and CT (E) images, respectively. Figure 7 Representative MRI images of ligament/tendon lesions at each site. (A): Mixed intensities are seen in both CCL and CdCL (sagittal plane, PDW fs). (B): Both MCL and LCL are mildly thickened and have hyperintensities within the ligament (dorsal plane, PDW fs). (C): Focal hyperintensity is noted within the LDET with fluid accumulation surrounding the tendon (transverse plane, T2W fs). (D): Mixed intensity is noted adjacent to the distal patella at PL (sagittal plane, PDW fs). Abbreviations: CCL, cranial cruciate ligament; CdCL, caudal cruciate ligament; LCL, lateral collateral ligament; LDET, long digital extensor tendon; MCL, medial collateral ligament; PL, patellar ligament. Figure 8 Comparison of medial fabella synovial effusion on DR (A), CT (B), and MRI (C), and infrapatellar synovial membrane thickening on DR (D), CT (E), and MRI (F) in the same leg. Obvious synovial effusion (arrowhead) is seen at the medial fabella region on MRI image (C) but cannot be observed on DR (A) or CT (B) images. Synovial membrane thickening is seen as low intense signal inside the synovial cavity (arrow) on MRI (F) but cannot be observed on DR (D) or CT (E) images. Figure 9 Comparison of subchondral bone defects, cysts, and bone-marrow-edema-like lesions of caudocranial view in caudocranial view on DR (A), bone window on CT (dorsal plane) (B), and PDW fs (dorsal plane) on MRI (C) in the same leg. Subchondral bone defects and cysts are mostly seen in two areas on DR (A, arrow), in three areas on CT (B, arrow), and in two areas on MRI (A, arrow). Subchondral bone marrow edema-like lesions surrounding the intertrochlear fossa are only identified on MRI (C, arrowheads). Subchondral bone defect-like lesion is suspected at the femoral trochlear region on DR image (D, asterisk), but not identified on CT (E) nor MRI (F). Figure 10 Meniscal maceration lesion noted on the dorsal plane, PDW fs MRI image. Chronic degenerative meniscal changes resulting the separation of the meniscus into constituent fragments (arrow). Synovial effusion accumulation is noted between the femoral condyle and tibial plateau, where the meniscus normally exists. animals-13-00849-t001_Table 1 Table 1 Clinical lameness scoring system. Adapted with the permission from Ref. . 2013, the Korean Society of Veterinary Science. Criterion Grade Clinical Evaluation Lameness 1 Walks normally 2 Slightly lame when walking 3 Moderately lame when walking 4 Severely lame when walking 5 Reluctant to rise and will not walk more than five paces Pain on palpation 1 None 2 Mild signs; dog turns head in recognition 3 Moderate signs; dog pulls limb away 4 Severe signs; dog vocalizes or becomes aggressive 5 Dog will not allow palpation Weight-bearing 1 Equal on all limbs standing and walking 2 Normal standing; favors affect limb when walking 3 Partial weight-bearing standing and walking 4 Partial weight-bearing standing; non-weight-bearing walking 5 Non-weight-bearing standing and walking animals-13-00849-t002_Table 2 Table 2 The MRI scanning parameters of the canine stifle joint. Sequence Plane TR (msec) TE (msec) NEX ET Slice (mm) Gap (mm) FOV (cm) Matrix T1 FSE Sagittal 605 Min Full 5 3 2.2 0 10 256 x 200 PD FSE Sagittal 2500 34 4 7 2.2 0 10 224 x 200 T2 FSE Sagittal 3000 60 5 16 2.2 0 10 248 x 188 PD FSE FS Sagittal 2500 34 4 7 2.2 0 10 256 x 200 3D FSPGR FS Sagittal 21.5 Min Full 3 n/a 2 0 10 256 x 200 T2 FSE FS Transverse 3865 60 5 16 2.5 0 10 240 x 200 T2 FSE Transverse 3825 60 5 16 2.5 0 10 240 x 200 PD FSE Dorsal 2500 34 4 7 2.2 0 10 252 x 200 T2 FSE Dorsal 3045 60 5 16 2.2 0 10 252 x 200 PD FSE FS Dorsal 2500 34 4 7 2.2 0 10 200 x 200 Abbreviations: ET, echo train length; FOV, field of view; NEX, number of excitations; TE, echo time; TR, repetition time. animals-13-00849-t003_Table 3 Table 3 Comparison of the osteophytes/enthesophytes lesion on DR, CT, and MRI images at different anatomic subregions. Compartments Modality Incidence Scales p-Value Compartments Modality Incidence Scales p-Value ME DR 5/5 (100%) 2 (1-3) >0.999 FT DR 4/5 (80%) 2 (0-3) >0.999 CT 5/5 (100%) 3 (1-3) CT 5/5 (100%) 3 (1-3) MRI 5/5 (100%) 3 (1-3) MRI 5/5 (100%) 3 (1-3) LE DR 5/5 (100%) 2 (1-3) >0.999 TT DR 1/5 (20%) 0 (0-2) >0.999 CT 5/5 (100%) 3 (1-3) CT 1/5 (20%) 0 (0-3) MRI 5/5 (100%) 3 (1-3) MRI 1/5 (20%) 0 (0-3) MC DR 5/5 (100%) 2 (1-3) >0.999 MF DR 3/5 (60%) 2 (0-3) >0.999 CT 5/5 (100%) 2 (1-3) CT 5/5 (100%) 2 (1-2) MRI 5/5 (100%) 3 (1-3) MRI 3/5 (60%) 2 (0-3) LC DR 5/5 (100%) 2 (1-3) >0.999 LF DR 3/5 (60%) 2 (0-3) >0.999 CT 5/5 (100%) 2 (1-3) CT 5/5 (100%) 2 (1-2) MRI 5/5 (100%) 3 (1-3) MRI 3/5 (60%) 2 (0-3) MT DR 5/5 (100%) 2 (1-3) >0.999 DP DR 4/5 (80%) 2 (0-3) >0.999 CT 5/5 (100%) 2 (1-3) CT 4/5 (80%) 3 (0-3) MRI 5/5 (100%) 2 (1-3) MRI 5/5 (100%) 3 (1-3) LT DR 5/5 (100%) 2 (1-3) >0.999 PP DR 3/5 (60%) 1 (0-3) 0.2222 CT 5/5 (100%) 2 (1-3) CT 2/5 (40%) 0 (0-3) MRI 5/5 (100%) 2 (1-3) MRI 5/5 (100%) 1 (1-3) Abbreviations: DP, distal patella; FT, femoral trochlear ridges; LC, lateral femoral condyle; LE, lateral femoral epicondyle; LF, lateral fabella; LT, lateral proximal tibial plateau to edge; MC, medial femoral condyle; ME, medial femoral epicondyle; MF, medial fabella; MT, medial proximal tibial plateau to edge; TT, tibial tuberosity; PP, proximal patella. animals-13-00849-t004_Table 4 Table 4 Comparison of the subchondral bone lesions on DR, CT, and MRI images at different anatomic subregions. Compartments Modality Incidence Scores p-Value FT DR 2/5 (40%) 0 (0-1) 0.3333 CT 0/5 (0%) 0 (0-0) MRI 0/5 (0%) 0 (0-0) ICF DR 4/5 (80%) 1 (0-1) 0.3333 CT 5/5 (100%) 1 (1-1) MRI 5/5 (100%) 1 (1-3) LC DR 1/5 (20%) 0 (0-1) 0.6667 CT 1/5 (20%) 0 (0-1) MRI 2/5 (40%) 0 (0-2) MC DR 1/5 (20%) 0 (0-1) 0.5185 CT 3/5 (60%) 0 (0-1) MRI 3/5 (60%) 1 (0-2) TP DR 1/5 (20%) 0 (0-1) 0.5556 CT 3/5 (60%) 1 (0-1) MRI 1/5 (20%) 0 (0-2) Abbreviations: FT, femoral trochlear ridges; ICF, femoral intercondylar fossa; LC, femoral lateral condyle; MC, femoral medial condyle; TP, tibia plateau. animals-13-00849-t005_Table 5 Table 5 Results of cartilage MRI T2 relaxation time. Cartilage Lesions Relaxation Time (ms) Reference MF 44.5 (range 40.0-52.3) 70.2 (range: 57.9-87.9) LF 43.8 (range 39.2-54.1) 57.5 (range: 46.8-66.9) MT 43.7 (range 37.4-55.2) 65.0 (range: 52.0-92.0) LT 42.4 (range 39.7-45.9) 57.0 (range: 49.0-66.2) Abbreviations: LF, lateral femoral condyle; LT, lateral tibia plateau; MF, medial femoral condyle; MT, medial tibia plateau. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000065

Acquiring sufficient data is imperative to accurately predict tumor growth dynamics and effectively treat patients. The aim of this study was to investigate the number of volume measurements necessary to predict breast tumor growth dynamics using the logistic growth model. The model was calibrated to tumor volume data from 18 untreated breast cancer patients using a varying number of measurements interpolated at clinically relevant timepoints with different levels of noise (0-20%). Error-to-model parameters and the data were compared to determine the sufficient number of measurements needed to accurately determine growth dynamics. We found that without noise, three tumor volume measurements are necessary and sufficient to estimate patient-specific model parameters. More measurements were required as the level of noise increased. Estimating the tumor growth dynamics was shown to depend on the tumor growth rate, clinical noise level, and acceptable error of the to-be-determined parameters. Understanding the relationship between these factors provides a metric by which clinicians can determine when sufficient data have been collected to confidently predict patient-specific tumor growth dynamics and recommend appropriate treatment options. patient-specific prediction logistic growth This research received no external funding. pmc1. Introduction Female breast cancer has the highest rate of cancer incidence in the world (11.7%), even surpassing lung cancer rates for both men and women combined (11.4%) . Rates of female breast cancer incidence have been on the rise in recent years due to societal lifestyle changes, including lower fertility rates as women bear children at later ages and increased obesity rates . In the United States, about one in eight women will receive a diagnosis of breast cancer in their lifetime, and breast cancer is the second leading cause of cancer deaths in females, even though survival rates have been on the rise . Timely diagnosis and treatment are essential to breast cancer survival. The American Cancer Society recommends that women aged 40-54 years conduct mammography screenings annually to ensure early detection of breast cancer . Whilst treatment guidelines have been established to effectively treat the cancer based on large clinical trials, how to tailor treatments to individual patients with comparable clinical features remains challenging. Clinical detection of a tumor offers only a static snapshot of the tumor growth history and future dynamics. Being able to accurately predict the growth dynamics of a patient's tumor may offer a novel tool to contribute to integrated multidisciplinary decision-making. This is where mathematical models come into the arena--these models can take existing tumor measurements and predict how the tumor will grow . One of the most commonly used models in mathematical oncology is the logistic growth model . Unlike exponential growth with a constant volume doubling time, logistic growth decelerates as it approaches limitations to tumor expansion, called the carrying capacity. The carrying capacity is defined as the maximum tumor volume that can be sustained given the patient's tumor biology, including immune status, tumor oxygenation, and the physical size of the breast. Two patients with identical tumor volumes can have vastly different growth dynamics based on their individual carrying capacities, and, thus, different responses to therapy . The motivation to use the logistic growth model for breast cancer comes from preceding literature which promotes the utility of logistic growth as a means to predict tumor growth. Atuegwu et al. used the logistic growth model to predict breast cancer patient responses to neoadjuvant chemotherapy using MRI data from before, during, and after treatment. Logistic growth has also been used for radiation therapy studies. Logistic growth and radiation response models were able to fit retrospective longitudinal non-small-cell lung cancer and head and neck cancer patient data across multiple institutions with high accuracy (R2 = 0.98). They defined the tumor-volume-to-carrying-capacity ratio as the proliferation saturation index (PSI), establishing it as the most important patient-specific parameter to determine treatment response . The logistic growth model has not only been used with treatment data but also applied to untreated breast cancer patients. Spratt et al. fit observational breast cancer data for 448 patients to various mathematical models, including exponential, logistic, and Gompertzian growth. Each patient data set contained between two and six measurements and logistic growth best described the breast cancer growth dynamics . In order to use tumor growth dynamics to help guide treatment decisions, we must be able to confidently derive the putative patient-specific carrying capacity. The aim of this study was to investigate the number of volume measurements necessary to predict tumor growth dynamics using the logistic growth model. The logistic model was calibrated to volumetric data from 18 untreated breast cancer patients. We found that without noise, three tumor volume measurements are necessary and sufficient to estimate patient-specific model parameters. More measurements were required as the level of noise increased. Estimating the tumor growth dynamics was shown to depend on the tumor growth rate, clinical noise level, and acceptable error of the to-be-determined parameters. To our knowledge, this is the first comprehensive analysis of the minimum number of measurements that are necessary to determine the growth rate and carrying capacity of a patient's breast tumor. By conducting error analyses to determine how many measurements are necessary to predict patient dynamics given a certain set of conditions, we hope to strike a balance between reducing wait times until treatment and improving accuracy in predicting the patient's tumor dynamics. 2. Methods 2.1. Clinical Data The data used herein was derived from the 1979 study by Heuser et al. of untreated breast cancer in 18 patients. Tumor dimensions were recorded at two time points to determine the tumor doubling time. Based on the given major and minor axes values for the tumor and the doubling times, we calculated the tumor radius and the tumor volume for each patient. The tumor radius was calculated by:r=2a+b6, where r is the radius, and a and b are the major and minor axes, respectively. With this value for the radius, the initial and second tumor volumes were calculated for three different shapes using the following equations: Sphere V=4pr33, Cylinder V=pr2h, Oblate Sphere V=4pa2b3, where V is the tumor volume, r is the tumor radius, h is the height of the cylinder, and a and b are the radii of the major and minor axes, respectively. After calculating each patient's initial (V0) and final (Vt) tumor volumes for each shape, the doubling time (DT) was calculated using:DT=ln(2)y, where y=ln(Vt)-ln(V0)t. The doubling times for each of the three shapes were compared to the actual doubling time provided by Heuser et al. to determine the most accurate volume calculation for each patient. We found that for all of the patients, the cylinder volume calculations were the most accurate. Patients for whom doubling time was not listed were omitted from this study. 2.2. Mathematical Model Tumor volume V dynamics were described using the logistic growth model given by the ordinary differential equation:(1) dVdt=aV(1-VK), where a is the intrinsic, exponential tumor growth rate and K is the tumor carrying capacity. Logistic growth takes on a sigmoidal, S-shaped curve. Initially, when the tumor volume is far from its carrying capacity, growth resembles exponential growth . In the presence of limited resources, the volume growth slows and approaches the carrying capacity of the population K. Mathematically, with increasing volume, the volume-to-carrying capacity ratio, VK, increases and the net growth rate declines linearly until the volume approaches K. This growth can be characterized into four phases: initiation, acceleration, deceleration, and saturation . The values of the growth rate a and carrying capacity K determine the model dynamics. A larger growth rate will result in the volume reaching its carrying capacity sooner, while a smaller a will increase the time it takes to reach K ; similarly, a larger K (i.e., more resources) will allow for a larger end volume, whereas a smaller K has opposing effects . 2.3. Parameter Optimization Parameter optimization was used to determine the growth rate a and carrying capacity K to accurately describe the patient data. Nested optimization was used to simultaneously fit the model to the data from all 18 patients, whereby a was uniform among all patients and K was patient-specific. Given that each patient only had two volume measurements, the uniform a and patient-specific K values were used to simulate the model and obtain "ground truth" tumor volumes at different time steps along the individual growth curves for each patient. The curve was sampled at clinically relevant timepoints corresponding to typical radiological scan times prior to treatment. The initial point represents the volume at the first abnormal mammogram, which was taken directly from Heuser et al. . The remaining sampled points occurred on days 7, 30, 60, 90, and 120, which may correspond to a follow-up screening, second opinion, and potential wait times until treatment . Parameter optimization was used to fit the model to the data using three, four, five, and six measurements. The nominal carrying capacity to initialize the optimization procedure was set to be the last interpolated data point. That is, for three, four, five, and six measurements, the nominal carrying capacity would be the patient-specific tumor volume at days 30, 60, 90, and 120, respectively. Both the growth rate and the carrying capacity were optimized. For each patient, we recorded the optimizer-derived growth rate, the percent error to the original growth rate, the optimizer-derived carrying capacity, the percent error to the original carrying capacity, and the relative mean squared error to the original interpolated data points and the final tumor volume. 2.4. Adding Uncertainty (Noise) to the Data It is generally accepted that clinical data contains noise, due to a combination of factors including, but not limited to, radiographic imaging resolution and physician contouring preference ; to account for this, we added noise to the data points interpolated from the original curves with noise levels of 5%, 10%, and 20%. Note, the initial and final tumor volume measurements were taken to be without error. To add noise, we used the following equations:noise=a+(b-a)r,VN=V+(V x level x noise), where noise is the amount of uncertainty added to the data, with a=1, b=-1, r is a random number from 0 to 1, V is the original tumor volume, VN is the tumor volume with noise, and level is the noise level (either 0.05, 0.1, or 0.2 for respectively 5%, 10%, or 20%). We conducted 10 simulations per patient to account for the stochasticity in r. 2.5. Error Calculation and Comparison to Data For each noise level, we determined the error between the optimization-derived parameter values and the original parameter values. The relative mean squared error to the interpolated data points is calculated using:rMSE=(1N)i=1N(Vm-Vd)2, where N is the number of interpolated data points (including the final tumor volume for each patient), Vm is the tumor volume simulated by the model at each time point, and Vd is the interpolated tumor volume at each time point. This was repeated across all the data sets with the different number of measurements and across all of the noise levels. The Mann-Whitney U test was used to evaluate the comparisons between distributions of error values across the different numbers of measurements. This test was used because it compares the median ranks between paired data. Since all the error distributions were for the same patient set, the data could be considered paired. The comparisons were made for three versus four points, four versus five points, and five versus six points. 3. Results 3.1. Parameter Fits with Uniform Growth Rates Yield Patient-Specific Carrying Capacities Using the logistic growth model, we first sought to determine the growth rate and carrying capacity values that could accurately describe patient-specific dynamics from 18 untreated breast cancer patients (see Methods). Given the limitation of only two available data points per patient, as well as the model being governed by just two model parameters, multiple growth rates could be used to fit the data equally well. We chose three growth rates (a0.0125, 0.025, and 0.0545 day-1) to fit the model and used parameter optimization to find the corresponding carrying capacities for each patient individually . These curves and their associated growth rate and carrying capacity parameter values served as the "ground truth" upon which all further analyses were compared. 3.2. Perfect Data Yield Accurate Patient-Specific Carrying Capacity Estimates Without noise in the clinical data and with a known growth rate a, the carrying capacity of Equation (1) can be estimated from the analytical solution to the logistic equation. If both model parameters are unknown, a priori, fitting the solution of Equation (1) to three ground truth data points at times 0, 7, and 30 days yields sufficient fits to the last data points . Increasing the number of measurements to four, five, and six does not noticeably further increase the fit to the last data points. A comparison of the relative mean squared error (MSE) shows that increasing the number of data points decreases the relative MSE, and the difference is statistically significant when comparing three versus four measurements for a=0.0125 . Further comparison of the error to the parameters shows that increasing the number of measurements significantly decreases the errors . Model fits to tumor volume dynamics with the slowest ground truth growth rate, a=0.0125 day-1, yields the highest average percent error to parameters a (5.056%) and K (0.851%). As the growth rate increases, the model reaches carrying capacity in a shorter time; therefore, at a larger growth rate, more of the dynamics of the curve are known in a shorter time period and, therefore, fewer measurements may be necessary. 3.3. Increasing Noise in the Data Requires Increasing the Number of Data Points to Estimate Patient-Specific Carrying Capacity To account for measurement error depending on the imaging method used , we added noise to the sampled data points and determined whether the model could accurately recapitulate the data or not. With as little as 5% noise added to the data, three measurements are no longer sufficient across all of the ground truth growth rates to train the logistic growth parameters to accurately predict the last data point. For data generated at the lower growth rates (a=0.0125, 0.025 day-1), the tumor volume at the last data point is estimated to be much higher than the patient data . With the addition of a fourth measurement, the logistic model parameters can be correctly identified to accurately predict the tumor volumes at the last data point for all considered ground truth growth rates. Larger noise levels of 10% or 20% require additional data points for up to five and six measurements, respectively, to adequately forecast future tumor volumes . For all noise levels ; 10% , 20% ), the relative MSE between model prediction and ground truth data reduces with additional measurements included in model fitting and parameter estimation. Regardless of which noise was added to the data, including four measurements instead of three significantly reduced the relative MSE for data generated with a high growth rate a=0.0545 day-1. Of interest, while additional measurements yielded visually improved fits, the improvement in fitting accuracy did not reach statistical significance. Analysis of the percent error of the estimated to the ground truth logistic growth model parameters, however, showed significant and clinically meaningful reductions in parameter errors with additional measurements . We have shown that the number of points necessary to accurately predict patient tumor growth, determined by the minimal error to the original data and parameters, is a function of the uniform growth rate, data noise level, and desired error range. The number of measurements necessary to predict a patient's tumor growth may further depend on the clinician's (un)certainty in data collection and acceptable error in model dynamics forecasts. From this, we can evaluate how many measurements should be taken to predict the patient's tumor growth with acceptable relative MSE, or percent error to logistic growth rate, a, or carrying capacity, K. We evaluated the median values for the error distributions against the number of measurements used for the different noise levels . Dependent on the clinicians' accepted level of uncertainty, this can be used to determine how many measurements are sufficient to confidently predict responses. 4. Discussion The aim of this study was to determine the minimum number of screenings that would be necessary in order to determine a cancer patient's tumor dynamics using the logistic growth model. Using tumor volume data from untreated breast cancer patients, we fitted all of the patient trajectories to the logistic growth model and interpolated data at clinically relevant time points. Using these data, we were able to produce tumor growth dynamic curves and compare the use of three, four, five, and six measurements along the ground truth data. We added varying levels of noise to the interpolated data in order to capture a variety of possible patient scenarios. Across growth rates and noise levels, we saw a trend where the median error decreases as the number of measurements increases. This is expected as having more measurements along the curve, especially further along the curve, provides more information into the dynamics of the logistic growth curve; however, the cost associated with having more measurements is time until treatment. This study aimed to provide the first insights into how many data points are necessary to estimate logistic growth parameters and predict tumor dynamics, dependent on uncertainty in clinically collected data and the clinician's acceptable error in model prediction. Across all of the noise levels, as the growth rate increases, the number of measurements necessary decreases. This could be due to the fact that at higher growth rates, the curves reach their carrying capacity in less time than at slower growth rates, and, thus, are able to capture more of the patient's tumor dynamics in less time. At varying noise levels, we also found that as the noise level increased, the number of necessary measurements also increased to balance out the uncertainty in the data collected. This is the first study, to our knowledge, that uses untreated patient-specific data to investigate when sufficient data have been collected to determine growth dynamics. Studies conducted in the past 10 years have primarily focused on predicting patient-specific responses to treatment to determine when and how to treat patients more effectively. While this is important, understanding untreated growth dynamics provides important information about the patient's system unperturbed by treatment that oncologists might use to determine when to begin treatment or how best to administer it. The presented study has focused on the logistic growth model and its two parameters, the growth rate and carrying capacity. If using an alternative model to predict tumor growth, for example, Gompertzian growth, the number of measurements necessary may vary, based on the parameters and dynamics of the model. When comparing models, determining the minimum number of measurements needed by each model for each distinct cancer type and patient cohort is likely a function of the specific model chosen, and the number of parameters being estimated. This will be explored in a future study. It is also worth noting that tumor volume alone may not be fully indicative of a breast cancer patient's condition. Additional prognosis factors such as the proliferation marker Ki67 or overexpression of HER2, as well as the mutational capacity of the tumor, might be useful in determining patient-specific carrying capacities. These should be considered in future studies; additionally, socio-economic factors might contribute to longer or shorter wait times and are, thus, important to consider when determining when sufficient data have been collected to determine patient-specific tumor growth dynamics. 5. Conclusions In conclusion, this study found that the number of measurements necessary to predict the tumor growth dynamics for breast cancer patients is reliant upon multiple factors. These factors include the growth rate of the patient's tumor, the uncertainty in data collection, and the desirable error range for model prediction. Though we demonstrated this in untreated breast cancer, we are confident that the methods described here can be expanded to other cancer types. We hope that this study can help inform future data collection for accurate prediction of patient-specific tumor growth dynamics. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Distribution of Patient-Specific Carrying Capacities; Figure S2: Error distributions of model fit to data with 10% noise; Figure S3: Error distributions of model fit to data with 20% noise. Click here for additional data file. Author Contributions Conceptualization, methodology, validation, formal analysis, investigation I.H., H.E. and R.B.-N.; software, resources, H.E.; writing--original draft preparation, writing--review and editing, I.H., H.E. and R.B.-N.; visualization, I.H., H.E. and R.B.-N.; supervision, H.E. and R.B.-N.; project administration, H.E.; funding acquisition, H.E. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data are available upon reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The Logistic Growth Model. (A) Volume grows uninhibited at rate a in the presence of unlimited resources. As the available resources decrease, growth slows and approaches the carrying capacity K. (B) Increasing (decreasing) the value of a allows the population to reach K faster (slower). (C) Increasing (decreasing) the value of K allows for the volume to grow larger (smaller). Figure 2 Logistic tumor growth curves of 18 untreated breast cancer patients. The black x markers denote the initial and final tumor volumes abstracted from clinical measurements. Three different curves were fitted through these two data points; each corresponds to the different growth rates of a = 0.0125 day-1, 0.025 day-1, and 0.0545 day-1 (red, green, and yellow curves, respectively) uniformly for all 18 patients. For easy comparison across the patient population, all curves are plotted on the same log-log scale with tumor volumes ranging from 80 to 11,000 mm3, and time ranging from zero to 750 days. Figure 3 Tumor growth trajectory predictions from optimized model fits to ground truth data for representative patient 9. (A) Model fit to data using three, four, five, or six measurements (solid curve) to predict the tumor growth dynamics forward in time (dashed curve) for data generated with the different ground truth growth rates (a= 0.0125 (red), 0.025 (green), or 0.0545 day-1 (yellow)). The black markers are the original tumor volumes taken from Heuser et al., whereas the colored markers represent the interpolated tumor volumes at days 7, 30, 60, 90, and 120. Since the interpolated data points are specific to each growth rate, the color of the marker matches the color of the curve from which the point was interpolated. (B) Relative mean square error (MSE) between model fit and prediction and data generated as a function of number of measurements included in model fit, with the different ground truth growth rates, a. (C) Percent error of model estimated growth rates, a, and carrying capacities, K, as a function of the number of measurements included in model fit for the different ground truth growth rates. In B-C, statistical significance is denoted by asterisks (* for p < 0.01, ** for p < 0.005, and *** for p < 0.001). Figure 4 Logistic growth training (solid curves) and predictions (dashed curves) for Patient 9 with different percent noise in the training data. Figure 5 Error distributions of model fit to data with 5% noise. (A) Relative mean squared error (MSE) of model fit to ground truth data with different growth rates as a function of different measurements included in parameter estimation. (B) Percent error of model fit-derived logistic growth parameters (growth rate, a, and carrying capacity, K) to ground truth parameters as a function of different measurements included in parameter estimation. Statistical significance is denoted by asterisks (* for p < 0.01, ** for p < 0.005, and *** for p < 0.001). Figure 6 Comparison of error analyses for model fit and percent error across the different noise levels. (A) Relative mean squared error (MSE) between model prediction and ground truth data for different growth rates and noise levels in the data. (B) Comparison of percent error of model parameters (growth rate, a, and carrying capacity, K) to ground truth for different growth rates and noise levels in the data. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000066

The rumen fluids from ten cows at Day 3~5 before calving and Day 0 after calving were collected to analyze the composition and quantity of bacterial communities and concentrations of SCFAs. The results showed that the relative abundances of unidentified Lachnospiraceae, Acetitomaculum, Methanobrevibacter, Olsenella, Syntrophococcus, Lachnospira, and Lactobacillus genera were significant increased (p < 0.05), while that of unidentified-Prevotellaceae was notably decreased after calving (p < 0.05). In addition, the concentrations of acetic acid, propionic acid, butyric acid, and caproic acid obviously decreased after calving (p < 0.01). Our findings show that parturition altered the rumen microbiota and their fermentation ability in dairy cows. This study defines a rumen bacteria and metabolic profile of SCFAs associated with parturition in dairy cows. parturition dairy cows rumen bacterial communities short-chain fatty acids National Natural Science Foundation of China32160848 This research was subsidized by the National Natural Science Foundation of China (NSFC, Grant No. 32160848). pmc1. Introduction Parturition is a complex physiological process modulated by the endocrine, nervous, and immune systems, and other factors, during which the metabolism of hormones, sugars, proteins, and lipids is severely disturbed. Among these factors, alterations in hormones play a crucial role in the loss of appetite during late gestation in dairy cows . On Days 3~5 before parturition, DMI is reduced sharply so that the daily feed intake is only approximately 8~9 kg per dairy cow . Due to the limited feed intake, rumination time has been observed to be largely decreased about 8 h before delivery and gradually recovered about 6 h later . Hence, rumination time has been well recognized as a means of detecting parturition in dairy cows . Rumen microbiota, consisting of anaerobic bacteria, archaea, protozoa and fungi, ferment fibrous and nonfibrous sources of carbohydrates in the feed into SCFAs to supply energy . Rumination is universally acknowledged as the primary means by which the size of feed particles consumed is decreased in dairy cattle . More sites for microbial attachment are exposed when the feed particle is ruminated. During rumination, saliva is added and the accumulated CO2 and VFA are released via chewing, which can make the microenvironment more beneficial to bacterial growth . Although the importance of rumination for microbial activities by reducing particle size is well recognized, the detailed effect of parturition on the quantity and fermentation activity of rumen microbial communities are not well reported and require further study. High-throughput sequencing is a category of powerful technologies for obtaining high-coverage information on the classification and diversity of microbial communities without isolation and culture , among which 16S rRNA sequencing has been commonly applied to explore the composition of microbial communities in the rumens and intestines of dairy cows . Targeted GC-MS/MS metabolomics are suitable for identifying and quantifying small molecule acids, amino acids, sugars, and fatty acids, and have also been successfully used to check the concentrations of these metabolites in the serum, feces, and rumen fluid of ruminants . Consequently, we collected the rumen fluids from ten cows on Days 3~5 before calving and Day 0 after calving to reveal the influence of parturition on rumen bacterial communities and SCFAs using 16S rRNA high-throughput sequencing and targeted GC-MS/MS metabolomics, attempting to provide more information on physiological process of parturition in Holstein dairy cows. 2. Materials and Methods 2.1. Ethical Statement The experimental scheme was approved by the Animal Ethics Committee of Ningxia University (authorization number: 025/22) and complied with the international guidelines for animal experiments. 2.2. Sample Collection At a large modern enterprise around the Yinchuan city of China, twenty healthy Holstein cows on Days 3~5 before calving (2~3 parity and 3.2~3.5 BCS) were selected to collect rumen fluids. The cows were fed three times each day with TMR diet (Supplemental Table S1) and DMI was 9.2 +- 0.6 kg*d-1 during calving. The concentrate-to-forage ratio of TMR diet was 3.6:1. Their body temperatures were normal and blood ketones values were around 1.0. Each cow had taken rumen fluid at 1 h after their second meal. Ultimately, the rumen fluids from ten cows were retained as "samples before parturition" because the calving date of the ten cows coincided with expected calving date. Then, the rumen fluids as "samples after parturition" were taken from the same ten cows at 1 h after calving without eating. Rumen fluids before parturition were labeled as E1~E10 (Group E), and those after parturition were marked as A1~A10 (Group A). The rumen fluids were taken using special collectors equipped with a metal filter at one end and a 50 mL syringe at the other end. Discarding the first tube of rumen fluid and the second tube of rumen fluid was used as the test sample. The rumen fluids were filtered by four layers of sterilized gauze, transferred to cryopreservation tubes and stored at -80 degC. 2.3. Targeted GC-MS/MS Metabolomics Analysis of Rumen SCFAs The rumen fluids were well mixed in maximum vortex frequency after slowly thawing at 4 degC. Fifty microliters of rumen fluid was vortexed with 100 mL of 36% chromatographic grade phosphoric acid solution for approximately 3 min in the eppendorf tube and then vortexed with 150 mL of chromatographic grade MTBE (methyl tertiary butyl ether) solvent containing internal standard to extract SCFAs from rumen samples with ultrasonication for approximately 5 min in an ice bath. The extracted solution was centrifuged at 12,000x g r/min and 4 degC for 10 min, and 90 mL of supernatant was absorbed into the sample bottle with a glass liner for later targeted GC-MS/MS analysis. An Agilent 7890A-5975C gas chromatograph-mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with a quadrupole analyzer was used for the qualitative and quantitative analysis of rumen SCFAs. According to previous literature , the GC-MS/MS conditions were developed (Supplemental Table S2). The high reliability of the GC-MS/MS conditions was confirmed with intraday precisions, interday precisions, and recovery rates of SCFAs with different concentrations of QC samples (Supplemental Table S3), and good stability of the instrument was observed through the total ion current overlap of QC sample mass spectrometry . The chromatographic peaks representing acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and caproic acid were ensured according to the ion pair information, secondary spectrum and retention time . The linear regression equations of the seven SCFAs were established based on the peak intensities of SCFA standard solutions with serial concentrations (Table 1). The concentrations of SCFAs in rumen fluid were calculated based on linear regression equations (Supplemental Table S4). The file in the csv format containing names of SCFAs, concentrations and samples information was imported into MetaboAnalyst 5.0 accessed on 8 October 2021) to carry out the following metabolomics analysis. Principal component analysis (PCA) was used to visualize the changes in the metabolic profile of the seven rumen SCFAs before and after parturition. Variable importance in projection (VIP) values of the seven SCFAs were obtained by partial least squares-discriminant analysis (OPLS-DA). The p value and fold change (FC) were also acquired by t test. SCFAs with VIP >= 1, FC >= 1.5, and p < 0.05 were selected as biomarkers for differentiating dairy cows before and after parturition. 2.4. 16S rRNA High-Throughput Sequencing of Rumen Bacteria Communities The SDS method was used to extract the genomic DNA of rumen bacterial communities, and the purity and concentration of genomic DNA were evaluated with 1% AGE. Next, 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT) primers with barcodes were designed to amplify the V3~V4 hypervariable regions of the rumen bacterial 16S rRNA gene . The PCRs were carried out using a thermal cycle PCR system (Gene Amp 9700, ABI, Waltham, MA, USA) according to the published literature . The amplified products were validated using 2% agarose gel electrophoresis and further purified using the Qiagen gel extraction kit (Qiagen, Hilden, Germany). After amplification, DNA libraries were constructed with a TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) and quantified with Qubit and Q-PCR methods. The qualified libraries were subsequently sequenced with a PE250 strategy via the NovaSeq6000 platform (Illumina Inc., San Diego, CA, USA). The reads of each sample were separated after removing the barcode and the primer sequences and then spliced into raw tags using FLASH (V1.2.7, accessed on 9 January 2023) . The raw tags were quality-filtered to obtain high-quality tags (clean tags) . The clean tags were truncated and filtered via QIIME (V1.9.1, accessed on 9 January 2023) , and chimeras were further removed with VSEARCH accessed on 9 January 2023) . The effective tags of all samples were finally obtained after the above data processing and then clustered into operational taxonomic units (OTUs) at 97% identity using UPARSE (v7.0.1001, accessed on 9 January 2023) . Meanwhile, the representative sequence of OTUs was selected according to the UPARSE algorithm. Species annotation and taxonomic analysis of each 16S rRNA gene sequence was performed with Mothur and the SSUrRNA database of SILVA132 accessed on 9 January 2023) with a threshold of 0.8~1 . The bacterial community composition of each sample was assessed at the levels of kingdom, phylum, class, order, family, genus, and species. Alpha diversity indices of the groups, including Shannon, Simpson, Chao1, and ACE, were calculated with QIIME after homogenizing the data of each sample. The program R (Version 2.15.3) was used to generate the rarefaction curve and t test of alpha diversity between groups. Principal coordinate analysis (PCoA) based on unweighted UniFrac distance was selected to visualize beta diversity using WGCNA, stats, and ggplot2 packages in R. Anosim analysis based on Bray-Curtis distance were used to determine the difference in bacterial communities between two groups with the anosim function of the vegan package in R. The t test in R was used to search and visualize differential species between the two groups at the genus level. LEfSe (LDA Effect Size) software was used to find biomarkers for the two groups and generate an LDA score cladogram. Spearman correlation analysis between rumen bacterial genera with the top 30 abundances and SCFAs was carried out using MicrobiomeAnalyst 2.0 accessed on 9 January 2023). 3. Results 3.1. Changes in the Metabolic Profile and Concentrations of Rumen SCFAs The 2D scatter plots of PCA and OPLS-DA exhibited a clear separation of the metabolic profiles of the seven SCFAs between Groups E and A , indicating that rumen fermentability in dairy cows obviously changed during parturition. Acetic acid, propionic acid, butyric acid, and caproic acid could be metabolic biomarkers for differentiating the rumen fluids before and after calving according to their VIP values (>=1), FC (>=1.5) p values (<0.05), and their concentrations in rumen fluid presented a significant decreasing trend during parturition. The average concentrations, pH of rumen liquids, p values, FC, and VIP values are shown in Table 1. 3.2. Alteration in Rumen Bacteria Communities An average of 1,399,432 raw tags were detected by 16S rRNA gene sequencing from the 20 rumen fluid samples of dairy cows before and after parturition, and 837,879 effective tags with an average length of 413 bp were ultimately screened after quality control and filtration (Supplemental Table S5). A total of 2590 OTUs were acquired by clustering at 97% identity, of which 1965 OTUs were shared by both groups. The species accumulation curve was prone to be flat when the number of samples were higher than 20, showing that the number of samples in this study was reliable for estimating species richness . The rarefaction curve of alpha diversity tended to be flat as the sequencing depth increased, indicating that the sequencing results could reflect the diversity of bacterial communities in rumen fluid samples and that there would be no large number of new OTUs to appear even if the sequencing depth was further increased . The Shannon, Simpson, Chao1, and ACE indices of alpha diversity reflecting the within-group diversity and abundance of the bacterial community are listed in Table 2. The Shannon, Chao1, and ACE indices in Group A were significantly higher than those in Group E (p < 0.05). The 2D scatter plot of PCoA by unweighted UniFrac distance of beta diversity showed a visible separation between Groups A and E . As shown in Figure 3b, the nonparametric test of anosim by the Bray-Curtis distance proved that the difference between groups was significantly greater than that within groups (R = 0.399, p = 0.001). Five kinds of bacterial communities from phylum to species were identified as biomarkers for Groups E and A with LEfSe analysis, including f_Methanobacteriaceae, o_Methanobacteriales, c_Methanobacteria, f_Prevotellaceae, and f_Lachnospiraceae . Furthermore, genera with the top 30 abundances in the two groups were selected for the t test, and eighteen rumen bacterial genera were ascertained to be significantly different in relative abundance between the two groups . Eight major abundant genera (accounting for 0.05% of the total sequences in at least one sample) with differences multiple >=2 were further confirmed as differential species between the two groups (Table 3). Among these eight genera, the relative abundances of unidentified_Lachnospiraceae, Acetitomaculum, Methanobrevibacter, Olsenella, Syntrophococcus, Lachnospira, and Lactobacillus in Group A were significantly higher than those in Group E. In contrast, the relative abundance of unidentified_Prevotellaceae in Group A was significantly lower than that in Group E. 3.3. Correlation between Rumen Bacteria and SCFAs Correlation analysis was performed on rumen bacterial genera in group A and group E. The bacterial genera and SCFAs with |r| > 0.4 and p <= 0.05 were listed in Table 4. The results showed that acetic acid was significantly negatively correlated with Syntrophococcus, Acetitomaculum, Lactobacillus, unidentified_Enterobacteriaceae, and Methanobrevibacter (p < 0.05 or p < 0.01). Propionic acid was significantly negatively correlated with Olsenella, unidentified_Lachnospiraceae, Desulfobulbus, unidentified_ Enterobacteriaceae, and Lachnospira (p < 0.05 or p < 0.01), and significantly positively correlated with unidentified_ Prevotellaceae (p < 0.05). Butyric acid was significantly negatively correlated with Subdoligranulum, Lactobacillus, Acetitomaculum, Syntrophococcus, Solobacterium, Desulfobulbus, and unidentified_ Enterobacteriaceae (p < 0.05 or p < 0.01). 4. Discussion The current study investigated the influence of parturition on the phenotype composition and quantity of bacteria in ruminal liquid of dairy cows using 16S sequencing technique. At present, either liquid or solid fractions in rumen are widely used for microbiome analysis. The two fractions have differentiated ecological niches , however, the high degree of similarity in microbial community composition, diversity, and relative abundance profiles between the two fractions in cattle and sheep have also been affirmed by a series of studies . Considering this, the ruminal liquid is selected for the current study, which is easy to collect and poorly contaminated. Ruminal SCFAs, originating from microbial fermentation of feed carbohydrates, are absorbed by the rumen epithelium as a dominant source of energy for ruminants. Acetic acid is the most-produced compound, accounting for approximately 70~75% of the total production of SCFAs and supplying energy through the tricarboxylic acid cycle . Propionic acid generates approximately 50~60% of glucose via hepatic gluconeogenesis . Butyric acid accounts for 10~20% of the total production of SCFAs and is transformed into b-hydroxybutyric acid to provide energy for muscle tissue , and it also plays an important role in the regulation of innate and adaptive immune cell generation and function . An intense negative energy balance at parturition is most likely induced by depressed appetite and the initiation of milk synthesis in dairy cows . Although dairy cows can mobilize adipose tissue into fatty acids to remedy the energy deficit, excessive lipolysis heightens the risk for metabolic and inflammatory diseases . The parturition period is critical for determining the potential of the cow mammary gland to synthesize milk, and the degree of that potential depends on how much nutrients are received by the gland . In this study, the concentrations of acetic acid, propionic acid, and butyric acid in the rumen fluid after parturition were significantly lower than those before parturition, indicating that parturition probably aggravated negative energy balance, which in turn negatively affected milk production performance and increased the risk for postpartum metabolic and inflammatory diseases. Bacteria population acts as a key role in digestive and metabolic activities of rumen, obtaining energy from fiber, starch, sugars, and protein of feed. In our study, alpha diversity and beta diversity of rumen bacteria are distinctly altered before and after calving, indicating rumen bacterial community composition and its digestive and metabolic activities were affected by parturition. The relative abundances of rumen bacteria in genera level was further observed in our study to characterize their change feature during parturition, showing that seven bacteria abundances increased including Lachnospiraceae, Acetitomaculum, Methanobrevibacter, Olsenella, Syntrophococcus, Lachnospira and Lactobacillus, and Prevotellaceae abundances decreased after calving. Prevotellaceae is the dominant flora, accounting for approximately 60%~70% of rumen microbes of dairy cows, which can decompose the protein and carbohydrates in the feed into propionic acid, lactic acid, and succinic acid . Approximately 90% of glucose in dairy cows is generated by gluconeogenesis, of which 50-60% originates from propionic acid through hepatic gluconeogenesis . In this study, propionic acid shows a positive correlation with Prevotellaceae in concentration before and after calving. Numerous studies have shown that dairy cows are prone to negative energy balance after calving . Therefore, the decreasing in Prevotellaceae abundance is most likely be one of reasons for the negative energy balance in postpartum dairy cows. In the rumen, methane production is beneficial to the microbial growth and digestion by regulating the partial pressure of hydrogen . Methanobrevibacter can utilize some metabolites, such as acetic acid, propionic acid, and H2, to generate methane , and a higher levels of methane emissions is correlated with high abundance of Methanobrevibacter . In this study, the abundance of Methanobrevibacter is lower before than after calving, which is speculated to be related to the inhibition of rumination during parturition. As the abundance of Methanobrevibacter decreases, several acetic acid-producing bacteria (Lachnospiraceae, Acetitomaculum, Lactobacillus, Olsenella, Syntrophococcus, and Lachnospira) have declined before calving in this study. Family Lachnospiraceae has been verified as the predominant acetogen in the rumen fermentation system of dairy cows . The genus Acetitomaculum exhibits a significant positive association with lower feed efficiency , which can ferment monosaccharides into acetic acid . Lactobacillus can hydrolyze starch and other sugars to produce acetic acid, butyric acid, and lactic acid . Olsenella has been found in GIT of human and animals, fermenting starch and glycogen substrates and producing lactic, acetic, and formic acid . Syntrophococcus produces acetate only from pyruvate and various carbohydrates although its role is not well-understood in the rumen metabolism . Lachnospira can produce pectin lyases and release into extracellular environment to decompose pectin to oligogalacturonides, which are metabolized as acetic acid within the cell . However, we find that the concentrations of acetic acid (including butyrate) in rumen are significantly higher before calving than after calving, which is inconsistent with these bacterial changes. The reason for this inconsistency is most likely related to the large amount of energy required for postpartum lactation initiation in dairy cows. 5. Conclusions Our study investigated the composition of the bacterial communities and the concentrations of SCFAs in the rumen of dairy cows before and after calving with 16S rRNA high-throughput sequencing and targeted GC-MS/MS metabolomics. Distinct alteration in the composition of rumen bacteria before and after calving could attributed to the sharply decreasing feed intake during parturition. As a dominant flora, the decreasing in levels of Prevotellaceae of propionic acid-producing bacteria are most likely be one of the reasons for the negative energy balance in postpartum dairy cows. Lower Methanobrevibacter abundance before calving may be related to the inhibition of rumination during parturition. On the other hand, the change in abundance of several acetic acid-producing bacteria is inconsistent with that of acetic acid before and after calving, which is most likely related to the large amount of energy required for postpartum lactation initiation in dairy cows. Our current study only focused on rumen bacterial populations; it provided a more comprehensive understanding of parturition on the dairy cow microbiota and as to how scientists would investigate all major microbial populations within therumen, including protozoa and fungi. Acknowledgments We appreciate Wuhan MetWare Co. Ltd. (Wuhan, China) for 16S rRNA sequencing and targeted GC-MS/MS metabolomics analysis. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Total ion current (TIC) overlap of quality control sample (QC) mass spectrometry; Table S1: The TMR diet formula of dairy cows before parturition; Table S2: GC-MS/MS testing conditions for short chain fatty acids (SCFAs) in rumen fluid sample; Table S3: Precision and recovery rate of QC (quality control) samples; Table S4: The concentrations of each SCFA in each rumen fluid sample (mg/mL); Table S5: Tags and average length of rumen bacteria communities. Click here for additional data file. Author Contributions Conceptualization, Y.G. and G.Z., Methodology, F.W., Formal Analysis, Y.M., Software, W.K., Data curation, J.W., Writing--Original Draft, Y.G., Writing--Review and Editing, G.Z., Project Administration, Y.G., Funding Acquisition, Y.G. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The experimental scheme was approved by the Animal Ethics Committee of Ningxia University (authorization number: 025/22) and complied with the international guidelines for animal experiments. Informed Consent Statement Not applicable. Data Availability Statement None of the data were deposited in an official repository. Data are available upon request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 2D scatter plots of principal component analysis (PCA) and partial least squares-discriminant analysis (OPLS-DA) of rumen SCFAs in dairy cows. Note: (a): 2D scatter plot of PCA; (b): 2D scatter plot of OPLS-DA. E: before parturition; A: after parturition. Figure 2 Species accumulation curve (a) and rarefaction curve of alpha diversity (b). Figure 3 2D scatter plot of principal coordinate analysis (PCoA) based on unweighted UniFrac distance (a) and boxplot of ANOSIM based on Bray-Curtis distance (b) of rumen bacterial communities. A, after parturition; E, before parturition. The y-axis is the order of the distance between the samples. On the x-axis, A and E means within Groups A and E. An R-value > 0 indicates a difference between groups greater than that within groups, and p < 0.05 indicates a significant difference. Figure 4 Cladogram of LDA effect size analysis. E, before parturition; A, after parturition. Each small circle represents a species at taxonomic levels (phylum, class, order, family, genus and species, represented by large circles from inside to outside in turn). Yellow circles show species with no significant difference abundance between groups; red (or green) circles show the species with significantly higher abundance in the red group (or green group). Figure 5 Plot of the t test for the relative abundance of the top 30 genera. The left section shows the relative abundance of differential sections between Groups E and A, and each bar represents the mean in the corresponding group of the genera with significant abundance differences between groups. The right section shows the confidence degree of the difference between the groups, and the color in the circle is the same as the color of the group with a high mean. animals-13-00782-t001_Table 1 Table 1 Regression equations, concentrations, and statistical parameters of each SCFA in the rumen fluid. Component Retention Time (min) Regression Equation Concentrations (Mean +- SD) (mg/mL) Statistical Parameters (Before Parturition vs. After Parturition) Before Parturition (n = 10) After Parturition (n = 10) p Value FC VIP Acetic acid 3.36 y = 0.64x + 0.03 9575 +- 117.54 6340 +- 294.36 0.01 1.51 | 1.21 Propionic acid 3.97 y = 0.46x + 0.001 6109 +- 72.89 3236.8 +- 165.61 0.0004 1.89 | 1.36 Isobutyric acid 4.14 y = 1.00x - 0.001 339.4 +- 8.47 366.4 +- 23.23 0.75 0.93 1.18 Butyric acid 4.55 y = 5.57x + 0.20 5218 +- 75.27 3281.7 +- 149.00 0.004 1.59 | 1.01 Isovaleric acid 4.8 y = 6.49x - 0.001 345.5 +- 7.17 349.6 +- 20.46 0.96 0.99 1.36 Valeric acid 5.22 y = 7.12x + 0.13 716.7 +- 12.10 416.8 +- 21.69 0.003 1.72 0.63 Caproic acid 5.79 y = 3.39x + 0.23 310.8 +- 8.51 141.7 +- 8.78 0.0006 2.19 | 1.15 pH 6.62 +- 0.0624 6.82 +- 0.03 0.0081 0.97 | Note: | indicates down and | indicates up. animals-13-00782-t002_Table 2 Table 2 Alpha diversity indices of rumen bacterial communities. Items Before Parturition, n = 10 After Parturition, n = 10 p Value Shannon 7.25 +- 0.48 7.86 +- 0.32 0.0042 Simpson 0.96 +- 0.02 0.97 +- 0.008 0.1762 Chao1 1495.27 +- 115.85 1673.59 +- 157.62 0.0205 ACE 15,224.87 +- 84.09 1701.15 +- 164.32 0.0177 animals-13-00782-t003_Table 3 Table 3 Percentage and difference multiples of the relative abundance of eight major genera before and after parturition (n = 10 cows per group). Taxonomy Percentage of the Relative Abundance SEM p Value Trend Difference Multiple Before Parturition After Parturition #AVE Unidentified_Lachnospiraceae 2.01 3.99 2.99 0.99 0.002 up 2.00 Acetitomaculum 0.23 0.50 0.37 0.14 0.007 up 2.16 Methanobrevibacter 0.29 2.46 1.37 1.08 0.025 up 8.43 Unidentified_Prevotellaceae 3.34 1.05 2.19 1.14 0.009 down 3.18 Olsenella 0.08 0.16 0.12 0.04 0.011 up 2.08 Syntrophococcus 0.09 0.23 0.16 0.07 0.005 up 2.63 Lachnospira 0.11 0.22 0.16 0.06 0.022 up 2.03 Lactobacillus 0.0004 0.24 0.12 0.11 0.030 up 600.25 animals-13-00782-t004_Table 4 Table 4 Spearman Correlation coefficients and p values of rumen bacterial genera and SCFAs. SCFAs Bacterial Genera Correlation Coefficient (r) p-Value AdjPvalue Acetic acid Methanobrevibacter -0.601 0.005 ** 0.013 Acetitomaculum -0.469 0.037 * 0.037 Syntrophococcus -0.472 0.036 * 0.036 Lactobacillus -0.763 0.000 ** 0.000 Unidentified_Enterobacteriaceae -0.680 0.001 ** 0.001 Propionic acid Unidentified_Prevotellaceae 0.477 0.035 * 0.144 Unidentified_Lachnospiraceae -0.567 0.010 * 0.052 Lachnospira -0.447 0.048 * 0.241 Lactobacillus -0.807 0.000 ** 0.000 Olsenella -0.629 0.003 ** 0.008 Desulfobulbus -0.515 0.020 * 0.020 Unidentified_Enterobacteriaceae -0.730 0.000 ** 0.001 Butyric acid Acetitomaculum -0.476 0.034 * 0.037 Syntrophococcus -0.489 0.029 * 0.036 Lactobacillus -0.746 0.000 ** 0.000 Desulfobulbus -0.642 0.002 ** 0.006 Solobacterium -0.716 0.000 ** 0.000 Unidentified_Enterobacteriaceae -0.673 0.001 ** 0.001 Subdoligranulum -0.786 0.000 ** 0.000 Note: * indicates p < 0.05, ** indicates p < 0.01. |r| > 0.7 means a very tight correlation is very close, |r| between 0.4 and 0.7 means a tight correlation. 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PMC10000067

A major problem faced by the agricultural industry is the resistance of Haemonchus contortus to anthelmintic drugs. For a better understanding of the response of H. contortus to IVM and for the screening of drug-resistance-related genes, we used RNA sequencing and isobaric tags for relative and absolute quantification (iTRAQ) technology to detect the transcriptomic and proteomic changes in H. contortus after ivermectin treatment. An integrated analysis of the two omics showed that the differentially expressed genes and proteins were significantly enriched in the pathways of amino acid degradation, the metabolism of xenobiotics by cytochrome P450, the biosynthesis of amino acids, and the tricarboxylic acid cycle. We found that the upregulated UDP-glycosyltransferases (UGT), glutathione S-transferase (GST), cytochrome P450 (CYP), and p-glycoprotein (Pgp) genes play important roles in drug resistance in H. contortus. Our work will help in the understanding of the transcriptome and proteome changes in H. contortus after IVM and will facilitate the discovery of genes related to drug resistance. This information can be further applied to increase the understanding of the response of IVM in relation to H. contortus. ivermectin Haemonchus contortus transcriptomics proteomics National Natural Science Foundation of China31660710 This research was funded by the National Natural Science Foundation of China, grant number 31660710. pmc1. Introduction Haemonchus contortus is one of the most pathogenic gastrointestinal nematodes infecting small ruminants worldwide. It feeds on the blood of ruminant abomasum, especially that of sheep and goats. Infection by this blood-sucking nematode causes the symptoms of haemonchosis, which include anemia, diarrhea, weight loss, and even death in cases of severe infection . Furthermore, milk and meat production can be reduced in infected animals, causing tremendous economic losses to the agricultural industry worldwide. However, no vaccines protecting against these parasites are currently available; the primary form of nematode control is the use of anthelmintic drugs . Ivermectin (IVM) is the first commercially available macrocyclic lactone endectocide. Since its introduction into the agricultural market in the early 1980s, it has been used to treat a wide variety of nematode parasites, including H. contortus, and it has quickly proven to be a very effective drug . However, despite its benefits, the widespread usage of IVM has resulted in serious problems related to drug resistance worldwide, especially in countries where livestock husbandry is the dominant industry. In a vicious cycle of use and resistance, as IVM use increases, parasite resistance increases accordingly, which necessitates the use of yet more drugs . Therefore, the problem of drug resistance has become an international issue that urgently requires a solution. Drug resistance is the tolerance of a parasite to a given drug. This problem arose in conjunction with IVM five years after its initial use in the control of parasitic diseases . It can therefore be inferred that the development of drug resistance is a gradual process. According to reports, IVM can be used as an agonist of glutamate to enhance the opening frequency of glutamate-gated chloride (GluCl). A low concentration of ivermectin can enhance the action of neurotransmitters, and a high concentration of ivermectin can enhance cell membrane permeability to chloride ions, leading to blocked nerve conduction and muscle relaxant paralysis. After the peristalsis of the pharyngeal muscles of H. contortus is blocked, the feeding of H. contortus is disturbed or hindered, which eventually leads to the starvation and death of H. contortus . Many studies have shown that nematode ATP-binding cassette (ABC) transport proteins, including P-glycoproteins (Pgps), play an important role in anthelmintic resistance . One study showed that Pgps act as efflux pumps to expel hydrophobic xenobiotics from cells . According to the report, the ability of multidrug-resistant Teladorsagia circumcincta to survive IVM exposure may be associated with the increased expression of Pgp-9 and gene sequence polymorphism . Similarly, the expression levels of Pgps in Caenorhabditis elegans after exposure to IVM and the sensitivity of Pgp knock-out strains of C. elegans to IVM are both increased . Furthermore, Pgp-9.2 may be one of the most relevant candidates contributing to the multi-genic nature of the IVM resistance trait . Cwiklinski et al. (2013) found that glc-3 of Cylicostephanus goldi is one of the primary targets of macrocyclic lactone anthelmintics through a transcriptome analysis . Anthelmintic resistance can be inherited, as its development requires the existence of resistance genes. The study of these drug-resistant genes is therefore the first step in understanding parasite resistance. Drug resistance is the result of the common regulation of multiple genes, rather than being attributable to a single gene . Studying drug resistance is therefore vital for monitoring and controlling its further evolution and for delaying the accumulation of drug-resistance-related genes. However, the study of drug resistance is still in relatively early stages; drug-resistance-related genes are not comprehensively understood, and there are many such genes that need to be explored. It is urgent to identify genes that are potentially related to drug resistance. The objective of the present study was to use high-throughput sequencing combined with a bioinformatics analysis to investigate the changes in gene expression in H. contortus in both resistant and sensitive strains before and after IVM treatment. This study shows that IVM can cause transcriptional and proteomic changes in H. contortus. 2. Materials and Methods 2.1. Ethics Approval The study design was reviewed and approved by the Animal Ethics Committee of Ningxia University (Permit No. 22-031). The procedures involving animals were carried out in accordance with the Animal Ethics Procedures and Guidelines of the People's Republic of China. All efforts were made to minimize suffering and to reduce the number of sheep used in the experiment. 2.2. Sample Collection The IVM-susceptible and -resistant H. contortus strains were isolated in our laboratory at Inner Mongolia Agricultural University, where they have been maintained for several years . All experimental sheep were newborn lambs from our laboratory in Hohhot. Each animal was housed in a single pen and had free access to food and water. Fecal samples were collected and examined using the McMaster technique at regular intervals to ensure that the nematode egg counts of all sheep showed negative values (mean fecal egg count = 0 eggs per gram). After a week of feeding, the sheep were infected with approximately 104 of either susceptible or resistant H. contortus in the L3 stage. The health of all infected sheep was monitored closely. After 20 days, feces were collected from each infected animal and placed into corresponding boxes with small holes, which were marked with the collection date and strain number. For the recovery of H. contortus, the boxes were incubated at 27 degC for approximately one week, and the fecal samples were slightly moistened with tap water as necessary (e.g., under dry conditions). After approximately one week, H. contortus at the L3 stage were collected with a self-made funnel separator, rinsed thoroughly with deionized water, and stored at 15 degC for further use. 2.3. Ivermectin Treatment The two treatment groups were susceptible larvae treated with IVM (S1) and resistant larvae treated with IVM (R1). The two control groups, S0 and R0, were L3 larvae from susceptible and resistant strains, respectively, that had not been treated with IVM. The final concentration of IVM in the treatment groups (S1 and R1) was 0.28 mM, while the control groups (S0 and R0) were treated with a fresh medium without IVM. Each group included three biological replicates. All samples were cultured for 24 h and then harvested and stored in liquid nitrogen until used for RNA extraction, RNA-seq, and iTRAQ. 2.4. Transcriptomics 2.4.1. RNA Extraction, Sequencing, and Identification of Differentially Expressed Transcripts (DETs) Lysis Buffer (600 mL) was added to the sequencing sample, and then the total RNA was individually extracted from each sample using a mirVana miRNA Isolation Kit (Ambion, Shanghai, China) following the manufacturer's protocol (Supplementary Materials Table S1). RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The samples with an RNA integrity number >= 7 were subjected to subsequent analyses. Libraries were constructed using a TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. The libraries were sequenced, performed on an Illumina sequencing platform (HiSeqTM 2500, Illumina, San Diego, CA, USA), and 150 bp paired-end reads were generated. 2.4.2. Sequence Filtering, Functional Annotation, and Analysis of Differentially Expressed Genes Raw data were processed using Trimmomatic to remove low-quality reads and those containing ploy-N in order to obtain clean reads. The clean reads were assembled into expressed sequence tag clusters (contigs) and assembled de novo into transcripts with Trinity using the paired-end method. The longest transcript was chosen according to similarity for subsequent analyses. All downstream analyses were based on the clean, high-quality reads. Blastx was used to annotate the unigenes by aligning these with the following NCBI databases: nonredundant protein (NR), SwissProt, and Clusters of Orthologous Groups (COG) for C. elegans complete genomes. In addition, the proteins with the highest number of unigene hits were assigned functional annotations. Based on the SwissProt annotation, Gene Ontology (GO) classification was performed by mapping the associations between the SwissProt sequences and the GO terms; to annotate potential metabolic pathways, the unigenes were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database . Fragments per kilobase of exon per million fragments mapped (FPKM) and the read count values of each unigene were calculated using Bowtie 2 and eXpress . The differentially expressed genes (DEGs) of each group (three replicates per group) were identified using the DESeq R package functions estimateSizeFactors and nbinomTest. A gene with a p-value < 0.05 and log2foldchange >= 1 or <=-1 was considered to be differentially expressed. GO and KEGG pathway enrichment analyses of DEGs were performed using R based on the hypergeometric distribution. RNA-seq, read alignment, and DEG identification were carried out at OE Biotech (Shanghai, China). 2.5. Quantitative Proteomics (iTRAQ) 2.5.1. Protein Extraction, Quantization, and SDS-PAGE Electrophoresis The frozen samples were removed and ground thoroughly in the presence of liquid nitrogen. A mixture of phenol extraction solution and PMSF (600 mL) was added to attain a final concentration of 1 mM. The samples were further lysed via sonication (1 s ntervals, 3 min, 80 W). A phenol-Tris-HCl (pH 7.8) saturated solution was added and shaken (30 min, 4 degC). The mixtures were centrifuged (7100x g, 10 min, 4 degC) to collect phenol supernatants. The supernatants were added to five volumes of 0.1 M cold ammonium acetate-methanol buffer and precipitated at -20 degC overnight. The precipitate was washed with five volumes of cold methanol and centrifuged again (12,000x g, 10 min, 4 degC) to remove more precipitate. This process was then repeated. Methanol was replaced with acetone, and the wash step was performed twice. The samples were centrifuged (12,000x g, 10 min, 4 degC) to collect the precipitate, which was dried at room temperature and dissolved in lysis buffer for 3 h. The samples were centrifuged, and the supernatants were collected. The supernatants were centrifuged again to remove precipitates completely. Protein concentration was determined using the BCA method , and the proteins were then stored at -80 degC. Additionally, 7 mg samples were subjected to 12% SDS-PAGE, visualized, and scanned according to Candiano's protocol . 2.5.2. Proteolysis and ITRAQ Labeling The FASP method was adopted for the enzymatic hydrolysis of the proteins (100 mg), and the labeling peptide solutions were lyophilized and stored at -80 degC. 2.5.3. Reversed-Phase Liquid Chromatography (RPLC) Reversed-phase liquid chromatography was performed on an 1100 HPLC System (Agilent) using an Agilent Zorbax Extend RP column (5 mm, 150 mm x 2.1 mm). The elution buffer was collected every 1 min and placed in turn into a 1-15 centrifuge tube (Thermo Fisher Scientific, Waltham, MA, USA); samples were harvested from 8 min to 60 min. After collection, the samples were vacuum freeze-dried and cryopreserved for MS detection. 2.5.4. LC-MS/MS Analysis and Data Processing The samples were loaded using a capillary C18 trap column (3 cm x 100 mm) and separated using a C18 column (15 cm x 75 mm) on an Eksigent nanoLC-1D Plus System (SCIEX, Framingham, MA, USA). An analysis was performed using a TripleTOF 5600 mass spectrometer (SCIEX, Framingham, MA, USA) equipped with a Nanospray III source (SCIEX, Framingham, MA, USA). All raw LC-MS/MS data were searched against the sample protein database using Proteome DiscovererTM 2.2 software (Thermo, USA). At least two peptides are required for a peptide group to be considered for the purpose of quantification; the false positive rate of peptide identification was controlled below 1%. 2.6. Validation of RNA-Seq Results Using Quantitative Real-Time PCR (q-PCR) The expressions of DEGs in different groups were detected using quantitative real-time PCR (q-PCR) to confirm the RNA-seq-based transcriptional response of the susceptible and resistant strains of L3-stage H. contortus before and after IVM treatment. Genes that were upregulated or downregulated were identified by performing a sequencing analysis. The RNA samples were reverse-transcribed to single-stranded cDNA using a PrimeScriptTM RT Reagent Kit (TaKaRa, Dalian, China). The same samples were used for sequencing and q-PCR. b-tublin was used as the reference gene, and the DEGs used for q-PCR verification were randomly selected. TB Green(r) Premix Ex TaqTM II (TaKaRa, Dalian, China) was used to perform q-PCR according to the manufacturer's instructions. The selected genes were analyzed in triplicate; the forward (F) and reverse (R) primers used in q-PCR are listed in Table S2. The q-PCR cycling was performed under the following conditions: 95 degC for 30 s, followed by 40 cycles of 95 degC for 5 s, 55 degC for 30 s, 95 degC for 10 s, and a melting curve analysis ranging from 65 degC to 95 degC. The 2-DDCT relative expression method was used to calculate the expression of each gene. 3. Results We used the Illumina HiSeqTM 2500 platform with the cDNA libraries from IVM-treated H. contortus and obtained over 48,000,000 raw reads from each sample and more than 47,000,000 clean reads after processing (Supplementary Materials Table S3). A total of 69,728 unigenes were spliced, with a total length of 62,243,154 bp and an average length of 892 bp. The correlation coefficient of the unigene expression level among the different samples was close to 1, which indicates a high similarity of expression patterns between the samples . The sequencing data determined in this work have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database accessed on 3 April 2022) under accession no. PRJNA663203. 3.1. Differentially Expressed Genes (DEGs) and Differentially Expressed Proteins (DEPs) The transcriptome analysis detected 3301 upregulated and 1227 downregulated genes in the susceptible H. contortus strain after IVM treatment (S0-vs-S1), while 1606 upregulated and 1432 downregulated genes were detected after IVM treatment in the resistant strain (R0-vs-R1). Additionally, 2058 upregulated and 2757 downregulated genes were detected in the non-treated resistant strain compared with the non-treated susceptible strain (S0-vs-R0), while 1406 upregulated and 3493 downregulated genes were detected in the IVM-treated resistant strain compared with the IVM-treated susceptible strain . Regarding proteomics, a total of 1549 proteins were identified, of which 354 (226 upregulated and 128 downregulated), 89 (11 upregulated and 78 downregulated), 655 (438 upregulated and 217 downregulated), and 236 (81 upregulated and 155 downregulated) proteins were differentially expressed in the four comparison groups, . We also found that the total number of DEPs was much lower than the total number of DEGs. The expressions of the genes obtained through the RNA-seq were confirmed using qPCR, and the validation results are shown in Figure 3. 3.2. Integrated Analysis of Transcriptome and Proteome To identify the genes and proteins associated with drug resistance, we integrated the differentially expressed transcripts and proteins. As shown in Figure 4, almost all of the log2 mRNA:log2 protein ratios are concentrated in the center of the plot; these genes and proteins were filtered out, while the DEPs and DEGs were left in place. An integrated analysis of the transcriptome and proteome data revealed that the expressions of one gene and the corresponding protein (TRINITY_DN33068_c0_g1_i6_4) were upregulated and that two other genes (TRINITY_DN29476_c0_g1_i7_3 and TRINITY_DN42604_c4_g1_i6_1) were downregulated in the S0-vs-S1 group; one gene and the corresponding protein (TRINITY_DN35814_c0_g1_i8_2) were downregulated in the R1-vs-R0 group; one gene and the corresponding protein (TRINITY_DN43412_c0_g1_i1_3) were upregulated and nine were downregulated in the R0-vs-S0 group; and three genes and the corresponding proteins (TRINITY_DN30909_c0_g1_i3_1, TRINITY_DN38401_c0_g1_i1_1, and TRINITY_DN3919_c0_g1_i1_1) were downregulated in the R1-vs-S1 group . 3.3. Gene Ontology (GO) Analysis of DEGs and DEPs The GO enrichment analysis showed dynamic differences in the biological processes, cellular components, and molecular functions of H. contortus before and after IVM treatment. Based on the -log10 p-value, we list the top 30 GO terms for DEGs and DEPs of different comparison groups . Most of the GO terms in the biological process category for DEGs were associated with metabolic processes and catalytic activity, while those for DEPs were mainly involved in redox and catabolism. In the cellular component category, DEGs showed significant enrichment in the extracellular region (GO:0005576), ribosome (GO:0005840), and cytosolic large ribosomal subunit (GO:0022625), while DEPs were mainly classified in the intracellular organelle region (GO:0044446), cytoplasm (GO:0005737), and mitochondrial membrane (GO:0044455). In the molecular function category, DEGs were enriched in metalloendopeptidase activity (GO:0004222), structural constituents of ribosomes (GO:0003735), and cysteine-type peptidase activity , while enriched DEPs were mainly related to oxidoreductase activity (GO:0016635), structural molecule activity (GO:0005198), and structural constituents of ribosomes . The other enriched terms shared by DEGs and DEPs (not shown in the figure) were response to drug (GO:0042493), drug transmembrane transport (GO:0006855), negative regulation of response to drug (GO:2001024), drug binding (GO:0008144), and drug transmembrane transporter activity (GO:0015238). In summary, the GO enrichment analysis further showed that the orderly cooperation of biosynthesis, decomposition, metabolism, and transmembrane transport collectively maintained the metabolism and homeostasis of H. contortus. 3.4. KEGG Pathway Analysis of the DEGs and DEPs The KEGG enrichment analysis of the DEGs and DEPs revealed 4528 DEGs in the 186 KEGG pathways of the S1-vs-S0 group; 3038 DEGs were enriched in the 170 KEGG pathways of the R1-vs-R0 group; 4815 DEGs were enriched in the 231 KEGG pathways of the R1-vs-R0 group; and 4899 DEGs were enriched in the 230 KEGG pathways of the R1-vs-R0 group. We selected the 20 most significantly enriched KEGG pathways according to the enrichment scores. These DEGs were significantly enriched in the pathways related to xenobiotic metabolism by cytochrome P450, amino acid degradation, the biosynthesis of amino acids, drug metabolism-cytochrome P450, carbon metabolism, and the tricarboxylic acid (TCA) cycle . The DEPs were significantly enriched in the pathways related to carbon metabolism, the TCA cycle, endocytosis, ABC transporters (MRPs, Pgps), and drug metabolism-other enzymes (UGT, GST) . Overall, these DEGs and DEPs were enriched in pathways related to the decomposition, metabolism, and synthesis of substances; this result is consistent with that of the GO enrichment analysis. These genes and proteins may play an important role in the response to the metabolism of anthelmintics in susceptible and resistant strains. 4. Discussion Drug resistance is usually defined as the ability of an organism to survive a given dose of drugs. Partly because of its large impact on economic development in most parts of the world, H. contortus is the most widely studied nematode in terms of drug resistance. The focus on this topic in this species has led to an upsurge in research into drug resistance. At the same time, due to its biological and physiological factors, such as its high fecundity, relatively large body size, and simple larval storage conditions, H. contortus is regarded as a good experimental model . Omics has become an important research tool for exploring the molecular mechanisms related to drug resistance and for identifying genes related to drug resistance . We therefore used omics sequencing techniques to analyze and investigate the expressions of different genes and proteins in resistant and sensitive strains of H. contortus. In this study, transcriptomic and proteomic sequencing techniques were used to evaluate the global transcriptomic and proteomic changes in H. contortus after IVM treatment. We found that 4528 genes in susceptible strains and 3038 genes in resistant strains were significantly regulated after IVM treatment , which indicates that IVM had significant effects on the gene and protein expressions in H. contortus, especially in susceptible strains. Conversely, 354 and 89 proteins were significantly regulated in the susceptible and resistant strains, respectively, after IVM treatment. The apparent quantitative contrast between the genes and proteins reflects the proteome-transcriptome complexity, and the result of this comparison may be due to the fact that most of the genes encoded either hypothetical or non-functional annotated proteins. Thus, there is still a large knowledge gap in our understanding of transcriptional responses under IVM treatment. GO and KEGG pathway enrichment analyses are downstream procedures that are commonly used to interpret differential expression results . Some of the genes we were interested in were upregulated or downregulated; we used annotation as part of our GO and KEGG analyses in order to determine which terms or pathways were significantly enriched . The research objectives of the two omics are the same, and there must be a certain correlation between the groups. The transcriptome and proteome association analyses showed that the genes and proteins that are associated with GTP, RNA, proteolysis, synthesis, and catabolism terms were upregulated under IVM treatment, indicating that the energy and protein production rates were increased in H. contortus after IVM treatment. It has been reported that the genes involved in these functions are also upregulated in H. contortus after albendazole (ABZ) treatment and in Acinetobacter baumannii after antibiotic treatment . This phenomenon may be caused by the drug delivery screening that the organisms are subjected to in determining resistant strains, with selective pressure leading to the upregulation of resistance-related gene expression in the presence of the drug. In addition, we found that a single drug-resistant gene did not necessarily lead to IVM resistance in H. contortus. An extensive network of resistance-related genes, such as MRPs and Pgps, play a protective role in response to the efflux of anthelmintics in susceptible and resistant strains. In this study, these genes and proteins were significantly regulated in both susceptible and resistant strains. The transcriptome and proteome KEGG enrichment analyses showed that some pathways associated with drug metabolism, such as that of xenobiotics by cytochrome P450 (cel00980) and drug metabolism-cytochrome P450 (cel00982), were activated after IVM treatment. Cytochrome P450 (CYP) is involved in a variety of biosynthetic, catabolic, and xenobiotic detoxification functions . The relationship between CYP expression and drug resistance has been demonstrated in insects. It has been reported that multi-insecticide resistance in Drosophila simulans is associated with the overexpression of CYP6g1 and that pyrethroid resistance is associated with the overexpression of CYP6P9 in Anopheles funestus . Additionally, some studies suggest the opposite. For example, some proteins of C. elegans, especially members of the CYP35 family, have been shown to be inducible by exogenous organisms . ABZ can induce the expression of multiple CYP genes in C. elegans , while the inhibitor of CYP, piperonyl butoxide (PBO), increases the toxicity of the insecticide rotenone to H. contortus larvae and adults . In this study, a comparative analysis showed that the CYPs of H. contortus were significantly regulated after IVM treatment. Differences at the molecular level in susceptible strains before and after IVM treatment play a role in the study of drug resistance. The transcriptome and proteome association analysis of the expressions of differential genes and proteins in the S1-vs-S0 group revealed that the expression of only one gene, glutathione S-transferase (GST; TRINITY_DN33068_c0_g1_i6_4), in the IVM-treated susceptible strains showed the same trend (upregulation) as the corresponding protein . This gene was enriched in the pathways of xenobiotic metabolism by cytochrome P450 (cel00980), drug metabolism-cytochrome P450 (cel00982), and glutathione metabolism (cel00480). It has been reported that the activity of glutathione S-transferase (GST) is 1.5-1.8 times higher in the cambendazole-resistant strains of H. contortus than in susceptible strains . The activity and expression of GST genes are upregulated in a dose-dependent manner in Helicoverpa armigera larvae after pesticide exposure . Pugazhendhi et al. (2017) found that the antibiotic-induced GST activity of bacteria from a poultry litter was 3-4 times higher than that of the control, which led to the speculation that GST plays an important role in antibiotic resistance . In addition, GST is involved in drug resistance in organisms such as Musca domestica, Bombyx mori, and Aedes aegypti . In this study, the expression of GST in the drug-resistant strains was three times higher than that in the susceptible strains. This gene upregulation may enable resistance to the pressure of drug selection before the susceptible strain becomes resistant to the drug. In addition, we found that the UGT (TRINITY_DN44820_c0_g1_i7_2) gene, which is related to detoxification in organisms, was significantly enriched in transcriptome and proteome association pathways. According to one study, the UDP-glycosyltransferases (UGT) inhibitor chrysin reduces ABZ biotransformation in C. elegans . Matouskova et al. (2018) found that the expression of UGT in drug-resistant strains of H. contortus was significantly higher than that in susceptible strains . In this study, the expression of UGT was upregulated in drug-resistant strains before and after treatment and in both strains after IVM treatment; the expression was higher in resistant strains than in susceptible strains. The significant differences in the transcription and protein levels support the possibility of important roles for UGT and GST in general drug resistance; however, the role played by this upregulated transcription is currently unknown. Additionally, it remains to be elucidated whether the UGT and GST genes are involved in the biotransformation of IVM anthelmintics and how significant a role they play in resistance. The genes and pathways that we identified here may serve as therapeutic targets to control the further development of drug resistance. Further research will provide insights into the function of the various DEGs, elucidating the mechanisms of drug resistance, thereby potentially enabling these to be overcome through targeted therapy. 5. Conclusions In this study, we identified and evaluated the genes and pathways related to IVM resistance in H. contortus using an integrated transcriptomic and proteomic analysis. We found that 1432 and 1227 genes were downregulated in the IVM-treated resistant and susceptible strains, respectively, suggesting that IVM inhibits the expression of some of the genes in H. contortus. Among the many upregulated genes that we uncovered, we focused on the changes in UGT, GST, and the CYP genes. These DEGs and their associated DEPs were significantly enriched in RNA, proteolytic synthesis, catabolic functions, and some metabolism-related pathways. To sum up, our study provides useful information for a better understanding of the response of H. contortus to IVM and for the screening of genes that may be associated with drug resistance. Acknowledgments We would like to thank OE biotech Co., Ltd. and Lu-Ming biotech Co., Ltd. for sequencing services. Supplementary Materials The following supporting information can be downloaded at: Figure S1: A heatmap showing the magnitude of the Pearson's correlation coefficient matrix among different groups, Figure S2: Venn diagram showing the co-upregulated and co-downregulated DEGs and DEPs in four groups, Table S1: RNA extraction procedure, Table S2: Primer sequences used for q-PCR analysis, Table S3: Quality metrics of the clean reads. Click here for additional data file. Author Contributions Conceptualization, X.Y.; methodology, Y.L., P.W. and X.L.; investigation, P.W.; data curation, Y.L. and B.Z.; writing--original draft preparation, Y.L. and X.Y.; writing--review and editing, Y.L., X.Y. and X.W.; visualization, Y.L.; supervision, X.Y.; project administration, X.Y., R.W. and J.L.; funding acquisition, X.Y. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study design was reviewed and approved by the Animal Ethics Committee of Ningxia University (Permit No. 22-031, 10 March 2022). Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Volcano plots showing the transcriptional response of the four comparison groups. Differentially expressed genes (DEGs) are shown as red (upregulated) and green (downregulated) dots. Non-significantly expressed genes are shown as gray dots. The X-axis represents the value of log2 (fold change), and the Y-axis shows the value of -log10 (p-value). Figure 2 Volcano plots showing the differentially expressed proteins (DEPs) of the four comparison groups. DEPs are shown as red (upregulated) and green (downregulated) dots. Non-significantly expressed proteins are shown as black dots. The X-axis represents the value of log2 (fold change), and the Y-axis shows the value of -log10 (p-value). Figure 3 Validation of RNA-seq results using quantitative real-time PCR (qRT-PCR). The X-axis shows the genes that we tested, and the Y-axis represents the relative expressions of those genes. Figure 4 Relationship patterns of total mRNA and protein. The X-axis shows protein expression, and the Y-axis represents gene expression. Gray dots indicate genes and proteins with no significant difference, red dots represent upregulated genes and proteins, green dots represent downregulated genes and proteins, purple dots represent differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) that show opposite regulation, and blue dots represent that one of the genes and proteins differ. Figure 5 The 30 most significantly enriched gene ontology (GO) terms for the DEGs in the four comparison groups. The GO analysis results are summarized in three categories: biological processes (green), cellular components (blue), and molecular functions (red). The X-axis indicates different GO terms, and the Y-axis represents the corresponding number of genes in each GO term. Figure 6 The 30 most significantly enriched gene ontology (GO) terms for the DEPs in the four comparison groups. The GO analysis results are summarized in three categories: biological processes (green), cellular components (blue), and molecular functions (red). The X-axis indicates different GO terms, and the Y-axis represents the corresponding number of genes in each GO term. Figure 7 Scatterplot of the top 20 most enriched KEGG enrichment pathways for the DEGs in the four comparison groups. The X-axis indicates enrichment score, and the Y-axis represents the distinct KEGG pathways. Figure 8 Scatterplot of the top 20 most enriched KEGG enrichment pathways for the DEPs in the four comparison groups. The X-axis indicates enrichment score, and the Y-axis represents the distinct KEGG pathways. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000068

Chimeric antigen receptor (CAR)-T-cell therapy is a kind of adoptive T-cell therapy (ACT) that has developed rapidly in recent years. Mesothelin (MSLN) is a tumor-associated antigen (TAA) that is highly expressed in various solid tumors and is an important target antigen for the development of new immunotherapies for solid tumors. This article reviews the clinical research status, obstacles, advancements and challenges of anti-MSLN CAR-T-cell therapy. Clinical trials on anti-MSLN CAR-T cells show that they have a high safety profile but limited efficacy. At present, local administration and introduction of new modifications are being used to enhance proliferation and persistence and to improve the efficacy and safety of anti-MSLN CAR-T cells. A number of clinical and basic studies have shown that the curative effect of combining this therapy with standard therapy is significantly better than that of monotherapy. chimeric antigen receptor T cells (CAR-T cells) mesothelin clinical trial solid tumor immunotherapy National Key Research and Development Program of China2022YFA1104704 National Natural Science Foundation of China92059204 Technological Innovation and Application Development Foundation of ChongqingCSTB2022TIAD-STX0012 This research was funded by the National Key Research and Development Program of China (grant number 2022YFA1104704); the major projects of National Natural Science Foundation of China (grant number 92059204); the major projects of Technological Innovation and Application Development Foundation of Chongqing (grant number CSTB2022TIAD-STX0012). pmc1. Introduction The chimeric antigen receptor (CAR)-T-cell technique is a kind of cancer immunotherapy that has attracted much attention in recent years. It has achieved good curative effects in hematological malignancies, but many obstacles remain in the treatment of solid tumors. Mesothelin (MSLN) is a tumor-associated antigen (TAA) that is usually expressed only on the mesothelial surface of the body, but is significantly overexpressed in most solid tumors. This article reviews the clinical research status, difficulties and challenges of anti-MSLN CAR-T cells to provide new ideas for their application in the treatment of solid tumors. 1.1. CAR-T Cells CAR is a protein composed of three parts: an extracellular antigen-binding domain, an intracellular signaling domain and a hinge region. Extracellular single-chain variable fragment (scFv) antibodies that specifically identify the surface antigen of cancer cells make up the extracellular antigen-binding domain. The target-binding domain of the CAR is composed of changeable heavy and light chains that are joined together by adaptable peptide linkers in the scFv fragment. The hinge region connects the scFv fragment to intracellular components. Through its glycine and serine sequences, the linker residue's hydrophilicity improves flexibility, while the intermittent glutamine and lysine sequences improve solubility. The signal transduction components of T cells or natural killer cells, such as 4-1BB and CD28, which transduce extracellular binding signals to start downstream cascade stimulation signals are often the source of intracellular signaling domains. The immunoreceptor tyrosine-based activation motif (ITAM) found in the cytoplasmic CD3 domain is required for T-cell activation . Four generations of CARs are currently undergoing experimental and clinical research. The difference lies in their intracellular signaling domains linked to the scFv receptor. Theoretically, third-generation CAR-T cells should have more activation and killing capacities than second-generation CAR-T cells. In addition, because of the heterogeneity of tumor cells, some tumor cells do not have antigens that can be specifically recognized by T cells and cannot be recognized and cleared by traditional CAR-T cells. This problem may be solved by fourth-generation CAR-T cells, which can recruit immune cells other than T cells to the tumor area . To exert antitumor effects in vivo, CAR-T cells must be persistent, proliferative and able to infiltrate tumor tissue. In general, antigens and costimulatory signals in major histocompatibility complex (MHC)-dependent complexes, which involve the recruitment of T-cell surface CD28 and the costimulatory molecules CD80 or CD86 on antigen-presenting cells (APCs), are required for T-cell-mediated immune responses. CAR-T cells recognize specific tumor antigens independent of MHC molecule restriction to perform their antitumor functions. Once CAR binds specifically to TAA, ITAM phosphorylation activates CAR-T cells and induces cytokine secretion, CAR-T-cell proliferation and cytotoxicity. CAR-T cells exert cytotoxic effects by secreting perforin and granzymes and activating death receptor signaling through Fas/Fas ligand (FasL) or TNF/TNF-a . 1.2. MSLN MSLN is a glycosylated phosphatidylinositol-anchored protein that is usually expressed in small amounts on the surface of mesothelial cells in the pleura, pericardium, peritoneum and sheath (in males). However, it has been found that MSLN is overexpressed in a variety of cancers , including malignant mesothelioma , ovarian cancer , breast cancer , pancreatic cancer , lung cancer , gastric cancer , cervical cancer , uterine serous cancer and cholangiocarcinoma . The overexpression of MSLN in triple-negative breast cancer (TNBC) , ovarian cancer , lung adenocarcinoma , cholangiocarcinoma and pancreatic cancer is related to poor prognosis. MSLN is related to chemotherapy resistance, and downregulation of MSLN can restore cell sensitivity to cisplatin in malignant pleural mesothelioma . The MSLN precursor is a 71-kDa glycoprotein that is cleaved by enzymes to release the 31-kDa megakaryocyte-enhancing factor (MPF) and 40-kDa mature MSLN. Mature MSLN can generate soluble mesothelin-related peptide (SMRP). The biological effect of SMRP is limited, but it can be quantified by detection in serum and pleural effusion. At present, some clinical studies have identified it as an observation index . The extracellular domain of MSLN consists of region I (N-terminal region; residues 296-390), region II (residues 391-486) and region III (C-terminal region; residues 487-598) . Region I is the membrane-distal region (MDR), which can bind to the mucin MUC16 (also known as CA125). The mucin MUC16 is also expressed in most malignant mesothelioma cells and is associated with tumor aggressiveness. Compared with region I, region III mediates stronger T-cell activation and cytotoxicity and is a better target . The mechanism may be that anti-MSLN CAR-T cells targeting MSLN region I must compete with CA125/MUC16 for the MSLN antigen interaction, which may weaken the binding and function of anti-MSLN CAR-T cells. However, MSLN region III bridges the extracellular domain and transmembrane region of MSLN, which might have a rigid structure or be responsible for a specific function, to provoke a stronger antitumor response . 2. Clinical Trial Progress of Anti-MSLN CAR-T-Cell Therapy As of December 2022, a total of 41 clinical trials have been registered on the clinicaltrials.gov website ). Six studies (NCT02159716, NCT01355965, NCT01897415, NCT02414269, NCT03545815 and NCT01583686) have been published with all but one obtaining clinical outcome events. NCT01583686 did not enter phase II because of the 15 patients enrolled only 1 achieved stable disease (SD) with the remaining patients having progressive disease (PD) or succumbed to their disease . (Table 1). 2.1. Pretreatment Pretreatment with radiotherapy and chemotherapy prior to CAR-T-cell therapy can alter the tumor microenvironment (TME) and host immune response through several potential mechanisms: immunogenic cell death, decreased regulatory T cells (Tregs) , localized T-cell infiltration and activation of various proinflammatory factor pathways. Common medications used in preclinical studies include cyclophosphamide, oxaliplatin, fludarabine and albumin-bound paclitaxel before CAR-T-cell therapy . Preclinical studies showed that pretreatment using oxaliplatin combined with cyclophosphamide (Ox/Cy) could improve the migration of CAR-T cells to the tumor, increase the infiltration of CAR-T cells into the tumor and enhance the sensitivity of the tumor to immune checkpoint blockade . The mechanism is as follows: Ox/Cy activates multiple proinflammatory pathways, including the expression of T-cell recruitment chemokines in multiple cells in the TME, which helps CAR-T cells recruit tumors expressing chemokine receptor 5 (CCR5) and C-X-C chemokine receptor type 6 (CXCR6). Upon CXCR3-dependent CAR-T-cell recruitment into tumors, infiltrating CAR-T cells release interferon-g (IFN-g), modify the TME to activate M1 macrophages that express the CXCR3 ligands CXCL9 and CXCL10, and then initiate a positive feedback loop. Cyclophosphamide combined with fludarabine (Cy/Flu) is another common pretreatment used in preclinical studies . Adding fludarabine can improve the proliferation of CAR-T cells and the disease-free survival rate of acute B-cell lymphoblastic leukemia . In neuroblastoma, Cy/Flu-induced lymphoid depletion increases the circulation level of the steady-state cytokine interleukin-15 (IL-15) and increases CAR-T-cell expansion by up to 3 logs . Currently, in clinical trials of anti-MSLN CAR-T cells, most of the conditioning regimens are cyclophosphamide alone (NCT03608618 and NCT02414269) and cyclophosphamide combined with fludarabine (NCT03814447, NCT03799913, NCT01583686 and NCT05531708). One clinical trial (NCT02159716) evaluated the feasibility and safety of anti-MSLN CAR-T cells with and without cyclophosphamide pretreatment in 15 patients, and the results showed that the maximum tolerable dose of anti-MSLN CAR-T cells was 3 x 108 cells/m2 and that there were no targeted toxic reactions, such as pleurisy, pericarditis, and peritonitis. However, CAR-T cells did not exert significant clinical efficacy on SD. 2.2. Programmed Cell Death Protein-1 (PD-1) and Its Ligand (PD-L1) There is strong evidence suggesting that the PD-1/PD-L1 interaction between T cells and tumor cells causes the inhibition of T-cell function and depletion of T cells . Therefore, combination with PD-1 or PD-L1 monoclonal antibodies or editing out PD-1 (PD-1 KO) may noticeably enhance the antitumor activity of cytotoxic T lymphocytes (CTLs), enable T cells to recognize tumor cells, promote tumor cell eradication and reduce T-cell exhaustion. Some studies have shown that PD-1KO CTLs are more effective in killing tumor cells than normal CTLs in vitro and in vivo . A mouse xenograft tumor model was used to confirm the anticancer activity of PD-1KO CTLs . The therapeutic impact of CAR-T cells may be enhanced by PD-1/PD-L1 inhibition, particularly in the treatment of solid malignancies. Mechanistically, PD-1KO can enhance the antitumor activity of CAR-T cells by blocking PD-1/PD-L1 and PD-1/PD-L2 signaling in these gene-edited T cells. As a technology to destroy the PD-1/PD-L1 interaction, gene modification has certain advantages in knocking out the PD-1 gene in T cells . The question of whether PD-1 gene editing in host T cells is better than or equally effective to PD-1 mAb treatment is still unanswered due to a lack of solid evidence. However, from recent research results, gene editing of T cells for intracellular intrinsic immune checkpoint blockade may be safer than systemically injecting blocking antibodies into the body . Hu et al. employed CRISPR--Cas9-mediated gene editing to interfere with the PD-1 gene locations in anti-MSLN CAR-T cells to circumvent the inhibitory activity of PD-1 on CAR-T cells. Compared to control CAR-T cells, the release of cytokines (IFN-g and IL-2) was significantly increased in PD-1 KO CAR-T cells. The results from experiments conducted in vitro and in vivo demonstrated that PD-1 KO CAR-T cells had potent anticancer efficacy against TNBC. The anticancer effect of CAR-T cells was improved in this model by PD-1 KO over PD-1 antibody. The aforementioned preclinical trials demonstrated the advantages of PD-1 inhibition and CAR-T-cell treatment in combination. To date, the results are very promising and have spurred a growing number of clinical studies. However, a study showed that PD-1 knockout can be problematic, with PD-1 KO T cells exhibiting advantageous short-term proliferation and cytotoxicity, but lacking long-term tolerance and showing vulnerability to T-cell depletion . The therapeutic potential of PD-1 KO or PD-1 antibodies must therefore be completely clarified by additional, carefully conducted follow-up research and clinical studies . In patients with solid tumors, PD-1 inhibition in combination with anti-MSLN CAR-T cells is being studied in terms of both safety and effectiveness. The relevant clinical trials are as follows: NCT05089266, NCT04503980, NCT04489862, NCT03615313, NCT03030001, NCT04577326, NCT03747965, NCT03545815, NCT03182803, NCT02414269 and NCT05373147. Of all these registered trials, only two have been published. Wang et al. used CRISPR--Cas9 technology to knock down PD-1 and T-cell receptor (TCR) in anti-MSLN CAR-T cells, thereby affecting the TME, in order to treat MSLN-positive solid tumors. A total of 15 patients were included, and CAR-T cells were identified by qPCR in biopsy specimens, which proved that CAR-T cells could effectively infiltrate tumors. The median progression-free survival (PFS) of the seven patients with SD was 7.1 weeks, and most of the eight patients with PD died within 2 months after CAR-T-cell therapy. Adusumilli et al. studied 23 patients who received cyclophosphamide combined with anti-MSLN CAR-T cells followed by at least three doses of pembrolizumab, and the overall survival (OS) following CAR-T-cell therapy was 23.9 months (95% CI, 14.7 months to NE). The one-year OS rate was 83% (95% CI, 68-100%). Among the combined immunotherapy patients (N = 16) with mRECIST measurable disease, two patients (12.5%) had partial response (PR), nine patients (56.3%) had SD, and five patients (31.3%) had PD. The application of combined CAR-T cells and an anti-PD-1 antibody in solid tumors is supported by these data. From this, a phase II study is now underway with a fixed dose of anti-MSLN CAR-T-cell infusion (6x107 CAR-T cells/kg) and pembrolizumab administration four weeks after CAR-T-cell infusion. After infusion, CAR-T cells can proliferate in the host and further differentiate into memory cells, potentially lasting up to 4 years . However, the presence of these memory cells also increases the likelihood of autoimmune disease. To switch off potential toxicities of PD-1 KO CAR-T-cell therapy, suicide genes can be introduced into the CAR-T structure. Adusumilli et al. , Minagawa et al. and Monica et al. linked inducible suicide genes (icaspase9, TK suicide gene) to MSLN-CAR, CD33-CAR, CD44v6-CAR, CD19-CAR and CEA-CAR in their experiments. 2.3. Local Administration The barrier effect of the TME and extracellular matrix (ECM) limits the tumor invasion rate of CAR-T cells. Local administration can cause CAR-T cells to directly enter tumor cells, which greatly improves the tumor infiltration rate. Mayor et al. and Adusumilli et al. showed that intrapleural injection of anti-MSLN CD28 costimulatory (M28z) CAR-T cells eradicated established pleural tumors, even at doses that were lower than intravenous injection of CAR-T cells by 30 times. Furthermore, locally injected CAR-T cells also demonstrated systemic, long-lasting antitumor immunity and were capable of easily traveling from the thoracic cavity to the flanking and peritoneal tumor locations. T-cell imaging revealed that local administration resulted in the accumulation of more CAR-T cells in the tumor at earlier time points. Several clinical trials (NCT04577326, NCT03608618, NCT03323944, NCT03267173, NCT03198052, NCT03054298, NCT02959151, NCT02706782 and NCT02414269) have been conducted to assess the effectiveness and safety of anti-MSLN CAR-T cells in cancer patients with positive results for MSLN administered via the intrathoracic, intraperitoneal, intratumoral or vascular routes. 3. Challenges of Anti-MSLN CAR-T-Cell Therapy Although anti-MSLN CAR-T-cell therapy offers new hope for patients with solid tumors, many challenges remain. Next, we identify solutions from two aspects: toxicity and technical challenges. 3.1. Toxicity The target antigen MSLN is not specifically expressed on the surface of tumor cells, which may lead to off-target effects. Anti-MSLN CAR-T cells recognize MSLN target antigens and are activated to release cytokines or trigger macrophages to release inflammatory cytokines , which may lead to cytokine release syndrome (CRS), neurotoxicity and other adverse reactions. 3.1.1. Off-Target Effects In a phase I/II clinical trial (NCI-09-C-0041), a patient experienced respiratory discomfort and a decline in blood oxygen saturation within 15 min after completing HER2-CAR-T-cell infusion. Approximately 40 min later, chest X-ray showed pulmonary edema, and he died. It is believed that after the first clearance of HER2-CAR-T cells in the lungs, inflammatory cytokines are subsequently released once the body detects HER2 expressed by healthy lung cells, causing lung toxicity and edema that leads to multiorgan dysfunction and death . MSLN is also expressed in normal mesothelial tissue. While targeting tumor cells, anti-MSLN CAR-T cells may also kill normal tissue cells expressing MSLN, resulting in off-target effects. However, from the results of the five published clinical studies of anti-MSLN CAR-T-cell therapy, no obvious off-target effects have been observed . Of course, this may be related to the small number of patients, and it remains to be further investigated. In addition, researchers have developed a variety of methods to reduce off-target effects: (1) The construction of bispecific antibodies . (2) Trans-signaling CARs, which means that the two CARs target different TAAs. CAR1 contains only the CD3z signaling domain, and CAR2 contains only the CD28 or other costimulatory factor signaling domain. They are transduced to construct T cells that coexpress two CARs. These CAR-T cells only target cells expressing both TAAs . (3) T cells designed using the synthetic Notch (synNotch) receptor, a new class of synthetic receptors based on the Notch receptor. Antigen A is on the synNotch receptor, and antigen B is on the CAR. Only cells expressing both antigens can be targeted . (4) Simultaneous introduction of two CARs, one targeting TAA and one targeting inhibitory receptors for antigens present on normal cells rather than tumor cells. CAR-T cells express inhibitory signals when they bind to normal cells and have tumor-killing effects when they bind to tumor cells . The above strategies can also be considered in the anti-MSLN CAR-T cell process in the future. 3.1.2. Cytokine Release Syndrome (CRS) CRS is one of the serious adverse reactions of CAR-T-cell therapy. At present, most studies on CRS are focused on hematological malignancies. Fever, weariness, headache, rash, joint discomfort and myalgia are some of the milder signs of CRS. Hypotension and a high fever are serious symptoms that can worsen and lead to circulatory shock, vascular leakage, disseminated intravascular coagulation and multiple organ failure . An anti-IL-6 receptor antibody (tocilizumab) and symptomatic support therapy (high-dose steroids, vasopressors, ventilatory support, etc.) are all included in the treatment . Some studies suggest that blocking IL-1 may be a new method for treating CRS . The pathogenesis of CRS remains unclear. It has been reported that CAR-T cells release granzyme B, activate caspase 3 and cleave GSDME in target tumor cells, leading to pyroptosis, thereby activating caspase 1 and GSDMD in macrophages and triggering CRS. Elevated GSDME levels in cancer patients were positively correlated with CRS severity. The type of therapy, the underlying condition and the features of the patient all impact the risk of CRS . The degree of T-cell growth and T-cell activation were linked with CRS severity . The nature of the CAR structure affects the clinical presentation, severity and occurrence time of CRS . The incidence rates of CRS in CAR-T-cell therapies containing CD28 and 4-1BB were 93% and 57%, respectively . The development of CRS is impacted by lymphoid depletion before CAR-T-cell injection. Following cyclophosphamide or fludarabine lymphodepletion, the likelihood of developing CRS increases . This may be a result of the increased rate of CAR-T-cell proliferation due to the more significant lymphoid depletion achieved by the combination therapy . Fortunately, no severe CRS has occurred in any published clinical trials of anti-MSLN CAR-T-cell therapy , which may be related to the small number of published clinical trials. The observations of follow-up clinical trials are highly anticipated. 3.1.3. Neurotoxicity Similar to CRS, mild to severe neurological dysfunction within days and weeks following CAR-T-cell infusion is termed CAR-T-cell-induced neurotoxicity, which often includes epileptic activity as well as specific deficits such as aphasia, altered eyesight, shaking and facial drooping. Symptomatic support therapy is the main treatment, except for the prophylactic use of levetiracetam during CAR-T-cell infusion, and data on other interventions are limited . At present, little is known about the mechanism of neurotoxicity. According to some studies , inflammatory mediators released by macrophages trigger the release of von Willebrand factor and angiopoietin-2 from the Weibel-Palade bodies of endothelial cells in the central nervous system. This replacement of angiopoietin-1 results in the inhibition of TIE receptor tyrosine kinase signal transduction. The integrity of the blood-brain barrier is compromised by endothelial cells, which also become more porous. Coagulopathy is caused by high-molecular-weight von Willebrand factor. As cytokines and activated inflammatory cells continue to cross the blood-brain barrier, this positive feedback loop continues. Because of the pathophysiology's resemblance to thrombocytopenic purpura, plasmapheresis is being studied as a treatment for neurotoxicity. It has also been postulated that the mechanism may involve NK-cell populations. NK cells secrete IL-2 and IL-15, both of which were found to be elevated in patients with neurotoxicity. This high level of NK cell activation triggers the activation of microglia and mediates a strong pathogenic inflammatory environment in the central nervous system. Fortunately, no serious neurotoxicity has been observed in published clinical trials of anti-MSLN CAR-T-cell therapy . Of course, the results of more clinical studies are awaited. 3.1.4. Human Anti-Mouse Antibody (HAMA) Immune Response The chimeric antibody substitutes the mouse constant region with the constant region of the human antibody-producing gene, which greatly reduces the immunogenic reaction produced by the mouse-derived antibody so that 70% of the antibody components are human components. The murine gene is still present in the chimeric antibody, even though it only makes up a very minor portion of the antigen-recognition sequence of the variable region of the antibody. Clinical trials have shown that chimeric antibodies can also generate HAMA immune responses when applied. The therapeutic efficacy of murine antibodies in humans are constrained by their immunogenicity. The use of murine-derived CAR limits the persistence of CAR-T cells in humans and raises the possibility of allergic responses. The construction of a fully human scFv CAR is one solution. A clinical trial (NCT01355965) conducted by Maus et al. included a total of four patients who received multiple intravenous infusions of MSLN-targeted mRNA transiently transduced second-generation CAR-T cells. One patient developed anaphylaxis during treatment; he received anti-MSLN CAR-T-cell infusions on days 0, 7 and 49, and anaphylaxis occurred within minutes of infusion on day 49. After exclusion, anti-MSLN CAR-T cells most likely triggered allergic responses by inducing murine antibody sequence-specific IgE antibodies present in CAR-T cells. It is suggested that a single infusion of CAR-T cells is sufficient to achieve efficacy. In this case, with continuous exposure to the product, CAR-T-cell infusion would not induce IgE antibodies. At present, the results of two clinical trials (NCT02414269 and NCT03545815 ) of fully human scFv anti-MSLN CAR-T cells have been published, and no new allergic reactions have been found. 3.2. Technical Barriers The technology related to CAR-T-cell therapy has been continuously improved, but there are still many obstacles that limit its further clinical promotion and application. In the following subsections, we introduce and summarize the corresponding solutions proposed by relevant basic research from the aspects of an immunosuppressive TME, insufficient transport into the tumor, target antigen heterogeneity, proliferation and persistence. 3.2.1. Immunosuppressive TME The glycolytic metabolism of tumor cells induces hypoxia within the TME, and the accumulation of metabolic waste leads to a decrease in pH and low nutrient content, resulting in oxidative stress . The TME can upregulate immune checkpoint molecules (e.g., PD-1, CTLA-4, TIM3 and LAG3), thereby limiting T-cell function. The TME contains a large number of stromal cells, such as cancer-associated fibroblasts (CAFs) and immunosuppressive cells, including myeloid-derived suppressor cells (MDSCs) , tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), mast cells and Tregs . M2 TAMs and Tregs can produce transforming growth factor-b (TGF-b) and prostaglandin E2 (PGE2) to protect tumors from immune surveillance and attack. CAR-T cells are sensitive to immunosuppressive mechanisms in the TME. An immune checkpoint molecular blockade can enhance the antitumor activity of CAR-T cells and is expected to enhance their functional persistence in solid tumors. At present, clinical trials mainly involve knockout of PD-1 by CRISPR-Cas9, PD-1 antibody and anti-PD-1 nanoantibody to combat this immunosuppressive mechanism. Preclinical studies have reported improvement in the activity of anti-MSLN CAR-T cells by knocking down TIM3 via shRNA or by applying TIM3 immune checkpoint inhibitors . TIM3 blockade combined with anti-MSLN CAR-T cells significantly improved their killing potency, cytokine secretion and proliferation. In addition, simultaneous downregulation of the inhibitory receptors PD-1, TIM-3 and LAG-3 on CAR-T cells can enhance the antitumor ability of anti-HER-2 CAR-T cells by upregulating the expression of CD56 . However, this approach has not been applied in anti-MSLN CAR-T cells. In addition to immune checkpoint inhibitors, oncolytic adenoviruses (OAds) are another immunotherapeutic approach for the treatment of solid tumors. The combination of OAd therapy and CAR-T-cell therapy is a new development direction. OAds expressing tumor necrosis factor-a (TNF-a) and IL-2 enhance and maintain T-cell function, promote the infiltration of anti-MSLN CAR-T cells in tumors, overcome the heterogeneity of tumor target antigen expression and reduce tumor immunosuppression, thereby improving the efficacy of anti-MSLN CAR-T cells in pancreatic cancer . In addition, a study evaluated the combination of anti-MSLN CAR-T-cell and OAd therapies in a TNBC model. It was found that OAds targeting TGF-b could directly lyse tumor cells, with an obvious antitumor response in the early stage and weakened antitumor activity in the later stage. However, anti-MSLN CAR-T-cell therapy has a sustained antitumor effect, with a stronger antitumor response detectable in the later stage. The combination of the two treatments produced a stronger antitumor response. CD40 is mainly expressed on APCs, including dendritic cells (DCs) and macrophages. CD40L expressed on CD4+ T cells plays a key role in the immune response of DCs and activation of antitumor CD8+ T cells . Studies have demonstrated that CD40 is expressed on activated CD8+ T cells and that CD40+CD8+ T cells can communicate with CD4 through CD40. The direct interaction between CD4+ T cells and CD8+ T cells can encourage CD8+ T-cell cytokine release and cell proliferation . CAR-T cells targeting MSLN region III (MSLN3) were designed to secrete anti-CD40 antibodies. The ratio of cytokines and central memory T cells (TCM) secreted by MSLN3 CD40 CAR-T cells was greater than that secreted by MSLN3 CAR-T cells. MSLN3 CD40 CAR-T cells elicited a stronger antitumor response in vitro and in vivo . To avoid immunosuppressive effects, genetically modifying the CAR can help increase the resistance of T cells to immunosuppression. The introduction of costimulatory molecules and inhibitory cytokines into the CAR can help T cells develop stronger resistance to Tregs, TGF-b and other related immunosuppressive molecules . Knockdown of TGF-b receptor II (TGFBR2) by CRISPR-Cas9 technology enabled anti-MSLN CAR-T cells to withstand the negative effects of TGF-b signaling . Adenosine and PGE2 can inhibit the immune system by triggering protein kinase A (PKA). Targeting the adenosine 2A receptor (A2AR) can diminish the inhibitory action of adenosine in vitro. Blocking the localization of PKA to the immune synapse increased the migration of anti-MSLN CAR-T cells to the tumor and enhanced the antitumor effect in a mouse model of melanoma . Additionally, knockdown of A2AR by shRNA resulted in enhanced proliferation, cytokine production and cytotoxic function of anti-MSLN CAR-T cells in a simulated TME . In addition, CAR-T cells can be further modified to express cytokines such as IL-2, IL-12, TNF-a and IFN-g to escape immunosuppression . 3.2.2. Insufficient Trafficking into the Tumor Tumor cells and CAFs form the ECM, which is essential for the progression of cancer. The physical barrier that prevents certain anticancer medicines from penetrating tumor cells is represented by the ECM. In addition, ECM collagen fibers around the tumor restrict T cells from entering the tumor . One method to increase the effectiveness of CAR-T-cell treatment is to use matrix degraders. CAR-T cells were engineered to express heparanase (HPSE), which can degrade heparan sulfate proteoglycans, allow CAR-T cells to better infiltrate tumors and increase antitumor activity in mouse models . Another strategy is to exploit the ability of macrophages to secrete matrix metalloproteinases (MMPs) to remodel the ECM so that CAR-T cells can infiltrate tumors . The ECM contains hyaluronic acid (HA), which is broken down by hyaluronidase. The membrane protein PH20, which is naturally expressed by human sperm, has high hyaluronidase activity. Because the PH20 protein has a brief half-life, the IgG2 Fc fragment was incorporated to stabilize the structure of the protein. CAR-T cells expressing sPH20-IgG2 were constructed and showed a strong ability to degrade HA and inhibit tumor growth in a mouse model of xenogeneic gastric cancer . The CAR-T-cell trafficking process requires chemokines secreted by tumor cells to interact with chemokine receptors on CAR-T cells. Chemokines are involved not only in leukocyte recruitment, but also in tumor angiogenesis, cell multiplication and metastasis. It is possible to stimulate more chemokine receptors to be expressed on CAR-T cells; for example, anti-MSLN CAR-T cells can be genetically modified to express the chemokine receptor CCR2 combined with a CXCR4 antagonist. The chemokine receptor CCR2b was introduced into anti-MSLN CAR-T cells to enhance the transport of CAR-T cells to the tumor. Functional CCR2b on anti-MSLN CAR-T cells can significantly increase the number of T cells in tumors and improve the antitumor effect in vitro and in vivo . In addition, CCR2b and CCR4 are receptors for serum monocyte chemotactic protein 1 (MCP-1) and are expressed at low levels on activated T cells. Anti-MSLN CCR2b CAR and anti-MSLN CCR4 CAR-T cells have increased migration rates into tumor supernatants expressing high levels of MCP-1 in vitro. In a mouse model of non-small cell lung cancer, anti-MSLN CCR2b CAR-T cells had better tumor tissue infiltration and antitumor function . Local administration allows the drug to be directly injected into the tumor through the barrier, which can alleviate the obstacle of drug transport into the tumor. This was discussed in detail in Section 2.3. 3.2.3. Target Antigen Heterogeneity In view of the good efficacy of CAR-T-cell therapy in early clinical trials of hematological malignancies, the FDA approved four anti-CD19 CAR-T cells, namely Kymriah (tisagenlecleucel), Yescarta (axicabtagene ciloleucel), Tecartus (brexucabtagene autoleucel) and Breyanzi (lisocatagene maraluecel) . In addition, idecabtagene vicleucel (ide-cel), which targets BCMA, is awaiting FDA approval. However, subsequent follow-up showed that acute B-lymphocytic leukemia (B-ALL) had a high relapse rate after CAR-T-cell therapy . The results of a clinical study of anti-CD19 CAR-T cells (CTL019) revealed that among the 59 patients enrolled, 55 (93%) achieved CR at 1 month. However, during the 12-month follow-up, the recurrence-free survival rate was only 55% . Loss of target antigens, downregulation of target antigen expression and heterogeneous expression of target antigens are the main causes responsible for relapse after CAR-T-cell therapy . Target antigen heterogeneity is mainly manifested in two aspects, time and space, which we call temporal expression heterogeneity and spatial expression heterogeneity. Among them, the temporal expression heterogeneity of target antigens refers to the fact that antigen expression on the surface of tumor cells can change significantly over time, mainly in the form of antigen loss and antigen expression downregulation . The first published phase I trial of CD19-4-1BB-CD3z CAR-T cells for B-ALL at Children's Hospital of Philadelphia (CHOP) (NCT01626495 and NCT01029366) showed that 3 of 27 CR patients (11%) relapsed due to loss of CD19 in leukemia cells . Another clinical trial of CAR-T cells in the treatment of B-ALL (NCT02315612) showed that 8 of 12 patients (67%) who had CR after CD22 CAR-T-cell therapy relapsed, and 7 of these patients had downregulated CD22 expression. Downregulation of CD22 expression occurs at the posttranscriptional level, and CD22 CAR-T cells cannot effectively eliminate cells with downregulated CD22 expression . This has also been demonstrated in glioblastoma , non-Hodgkin lymphoma and multiple myeloma . However, there is no research on the target antigen MSLN thus far. Heterogeneity in the spatial expression of target antigens refers to the inconsistent expression of antigens on the surface of different tumor cells in the same patient . Rabilloud et al. performed single-cell sequencing analysis of leukemia cells from a patient with relapsed B-ALL, found that CD19-negative leukemia cells were present before CAR-T-cell therapy and confirmed that relapse was due to the cloning of these CD19-negative leukemia cells. A phase I clinical study conducted by Haas et al. investigated the safety and activity of anti-MSLN CAR-T cells in patients with malignant pleural mesothelioma, ovarian cancer and pancreatic ductal adenocarcinoma, (NCT02159716) and found that in only 3 of 15 patients the expression of MSLN on tumor cells was >75%. Thus, clinical trials focusing on efficacy are recommended to prospectively screen MSLN expression to reduce recurrence in the future. By downloading immunohistochemical (IHC) staining data from The Human Protein Atlas website (accessed on 18 August 2022)) to quantitatively analyze the IHC results of MSLN expression in various solid tumors, we found that there were some malignant cells with negative MSLN expression in solid tumors defined as having positive MSLN expression. In different IHC samples, the positive rates of MSLN expression were approximately 0-99%. In addition, we analyzed the single-cell sequencing results of TNBC (GSE75688 and GSE118389), ovarian cancer (GSE118828) and pancreatic cancer (GSE111672) downloaded from the Gene Expression Omnibus (GEO: (accessed on 28 September 2022)) database and found that approximately 70-97% of malignant cells had negative MSLN expression . The heterogeneous expression of MSLN significantly affects the efficacy and recurrence rate of solid tumors after treatment with anti-MSLN CAR-T cells . In view of this, a variety of measures against target antigen heterogeneity have been developed. Among them, enhancing the "bystander effect" of tumor cells is an important strategy. The bystander effect suggests that CAR-T cells can induce tumor killing, even though these malignant cells negatively express the target antigen. To dissect the mechanism of the bystander effect, Upadhyay et al. used CRISPR--Cas9 technology to screen and determine the essential role of Fas-FasL in antigen-specific T-cell killing. They discovered that Fas-FasL mediated the off-target bystander killing effect on antigen-negative malignant cells: the killing effect of CD19 CD3 z-CD28 CAR-T cells on target cells showed moderate Fas dependence, and Fas-expressing mice were more sensitive to bystander killing effects. An analysis of pretreatment tumor RNA-sequencing data from the ZUMA-1 trial (NCT02348216), which included patients with CAR-T-refractory diffuse large B-cell lymphoma, showed that patients with a durable response to treatment had significantly increased tumor Fas expression. Similarly, an analysis of a diffuse large B-cell lymphoma patient cohort receiving standard treatment in the TCGA database showed that tumor patients with high expression of Fas had a poorer prognosis; in contrast, patients with high expression of Fas treated with CAR-T cells had significantly longer survival times. This mechanism also applies to anti-MSLN CAR-T cells. Klampatsa et al. designed a model in which anti-MSLN CAR-T cells could cure xenografts in mice inoculated with 100% MSLN-positive tumor cells. However, even if the positivity rate of MSLN dropped to 90%, anti-MSLN CAR-T cells could not cure the tumor. Nevertheless, up to 25% of MSLN-negative xenografts could be cured if pretreatment with a nonlymphoid-depleting dose of cyclophosphamide prior to CAR-T-cell therapy induces a bystander effect. Therefore, exploring the application of multidrug combinations and the application of small-molecule Fas signaling modulators may solve the poor curative effect caused by target antigen heterogeneity to a certain extent. Park et al. introduced a new method for the exogenous introduction of a homogeneous antigen. Oncolytic vaccinia virus (OV19t) encoding CD19t was used to infect various solid tumor cells. Before the virus-infected tumor is lysed, its cell surface produces new CD19. The expression of CD19t in tumors is encouraged by CAR-T-cell-mediated tumor lysis, which can also cause the release of the virus from tumor cells that are on the verge of death. At this point, all tumor cells are loaded with the new target antigen CD19, potentially extending the application of clinically approved anti-CD19 CAR-T cells to the treatment of solid tumors and overcoming the problem of target antigen expression heterogeneity. In addition, to address target antigen heterogeneity, multiantigen-targeted CAR constructs are currently being developed, thereby reducing the risk of tumor recurrence. There are numerous methods for creating CARs that target several antigens : (1) dual-targeted CAR-T cells, including bicistronic transgenes and cotransduction and tandem single-chain antibodies ; (2) coadministered monospecific CAR-T cells ; and (3) sequential monospecific CAR-T cells . In addition, some studies have designed aptamer CAR-T-cell technology. The construction of universal CAR-T cells expressing nontumor-associated antigens, such as fluorescein isothiocyanate (FITC) CAR-T cells, targets multiple target antigens on the surface of tumor cells through a mixture of bispecific aptamers . Kailayangiri et al. found that inhibition of enhancer of zeste homolog 2 (EZH2) could induce the surface expression of GD2 in Ewing sarcoma cells, thereby enhancing the killing ability of anti-GD2 CAR-T cells against Ewing sarcoma cells. These studies suggest that our similar mechanism may also apply to anti-MSLN CAR-T cells. In a tissue microarray of 107 excised pancreatic cancers, Tholey et al. stained tissues with antibodies against MUC1, MSLN or both. At the protein level, they verified that when MUC1 and MSLN were labeled separately and simultaneously, the percentage of tumor cells with a high labeling pattern (2+ or 3+) increased from 56% and 62% to 82%. To increase the effectiveness, infiltration, persistence and proliferation of CAR-T cells in ovarian cancer, Liang et al. created a unique tandem CAR expressing an anti-FOLR1 scFv, an anti-MSLN scFv and two peptide sequences of IL-12. KRAS mutations were shown to be positively associated with MSLN expression in pancreatic cancer and lung cancer, as demonstrated by Fukamachi et al. and Bauss et al. . It is reasonable to infer that the use of bispecific CAR-T cells (targeting both MSLN and MUC1 or targeting both FOLR1 and MSLN) and CAR-T-cell therapy combined with mutated gene targeting drugs could improve the efficacy of anti-MSLN CAR-T-cell therapy. However, these therapies are still in the preclinical stage, and whether they can solve the issue of tumor recurrence caused by the heterogeneity of the target antigen requires further observation. 3.2.4. Proliferation and Persistence There are four factors that affect the proliferative capacity and persistence of CAR-T cells: preconditioning, the intracellular signaling domain of CAR-T cells, the immunogenicity of CAR-T cells, and T-cell depletion. The current commonly used conditioning regimens are cyclophosphamide and cyclophosphamide combined with fludarabine. A clinical trial (NCT02159716) demonstrated that preconditioning with cyclophosphamide before CAR-T infusion can increase the proliferation of CAR-T cells, but has little effect on durability. Preclinical and clinical studies conducted by Goto et al. and Pang et al. showed that the secretion of IL-7 and CCL19 promoted the proliferation and persistence of anti-MSLN CAR-T cells in vivo. CD28 and 4-1BB are commonly used intracellular signaling domains of CAR-T cells, but their functions are quite different. CD28 CAR-T cells have a better tumor-killing ability, while 4-1BB can better prolong the persistence of CAR-T cells. The reason may be that CD28 CAR-T cells have higher basal activity of nuclear factor of activated T cells (NFAT) and unique sensitivity to PD-1/PD-L1-mediated checkpoint inhibition, while 4-1BB CAR-T cells have stronger nuclear transcription factor kappa B (NF-kB) activity and are not affected by the PD-1/PD-L1 checkpoint . Guedan et al. suggested that a single amino acid residue in CD28 causes T-cell depletion and may hinder the persistence of CD28-based anti-MSLN CAR-T cells. Another preclinical study demonstrated that the conversion of asparagine to phenylalanine enhanced the in vivo persistence of anti-MSLN CAR-T cells containing the CD28 costimulatory domain, thereby enhancing their antitumor efficacy . CAR is a protein that also has immunogenicity, so an immune response against CAR is generated in the body, thereby reducing the persistence of anti-MSLN CAR-T cells. Clinical trials (NCT01355965 , NCT02159716 and NCT01897415 ) have reported the detection of human anti-chimeric antibody (HACA) during CAR-T-cell infusion. Constructing a CAR fully targeting human scFv MSLN can reduce the immunogenicity of anti-MSLN CAR-T cells. The scFv in the CAR structure is unstable and has an inherent tendency to self-aggregate, which may lead to depletion of anti-MSLN CAR-T cells in vivo, thereby reducing their persistence. Studies have shown that replacing the scFv with a fully human VH domain or linking the TCR constant region to the heavy and light chain variable regions of monoclonal antibodies produces synthetic T-cell receptors and antigen receptors (STAR). This approach can reduce T-cell exhaustion and results in better or equal cytotoxicity, proliferation and persistence than CAR-T cells. At present, for anti-MSLN CAR-T-cell therapy, the above four methods are mainly used to improve proliferation and persistence, which are key factors affecting efficacy. In addition, local administration and induction of T-cell costimulators to improve the structure of CAR contribute as well . 4. Conclusions Clinical trials of CAR-T-cell therapy in solid tumors have begun in recent years as a result of the approval of the treatment for hematological malignancies. The present state of anti-MSLN CAR-T-cell treatment in patients with solid tumors was discussed in this study. Its toxicity, including off-target effects, CRS, neurotoxicity and immune response, was analyzed. Finally, technical hurdles that may affect its safety and efficacy, including the immunosuppressive TME, trafficking into tumor tissue, target antigen heterogeneity, proliferation and durability, were defined. At present, the persistence and efficacy of the intravenous infusion of anti-MSLN CAR-T cells still has much room for development. Novel strategies of combination therapy with immune checkpoint inhibitors and/or chemotherapeutic drug pretreatment have improved the antitumor ability of anti-MSLN CAR-T cells to some extent. The application of topical, fully human anti-MSLN scFv, which has entered clinical trials, is also expected to improve the efficacy and reduce the toxicity of anti-MSLN CAR-T-cell therapy. The next steps in the development of new CAR-T-cell therapies for solid tumors will involve multidisciplinary collaboration, focusing on combination therapy and new clinical study designs, and anti-MSLN CAR-T-cell therapy is expected to impact clinical outcomes in patients with various solid tumors. Author Contributions Conceptualization, X.Z., L.M., M.W., J.L. and S.Y.; methodology, X.Z.; software, X.Z.; validation, X.Z. and L.M.; formal analysis, X.Z.; investigation, X.Z.; resources, X.Z.; data curation, X.Z.; writing--original draft preparation, X.Z.; writing--review and editing, X.Z., L.M. and S.Y.; visualization, X.Z.; supervision, M.W.; project administration, J.L. and M.W.; funding acquisition, S.Y. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Chimeric antigen receptor (CAR) is a protein composed of three parts: an extracellular antigen-binding domain, an intracellular signaling domain, and a hinge region. The single-chain variable region (scFv) of the extracellular antigen-binding domain of CAR-T cells originates from the VH and VL of monoclonal antibodies. The intracellular signaling domain consists of a costimulatory factor and CD3 signal domain. The CD3 signal domain originates from the immunoreceptor tyrosine-based activation motif (ITAM) of the TCR/CD3 subunit structure. The scFv of anti-MSLN CAR-T cells binds specifically to MSLN on the surface of tumor cells to induce an antitumor immune response. Illustration was created with Biorender.com. Figure 2 Structural differences in the four generations of CARs. First-generation CARs had only CD3z as the intracellular signaling domain. Second-generation CARs contain CD3z and a costimulatory domain, which may be CD27, CD28, CD137 (4-1BB) or OX40. Third-generation CARs contain CD3z and two costimulatory domains, such as CD28 and CD137 (4-1BB), or other costimulatory molecules. On the basis of the third generation, the latest fourth-generation CARs can additionally secrete cytokines or other effector molecules, such as IL-12, IL-15, IL-7, CCL19 or aPD-1, to modulate the tumor immune microenvironment. Illustration was created with Biorender.com. Figure 3 Protein structure model of human mesothelin (MSLN). MSLN is a membrane-binding protein anchored to the cell membrane by glycosylated phosphatidylinositol. The extracellular domain of MSLN consists of region I (N-terminal region; residues 296-390), region II (residues 391-486) and region III (C-terminal region; residues 487-598). Illustration was created with Biorender.com. Figure 4 Heterogeneous expression of MSLN in solid tumors. (A) Calculation of the expression rate of MSLN in malignant cells of different solid tumors at the protein level with ImageJ. The percentage of MSLN-positive tumor cells among the total number of tumor cells was calculated by IHC. The result of IHC verified by two pathologists. (B) Calculation of the expression rate of MSLN in malignant cells of triple-negative breast cancer (GSE75688 and GSE118389), pancreatic cancer (GSE111672) and ovarian cancer (GSE118828) using single-cell sequencing data downloaded from the GEO database (accessed on 28 September 2022)). The percentage of tumor cells with MSLN expression >0 of the total number of tumor cells in the single-cell data was calculated. Figure 5 Dual-target CAR-T-cell therapy strategies. Dual-targeting CAR-T cells, including bicistronic transgenic (A), cotransduced (B), tandem single-chain antibodies (C) and coadministered or sequentially administered monospecific CAR T cells (D). Illustration was created with Biorender.com. Figure 6 Aptamer CAR-T-cell technology. Construction of universal CAR-T cells expressing nontumor-associated antigens, such as fluorescein isothiocyanate (FITC)-CAR-T cells, targeting multiple target antigens on the surface of tumor cells through a mixture of bispecific aptamers. Illustration was created with Biorender.com. cancers-15-01357-t001_Table 1 Table 1 Clinical trials using anti-MSLN CAR-T therapy for solid tumors. NCT Number Title Status Type of Cancer Phase Enrollment Start Date Publication NCT05531708 Exploratory Study of Novel MSLN CAR-T Cell Therapy in Patients With MSLN-positive Advanced Refractory Solid Tumors Recruiting Refractory Solid Tumors Phase 1 20 8 September 2022 NCT05373147 aPD1-MSLN-CAR T Cells for the Treatment of MSLN-positive Advanced Solid Tumors Recruiting Solid Tumor Early Phase 1 21 13 May 2022 NCT05623488 CAR T Cells in Mesothelin-Expressing Breast Cancer Not yet recruiting Breast Cancer Phase 1 12 21 November 2022 NCT05089266 Study of aPD1-MSLN-CAR-T Cells to Evaluate the Safety, Tolerability, and Effectiveness for Patients with MSLN-Positive Advanced Solid Tumors Not yet recruiting Colorectal Cancer Phase 1 30 30 November 2021 NCT05057715 huCART-meso + VCN-01 in Pancreatic and Ovarian Cancer Not yet recruiting Pancreatic Cancer, Serous Ovarian Cancer Phase 1 12 1 December 2021 NCT04981691 Anti-Mesothelin CAR-T Cells with Advanced Refractory Solid Tumors Recruiting Refractory Malignant Solid Neoplasm Phase 1 12 1 October 2021 NCT04577326 Mesothelin-Targeted CAR-T-cell Therapy in Patients with Mesothelioma Recruiting Malignant Pleural Mesothelioma (MPM) Phase 1 30 30 September 2020 NCT04562298 A Phase I Clinical Study to Evaluate the Safety, Tolerability, and Efficacy of LCAR-M23, a CAR-T-Cell Therapy Targeting MSLN in Patients with Relapsed and Refractory Epithelial Ovarian Cancer Recruiting Epithelial Ovarian Cancer Phase 1 34 21 October 2020 NCT04503980 aPD1-MSLN-CAR-T Cells for the Treatment of MSLN-Positive Advanced Solid Tumors Recruiting Colorectal Cancer, Ovarian Cancer Early Phase 1 10 26 March 2020 NCT04489862 aPD1-MSLN-CAR-T Cells for the Treatment of MSLN-Positive Advanced Solid Tumors Recruiting Non-Small Cell Lung Cancer, Mesothelioma Early Phase 1 10 13 May 2020 NCT04203459 The Mechanism of Enhancing the Antitumor Effects of CAR-T on PC by Gut Microbiota Regulation Recruiting Pancreatic Cancer 80 20 October 2019 NCT03941626 Autologous CAR-T/TCR-T-Cell Immunotherapy for Solid Malignancies Recruiting Esophageal Cancer, Hepatoma, Glioma, Gastric Cancer Phase 1 Phase 2 50 1 September 2019 NCT03916679 MESO-CAR-T-Cell Therapy Relapsed and Refractory Epithelial Ovarian Cancer Recruiting Ovarian Cancer Phase 1 Phase 2 20 20 April 2019 NCT03814447 Fourth-Generation CAR-T-Cell Therapy for Refractory-Relapsed Ovarian Cancer Recruiting Ovarian Cancer Early Phase 1 10 16 August 2019 NCT03799913 meso-CAR-T-Cell Therapy Relapsed and Refractory Ovarian Cancer Recruiting Ovarian Cancer Early Phase 1 20 10 April 2019 NCT03747965 Study of PD-1 Gene-Knocked Out Mesothelin-Directed CAR-T Cells with the Conditioning of PC in Mesothelin-Positive Multiple Solid Tumors Unknown status Solid Tumor Phase 1 10 1 November 2018 NCT03638193 Study of Autologous T Cells in Patients with Metastatic Pancreatic Cancer Recruiting Pancreatic Cancer Not Applicable 10 11 July 2018 NCT03615313 PD-1 Antibody-Expressing Meso-CAR-T Cells for Mesothelin-Positive Advanced Solid Tumor Unknown status Advanced Solid Tumor Phase 1 Phase 2 50 6 August 2018 NCT03608618 Intraperitoneal MCY-M11 (Mesothelin-Targeting CAR) for Treatment of Advanced Ovarian Cancer and Peritoneal Mesothelioma Terminated Peritoneal Mesothelioma, Fallopian Tube Adenocarcinoma, Adenocarcinoma of the Ovary, Primary Peritoneal Carcinoma Phase 1 14 27 August 2018 NCT03545815 Study of CRISPR--Cas9-Mediated PD-1 and TCR Gene-Knocked Out Mesothelin-Directed CAR-T Cells in Patients with Mesothelin-Positive Multiple Solid Tumors Recruiting Solid Tumor Phase 1 10 19 March 2018 PMID: 34381179 NCT03497819 Autologous CART-meso/19 Against Pancreatic Cancer Unknown status Pancreatic Cancer Early Phase 1 10 1 October 2017 NCT03356808 Antigen-Specific T Cells Against Lung Cancer Unknown status Lung Cancer Phase 1 Phase 2 20 15 December 2017 NCT03356795 Intervention of CAR-T Against Cervical Cancer Unknown status Cervical Cancer Phase 1 Phase 2 20 15 November 2017 NCT03323944 CAR-T-Cell Immunotherapy for Pancreatic Cancer Active, not recruiting Pancreatic Cancer Phase 1 8 15 September 2017 NCT03267173 Evaluation of the Safety and Efficacy of CAR-T in the Treatment of Pancreatic Cancer Unknown status Pancreatic Cancer Early Phase 1 10 15 June 2017 NCT03198052 HER2/Mesothelin/Lewis-Y/PSCA/MUC1/GPC3/AXL/EGFR/B7-H3/Claudin18.2-CAR-T Cell Immunotherapy against Cancers Recruiting Lung Cancer Phase 1 30 1 July 2017 NCT03182803 CTLA-4 and PD-1 Antibodies Expressing Mesothelin-CAR-T Cells for Mesothelin-Positive Advanced Solid Tumor Unknown status Advanced Solid Tumor Phase 1 Phase 2 40 7 June 2017 NCT03054298 CAR-T Cells in Mesothelin-Expressing Cancers Recruiting Lung Adenocarcinoma, Ovarian Cancer, Peritoneal Carcinoma, Fallopian Tube Cancer, Pleural Mesothelioma, Peritoneal Mesothelioma Phase 1 27 1 March 2017 NCT03030001 PD-1 Antibody-Expressing CAR-T Cells for Mesothelin-Positive Advanced Malignancies Unknown status Solid Tumor Phase 1 Phase 2 40 15 February 2017 NCT02959151 A Study of Chimeric Antigen Receptor T Cells Combined with Interventional Therapy in Advanced Liver Malignancy Unknown status Hepatocellular carcinoma, Metastatic Pancreatic Cancer, Metastatic Colorectal Cancer Phase 1 Phase 2 20 1 July 2016 NCT02930993 Anti-Mesothelin CAR-T Cells for Patients with Recurrent or Metastatic Malignant Tumors Unknown status Mesothelin-Positive Tumors Phase 1 20 1 August 2016 NCT02792114 T-Cell Therapy for Advanced Breast Cancer Active, not recruiting Metastatic HER2-Negative Breast Cancer Phase 1 186 1 June 2016 NCT02706782 A Study of Mesothelin Redirected Autologous T Cells for Advanced Pancreatic Carcinoma Unknown status Pancreatic Cancer Phase 1 30 1 March 2016 NCT02580747 Treatment of Relapsed and/or Chemotherapy Refractory Advanced Malignancies by CART-meso Unknown status Malignant Mesothelioma, Pancreatic Cancer, Ovarian Tumor, Triple-Negative Breast Cancer, Endometrial Cancer, Other Mesothelin-Positive Tumors Phase 1 20 1 October 2015 NCT02465983 Pilot Study of Autologous T cells in Patients with Metastatic Pancreatic Cancer Terminated Pancreatic Cancer Phase 1 4 1 May 2015 NCT02414269 Malignant Pleural Disease Treated with Autologous T Cells Genetically Engineered to Target the Cancer-Cell Surface Antigen Mesothelin Active, not recruiting Malignant Pleural Disease, Mesothelioma, Metastases, Lung Cancer, Breast Cancer Phase 1 Phase 2 113 1 May 2015 PMID: 34266984 NCT02388828 CART-meso Long-Term Follow-up Completed Subjects Who Have Received Lentiviral-Based CART-meso Therapy 10 1 March 2015 NCT02159716 CART-meso in Mesothelin-Expressing Cancers Completed Metastatic Pancreatic (Ductal) Adenocarcinoma, Epithelial Ovarian Cancer, Malignant Epithelial Pleural Mesothelioma Phase 1 19 1 June 2014 PMID: 31420241 NCT01897415 Autologous Redirected RNA Meso-CAR-T Cells for Pancreatic Cancer Completed Metastatic Pancreatic Ductal Adenocarcinoma (PDA) Phase 1 16 1 July 2013 PMID: 29567081 NCT01583686 CAR-T-Cell Receptor Immunotherapy Targeting Mesothelin for Patients with Metastatic Cancer Terminated Cervical Cancer, Pancreatic Cancer, Ovarian Cancer, Mesothelioma, Lung Cancer Phase 1 Phase 2 15 4 May 2012 NCT01355965 Autologous Redirected RNA Meso-CAR-T Cells Completed Malignant Pleural Mesothelioma Phase 1 18 1 May 2011 PMID: 24579088 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000069

This paper presents and illustrates, with a working example, a hypothesis for the assessment of ongoing severity before and during an experiment that will enable humane endpoints and intervention points to be applied accurately and reproducibly, as well as helping to implement any national legal severity limits in subacute and chronic animal experiments, e.g., as specified by the competent authority. The underlying assumption of the model framework is that the degree of deviation from normality of specified measurable biological criteria will reflect the level of pain, suffering, distress and lasting harm incurred by or during an experiment. The choice of criteria will normally reflect the impact on an animal and have to be chosen by scientists and those caring for the animals. They will usually include measurements of good health such as temperature, body weight, body condition and behaviour, which vary according to the species, husbandry and experimental protocols and, in some species, unusual parameters such as time of the year (e.g., migrating birds). In animal research legislation, endpoints or severity limits may be specified so that individual animals do not suffer unnecessarily or endure severe pain and distress that is long-lasting (Directive 2010/63/EU, Art.15.2). In addition, the overall severity is estimated and classified as part of the harm: benefit licence assessment. I present a mathematical model to analyse the measurement data to determine the degree of harm (or severity) incurred. The results can be used to initiate alleviative treatment if required or if permitted during the course of an experiment. In addition, any animal determined to have breached the severity classification of a procedure can be humanely killed, treated or withdrawn from the experiment. The system incorporates the flexibility to be used in most animal research work by being tailored to the research, the procedures carried out and the species under investigation. The criteria used to score severity can also be used as additional scientific outcome criteria and for an analysis of the scientific integrity of the project. assessment adverse effects mental pain distress severity limits This research received no external funding. pmc1. Introduction Morton and Griffiths pointed out that the very first step in any assessment for avoidance or alleviation of pain and distress in animals is to recognise when they are suffering or experiencing adverse states in some way. (I am using the term suffering in a broad way to encompass all adverse physical, physiological and mental states in animals, which may be exacerbated further through some form of cognitive reflection and memory ('mentation' as termed by De Grazia ). Other ways of looking at suffering include the notion of an impaired wellbeing and measuring the impact of the procedures on an animal. While pain and mental distress may be the commonest adverse states for most experimental animals, there are other ways in which animals experience adverse states that can lead to poor physical, physiological and mental welfare and, consequentially, a poor quality of life. In animal research, the impact of an experiment on an animal's homeostasis (as measured by deviation from 'normality' that is not transient) will most likely reflect the harmful consequences of experimental procedures, as well as the extent to which the animal is suffering . As we humans cannot verbally communicate with animals, this approach is as good as (and probably better than) most other scientific measures. It should be appreciated that all scientific measures of welfare, whether physiological, biochemical, hormonal, behavioural or genomic, need to be interpreted for assessment of the degree of harm that an animal is experiencing, which can be further categorised as a mental or physical state (and the possibility and need to provide some form of alleviation). An assessment may be carried out for the purposes of avoidance of suffering (through refinement of the experimental design), to determine the effectiveness of alleviative therapies (e.g., analgesics for pain or anxiolytics for fear) or, in the case of animal research, for assessment of the level of severity (adverse effects) for ethical and legal reasons. Severity comprises two components: the intensity of the adverse state such as pain or distress and, secondly, its duration. Another factor to consider when calculating the overall animal welfare impact of an experiment is any cumulative suffering, for example, inadequate husbandry, in addition to the experimental procedures, the number of times an animal is subjected to a repeated technique (e.g., injections) or procedure or any additional procedures in more complex protocols. Short-term intense stressors may also have long-term impacts, e.g., on the immune system. It is possible to determine the overall animal 'cost' in terms of harms in a series of experiments by including the number of animals affected. In competent humans, the assessment of suffering usually relies on verbal reporting of some sort, whereas in babies and other non-verbal or non-communicating humans and non-human animals, other methods have to be used. In animals, an estimate of the intensity of suffering can be made by measuring the impact of an experimental protocol on an animal, i.e., the degree to which it has deviated from normality compared with a control naive group kept in a similar or the same way. The duration of any suffering is the second most important component of severity and is far easier to measure objectively, although it is often not specified. The combination of intensity and duration is used to estimate 'severity'. The advantage of measuring impact by deviation from normal in this way is that it does not require the type of adverse state experienced to be explicit, that is, it does not need to be defined as pain, physical dystress (a state of stressor overstimulation of the endocrine system as first described by Moberg 1985) or mental distress, such as fear, anxiety, boredom, frustration, grief, etc. If there is no measurable impact, then it is unlikely that the animal will be suffering to any serious extent. That said, short-term suffering is very difficult to assess, as many of the usual markers such as weight loss or appetite will not be measurable, and other, usually behavioural, measures need to be used. Consequently, the approach outlined in this paper is not applicable to acute/hyperacute suffering and is more applicable when the duration extends over a day or more. Scorable clinical signs have to be selected in accordance with the cause and the type of suffering involved, which are partly determined by the experimental procedures being carried out and the species. These clinical signs (sometimes mistakenly referred to as symptoms, which are feelings reported by patients to doctors and not observable) in animals must be observable and assessed as to whether they are within the range of normality or whether they are 'abnormal'. Recognition can be grouped into five categories: relating to appearance, posture, natural behaviour, provoked behaviour and clinical measurements . Considerable advances have been made in the details of these sorts of abnormalities and their observational robustness in many areas of research. For example, in one year in one laboratory animal journal, four papers were published in the fields of rat models of osteoarthritis . Three or four decades ago, no mention would have been made of the adverse effects on an animal in an experiment, let alone any measurement of them . This serves to emphasise that with the increasing awareness that pain and distress in animals is important, the measurements used are become increasingly more accurate, reliable and generally accepted. Moreover, the Federation of European Laboratory Animal Science Associations (FELASA) provides comprehensive guidelines on the classification and reporting of severity experienced by animals used in scientific procedures . What to Measure? In a research setting, it has to be decided what measurements to take to assess any suffering. It is always better to use several criteria to ensure a better estimate, as just one or two may be misleading. It may be possible to carry out measurements of the body's reaction to adverse effects such as effects on the sympathetic nervous system (catecholamine levels and their corresponding end-organ responses, e.g., heart rate, blood pressure and acute-phase proteins) or the pituitary-adrenal cortex axis (particularly adrenal corticosteroids). However, these tests can be expensive and may themselves require invasive blood sampling methods, whereas the behaviour of an animal can be simply observed without cost, including how an animal responds to a particular stimulus (provoked behaviour). Furthermore, some of the affected parameters may be compounded by an inherent variation and vary according to the time of day, the time at which an animal is observed or sampled and the specific experimental procedures being carried out. The time and method of sampling, as well as behavioural observation, have to be practical. For example, in terms of time, one would expect a greater change in pain and distress parameters around the time of surgery rather than 2-4 weeks later. For experimental arthritis, adverse feelings of pain such as dull aches may persist for weeks and not just hours or days as for a surgical procedure; therefore, measuring body weight may be more appropriate. For an experiment with several techniques or procedures being carried out over time, several time points within the whole experimental period may have to be scored to develop an accurate picture of the overall 'suffering' from the animal's viewpoint. In conclusion, the time of observation and the key criteria to be measured have to be carefully selected. In EU Directive 2010/63/EU , harm or the adverse effects are defined as "any pain, suffering, distress or lasting harm equivalent to or higher than that caused by the introduction of a needle in good veterinary practice" experienced by an animal as a result of the scientific procedures performed ( Art. 3.1). There is an obligation for an assessment of the harm likely to occur as a result of the experimental protocols to be conducted at the outset of a research project and for this to be recorded on the project licence as the severity classification for a procedure or series of procedures, i.e., for the whole project. Severity may also have to be recorded in hindsight as the harm that actually occurred, referred to in the Directive as a "Retrospective assessment" ( Art. 39); in specific cases, this is a legal requirement. If an animal is to be reused, then the severity of any previous use has to be determined ( Art. 16.1. (a)). Finally, almost all international legislative measures to control animal experimentation worldwide require the successful application of the Three Rs. This paper deals with the aspect of refinement and provides a model whereby animal suffering can be recognised, reduced, reviewed and even terminated on objective grounds when necessary. This model (first drawn up in the UK prior to the revision of the Directive in 2010 and presented at various seminars) concerns measuring severity retrospectively and comparing it with the predicted severity in order to determine if the severity classification is being or has been exceeded. It is based on the author's practical experience as a laboratory animal veterinarian and close observation of experimental animals in a variety of research settings. In addition, FELASA has produced some valuable and complementary guidelines (12) that expand further on this theme and provides several more comprehensive and complicated examples. In this paper, I have tried to provide a more practical and accessible approach that is simpler and easier to apply through the selection of the key criteria that fit the purpose of assessing the level of harm and that can be applied to all vertebrates and even invertebrates. 2. Methodology and Results The first stage is to identify the criteria that can be used to assess any harm and then to select from those the ones that commonly occur and are easy to score. The second stage is to select the time at which to score those criteria. There is no limit to the number of criteria scored or the number of time points chosen, but in order to reduce workload, only the key and most relevant criteria should be used. These key criteria should also best reflect an animal's adverse state and not simply repeat other closely correlated signs. In addition, the measured signs should reliably indicate severity, be robust, subject to little inter- or intraobserver variation, and be economical and easy to measure. Furthermore, the criteria measured should be independent of each other, for example, a failure to eat and body weight are usually closely interdependent. The aim of this semiquantitative assessment is to be able to compare predicted and actual suffering and to express these estimates mathematically. In the following example, the deviation from normality or intensity is determined for each independent criterion for each animal in the group at a set time. This figure is then averaged to obtain a score for that criterion. The average score per animal is made up of the averages for each criterion. It is then possible to compare, for example, different experimental groups at the same time point, as well as at different time points in an experiment. The easiest way to understand this is to provide a simplified working example. Example: A Hypothetical Comparison of Two Dose Groups in a Toxicity Test in Rats In a study of drug safety and effectiveness, several doses were given, and the rats were observed for adverse effects. Pilot studies and previous data with respect to closely related compounds had shown the chemical's likely adverse effects, and the relevant criteria were chosen according to the criteria described above. In this example, only two dose groups and two criteria were compared at the same time point. The project licence for the work had predicted the severity classification, and severity limits had been set for individual animals in a dose-level group. For the Low-dose group, the severity limit was mild, and for the High-dose group, the severity limit was severe. The accuracy of these severity limit predictions can be determined by assessing the actual severity outcomes in real time as illustrated below. 2.1. Choice of Criteria Affected animals were predicted to show signs, to varying degrees, of lethargy, fever, reduced appetite, a failure to groom or preen, lachrymal secretions from the eyes and nose, diarrhoea and occasional vomiting. The control group (in this case, drug vehicle only) kept under similar conditions was not expected to show any adverse effects and would be classified as the 'normal comparator' for the dosed groups. Among these clinical signs, body temperature (Criterion 1) and body weight (Criterion 2) were the easiest and most accurate to measure. 2.2. Scoring the Criteria The next step was to classify the criteria to reflect the 'intensity' of the adverse state, i.e., the impact on the animal based on the extent to which the two criteria deviated from normality (see Table 1 and Table 2). Three major bands were chosen to reflect the three recognised legal severity categories of mild, moderate and severe. However, in practice, there are two other recognisable and necessary categories: no change (normal variation) and excessive change that is beyond the upper severity limit (i.e., more than severe and long lasting, which has to be described and be quantitatively and qualitatively defined and would include death). These bands were also scored, resulting in a total of five bands. For body temperature, the five bands corresponded to no significant change (0-0.2C, i.e., normal), an increase of 0.2 to 1C (mild), and increase of 1 to 2C (moderate), an increase of 2 to 3C (severe) and an increase of more than 3C above normal (greater than severe). For body weight, the five bands are no significant change (0-5%, i.e., normal); a mild change of 5-10%, a moderate change of 10-20%, a severe change of 20-30% and a loss of more than 30% (greater than severe). Each of the five recognisable bands for each criterion is assigned a score of 0, 1, 2, 3 or 4, and in this way, the scores are converted into a mathematical model for analysis. Each animal is scored at the same time point for each criterion at the same time of day in an attempt to keep everything constant except for the measurement. This figure is then averaged to obtain a group average for that criterion at that specific time. Part A in Table 1 (body temperature, low dose) shows that two animals scored within the range considered normal, and three animals had a rise of between 0.2 and 1C. This resulted in a group score of 3 and a group average of 0.6 (i.e., total group score (3) divided by 5 animals) for that criterion. For body weight (body weight (low dose), Part B in Table 1) four animals lost between 5 and 10% body weight, and one animal lost between 10 and 20% body weight, resulting in a group score of 6 (0 + 4 + 2 + 0 + 0) and a group average of 1.2 (i.e., total group score (6) divided by 5 animals). The same process is now applied to the high-dose group for the two criteria (see Table 2). The scores for the calculation of actual overall impact assessment can now be performed by summing the two criteria to derive an average impact intensity score for each dose level. 2.3. Low-Dose Group Overall score:= Number of animals x (group average criterion 1 + group average criterion 2)Number criteria used =5 x (0.6 + 1.2)2=5 x 1.82=4.5 Therefore, the average impact score for each criterion for each animal in the low-dose group is 4.5/5 = 0.9. 2.4. High-Dose Group Overall score:= Number of animals x (group average criterion 1 + group average criterion 2)Number criteria used =5 x (3 + 3.2)2=5 x 6.22=5 x 3.1=15.5 Therefore, the average impact score for each criterion of each animal in the high-dose group is 15.5/5 = 3.1. 3. Interpretation of the Results The data obtained from this example can be used to determine whether the severity limit has been exceeded during the study, as well as whether the overall severity classification for the project has also been exceeded. 3.1. Severity Limit The severity limit is imposed to protect the individual animal; any animal that exceeds the severity limit on the average of the specific criteria should be reported to the project licence holder to take some sort of action. For example, the animal can be withdrawn from the study or, in some circumstances, attempts can be made to alleviate the suffering. An alternative is to contact the competent authority to change the limit and for the study to continue as planned. 3.2. Severity Classification of the Project A comparison can now be made with the prediction of mild severity in the low-dose group and severe in the high-dose group. In theory, the average score for each animal in a mild band should be 1; however, the actual average was 0.9. Therefore, in the final analysis for that time point, the suffering was less than predicted. Using the same approach, the average score for each animal in the high-dose group should have been 3 (severe) but it was actually 3.1. Therefore, in the high-dose case, the severity classification was exceeded. As the severity limit applies to an individual animal (see . Dir.Annex VIII), some of these animals should have been killed or withdrawn from the study, although their scores should still be recorded. 4. More Complex Experiments: Continued Use and Reuse (Directive 2010/63/EU, Art 16.1 (a)) If a project is more complex with several phases comprising a continued use, each phase can be scored separately and assigned a severity limit, e.g., surgery to implant a monitor followed by a treatment and removal of the monitor. The cumulative severity would then be the severity of each phase of the project, i.e., the severity classification for the whole project. This may be useful scientifically, as a grossly abnormal animal during one phase may not yield reliable data at a later stage because the results could have been confounded by physiological, homeostatic or behavioural responses during an earlier phase. In terms of reuse, no animal may be reused if it has experienced suffering of mild or moderate severity during a previous use; this scheme will be useful for such assessment. 5. Discussion 5.1. Legal Aspects Current EU and UK legislation requires project licence holders to conduct an ongoing assessment of the levels of pain, suffering, distress and lasting harm (severity) experienced by animals in an experiment. It also requires that any such suffering be reduced to the minimum necessary to achieve the scientific objective. The scheme described in this paper will help to do this for subacute and chronic experiments (i.e., more than 2 days) but not always for acute experiments, in which the intensity of suffering may exceed the severity limit but not result in any significant physiological or physical changes that can easily be observed and scored. These hyperacute changes, indicating pain and distress, require a different approach such as observing the behaviour of the animals (e.g., vocalisation, escape behaviours, focal attention to a particular body site, etc). It would also be possible to measure blood hormone hormones (e.g., a rise in corticosteroid and/or catecholamine levels) and end-organ responses (e.g., increases in heart rate, blood pressure and respiration); however, these are complicated by the measurement procedures involved, e.g., handling and blood sampling, which in and of themselves, can cause extra suffering . 5.2. Choice of Signs to Score The framework outlined above allows the licence holder to use whatever signs are most appropriate for the specific experimental procedures being carried out. They can be clinical signs, a change in normal behaviour, exhibition of abnormal behaviour or an experimental variable that itself may indicate an adverse effect. The key points are that the signs being scored should be robust in the sense that they reliably indicate a scorable change; any change is easy and convenient to observe; scoring does not cause any further suffering to the animals concerned, i.e., measurement should be non-invasive; and is economical to implement. It is best if the signs also reflect some biological significance; for example, a ruffled/harsh/starey coat is a more general sign of poor wellbeing than measuring tumour size or joint pain, which are specific to the experiment. General signs may be of help in diagnosing an unspecified adverse state, whereas specific signs can be used to make a better scientific severity assessment and may also be used to help with the scientific objectives. Some signs can relate more to mental distress than pain, and these two states, i.e., pain and distress, often go hand in hand; all painful states are likely to cause mental distress, but not all signs of mental distress indicate pain. 5.3. Number of Scorable Signs How many scorable signs are needed to make an assessment? In one sense, the fewer signs to score, the easier; therefore, it is useful to try to restrict the number to those that are most important or are most likely to reflect animal suffering. The signs to be scored can be evaluated using a statistical elimination/reduction analysis and the minimum number of signs that closely correlate with the final severity outcome. This may be a circular argument and open to bias, but the overall direction of changes and their magnitude make it less likely to be wildly inaccurate. Using the approach described above, any number of signs can be scored cumulatively and scored in pairs or trios, e.g., A + B, or A + B + C, etc., to provide the best fit; that is, A should normally always accompany B, B should not normally be measured in combination with C, etc. The final crucial assessment is to compare and rank the experimental group(s) with the control group. The control group may be a scientific control (e.g., vehicle only or sham-operated); the most valid type of control would probably be to compare the experimental group with a group of naive animals kept under the same conditions. Scoring the 'extra' severity or suffering is a true indication of the impact of the experimental procedures on an animal. It is worth noting that even the scientific control may sometimes experience some pain and distress, e.g., injection of vehicle only or sham surgery. It is therefore important to always measure and score the control group as well as the experimental group. A comparison with naive animals kept under the same housing and husbandry conditions provides the most objective indicator of all extra adverse effects experienced by the animals in the experiment, although it will not include any mental distress that may be caused by the housing and husbandry alone. 5.4. Categories of Adverse States Adverse states inevitably include both a physical and a mental component; for example, all painful states will also have a mental component of suffering. Distress, on the other hand, is more difficult to determine and quantify and can be broken down into two types. The classical type described by Moberg , where there is a marked perturbance in the pituitary-hypothalamic axis that affects end-organ responses such as the adrenal cortex, thyroid and reproductive organs can lead to a marked loss of homeostasis and, ultimately, a failure to thrive. This type of stress response is termed physiological dystress. The other sort of distress, which was originally described by Selye, involves behavioural changes and is more mental than physical. It can be caused by negative emotional states of, e.g., frustration, boredom, fear or anxiety, and is usually transient, although not always; this type of distress is termed mental distress. The choice of controls mentioned in the preceding paragraph dealing with naive animals will inevitably include a minimum of mental distress as a result of the housing and husbandry, e.g., frustration and boredom. Extra suffering as a result of the procedures is the difference between the control group and the experimental group(s), e.g., pain and fear. This further illustrates the difference between any physiological dystress and any mental distress. 5.5. Change in Score Direction Some changes in scorable signs may be positive or negative and can go in either direction depending on the experiment and the species being scored. For example, in experimental studies on infection, an increase in body temperature is normally seen in most species, but for those species with a high metabolic rate, e.g., small rodents such as mice, infection may lead to a decrease in temperature; exactly the same principle applies to scoring the severity deviation from normality . In terminally ill animals, the body temperature will gradually fall if they are left to die. When using body weight as a measurement, the age and maturity of an animal has to be taken into account. That is why the control should be chosen carefully. A failure to eat will lead to body weight loss depending on the level of intake, but in growing animals, the comparison should be age-matched, as a slowing of growth may be just as important. Using body weight to measure tumour growth may result in appetite being decreased or increased, whereas body weight may increase or decrease or not change. It has to be coupled with other signs such as tumour size. Body weight may decrease as a result of lowered intake or increased metabolic rate, or an increase in body weight may be observed as a result of fluid accumulation, e.g., ascites with liver tumours or decreased activity. All confounding factors need to be taken into account and interpreted biologically when selecting appropriate clinical signs of suffering. 5.6. Scoring of Death Death should always be scored as a criterion in its own right, as it represents major change (usually unexpected) and should considered a serious adverse effect regardless of deviation from normality. If death is expected, stringent efforts must be made to predict it and to apply a humane endpoint, as death often indicates prior hyperacute suffering of some sort . However, death can also occur very rapidly between inspections. The legislation permits death as an endpoint in exceptional circumstances . 5.7. What Is Normal? Sometimes it can be challenging to decide what is the normal range for a particular sign, as there is so much biological variation. Each animal is scored as an individual and not as a group; therefore, investigators look for a change in the individual rather than an absolute number or standardised qualitative estimate. Depending on the species, change relative to normal can vary in rodents from 0-10% for body weight depending on the time of day, whereas for other species, the range is much smaller. This is the reason why scoring should take into account the circadian rhythm of animals and ideally be carried out at the same time each day. An animal's reaction to its housing and husbandry vary as much as the husbandry, the housing and the personnel. That confounded by individual biological variation means that 'normal' will inherently have some variation, which is why +/-5% leeway is given for that category. In the examples I have chosen for this study, I have given a score for each level of change that correlates with severity classification in the legislation. The classification of normal can be difficult in some species due to the natural variation in some species, especially in non-mammals. Normal can vary with ambient temperature (poikilotherms such as retiles amphibia, fish, including and the mole rat) and season of the year (e.g., body weight in migratory birds); however, for laboratory mammals kept under research conditions and husbandry, 'normal' parameters are relatively stable for a given age and sex. The normal baseline forms the basis for assessing the impact of an experiment on an animal. The degrees of change above or below the normal threshold are set to reflect the severity classification from 'mild' to greater than 'severe', which includes death; although changes greater than severe are not permitted, they will inadvertently occur between scoring inspections unless an adequate safety margin is implemented (see the section above on scoring death). 5.8. Assessment of the Severity Limit and Severity Band The average score for an individual animal in a group at a particular time is the sum of all the scores for a specific sign divided by the number of animals. This score is then added to the scores for all the other signs, which provides an estimate of the actual severity being experienced by that animal at that time. For the total score in an experiment, all the scores for all the animals in that group are added together, and that sum can then be used to obtain the average score for each animal in that experimental group. This figure can them be used to determine any breach of the project severity classification, which can be an overestimate or an underestimate. However, for the application of the severity limit and the implementation of a humane endpoint, it is the estimated suffering of an individual animal that is important. The project severity classification, on the other hand, is based on the overall average for the worst-case scenario of the project. The estimation of the level of suffering (severity) can then be used to corroborate the estimated projected severity of the project. 5.9. Inspection Intervals It is a legal requirement that animals be inspected for health every day ( Art 13.1. (c)). Animals used in an experiment should be checked more regularly depending on to how likely they are to undergo a change in their severity classification; the frequency of inspection should be increased for higher severity levels to three to four times or more daily or if there is a likelihood of exceeding the upper severe category during the night and outside normal working hours (e.g., weekends) (see unpredicted endpoints in Ashall and Millar, 2014) . 5.10. Uses of Scoring Results Other than Severity Estimates Ongoing contemporaneous scoring of the signs can help to achieve the goals of ascertaining when an animal has exceeded an allocated individual severity classification or limit, in addition to helping to define a humane endpoint, scientific endpoint or intervention point depending on which is being sought. It will also help in any retrospective analysis of any suffering experienced by animals, as well as the effectiveness of any alleviating strategies. Scoring the adverse effects of scientific procedures during an experiment will help in the validation of the scientific data being measured, as confounding covariables may negate or modify the interpretation of the scientific results. It must always be remembered that an animal's physiological responses to the experimental procedures being carried out are likely to affect the scientific data being harvested and may confound the interpretation of the data. Furthermore, these physiological responses may even be of greater significance than any alleviative treatments; therefore, attempts should always be made to treat pain and distress. Training: For some clinical signs used for scoring, e.g., behaviours, appearance, stance and posture and provoked responses, it is often vitally important that there is consistency between those scoring the signs; this may require some serious training. Such training should be extended to all appropriate staff, including those staff working at weekends, holidays and out-of-hours during the evening and the night. Finally, it should not be forgotten that such an approach may be useful to assess positive mental states, as well as negative ones, although the choice of specific measures will obviously vary, e.g., use of any enrichment or attractions and time spent playing in young animals. 6. Conclusions This paper describes the principles of a simplified system of analysis of the impact of an experiment on an animal that is objective, robust and reproducible. It will permit a contemporaneous assessment of the adverse effects and a scheme for the implementation of the severity classification limit. It will also provide a mechanism for the retrospective assessment and accuracy assessment of the predicted severity classification. Institutional Review Board Statement Not applicable as theoretical and no animals were used. Informed Consent Statement Not applicable as theoretical and no humans were used. Data Availability Statement Not applicable as a theoretical concept. Conflicts of Interest The author declares no conflict of interest. animals-13-00800-t001_Table 1 Table 1 (A) The proposed scoring matrix for body temperature (low dose). (B) The proposed scoring matrix for body weight (low dose). A Change <0.2 >0.2 to 1C >1 to 2C >2 to 3C >3C Control Normal range Mild Moderate Severe >Severe or death SCORE: 0 1 2 3 4 1 X 2 X 3 X 4 X 5 X Total score (2 x 0) (3 x 1) = 3 0 0 0 B Change 0 to 5% >5 to 10% >10 to 20% >20 to 30% > 30% Control Normal range Mild Moderate Severe > Severe or death SCORE: 0 1 2 3 4 1 X 2 X 3 X 4 X 5 X Total score 0 (4 x 1) = 4 2 0 0 Group total = 0 + 3 + 0 + 0 + 0 = 3; group average = 3/5 = 0.6. animals-13-00800-t002_Table 2 Table 2 (A) The proposed scoring matrix for body temperature (high dose). (B) The proposed scoring matrix for body weight (high dose). A Change <0.2 >0.2 to 1C >1 to 2C >2 to 3C >3C Control Normal range Mild Moderate Severe >Severe or death SCORE: 0 1 2 3 4 1 X 2 X 3 X 4 X 5 X Total score 0 0 1 x 2 = 2 3 x 3 = 9 1 x 4 = 4 B Change 0 to 5% >5 to 10% >10 to 20% >20 to 30% >30% Control Normal range Mild Moderate Severe >Severe or death SCORE: 0 1 2 3 4 1 X 2 X 3 X 4 X 5 X Total score 0 0 1 x 2 = 2 2 x 3 = 6 2 x 4 = 8 Group total = 0 + 0 + 2 + 9 + 4 = 15; group average = 15/5 = 3.0. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000070

The construction of marine ranching is a concrete practice to fulfil the strategic objective of China's maritime power. The shortage of funds has turned into an important issue to be resolved urgently in the modernization of marine ranching. This study constructs a supply chain system, involving a leading enterprise of marine ranching with short funds and a retailer, and introduces the government guidance fund to solve the issue of capital shortage. Then, we discuss the supply chain financing decision under two different power structure modes, and analyze the product environmental attribute (the product's environmental friendliness and the environmental enrichment) and the guiding effect of government investment on the operation of different modes. The research shows that: (1) The wholesale price of products is mainly influenced by the dominant position of the marine ranching leading enterprise. Furthermore, the wholesale price and the marine ranching company's profits increase with the growth of the product environmental attribute. (2) The retailer's profit and the supply chain system's profit are mainly affected by the dominant power of the retailer and are positively correlated with the product environmental attribute. In addition, the supply chain system's overall profits are negatively related to the guiding effect of government investment. government guidance fund supply chain financing decision environmental enrichment marine ranching National Natural Science Foundation of China71901199 72273135 China Postdoctoral Science Foundation Funded Project2019M660170 Fundamental Research Funds for the Central Universities201913015 Postdoctoral Innovation Project of Shandong Province201902019 Special Program for Rural Revitalization Research of OUCZX2022002 "Youth Innovation Team Program" Team in Colleges and Universities of Shandong Province2022RW011 2022RW13 2022KJ042 2022KJ045 Special Funds for Fundamental Scientific Research Operation of Central Universities202113011 Guangxi Key Laboratory of Marine Environmental Science, Guangxi Academy of SciencesGXKLHY21-04 This research was funded by National Natural Science Foundation of China, grant number: 71901199, 72273135; the China Postdoctoral Science Foundation Funded Project, grant number: 2019M660170; the Fundamental Research Funds for the Central Universities, grant number: 201913015; the Postdoctoral Innovation Project of Shandong Province, grant number: 201902019; the Special Program for Rural Revitalization Research of OUC, grant number: ZX2022002; "Youth Innovation Team Program" Team in Colleges and Universities of Shandong Province, grant number: 2022RW011, 2022RW13, 2022KJ042, 2022KJ045; the Special Funds for Fundamental Scientific Research Operation of Central Universities, grant number: 202113011; Guangxi Key Laboratory of Marine Environmental Science, Guangxi Academy of Sciences, grant number: GXKLHY21-04. pmc1. Introduction The waste of ocean resources and the disruption of the ocean environment make traditional fisheries face the dual constraints of resources and environment . Marine ranching has attracted extensive attention from all countries since its appearance, and it has become a hot spot in the growth of the ocean economy as a new ecological fishery model . Compared with traditional mariculture, marine ranching pays more attention to environment and quality, which not only reduces the pollution to the ecological environment, but also improves production efficiency. The "14th Five-Year Plan" of China proposed that we should adhere to land and sea coordination, promote marine ecological protection, marine economic development, marine rights protection, and accelerate the construction of a maritime power. According to Announcement No. 515 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China, as of mid-January, 2022, 153 national marine ranching demonstration zones have been approved, which indicates that the marine ranching industry has begun to take shape. China's marine ranching has achieved a large-scale output in coastal provinces in recent years, but its construction and development are still in the primary stage. Due to marine ranching being a comprehensive system, its construction and development need to be considered comprehensively . Marine ranching has a long construction period , and it belongs to a capital-intensive industry. Its establishment and development need more time and money, and the capital demand is large. China's marine ranching leading enterprises are still in their infancy, and the financing difficulties caused by insufficient funds and broken a capital chain has become a bottleneck in the growth of marine ranching . Thus, there is no time to delay in solving the dilemma of capital shortage in China's marine ranching and building maritime power. In view of the fund shortage for marine ranching establishment, the leading enterprises can adopt a variety of ways and channels to grow capitals. Enterprises can adopt supply chain finance (SCF) to ease the fund shortage. In the past few years, academic circles have paid more attention to SCF and SCF has become a prospective solution to alleviate the financing problems of small and medium enterprises (SMEs) . Marine ranching construction and operation involves many stakeholders, such as the government, enterprises, and fishermen, and needs overall planning and comprehensive management. The government, as a key subject, also plays a crucial role in alleviating the shortage of funds for leading enterprises in marine ranching. The government can not only support the operation and development of leading enterprises in marine ranching by formulating relevant policies , but also solve the financial constraints faced by enterprises through direct government subsidies and government guidance fund. Among them, the government guidance fund is an efficient way for the government to provide relief programs for the leading enterprises of marine ranching. Since marine ranching in China is a new industry, and the establishment scale of most leading enterprises in marine ranching is small, it belongs to start-ups . The government's shareholding in marine ranching through the government guidance fund can not only effectively ease the financing issues faced by the marine ranching leading companies as start-ups , but also effectively deal with the financing risks of enterprises in the process of SCF. In addition, the government guidance fund can also guide social capital through its guiding effect to jointly invest in marine ranching with a fund shortage. However, the existing research on solving the problem of fund shortages at the government level mostly focuses on the method of direct government subsidies, while the research on government guidance funding is relatively less. There can be many diverse structures in the supply chain model. For the study of different power structures in a supply chain, scholars have conducted extensive discussions. For example, Luo et al. constructed a retail supply chain model including manufacturers and retailers, and researched the impact of different power structures on the decision-making and profit of the supply chain. Li et al. studied the impact of different power structures on products and their pricing decisions in the supply chain. Li and Mizuno revealed the optimal pricing and inventory control strategy of a dual-channel supply chain under three different power structures. In addition, Zhai et al. believed that the dominant power of manufacturers and retailers determines whether participants are leaders or followers in the market, which in turn affects the decision-making order. Therefore, how to make operational decisions based on different power structures to maximize their own benefits is an urgent problem for supply chain members. Motivated by this, this study focuses on the shortage of capital for marine ranching establishment. With "supply chain financing" and "government guidance fund" as the breakthrough points, a two-stage supply chain model including a leading enterprise of marine ranching and a retailer with a capital shortage is constructed. According to the dominant power of the marine ranching company and the retailer, the model can be divided into two modes, and the optimal solution under the two modes can be solved, respectively. In addition, the products' environmental friendliness can protect the marine environment and environmental enrichment can effectively improve the behavior and benefits of marine organisms. Therefore, this study introduces the environmental enrichment variable into the model and this, combined with a product's environmental friendliness variable, constitutes the product environmental attribute. Then, it analyzes the effect of the variable on the supply chain operation, and compares the decision outcomes for two different power structures. What is more, it provides a theoretical basis for the financing decision behaviors of the marine ranching supply chain, and offers relevant advice for solving the fund shortages of marine ranching. According to the above analysis, the contribution of this paper can be divided into the following three points:(i). The government guidance fund is applied to the marine ranching to study the influence of different power structures on supply chain operation. It broadens the research boundary of the government guidance fund. (ii). Environmental enrichment and product's environmental friendliness constitute the product environmental attribute of the marine ranching leading enterprise. The impact of this variable on the operation of the supply chain is analyzed. (iii). Through parameter sensitivity analysis and comparative analysis of the two modes, relevant management suggestions are put forward from the perspective of the marine ranching leading company and the retailer, respectively. 2. Literature Review This part expounds four kinds of literature flows based on the research themes and objectives: (1) marine ranching, (2) government guidance fund, (3) supply chain financing decision, and (4) environmental enrichment. 2.1. Marine Ranching The notion of marine ranching comes from the practical experience of the scientific utilization of the ocean, and has steadily improved with the growth of fisheries. Marine ranching was first proposed by Japanese scholars in 1971, and it was defined as a system that can continuously produce food from marine biological resources. In 1979, Zeng Chengkui put forward "marine farming, marine pasture", from which the notion of marine ranching in China originated . The 230th Shuangqing Forum made clear the most authoritative definition so far, that is, marine ranching is an ecosystem that has the function of environmental protection and can realize the sustainable production of fishery in suitable sea areas. As a new fishery model , marine ranching has brought great economic and ecological benefits . Marine ranching is a vital means for rich resources and ecological sustainable development . It can not only make fishery production efficient, protect ocean environments, and realize the sustainable utilization of fishery resources , but also be a crucial fishery carbon sink producer . Marine ranching's development in China mainly relies on the guidance of the government and the participation of enterprises , which has become a major strategic issue of concern to the government. Recently, there have been many bottlenecks in the growth of marine ranching. The establishment of marine ranching was stagnant due to faulty laws and management systems , the urgent breakthrough of key technology , the low allocation efficiency of marine related resources , and fund shortages . Modern marine ranching needs to consume more money in the construction process, so sufficient and sustainable funds are the necessary conditions to ensure the marine ranching establishment. Due to the long construction period of marine ranching , a large amount of funds need to be invested, and most of the marine ranching leading enterprises have a small construction scale, and their own funds and capital inflows are in a state of shortage. In the development process, enterprises will have the problem of capital chain rupture . The construction level of marine ranching in China lags behind the strategic goal of accelerating the construction of a maritime power. Therefore, it is urgent to provide a financial relief plan for marine ranching. 2.2. Government Guidance Fund The problem of fund shortages in marine ranching leading enterprises has many ways and channels to solve. SCF and other financing methods can raise more funds for enterprises . The SCF is a novel financing model, which provides flexible financial products and services for upstream and downstream SMEs based on trust and the fair distribution of benefits around the core enterprises . It can not only effectively solve problems such as liquidity pressure existing in traditional financing methods , but also optimize the financing performance of supply chain companies and improve the benefits of all participants in the supply chain system . However, SCF has some financing risks in the financing process, such as credit risk , trust risk , pledge risk , and moral hazard . Furthermore, from the government level, government intervention is of great help to SMEs' financing ; the problem of capital shortage can be solved through the government's financial support for enterprises. Guo et al. believes that with the participation of the government, enterprises will obtain more funds for financing activities. The government's intervention supports the financing channels of enterprises and promotes opportunities for enterprises to obtain financing . There are two ways for the government to ease the dilemma of company capital shortage: direct government subsidy and a government guidance fund . On the one hand, marine ranching leading enterprises can use government subsidies to gain more economic benefits . Government subsidies, as a coordination mode , provide financial support to enterprises in the supply chain, thus solving the financing difficulty caused by capital constraints . It can also improve the output level and operation ability of enterprises , thereby improving the level of a supply chain system's overall performance . On the other hand, the government guidance fund is a valid way to guide the growth of start-ups. It is funded by the government and attracts investment from local governments, financial institutions, and social capital. It can not only provide financial support to start-ups, but also solve the financing risks (credit risk, pledge risk, etc.) generated in the process of supply chain financing . The government guidance fund needs policy support and Cui et al. constructed the evaluation indicator system of policy effectiveness from three dimensions, and believed that without the support of policy, the government could not guide the growth of funds. Compared with the method of direct government subsidies, the method of the government guidance fund tends to invest in start-ups. The government guidance funds' aim is to inspire the growth of emerging industries and stimulate the vitality of social capital to make venture capital investment. The government guidance fund is a new way for the government to resolve the financing difficulties in the initial stage of companies. In addition, the government guidance fund gathers the advantages of the government and the market and plays a guiding role, which can attract the investment of social capita and promote the upgrading and innovation of the industries . The advantage of the government guidance fund is that it operates in a market-oriented way, which drives social capital to invest in areas encouraged and supported by the state. The literature review shows that the government guidance fund can effectively solve the initial financing difficulties faced by leading enterprises in marine ranching. However, the existing research on the government guidance fund to alleviate corporate financial constraints still needs to be strengthened. 2.3. Supply Chain Financing Decision SCF is an effective way for companies to obtain operating capital by using supply chain cooperation relationships . How decision makers make optimal decisions is an important issue, and supply chain financing can combine financial problems with operational decision-making problems . Financing decision refers to the choice of the best solution for enterprises to raise funds and it is a key problem for the enterprises' survival and growth. Currently, the supply chain system has become increasingly complex. Each subject in this system should make reasonable decisions to create value, reduce risk, and strengthen cooperation with each other in the system . Many scholars have researched the financing decision of the supply chain from different situations. For example, Huang et al. studied supply chain financing decisions under capital constraints; Shi et al. analyzed the influence of reduced demand uncertainty on the financing decisions of supply chains constrained by capital; Zheng et al. studied the impact of market uncertainty on supply chain financing decisions. In addition, Xia et al. introduced cross-shareholding, and discussed the optimal decision behaviors of supply chain entities in diverse power structures. Also considering the effect of cross-shareholding on supply chain financing decisions are Fu and Ma , Zhang and Meng , etc. According to the literature review of supply chain financing decisions, we find that most scholars divided the supply chain system into two modes according to their different dominant positions, and discuss the optimal decision-making behaviors of different participants in diverse modes, for example Ren et al. , Xia et al. . To sum up, the paper considers the effect of diverse power structures on marine ranching supply chain financing decisions under government guidance fund, and discusses the effect of product environmental attribute variable on the optimal decision-making result. 2.4. Environmental Enrichment Environmental enrichment, which refers to a method of improving animal welfare and biological functions through a deliberate increase in environmental heterogeneity and complexity , has shown huge potential for meeting the requirements of both aquatic animals and human owners. It is an important requirement for improving farm animal welfare . Environmental enrichment provides complexity that can benefit poultry welfare, including health and behavior, as well as emotional states . Exposure to environmental enrichment affects various cognitive functions in animals . That is, environmental enrichment can improve the cognitive development of animals and induce positive emotions . In addition, environmental enrichment is also an experimental method, which refers to richer living conditions relative to the standard environment, including increasing sense, exercise, cognition, and social stimulation . For the past few years, scholars have studied all kinds of animals and explored the influence of environmental enrichment on animals. For example, van der Staay et al. explored the mechanism of environmental enrichment on pig decision-making behavior, and reached the conclusion that environmental enrichment can improve the learning ability and memory ability of animals. Alaniz et al. demonstrated behavioral evolution based on environmental enrichment, and believed that environmental enrichment could significantly improve the welfare of toucans by stimulating food consumption and promoting exercise. Sumon et al. explored the importance of environmental enrichment in animal reproduction, and believed that environmental enrichment will increase the reproduction rate of fish. The literature shows that the environmental enrichment can promote the improvement of animal behavior and increase animal benefits. In view of this, this study combines the environmental enrichment variable and the product's environmental friendliness variable into the product environmental attribute variable, and discusses the effect of this variable on the optimal decision results. In summary, based on the existing literature, this study introduced the government guidance fund to deal with the financing problem of marine ranching. Then, the effect of diverse power structures on supply chain operation were studied, which broadens the research boundary of the government guidance fund. In addition, the environmental enrichment variable is introduced into the study of the model to discuss the effect of this variable on the optimal decision results. Finally, the corresponding management enlightenment is put forward from the view of leading enterprises in marine ranching and retailers. 3. Methods 3.1. Problem Description The construction of marine ranching can not only develop and utilize marine biological resources reasonably and effectively, but also bring great social, economic, and ecological value. However, the marine ranching leading enterprises will face the shortage of funds in the progress of management and growth. As start-ups, the shortage of funds limits the operation and scale expansion of leading enterprises in marine ranching. The government, as a promoter of economic development, can take shares in marine ranching through government guidance funds, alleviate the constraints of the enterprise capital shortage, and then promote the continuous growth of marine ranching. Meanwhile, the government also promotes social capital into the marine ranching through its guiding effect, and then jointly solves the capital shortages of leading enterprises in marine ranching. Based on the description of the problem, this study constructs a supply chain system including a leading enterprise in marine ranching and a retailer. After the production process of the leading enterprise is completed, the enterprise sells the products related to marine living resources such as fish, shrimp, and shellfish to the retailer at a wholesale price o, and then the retailer sells them to consumers at a retail price p. The basic model of marine ranching supply chain under government guidance fund is shown in Figure 1. 3.2. Basic Hypotheses According to the difference of dominant power between the leading enterprise and the retailer in the supply chain system, two different modes are formed. In the two modes, there is a game relationship between the two sides. In the process of game, both sides constantly adjust their own strategies to seek optimal decisions. To facilitate analysis, the following hypothesis are put forward. Hypothesis 1. The private capital of the leading enterprise in marine ranching is m0, the government investment is Ig, and the initial investment of social capital is I0. Considering the guiding role of government investment, the final investment of social capital is Is=I0(1+s), where s is the guiding role of government investment. Considering that the government can drive social capital into the marine ranching leading enterprises through its guiding effect, the guiding role of government investment should meet s>=0. Hypothesis 2. The product demand of the leading enterprise in marine ranching is q=a-bp+aes, in which the market capacity is a, p is product price, and a, p are both >0. b is the product price coefficient, and b>0; es is the product environmental attribute, which are influenced by the product's environmental friendliness and the marine ranching enterprise's environmental enrichment , and meet es>=0; a is the influence coefficient of the product environmental attribute on demand, a>0; when the product ' s environmental friendliness and the marine ranching enterprise's environmental enrichment are improved, consumers will increase their purchase of the product. Considering the product demand should be greater than 0, so the product demand should meet a-bp+aes>0. Hypothesis 3. The production cost per unit product of the leading enterprise in marine ranching is c, the financing cost is cI, and c>0, cI>0. Hypothesis 4. The proportion of government-owned marine ranching leading enterprise is Igm0+Ig+Is, the proportion of social capital-owned marine ranching leading enterprise is Ism0+Ig+Is, and the private capital ratio of the marine ranching leading enterprise is m0m0+Ig+Is . Therefore, it can be seen that the shareholding in the leading enterprise of marine ranching includes two parties. One is that the government shares in the leading enterprise through the government guidance fund. The other is that the social capital driven by the government guidance effect shares in the leading enterprise of marine ranching, and it is satisfied that 0<=l1<=l , 0<=l2<=l . Where l represents the maximum proportion of equity that two entities can have. In order to ensure that both parties do not lose their control rights, it is necessary to meet the condition 0<=l <=0.5 , namely 0<=l1<=0.5, 0<=l2<=0.5. Hypothesis 5. The marine ranching company and the retailer are both risk-neutral. They all make their own deci-sions with the aim of maximizing their own benefits. Then, for the convenience of analysis, all the notations and the definition of the corresponding notations are listed in Table 1. According to the above hypotheses, the profit of the leading enterprise and the retailer can be expressed, respectively:(1) pm=m0m0+Ig+I0(1+s)(o-c)q-cI (2) ps=(p-o)q The profit of the supply chain system can be expressed as:(3) pt=pm+ps=m0m0+Ig+I0(1+s)(o-c)q-cI+(p-o)q 3.3. The Stackelberg Game Method In the Stackelberg game model, the latter player can choose his own strategy according to the strategy of the first player, and the latter player's strategy will also respond to the first player . In the supply chain model constructed in the paper, different supply chain models are formed because the leading enterprise and the retailer occupy different leading positions in the supply chain system. Therefore, the optimal decision under each mode is different, and the strategies adopted will be different to some extent. The two different supply chain models proposed in this paper include the mode dominated by the marine ranching company and the mode dominated by the retailer. Taking the mode dominated by the marine ranching enterprise as an example, in this mode, the leading enterprise of marine ranching is the leader and the retailer is the follower, which satisfies the master-slave relationship in the Stackelberg game. The backward induction is a method to solve the dynamic game equilibrium, which means that the actions of the participants in the game have a sequence, and the latter party can observe the actions of the former . Because the supply chain decision-making model constructed in this paper follows the Stackelberg game model, and the backward induction is a commonly used method to solve the equilibrium solution in the Stackelberg game model. However, the backward induction is only suitable for finite step dynamic equilibrium, so it also has some limitations. Two different supply chain modes proposed in this paper include the mode dominated by the enterprise of marine ranching and the mode dominated by the retailer. The fourth part of this paper makes specific calculation and analysis on different supply chain decision-making modes. For ease of distinction, the two modes are marked as "Mode 1" and "Mode 2", respectively. 4. Model Solving 4.1. Decision in Mode 1 In this mode, the leading company of marine ranching occupies a dominant position. Then, the marine ranching leading enterprise first decides the product wholesale price o, and then the retailer decides the product retail price p. The game sequence of the marine ranching leading enterprise and the retailer is shown in Figure 2. Based on the backward induction method, the optimal decision-making problem of the marine ranching leading company and the retailer under this mode is solved. From Equation (1) and q=a-bp+aes, the profit of the marine ranching leading company can be expressed as: (4) pm=m0(o-c)(a-bp+aes)m0+Ig+I0(1+s)-cI Similarly, the profit of the retailer can be expressed as:(5) ps=(p-o)(a-bp+aes) From Equation (5), the first and the second partial derivative of ps to p are calculated, respectively, to obtain psp=a-2bp+aes+bo, 2psp2=-2b<0. Therefore, ps is a strictly concave function about p, and based on psp=0, the reaction function can be as follows:(6) p=a+aes+bo2b Substitute Equation (6) into q=a-bp+aes, the product demand is:(7) q=a+aes-bo2b By substituting Equation (6) into Equation (4), the profit function of the leading enterprise in marine ranching is:(8) pm=m0(o-c)(a-bo+aes)2[m0+Ig+I0(1+s)]-cI From Equation (8), first order partial derivative and second order partial derivative of pm to o are calculated, respectively, to obtain pmo=m0(a+aes+bc)-2bm0o2[m0+Ig+I0(1+s)], 2pm2o=-bm0m0+Ig+I0(1+s)<0. Therefore, pm is a strictly concave function about o. Due to pmo=0, the optimal wholesale price of is:(9) o=a+aes+bc2b Substituting Equation (9) into Equation (7), the optimal of product's retail price can be obtained:(10) p=3a+3aes+bc4b Substituting Equation (10) into q=a-bp+aes, the product demand is:(11) q=a+aes-bc4b Equations (9) and (10) are substituted into Equations (4) and (5), respectively, to solve the optimal profits of the leading company and the retailer. 4.2. Decision in Mode 2 In this mode, the sales dominance is in the hands of the retailer, who occupies a dominant position in the supply chain system. The retailer's position is higher than that of the marine ranching leading enterprise, and it occupyies a dominant position in the supply chain system. Meanwhile, the retailer first decides the product's retail price p, and then the marine ranching leading enterprise decides the wholesale price o. The game sequence of the leading enterprise and the retailer is shown in Figure 3. Then, the optimal decisions of the leading enterprise and the retailer under this mode are solved by the backward induction method. For simplicity of calculation, we assume that the expected return of the retailer is th, then:(12) p=o+th By substituting the Equation (12) into the Equations (4) and (5), the profit of the leading enterprise and the retailer can be expressed, respectively:(13) pm=m0(o-c)(a-bo-bth+aes)m0+Ig+I0(1+s)-cI (14) ps=th(a-bo-bth+aes) According to Equation (13), the first order partial derivative and the second order partial derivative of pm to o are calculated, respectively, to obtain pmo=m0(a-2bo-bth+aes+bc)m0+Ig+I0(1+s), 2pm2o=-2bm0m0+Ig+I0(1+s)<0. Therefore, pm is a strictly concave function about o. In view of pmo=0, we can have:(15) o=a-bth+aes+bc2b Substituting Equation (15) into Equation (14), we can obtain:(16) ps=ath-bth2+aesth-bcth2 Based on Equation (16), the first order partial derivative and the second order partial derivative of ps to th are calculated, respectively, obtained psth=a+aes-bc2-bth, 2psth2=-b<0, so ps is a strictly concave function about th. Due to psth=0, we can obtain:(17) th=a+aes-bc2b Substituting Equation (17) into Equation (15), the optimal wholesale price of product is:(18) o=a+aes+3bc4b Substituting Equations (17) and (19) into Equation (12), the optimal retail price of product is:(19) p=3a+3aes+bc4b Substituting Equation (19) into q=a-bp+aes, the product demand is:(20) q=a+aes-bc4 Equations (17) and (18) are substituted into Equations (13) and (14), respectively, to solve the optimal profits of the leading company and the retailer. 5. Discussion 5.1. Conclusion of Equilibrium Results in Different Modes According to Section 4.1 and Section 4.2, we can separately obtain the optimal solution in two different dominant modes, obtained Conclusions 1 and 2. Conclusion 1. In the mode dominated by the marine ranching leading company: The optimal product demand is q1*=a+aes-bc4b. The optimal retail price of the product is p1*=3a+3aes+bc4b. The optimal wholesale price of the product is o1*=a+aes+bc2b. The optimal profits of the retailer is ps1*=(a+aes-bc)216b. The optimal profits of the leading enterprise in marine ranching is pm1*=m0(a+aes-bc)28b[m0+Ig+I0(1+s)]-cI. The overall optimal profits of the supply chain system is pt1*=ps1*+pm1*=(a+aes-bc)216b+m0(a+aes-bc)28b[m0+Ig+(1+s)]-cI. Conclusion 2. In the mode dominated by the retailer: The optimal product demand is q2*=a+aes-bc4b. The optimal retail price of the product is p2*=3a+3aes+bc4b. The optimal wholesale price of the product is o2*=a+aes+3bc4b. The optimal profits of the retailer is ps2*=(a+aes-bc)28b. The optimal profits of the leading enterprise in marine ranching is pm2*=m0(a+aes-bc)216b[m0+Ig+I0(1+s)]-cI. The overall optimal profits of the supply chain system is pt2*=ps2*+pm2*=(a+aes-bc)28b+m0(a+aes-bc)216b[m0+Ig+I0(1+s)]-cI. In order to facilitate the comparative analysis of diverse dominant modes, we list the equilibrium results for the two dominant modes in Table 2. 5.2. Comparison of Equilibrium Results in Different Modes This part compares and analyzes the optimal solutions of the two modes (Conclusion 1 and Conclusion 2) in the supply chain system, and draws Conclusions 3-8. Conclusion 3. In the two modes, the wholesale price of product has a relationship of o2*<o1*; o1*, o2* increase with the growth of es. Proof. From q=a+aes-bp>0, we know a+aes>bp>bc, and b>0. According to Equations (9) and (18), we have, o1*-o2*=a+aes-bc4b>0. In addition, the first partial derivatives of o1* and o2* to es are calculated, respectively, and we can obtain, o1*es=a2b>0; o2*es=a4b>0. # As seen in Conclusion 3, the products' wholesale price is mainly influenced by the dominant position of the marine ranching leading company in the supply chain system. By comparing the mode dominated by the leading enterprise and the mode dominated by the retailer, we can see that when the leading enterprise dominates the supply chain system, to effectively guarantee its own profits maximization, the wholesale price set in the decision-making is higher. Under the retailer-led model, the retailer makes its own best decisions to make the profits optimal, which will decrease the product's wholesale price as a retailer's cost. Under the two modes, with the growth of the product environmental attribute, the marine ranching leading company will increase wholesale price accordingly. This is because with the growth of es, the product's environmental friendliness and the environmental enrichment increase and then the retailer's demand for such products rises. Therefore, the leading enterprise of marine ranching will obtain profits by increasing wholesale prices. Conclusion 4. In the two modes, the retail price of product has a relationship of p1*=p2*; p1*, p2* increase with the growth of p2*. Proof. From Equations (10) and (19), we can obtain: p1*-p2*=0. In addition, the first partial derivatives of p1* and p2* to es are calculated, respectively, and we can obtain: p1*es=p2*es=3a4b>0. # From Conclusion 4, it can be concluded that the retail price of products has nothing to do with the dominant position of all participants in different supply chain modes. Under the mode dominated by the marine ranching leading company and the mode dominated by the retailer, the retail price of products set by the retailer is the same. This indicates that regardless of the retailer's position in the supply chain system, it will not affect the product retail price. In these two modes, the price formulated by the retailer is related to es. With the growth of es, the products' retail price rises. For one thing, the growth in the product environmental attribute will rise the wholesale price (from Conclusion 3), and the retailer's costs will rise. To reduce costs, the retailer passes on some costs to consumers by raising retail prices. For another thing, the growth of the product environmental attribute will increase consumer demand for the product. In a period of time, the retailer will raise retail prices due to the phenomenon of "oversupply". Conclusion 5. In the two modes, product demand has a relationship of q1*=q2*; q1*, q2* increase with the growth of es. Proof. From Equations (11) and (20), we can obtain: q1*-q2*=0. In addition, the first partial derivatives of q1* and q2* to es are calculated, respectively, and we can obtain: q1*es=q2*es=a4b>0. # As with Conclusion 4, the product demand has nothing to do with the dominant position of all participants in diverse supply chain modes. As shown in the two different modes, no matter which subject is dominant in the supply chain system, the product demand in optimal decision-making is always the same. This is because, in these two modes, the retail price of the product remains consistent (Conclusion 4 shows q1*=q2*), and consumers' demand for the product will not change. The change of product demand is connected with the change of es. With the growth of es, consumers' demand for the product will increase. This is because, when es growth, the product's environmental friendliness and the environmental enrichment will increase. When other factors remain unchanged, consumers will increase the purchase and use of the product. Conclusion 6. In the two modes, the profits of the retailer have a relationship of ps1*<ps2*; ps1*, ps2* increase with the growth of es. Proof. From Conclusion 1 and Conclusion 2, ps2*-ps1*=(a+aes-bc)216b>0. In addition, because a+aes-bc>0, the first partial derivative of ps1* to es are calculated, and we can obtain: ps1*es=a(a+aes-bc)8b>0; Similarly, the first partial derivative of ps2* to es are calculated, and we can obtain: ps2*es=a(a+aes-bc)4b>0. # From Conclusion 6, it is concluded that the profits of the retailer mainly depend on its position in the supply chain system. Comparing the two modes, it can be concluded that under the retailer leading supply chain mode, the retailer gains higher profits. This is because, although the retail price in the two modes is the same (Conclusion 4 shows p1*=p2*), the wholesale price of products in the retailer leading supply chain mode is lower (Conclusion 3 shows o2*<o1*). Therefore, for the retailer, they bear lower costs and gain higher profits. In these two modes, the profits of a retailer's optimal decision increases with the growth of es. This is because, with the growth of es, consumers' product demand increases, the retail price of products formulated by retailers rises, and then the profits of the retailer increase. Conclusion 7. In the two modes, the profits of the marine ranching leading enterprise have a relationship of pm2*<pm1*; pm1*, pm2* increase with the growth of es and decrease with the growth of s. Proof. From Conclusions 1 and 2, pm1*-pm2*=m0(a+aes-bc)216b[m0+Ig+I0(1+s)]>0. In addition, because a+aes-bc>0, the first partial derivative of pm1* to es are calculated, and we can obtain: pm1*es=m0a(a+aes-bc)4b[m0+Ig+I0(1+s)]>0; Similarly, the first partial derivative of pm2* to es are calculated, and we can obtain: pm2*es=m0a(a+aes-bc)8b[m0+Ig+I0(1+s)]>0; because Is=I0(1+s), so pm1*=m0(a+aes-bc)28b[m0+Ig+I0(1+s)]-cI, the first partial derivative of pm1* to s are calculated, and we can obtain: pm1*s=-m0(a+aes-bc)28bI0[m0+Ig+I0(1+s)]2<0; similarly, pm2*s=-m0(a+aes-bc)216bI0[m0+Ig+I0(1+s)]2<0. # From Conclusion 7, we can determine that the profits of the marine ranching leading company mainly depending on its dominance in the supply chain system. In the mode dominated by the marine ranching company, the optimal decision-making of the enterprise obtains higher profits. This is because, in this mode, the leading enterprise will take advantage of its dominance in decision-making, and the product wholesale price is higher (Conclusion 3 shows o2*<o1*). Moreover, with the growth of es, it will stimulate the rise of the retail price and the wholesale price, making the profits of the enterprise show an upward trend. In these two modes, the profits of the leading enterprise are also related to the guiding role of government investment s. With the growth of s, the profits of the enterprise decrease. This is mainly due to the government increasing the guidance of social capital and promoting social capital into marine ranching. The increase in social capital investment funds will make the proportion of the marine ranching leading enterprise's private capital m0m0+Ig+Is decreased, and then the financial level of the enterprise decreases, and eventually leads to the profits of the marine ranching leading company showing a downward trend. Conclusion 8. In the two modes, the profits of the supply chain system has a relationship of pt1*<pt2*; pt1*, pt2* increase with the growth of es and decrease with the growth of s. Proof. From Conclusions 1 and 2, pt2*-pt1*=(Ig+Is)(a+aes-bc)216b[m0+Ig+I0(1+s)]>0. In addition, because a+aes-bc>0, the first partial derivative of pt1* to es are calculated, to obtain: pt1*es=a(a+aes-bc)8b+m0a(a+aes-bc)4b[m0+Ig+I0(1+s)]>0; Similarly, pt2*es=a(a+aes-bc)4b+m0a(a+aes-bc)8b[m0+Ig+I0(1+s)]>0; because Is=I0(1+s), the first partial derivative of pt1* to s are calculated, to obtain: pt1*s=-m0(a+aes-bc)28bI0[m0+Ig+I0(1+s)]2<0; Similarly, pt2*s=-m0(a+aes-bc)216bI0[m0+Ig+I0(1+s)]2<0. # It can be seen from Conclusion 8 that the supply chain system's profits are mainly connected with the dominant position of the retailer. In the mode dominated by the retailer, the product's wholesale price is low (Conclusion 3 shows o2*<o1*), which will lead to more profits for the retailer under the premise that the retail price and the product demand are consistent (Conclusions 4 and 5 shows p1*=p2*, q1*=q2*). As a start-up enterprise, the shortage of capital restricts the operation and scale expansion of the enterprise. Therefore, for the supply chain system, the retailer's profits occupy a major part. In these two modes, the supply chain system's profits will rise with the growth of es. When es growth, the product demand will rise, making profits increase. In addition, the overall profits level of the supply chain system will also reduce with the growth of s. This is mainly because with the growth of s, the proportion of the leading company's private capital decreases, which makes the profits of the enterprise decrease, and thus leads to the overall profits of the supply chain system decrease. 6. Numerical Analysis To prove the above conclusions more intuitively, the optimal decisions under different modes are further analyzed. Through the application program of MATLAB R2021b, we simulated the decision-making behavior of all participants in two modes. In addition, the numerical values of the variables and parameters assigned to the model in this paper are consistent with the hypotheses. Assuming that the market capacity of the sales area a=110, the price coefficient of the product b=4, the impact coefficient of the product environmental attribute on demand a=80, the production cost of the unit product of the leading enterprise c=10, the financing cost of the unit product cI=5, the private capital of the marine ranching leading enterprise m0=150, the government investment Ig=250, and the initial investment of social capital I0=100. Let the guiding role of government investment s=0.5, and the product environmental attribute of the leading company es is a variable, which changes in the interval and makes the change images of each decision parameter and independent variable es. The comparison of a product's wholesale price, a product's retail price, product demand, the retailer's profits, the marine ranching leading enterprise's profit and the supply chain system's profit under different modes are shown in Figure 4, Figure 5, Figure 6, Figure 7, Figure 8 and Figure 9. From Figure 4, the product wholesale price is impacted by the dominant position of the leading company in the supply chain system. The product wholesale price made by enterprises is higher in the mode dominated by the marine ranching leading company, but lower in the mode dominated by the retailer. In addition, regardless of which mode, the wholesale price of products formulated by the leading enterprise will increase with the growth of es. This is consistent with Conclusion 3. From Figure 5, the retail price is consistent in the two modes, indicating that the product retail price p is not significantly influenced by the dominant position of either subject. Moreover, from Figure 5, with the increase in the product environmental attribute es, the product retail price will also increase. This is consistent with Conclusion 4. As can be seen in Figure 6, consumers' demand for the marine ranching leading enterprise's product is equal in either mode and consistent with the changing direction of es. This further illustrates that the product retail price and product demand size have a one-to-one correspondence. This is the same as in Conclusion 5. Seen from Figure 7, under different modes, the size of the retailer's profits meets the relationship of ps1*<ps2*, which further shows that the ps size of the retailer's profits is connected with the size of the retailer's dominant position in the supply chain system. Moreover, from Figure 7 that under any mode, the profits obtained by the retailer will increase with the growth of es. This is because, with the growth of es, consumers' demand for high environmental friendliness and high environmental enrichment products will rise, the retail price of the product will also rise, which eventually leads to the increase in profits obtained by the retailer ps. This is consistent with Conclusion 6. As can be seen in Figure 8, the size of the profits of the leading company under the two modes satisfies pm2*<pm1*, which further illustrates that the profits' size of the enterprise has a one-to-one correspondence with the size of the dominant power of the enterprise. Moreover, it can be seen in Figure 8 that the profits of the enterprise pm and the product environmental attribute es change in the same direction. Therefore, it is consistent with Conclusion 7. It can be seen from Figure 9 that, under the two modes, the relationship between the supply chain system's overall profits satisfies pt1*<pt2*, which indicates that the profits are related to the dominant position of the retailer in the supply chain system. This is mainly due to the shortage of funds faced by the leading company in the initial stage of establishment, which limits their own development of the enterprise itself. In addition, as shown in Figure 9, the supply chain system's profits rise with the growth of es. This is consistent with Conclusion 8. Let the enterprise's product environmental attribute es=0.5, and the guiding role of government investment as an independent variable change in the interval . Under different modes, the changes of the marine ranching leading enterprise's profits and the supply chain system's overall profits with the guiding role of government investment s are shown in Figure 10 and Figure 11. From Figure 10, the profits of the enterprise are also related to the guiding role of government investment s, and under the two modes, the profits of the marine ranching leading enterprise are inversely correlated with s. This is because, with the growth in the guiding role of government investment s, the ratio of the leading enterprise's private funds will decrease, and ultimately lead to a downward trend in profits. This is consistent with Conclusion 7. Figure 11 shows that the supply chain system's overall profits level is also in connection with s. With the growth of the government investment's guiding role s, the overall profits show a downward trend. This is because, s is inversely proportional to the profits of the marine ranching leading company, which consistent with Conclusion 8. To sum up, the results of numerical analysis in this section are consistent with the conclusions drawn by the model, which further supports the conclusions drawn in the study. 7. Conclusions and Implications 7.1. Conclusions This study constructs a two-level supply chain system, including a leading marine ranching enterprise short of funds and a retailer, and the government guidance fund is introduced. Considering the difference in the dominant positions of the marine ranching leading enterprise and the retailer in the supply chain system, the mode dominated by the leading enterprise and the mode dominated by the retailer are constructed. Using the backward induction method in the game model, the optimal decisions of each subject under the two modes are calculated, and the optimal values in two different game modes are compared. Furthermore, we analyze the optimization results of the two modes in the application of MATLAB R2021b by numerical analysis. The research shows that: (1) As far as the marine ranching leading companies are concerned, the wholesale price is mainly impacted by the dominance of the marine ranching leading company in the supply chain system. Generally speaking, there is a positive correlation between wholesale price and its dominant position. The products' wholesale price is higher in the mode dominated by the marine ranching enterprise and lower in the mode dominated by the retailer. In addition, the optimal profits of the leading company are also related to its dominant position, and they are positively correlated. The marine ranching leading enterprises' profits show the same trend with the environmental attributes of their products and decreases with the growth in the guiding role of government investment. (2) For retailers, the products' retail price is not significantly affected by the retailer's dominant power. The product demand and the retail price are one-to-one correspondence in these two modes. The retail price is consistent in the two modes, and the demand also has an equal relationship in the two modes. The retailer's optimal profits are positively related to its dominance in the supply chain system, which is higher in the mode dominated by the retailer and lower in the mode dominated by the marine ranching leading company. The product retail price, demand, and retailer's profits will increase with the growth of the product environmental attribute. In addition, the overall profits of the supply chain system will also be affected by the retailer's dominance. They are positively related to the environmental attribute of products and negatively related to the guiding role of government investment. 7.2. Management Implications According to the conclusions of the last section, this section puts forward corresponding management enlightenment from the perspective of the marine leading companies and retailers. First, on the one hand, the leading companies of marine ranching can effectively integrate the concept of environmental friendliness into the whole process of the product production line by using green environmental protection materials and then improving the production technology of products and developing the production process with more environmental protection concepts, so as to improve the products' environmental friendliness produced by the leading enterprises. It can also make reasonable and effective use of green technologies, such as environmental enrichment, to improve the environmental enrichment degree of enterprises and stimulate consumers to buy so as to obtain a higher profit level . In the meantime, if the leading enterprises of marine ranching can use a government guidance fund and social capital rational to enhance the environmental friendliness of products and the environmental enrichment, the willingness of governments to guide the fund will be improved. On the other hand, enterprises can also expand the sales of their products through a variety of marketing channels and increase the proportion of their own funds, so as to improve their financial level and operational capacity. Second, retailers, as the downstream enterprises of the supply chain system, can stimulate consumers' demand for highly environmental friendliness marine-related products by designing efficient marketing strategies to encourage consumers to purchase. In addition, retailers can also carry out product brand publicity activities through a variety of channels to convey information on the environmental friendliness of marine products and their environmental enrichment to potential consumers, and to enhance the brand image of products, so that consumers have a sense of identity and belonging to such products, and then the retailer's own profits reach a high level. Third, government, as a policy maker, can formulate relevant policies to enhance consumers' awareness of purchasing environmental products. Moreover, the government can also establish an effective supervision mechanism and use public opinion to enhance consumers' demand for environmental friendliness products produced by marine ranching leading enterprises, thereby enhancing the overall profit level of the leading enterprises, retailers, and supply chain systems. In addition, the government should effectively use the amplification effect of financial leverage and guide social capital to jointly invest in the start-up marine ranching leading companies so as to promote the operation of enterprises. 7.3. Future Research This study discusses decision-making under different dominant modes, the action mechanism of product environmental attribute on the decision-making of the marine ranching leading company, and the guiding role of government investment in decision-making. The application of this model will help study the establishment and development of marine ranching from a multi-disciplinary perspective. However, this research can be further developed. Firstly, in this model, we only consider the influence of the product environmental attribute of the leading enterprise in marine ranching on the decision-making of the supply chain system. In the progress of actual management operations, in addition to the impact of the product environmental attribute on decision-making, other elements (such as advertising and public opinion) will also affect the decision-making system . Secondly, the supply chain financing decision of marine ranching will also be influenced by potential factors such as citizen science initiatives and social media platforms. Citizen science can be clearly designed and incorporated into fishery decision-making management , which is helpful for the long-term sustainability of marine resources fishing. Similarly, as a supplementary form of the government, the social media can restrain and supervise the behavior of the marine ranching leading enterprises and enhance the fairness of marine ranching transactions . Finally, this study introduces the government guidance fund, and through the guiding role of government investment, promotes social capital to jointly solve the dilemma of funds shortage of leading enterprises in marine ranching. However, according to Zheng et al. , although some coastal governments have established a government guidance fund, most of these marine industry funds are scattered, and investment and industrial scope are clearly limited and the scale of financing cannot meet the financial needs of marine ranching enterprises. These problems are the future research directions that need to be further expanded and improved. Future research should be further expanded and explored from the above two aspects, and the model can be enriched by introducing more influencing variables, such as advertising investment effort level , and the boundary of the constructed model can be expanded by increasing or reducing hypotheses. In addition, we can also consider the role of other parties (such as banks and insurance) in solving the shortage of funds in marine ranching construction. Author Contributions Conceptualization, X.W.; methodology, X.W., Z.T., J.S., Y.Z. and K.Z.; software, Z.T.; validation, X.W. and K.Z.; formal analysis, Z.T.; investigation, X.W., J.S., Y.Z. and K.Z.; resources, X.W., J.S., Y.Z. and K.Z.; data curation, X.W.; writing--original draft preparation, Z.T.; writing--review and editing, X.W., Z.T., J.S., Y.Z. and K.Z.; visualization, X.W., Z.T., J.S., Y.Z., and K.Z.; supervision, X.W.; project administration, X.W., Z.T., J.S., Y.Z. and K.Z.; funding acquisition, X.W. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Basic model of marine ranching supply chain under government guidance fund. Figure 2 The game sequence of the leading enterprise and the retailer in Mode 1. Figure 3 The game sequence of the leading enterprise and the retailer in Mode 2. Figure 4 The relationship between the product environmental attribute and the product's wholesale price. Figure 5 The relationship between the product environmental attribute and the product's retail price. Figure 6 The relationship between the product environmental attribute and the product demand. Figure 7 The relationship between the product environmental attribute and the retailer's profits. Figure 8 The relationship between the product environmental attribute and the marine ranching leading enterprise's profits. Figure 9 The relationship between the product environmental attribute and the supply chain system's overall profits. Figure 10 The relationship between the guiding role of government investment and the marine ranching leading enterprise's profits. Figure 11 The relationship between the guiding role of government investment and the supply chain system's overall profits. animals-13-00897-t001_Table 1 Table 1 List of notations. Notations Definition Model Parameters m0 The private capital of the leading company in marine ranching Ig The government investment I0 The initial investment of social capital Is The final investment of social capital, Is=I0(1+s) s The guiding role of government investment, s>=0 a The market capacity, a>0 b The product price coefficient, b>0 es The product environmental attribute, es>0 a The influence coefficient of the product environmental attribute on demand, a>0 c The production cost per unit product cI The financing cost Decision Variables p1, p2 The product retail price o1,o2 The product wholesale price Functions q The product demand pm The profits of the enterprise ps The profits of the retailer pt The profits of the supply chain system animals-13-00897-t002_Table 2 Table 2 Equilibrium results. Mode Dominated Be the Enterprise (Mode 1) Dominated by the Retailer (Mode 2) Product demand q1*=a+aes-bc4b q2*=a+aes-bc4b Product's retail price p1*=3a+3aes+bc4b p2*=3a+3aes+bc4b Product's wholesale price o1*=a+aes+bc2b o2*=a+aes+3bc4b Profits of the retailer ps1*=(a+aes-bc)216b ps2*=(a+aes-bc)28b Profits of the enterprise pm1*=m0(a+aes-bc)28b[m0+Ig+I0(1+s)]-cI pm2*=m0(a+aes-bc)216b[m0+Ig+I0(1+s)]-cI Profits of the supply chain system pt1*=(a+aes-bc)216b-cI+m0(a+aes-bc)28b[m0+Ig+(1+s)] pt2*=(a+aes-bc)28b-cI+m0(a+aes-bc)216b[m0+Ig+I0(1+s)] Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000071

Colorectal cancer (CRC) is one of the most common malignancies and is associated with high mortality rates worldwide. The underlying mechanism of tumorigenesis in CRC is complex, involving genetic, lifestyle-related, and environmental factors. Although radical resection with adjuvant FOLFOX (5-fluorouracil, leucovorin, and oxaliplatin) chemotherapy and neoadjuvant chemoradiotherapy have remained mainstays of treatment for patients with stage III CRC and locally advanced rectal cancer, respectively, the oncological outcomes of these treatments are often unsatisfactory. To improve patients' chances of survival, researchers are actively searching for new biomarkers to facilitate the development of more effective treatment strategies for CRC and metastatic CRC (mCRC). MicroRNAs (miRs), small, single-stranded, noncoding RNAs, can post-transcriptionally regulate mRNA translation and trigger mRNA degradation. Recent studies have documented aberrant miR levels in patients with CRC or mCRC, and some miRs are reportedly associated with chemoresistance or radioresistance in CRC. Herein, we present a narrative review of the literature on the roles of oncogenic miRs (oncomiRs) and tumor suppressor miRs (anti-oncomiRs), some of which can be used to predict the responses of patients with CRC to chemotherapy or chemoradiotherapy. Moreover, miRs may serve as potential therapeutic targets because their functions can be manipulated using synthetic antagonists and miR mimics. microRNAs colorectal cancer chemoresistance radioresistance predictive biomarkers Ministry of Science and TechnologyMOST 109-2314-B-037-046-MY3 MOST110-2314-B-037-097 MOST 111-2314-B-037-070-MY3 MOST 111-2314-B-037-049 Ministry of Health and WelfareMOHW111-TDU-B-221-114014 Kaohsiung Medical University (KMU) HospitalKMUH110-0R37 KMUH110-0R38 KMUH110-0M34 KMUH110-0M35 KMUH110-0M36 KMUH-DK(B)110004-3 KMU Center for Cancer ResearchKMU-TC111A04-1 KMU Office for Industry-Academic CollaborationS109036 Taiwan Precision Medicine InitiativeTaiwan BiobankThis work was supported by grants from the Ministry of Science and Technology (MOST 109-2314-B-037-046-MY3, MOST110-2314-B-037-097, MOST 111-2314-B-037-070-MY3, MOST 111-2314-B-037-049) and the Ministry of Health and Welfare (MOHW111-TDU-B-221-114014, financed by the Tobacco Health and Welfare Surcharge), Kaohsiung Medical University (KMU) Hospital (KMUH110-0R37, KMUH110-0R38, KMUH110-0M34, KMUH110-0M35, KMUH110-0M36, KMUH-DK(B)110004-3), the KMU Center for Cancer Research (KMU-TC111A04-1), and the KMU Office for Industry-Academic Collaboration (S109036). In addition, this study was supported by a grant from the Taiwan Precision Medicine Initiative and Taiwan Biobank (Academia Sinica, Taiwan, R.O.C.). pmc1. Introduction Colorectal cancer (CRC) is a major public health problem among both sexes and has a worldwide mortality rate of 47.8% . According to the annual report of the Health Promotion Administration in USA, each year, more than 1.1 million patients receive diagnoses of CRC worldwide and approximately 608,000 CRC-related mortalities occur, making CRC the third most common cause of death . Approximately 20-30% of patients with stage I-III CRC who undergo a surgical resection eventually develop distant metastasis, which is associated with poor prognoses . Researchers must continue investigating biomarkers that can be used to more accurately identify patients with CRC who are at a high risk of recurrence. Adjuvant or neoadjuvant FOLFOX chemotherapy, which involves the use of 5-fluorouracil (5-FU), leucovorin (LV), and oxaliplatin, is widely used to reduce the risk of recurrence in patients with advanced-stage CRC . Even after undergoing radical surgical resection or oxaliplatin-based adjuvant chemotherapy, some patients ultimately develop recurrence or metastasis, indicating that current treatments for CRC are insufficient . Genomic and metabolomic analysis of right-sided and left-sided CRC are keystones in the study and treatment of subtypes of CRC . In patients with metastatic CRC (mCRC), two signaling pathways--the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) pathways--are involved in the proliferation and metastasis of CRC . FOLFIRI (5-FU, LV, and irinotecan) plus anti-EGFR monoclonal antibodies provides a survival benefit to patients with mCRC with distant metastases . For patients with right-sided mCRC, FOLFIRI or FOLFOXIRI (5-FU, LV, irinotecan, and oxaliplatin) plus bevacizumab (an anti-VEGF monoclonal antibody) are preferred first-line treatment options, irrespective of the patient's RAS and B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutational statuses . For patients with locally advanced rectal cancer (LARC), neoadjuvant chemoradiotherapy (NACRT) is a standard treatment that can improve the outcomes of radical resection, prevent local recurrence, ensure sphincter preservation and tolerable toxicity levels, and maintain postsurgical quality of life . However, patient responses to NACRT are highly variable , and resistance to chemoradiotherapy (CRT) is a major obstacle in the treatment of patients with LARC . In patients with CRC, epigenetic modifications, including DNA methylation and histone modifications, can be used as clinical biomarkers for diagnosis, prognosis, and the prediction of patient responses to adjuvant or neoadjuvant therapy . Liquid biopsies employ a wide range of technologies to acquire tumor information, including levels of carcinoembryonic antigen (CEA), circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), circular RNAs (circRNAs), and microRNAs (miRs) in body fluids . CEA is widely used as a surrogate biomarker in clinical practice for treatment response prediction and follow-ups in patients with CRC . Moreover, the serum levels of CEA and expression levels of EGFR and FGD5-AS1 (FGD5 Antisense RNA 1, an oncogenic long non-coding RNA) have been observed to be significantly elevated in 5-FU-resistant CRC cells . Using CTCs as individualized biomarkers can help healthcare providers develop effective treatment strategies for patients with CRC. The persistent presence of CTCs after adjuvant chemotherapy indicates chemoresistance and is often reflected in subsequent recurrence of CRC . In addition, the progression of CRC can be modulated via circRNAs, which can sponge and downregulate target miRs . In CRC, miRs have been determined through gene network analysis to act as both oncogenes and tumor suppressors of differentially expressed mRNAs and proteins . MiRs, small noncoding RNAs consisting of approximately 20 nucleotides, can posttranscriptionally regulate the expression of several target genes by directly binding to the 3' untranslated regions (3'-UTRs) of target mRNAs, thereby triggering mRNA degradation, suppressing mRNA translation, and subsequently regulating the protein expression . The heterogeneity of CRC makes its accurate classification and treatment challenging . The specific panel of miRNAs/mRNAs or miR clusters can contribute to the progression from adenomatous and carcinomatous lesions . Four consensus molecular subtypes (CMSs) were identified via the classification of patients with CRC in biologically homogeneous CRC subtypes, and mesenchymal CMS4 presented with a worse prognosis . Plasma miR profiling has been observed to be significantly associated with colorectal cancer CMS and validated for predicting both prognosis and treatment response . For example, the miR-200 family is the most powerful determinant of CMS4-specific gene expression, which is associated with epithelial-mesenchymal transition . The analysis of serum miRNAs is less invasive and still facilitates a real-time analysis of the disease course . To date, researchers have identified at least 250 miRs that may serve as diagnostic biomarkers as well as prognostic indicators of CRC . Therefore, miRs have the potential to serve as biomarkers for cancer detection, prognosis, CMS classification, and treatment response prediction . MiR panels have been reported to facilitate early detection of relapse in patients with CRC . MiRs are attractive as biomarker candidates because they can be identified through liquid biopsies, which makes them minimally invasive and more convenient for use in the early detection of relevant signals . Several reviews have discussed miRs as biomarkers in CRC , and even textbooks mention the potential cancer-sensitizing agents for CRC chemotherapy . Stiegelbauer et al. (2014) summarized the chemotherapeutic approaches for treating CRC and highlighted the role of miRNAs as predictive biomarkers for chemoresistance in patients with CRC . In their meta-analysis, Masuda et al. concluded that miRs have strong statistical confidence as biomarkers for the early detection of CRC and prediction of prognosis and chemoresistance, but they also indicated that most miR reports were small-scale studies until 2017 . Shirafkan et al. summarized the roles of miRs in CRC by emphasizing their importance in different signaling pathways, such as the EGFR, transforming growth factor beta (TGF-b), and tumor protein (TP53) pathways, and suggested miRs as predictive factors of chemotherapy . Elucidating the mechanisms underlying the development of resistance to chemotherapy and radiotherapy is crucial to the development of more effective cancer treatment strategies. However, no reviews have focused on miRs as predictors of response to chemoradiotherapy in patients with CRC. Numerous studies have explored the activation of regulatory pathways involving miRs in response to chemotherapy, radiotherapy, or antitumor agents, and miRs are attractive candidates for targeted therapy because their functions can be manipulated through the use of synthetic antagonists and miR mimics . No miRNA-based drugs are currently on the market; however, many RNA-based therapies, including antisense oligonucleotides, aptamers, small interfering RNAs, miRs, and mRNA, are currently undergoing clinical trials or have already received regulatory approval (including as treatments for liver cancer, lymphoma, and melanoma) . Identifying a reliable biomarker for predicting chemoresistance or radioresistance in patients with CRC may improve survival outcomes . In this review, we would discuss how these miRs can serve as clinical biomarkers in the early detection of CRC or mCRC and as predictive biomarkers in CRC treatment. 2. Methods We searched PubMed, the Cochrane Central Register of Controlled Trials, and the Cochrane Library for relevant studies. We did not apply any language or regional restrictions. We used the following MeSH terms in our search: ("Colorectal Neoplasm"[Title/Abstract]) AND (MicroRNA*[Title/Abstract]) AND ("Chemotherapy"[Title/Abstract] OR "Chemoradiotherapy"[Title/Abstract] OR "Neoadjuvant Radiotherapy [Title/Abstract] OR "Neoadjuvant Chemotherapy*"[Title/Abstract]). We thoroughly evaluated all the relevant studies and bibliographies to identify additional potentially eligible studies. 3. Results 3.1. MiRs Associated with the Detection of CRC and mCRC MiRs may act as oncogenes or tumor suppressors and may serve as biomarkers for the early diagnosis of CRC to facilitate efficient treatment . The expression levels and functions of miRs in various body extracts (including serum, tumor tissues, feces, or urine) have been analyzed in several case-control studies (Table 1). Expression levels of miRs in feces or urine can be used for noninvasive screening for the detection of CRC. Researchers developed a urinary biomarker panel combining miR-129-1-3p and miR-566 that could accurately detect stage 0/I CRC . The expression levels of miR-29a, miR-223, and miR-224 in fecal samples from patients with CRC were all significantly lower than those in fecal samples from healthy volunteers (all p < 0.001) . A systematic review revealed that the expression levels of miR-20a in the feces, serum, or tumor tissues of patients with CRC were upregulated relative to those in the control samples, indicating that miR-20a may serve as an accurate biomarker for CRC detection . MiR-106a and miR-125b are associated with the pathogenesis of CRC and may therefore also be used as significant prognostic markers of early-stage CRC . The overexpression of miR-21 in CRC tumor tissue was significantly associated with advanced CRC, but interestingly, the lower expression levels of serum miR-21 were associated with a higher mortality rate . MiRs are used not only in the diagnosis of primary CRC but also in the prediction of early relapse of CRC. In several case-control studies including patients with UICC stage II and III CRC, the miR-29c expression levels in the tumor tissues of the early relapse group were significantly lower than those in the tumor tissues of the non-early relapse group . Tsai et al. reported that serum miR-148a expression in the early relapse group was significantly lower than that in the non-early relapse group . In another study, lower miR-148a expression in CRC tissues was positively associated with an advanced TNM stage, poor tumor differentiation, lymph node metastasis, and distant metastasis . Researchers have identified some miRs that can be used to predict the development of metastasis in CRC. Chen et al. reported that plasma miR-96/miR-99b can be used as a biomarker for the early detection of mCRC . In another study, the circulating miR-762 levels of patients with CRC with distant metastasis were higher than those of patients with CRC without distant metastasis . Pidikova and Herichova discovered that the miR-17/92a-1, miR-106a/363, miR-106b/93/25, and miR-183/96/182 clusters were strongly associated with metastasis and poor patient survival . Hoye et al. analyzed various next-generation sequencing data sets of samples of primary CRC and mCRC (liver, lung, and peritoneal metastases) and tumor-adjacent tissues and identified five miRNAs--miR-210-3p, miR-191-5p, miR-141-3p, miR-1307-5p, and miR-155-5p--that were upregulated at multiple metastatic sites , which may serve as distinguishing biomarkers of mCRC. Several miR panels have been established on the basis of case-control studies. For example, Wang S. et al. developed a serum panel of miR-409-3p, miR-7, and miR-93 that could accurately discriminate between the plasma samples of patients with CRC and healthy controls . Another serum miR panel comprising miR-203a-3p, miR-145-5p, miR-375-3p, and miR-200c-3p could also discriminate between the plasma samples of patients with CRC and healthy controls with a sensitivity of 81.25% and a specificity of 73.33% . Another panel combining six clinicopathologic factors with six miRs (miR-7, miR-93, miR-195, miR-141, miR-494, and let-7b) could be used to detect early relapsed CRC with a sensitivity of 89.4% and a specificity of 88.9% . 3.2. MiRs Associated with the Prediction of Responses to Chemotherapy in CRC Resistance to chemotherapy is one of the most common reasons for treatment failure among patients with CRC, and many patients with advanced CRC are initially responsive to chemotherapy but ultimately develop chemoresistance . OncomiRs, which can increase chemoresistance, may reduce the levels of apoptosis proteins and silent apoptosis information and neutralize or reverse antiapoptotic signals . Table 2 lists oncomiRs that can enhance chemoresistance through specific regulatory pathways. The transcription factor p53 is the most thoroughly characterized tumor suppressor gene, and p53 variants appear in approximately 50% of patients with CRC . Loss or mutation of the p53 gene and its related pathways, such as those involving TP53INP1 and TP53INP2 or Bcl-2 and caspase proteins, is positively associated with therapeutic resistance in various cancers . Several oncomiRs, including let-7f-5p , and miR-96 , are overexpressed in patients with chemoresistant CRC (relative to patients with chemosensitive CRC) (Table 2). MiR-34a may serve as a predictor of 5-FU chemosensitivity in CRC, and a combination of miR-34a and 5-FU is effective as a treatment for CRC . Chemoresistance may be related to the AMP-activated protein kinase-mammalian target of the rapamycin (AMPK-mTOR) pathway, and several miRs, including miR-27a, miR-103, and miR-107, are overexpressed in patients with chemoresistant CRC (relative to patients with chemosensitive CRC) . Overexpression of miR-744 may mediate oxaliplatin chemoresistance in CRC by suppressing BIN1 expression . Chemoresistance was correlated with cancer cell stemness in patients with CRC , and patients expressing the CD44 variant appear to present with more aggressive phenotypes of CRC. Moreover, miR-1246 was overexpressed in CD44v6+ cells and was associated with poor overall survival and disease-free survival in patients with CRC . Table 3 presents anti-oncomiRs that can modulate patients' sensitivity to chemotherapy. Overexpression of miR-141-3p negatively regulates epithelial-mesenchymal transition (EMT) and can restore chemosensitivity; in a previous study, the 5-year overall survival of the high miR-141-3p expression group was superior to that of the low miR-141-3p expression group . Overexpression of miR-377-3p (miR-154 family) or miR-193b-5p also enhanced chemosensitivity to 5-FU in CRC cells by negatively regulating EMT or the forkhead box M1-ATP-binding cassette subfamily C member 5 (FOXM1-ABCC5/10) signaling pathway and decreasing cell stemness . Xiao et al. demonstrated that miR-1915-3p can improve the chemotherapeutic efficacy of oxaliplatin in CRC cells by suppressing EMT-promoting oncogenes, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), and ubiquitin specific peptidase 2 (USP2) . The mechanism of 5-FU-induced cytotoxicity in CRC cells involves the inhibition of thymidylate synthase (TS), a 5-FU target enzyme. In one analysis of clinical CRC samples, the miR-375 expression levels in the 5-FU-resistant group were much lower than those in the 5-FU-sensitive group . MiR-375 can enhance 5-FU cytotoxicity in CRC cells by suppressing TS and the Sp1 transcription factor (SP1) . High miR-218 and miR-330 expression levels had a positive prognostic value in 5-FU-based treatments for CRC, and miR-218 inhibits TS . In one study, lower miR-148a expression was associated with advanced CRC and distant metastasis, and overexpression of miR-148a suppressed the expression of stem cell markers and increased chemosensitivity by regulating the b-catenin signaling pathway . In a study by Huang et al., upregulated miR-148a enhanced chemoradiosensitivity and promoted apoptosis of CRC cells by targeting the MET proto-oncogene, receptor tyrosine kinase (c-Met), in vitro and in vivo . High miR-148a expression levels were associated with more favorable tumor responses to neoadjuvant CRT and survival outcomes . By conducting a Kaplan-Meier survival analysis of a sample of 62 patients, researchers determined that miR-27b-3p expression levels are positively correlated with disease-free survival . The MYC proto-oncogene, bHLH transcription factor (c-Myc), can downregulate miR-27b-3p expression, inducing oxaliplatin resistance in CRC cells by inhibiting autophagy . Diabetes mellitus and hyperglycemia have been demonstrated to affect chemoresistance in and the prognosis of CRC . In one study, miR-488 mimic decreased glucose uptake and increased oxaliplatin/5-FU-sensistivity in CRC cells by targeting the oncogene, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) ; the miR-488 expression levels of patients with metastatic/recurrent CRC were significantly lower than those of patients with CRC without metastasis/recurrence; and low miR-488 expression levels were associated with low 3-year survival rates, poor differentiation, and advanced-stage disease . Another study demonstrated that tumor-secreted miR-208b promotes Treg expansion by targeting programmed cell death factor 4 (PDCD4) and may be related to chemoresistance to oxaliplatin in CRC . In one study, the diagnostic accuracy (in terms of area under the curve [AUC]) of CEA and CA19-9 (for distinguishing between oxaliplatin-chemoresistant and oxaliplatin-chemosensitive patients) was 0.542 and 0.686, respectively, whereas a panel containing miR-100, miR-92a, miR-16, miR-30e, miR-144-5p, and let-7i achieved the highest diagnostic accuracy, with an AUC of 0.825 . Comparing plasma miR levels with CEA and CA19-9 in the follow-up of patients with CRC, CEA, and CA19-9 showed a higher specificity for CRC but a lower sensitivity than miRs in predicting disease recurrence . EGFR expression serves as a prognostic factor in patients with stage III CRC receiving metronomic maintenance therapy . In one study, the inhibition of EGFR by the specific inhibitor erlotinib effectively enhanced the antitumor toxicity of 5-FU though miR-330 directly targeting thymidylate synthase . For patients with mCRC with RAS variations, one therapeutic alternative is targeting and preventing angiogenesis by inhibiting VEGF. FOLFIRI plus bevacizumab can increase sensitivity to and enhance the antitumor effects of 5-FU (Table 3) . Upregulation of miR-1207-5p can suppress bevacizumab resistance in bevacizumab-resistant CRC cells by modulating the expression of ATP-binding cassette subfamily C member 1 (ABCC1) . Lower miR-1287-5p expression levels upregulate the expression of multifunctional Y-box binding protein 1 (YBX1) and are associated with cetuximab resistance in CRC . Circulating plasma levels of miR-20b, miR-29b, and miR-155 serve as predictors of bevacizumab efficacy in patients with mCRC (Table 1) . In one study, the serum miR-148a expression levels of patients with mCRC who exhibited partial responses to treatment were higher than those of patients with mCRC with disease progression . MiR-148a decreases angiogenesis and increases the apoptosis of CRC cells by downregulating hypoxia-inducible factor 1 subunit alpha (HIF-1a)/VEGF and the MCL1 apoptosis regulator, BCL2 family member (Mcl-1), and serum miR-148a levels have prognostic and predictive value in patients with mCRC receiving bevacizumab therapy . 3.3. MiRs Associated with the Prediction of Responses to Radiotherapy in CRC Radiotherapy induces various DNA lesions and unpaired double-strand breaks; therefore, biomolecules that inhibit DNA repair pathways usually improve radiosensitivity . Radiation-induced DSBs principally activate the intrinsic apoptotic pathway, and two principal methods are involved in DSB repair: homologous recombination (HR) and nonhomologous end joining (NHEJ) . MiRs are some of the most important mediators of radiosensitivity in CRC cells and are involved in the HR and NHEJ repair pathways . Huang et al. reviewed and discussed the biological functions of miRNAs in the regulation of radiosensitivity in CRC cells . However, some investigations of radioresistance in CRC cells were in vitro studies in which CRC cell lines were transfected with miR mimics or knocked out to modulate the expression of various miRs, such as miR-101-3p , miR-185 , miR-195 , and miR-5197 ; these miRs were omitted from Table 4. By analyzing the differentially expressed miRNA profiles of patients with CRC, researchers have identified several oncomiRs that enhance radioresistance as biomarkers of responses to radiotherapy (Table 4). p21 is a tumor suppressor gene that can protect cancer cells from DNA damage . The overexpression of several miRs, including miR-106b and miR-222, can inhibit p21 expression, thereby inducing radioresistance through the phosphatase and tensin homolog/phosphatidylinositol-3 kinase/AKT serine/threonine kinase 1 (PTEN/PI3K/AKT) pathway, in vitro and in vivo . MiR-93 and miR-106b are categorized into the same family, and upregulated miR-93 induces radioresistance by downregulating forkhead box A1 (FOXA1) and upregulating transforming growth factor beta 3 (TGFB3) . Exosomes containing miR-93-5p derived from cancer-associated fibroblasts can prevent CRC cells from undergoing radiation-induced apoptosis . MiR-96-5p also reportedly induces radioresistance in rectal cancer cells by inhibiting glypican 3 (GPC3) and abnormally triggering the canonical Wnt signaling pathway (Table 4) . MiR-19b targets the F-box and WD repeat domain containing 7 (FBXW7), thereby promoting CRC stem cell stemness and inducing radioresistance, and in one study, patients with LARC with low miR-19b expression levels had markedly longer overall survival and event-free survival . cancers-15-01358-t004_Table 4 Table 4 OncomiRs that enhance radioresistance in CRC and mCRC. Family MiRs Verified Targets in CRC or Other Cancers Sample Source Target for miRNA Ref. miR-17 miR-17, miR-18a/b, miR-106a/b, miR-20a/b, miR-93 MiR-106b induces cell radioresistance by targeting the PTEN/PI3K/AKT pathways and p21 in CRC. MiR-93 acts as a specific exosomal cargo that increases radioresistance. Inhibition of miR-93 suppressed radioresistance. Tumor tissue Tumor tissue PTEN/PI3K/AKT pathways and p21 FOXA1 BTG3 miR-19 miR-19a, miR-19b-1, miR-19b-2 The low miR-19b expression levels of patients with LARC had markedly longer OS and DFS. MiR-19b induces radioresistance, and the patients with higher miR-19b expression levels had a shorter survival time. Tumor tissue FBXW7 miR-96 miR-96 miR-96-5p induced radioresistance is upregulated in rectal cancer cells through the inhibition of GPC3 and abnormal triggering of the canonical Wnt signaling pathway. Tumor tissue GPC3 miR-103 miR-103a/b, miR-107 MiR-107 induces chemoresistance. Hsa-mir-107 and WDFY3-AS2 may serve as prognostic biomarkers in RC. Tumor tissue Through the CAB39-AMPK-mTOR pathway miR-221 miR-221, miR-222 MiR-222 induces radiation resistance. Serum PTEN. Likewise, high miR-125b expression levels in tissue and serum were associated with a poor treatment response in patients with LARC (Table 5). By analyzing receiver operating characteristic curves, D'Angelo et al. demonstrated that circulating miR-125b levels exhibited greater discriminatory power for treatment responses than serum CEA levels did; therefore, miR-125b may serve as a new noninvasive predictive biomarker in the treatment of LARC . Recent clinical findings have demonstrated that cancer stem cells and their inherent radioresistance are crucial to local control after radiotherapy . Differentially expressed miRs, such as miR-148a and miR-214, were observed in radiated CRC cells. MiR-214 promotes radiosensitivity by inhibiting autophagy-related 12 (ATG12)-mediated autophagy in CRC . Comparisons of the serum miR expression levels of patients with CRC before or after radiation therapy have revealed that radiation therapy can significantly reduce serum miR-296-5p expression. Moreover, miR-296-5p overexpression significantly reduced the survival fraction of CRC cells under ionizing radiation (IR) treatment by targeting the insulin-like growth factor 1 receptor (IGF1R) and musashi RNA binding protein 1 (MSI1) . Therefore, miRs serve as potential regulators of radioresistance in CRC cells and, in turn, may be a promising therapeutic target in the treatment of CRC. 3.4. MiRs Associated with the Prediction of Responses to Chemoradiotherapy in CRC The present study focused on oncomiRs and anti-oncomiRs modulating the sensitivity of chemoradiotherapy, and we noted that studies on chemotherapy- or radiotherapy-related miRs far outnumber those on chemoradiotherapy-related miRs (Table 6). Huang et al. demonstrated that alterations in miR-148a overexpression may enhance chemoradiosensitivity and promote apoptosis by directly targeting c-Met in vivo in both human CRC cells and mice . Moreover, miR-34a attenuates chemoradioresistance in CRC . MRX34, a miR-34a mimic, is the first synthetic miR to undergo clinical trials to restore the sensitivity of CRC cells to chemotherapeutic agents . 3.5. Current Undergoing Clinical Trials for miRs After the discovery of the dysregulation of miRNA expression itself being associated with human disease progress and therapy response, the introduction of a disease suppressor miRNA mimic to restore its functionality is one therapeutic approach under careful consideration . To date, dozens of miRNA molecules are in clinical trials , such as: miravirsen (miR-122) for the treatment of hepatitis C virus (HCV) infection, under phase II clinical trials in several countries, Remlarsen (miR-29) for the treatment of different type of fibrosis, under phase I clinical trials, and MRX34 (miR-34a) for the treatment of different types of cancers . MRX34 is a synthetic, double-stranded miR-34a mimic that encapsulated in a liposomal nanoparticle for the treatment of different types of advanced solid tumors . In the first-in-human Phase 1 study of MRX34-based cancer therapy, Hong et al. (2020) revealed that the need to anticipate toxic effects for this class of miR-based drug and the effective delivery of these RNA constructs also remains an unresolved challenge . 4. Conclusions As highlighted in this review, miRs have considerable potential as predictive biomarkers in patients with CRC receiving chemotherapy or chemoradiotherapy; therefore, miRs warrant the further investigation in prospective clinical studies. The clinical roles of miRs in the regulation of chemosensitivity and radiosensitivity in CRCs must be further explored. By interacting with other miRs, mRNA, DNA, or proteins, miRs can modulate responses to chemotherapy and radiotherapy. Although miRs have great potential as predictive biomarkers in guiding precision medicine or as therapeutic targets to improve chemosensitivity and radiosensitivity in the treatment of CRC and mCRC, the use of miRs in clinical practice warrants further investigation. Acknowledgments This work was supported by grants through funding from the Ministry of Science and Technology (MOST 109-2314-B-037-046-MY3, MOST 111-2314-B-037-070-MY3, MOST 111-2314-B-037-049) and the Ministry of Health and Welfare (12D1-IVMOHW02)and funded by the health and welfare surcharge of on tobacco products, and the Kaohsiung Medical University Hospital (KMUH111-1R31, KMUH111-1R32, KMUH111-1M28, KMUH111-1M29, KMUH111-1M31)and Kaohsiung Medical University. In addition, this study was supported by the Grant of Taiwan Precision Medicine Initiative and Taiwan Biobank, Academia Sinica, Taiwan, R.O.C. Author Contributions Conceptualization, J.-Y.W.; methodology, I.-P.Y., K.-L.Y. and Y.-T.C.; formal analysis, I.-P.Y., K.-L.Y. and Y.-T.C.; writing--original draft preparation, I.-P.Y.; writing--review and editing, Y.-C.C., C.-W.H., H.-L.T., Y.-S.Y. and J.-Y.W.; visualization, I.-P.Y., K.-L.Y. and Y.-T.C.; supervision, J.-Y.W.; funding acquisition, J.-Y.W. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest The authors declare no conflict of interest. Figure 1 MiRs associated with the detection of CRC and mCRC, the prediction of early relapse of CRC, and the prediction of responses to chemotherapy/radiotherapy/chemoradiotherapy in CRC and mCRC. Red words indicate the oncomiRs enhance chemoresistance/radioresistance and blue words indicate the anti-oncomiRs that enhance chemosensitivity/radiosensitivity in CRC and mCRC. cancers-15-01358-t001_Table 1 Table 1 MiRs used in the detection of CRC and mCRC. MiRs Used in the Diagnosis of Primary CRC Sample Source Ref. miR-20a MiR-20a was upregulated in patients with CRC (relative to controls) and may be a valid biomarker for CRC detection but may not be a strong prognostic indicator. Feces, serum, and tumor tissue miR-21 The high expression of miR-21 was significantly correlated with advanced clinical stage and poor cell differentiation. Tumor tissue miR-29a, miR-223, miR-224 The expression levels of miR-29a, miR-223, and miR-224 from patients with CRC were significantly lower than those from health volunteers. Feces miR-106a, miR-125b MiR-106a and miR-125b were associated with the pathogenesis and invasion of CRC and may be used as significant prognostic markers of early-stage CRC. Tumor miR-129-1-3p, miR-566 A urinary biomarker panel combining miR-129-1-3p and miR-566 could accurately detect stage 0/I CRC. Urinary samples MiRs Used in the Prediction of Early Relapse of CRC Sample Source Ref. miR-21 Lower serum miR-21 expression was associated with higher local recurrence (p = 0.025) and mortality (p = 0.029). Serum miR-29c miR-29c expression in the early relapse group was significantly lower than that in the non-early relapse group. Tumor tissue miR-93 The miR-93 expression levels of the early relapse group were significantly lower than those of the non-early relapse group. The in vitro and in vivo effects of miR-93 overexpression were inhibited by CRC proliferation and migration, and miR-93 decreased CRC recurrence. Tumor tissue miR-148a miR-148a expression levels in the early relapse group were significantly lower than those in the non-early relapse group. MiR-148a inhibits VEGF secretion by indirectly targeting hypoxia-inducible factor 1 subunit alpha (HIF-1a). Tumor tissue and serum Lower miR-148a expression was positively associated with advanced TNM stage, poor tumor differentiation, lymph node metastasis, and distant metastasis. Tumor tissue MiRs Used in the Diagnosis of mCRC Sample Source Ref. miR-17/92a-1, miR-106a/363, miR-106b/93/25, miR-183/96/182 clusters The miR-17/92a-1, miR-106a/363, miR-106b/93/25, and miR-183/96/182 clusters were strongly associated with metastasis and poor patient survival. Tumor tissue, blood, and feces miR-20b, miR-29b, miR-155 A multivariate analysis of patients with mCRC receiving bevacizumab-based treatment revealed that circulating expression levels of miR-20b, miR-29b, and miR-155 were significantly associated with progression-free survival (p < 0.05) and overall survival (p < 0.05). Serum miR-96/miR-99b Plasma miR-96/miR-99b expression levels may serve as a promising biomarker for the early detection of mCRC. Plasma miR-210-3p, miR-191-5p, miR-141-3p, miR-1307-5p, miR-155-5p Five miRNAs--miR-210-3p, miR-191-5p, mir-141-3p, miR-1307-5p, and miR-155-5p--were determined to be upregulated at multiple metastatic sites according to an analysis of new and previously published next-generation sequencing data sets of samples of primary CRC and mCRC (liver, lung, and peritoneal metastases) and tumor-adjacent tissues. Tumor tissue miR-762 The circulating miR-762 levels of patients with CRC with distant metastasis were higher than those of patients with CRC without distant metastasis. Serum cancers-15-01358-t002_Table 2 Table 2 OncomiRs enhance chemoresistance in patients with CRC or mCRC. Family miRNAs Verified Targets in CRC or Other Cancers Sample Target for miRNA Ref. Let-7 Let-7a/b/c/d /e/f/g/i, miR-98 Upregulation of let-7f-5p promotes chemoresistance in CRC by increasing the expression levels of the antiapoptotic proteins B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma extra-large (Bcl-xL) and decreasing the activity of caspase-3 and caspase-9 in CRC cells. Tumor tissue p53, p53-inducible nuclear protein 1, p53-inducible nuclear protein 2 and caspase-3. miR-27 miR-27a, miR-27b MiR-27a-overexpressing hampered AMPK, enhanced mTOR signaling, unrestricted cell growth, and enhanced chemoresistance. Tumor tissue AMPK miR-96 miR-96 Inhibition of miR-96 enhances the sensitivity of CRC cells to oxaliplatin. Serum TPM1 miR-103 miR-103a/b, miR-107 MiR-107 induces chemoresistance in CRC cells. Tumor tissue CAB39-AMPK-mTOR pathway MiR-103 and miR-107 enhance chemoresistance in CRC cells by promoting cell stemness. Wnt/b-catenin signaling miR-744 miR-744 The expression levels of miR-744 were significantly elevated in CRC tissues from patients who exhibited preoperative oxaliplatin chemoresistance. MiR-744 may positively mediate oxaliplatin chemoresistance. Tumor tissue BIN1 miR-1246 miR-1246 MiR-1246 was overexpressed in CD44v6+ cells and associated with poor overall survival and disease-free survival in patients with CRC. CD44v6+ cells exhibited elevated resistance to chemotherapeutic drugs and significantly higher tumor initiation capacity. Tumor tissue DENN/MADD Domain Containing 2D (DENND2D) cancers-15-01358-t003_Table 3 Table 3 Anti-oncomiRs enhance chemosensitivity in patients with CRC or mCRC. Family miRNAs Verified Targets in CRC or Other Cancers Sample Source Target for miRNA Ref. miR-8 miR-8, miR-141, miR-200a/b/c, miR-429 MiR-141-3p enhanced the cetuximab sensitivity of CRC cells Tumor tissue ZEB1-ZEB2 Expression of miR-200c and miR-141 was downregulated in oxaliplatin-resistant CRC cell lines. EGFR miR-27 miR-27a, miR-27b-3p MiR-27b-3p sensitizes CRC cells to oxaliplatin in vitro and in vivo, and miR-27b-3p expression was positively correlated with disease-free survival time in patients with CRC. Tumor tissue ATG10 miR-29 miR-29a, miR-29b, miR-29c Circulating miR-20b, miR-29b, and miR-155 expression levels were significantly associated with progression-free survival (p < 0.05) and overall survival (p < 0.05). Serum No data miR-34 miR-34a/b/c/d MiR-34a enhanced chemosensitivity to 5-FU. Serum E2F3; SIRT1. miR148 miR-148a/b, miR-152 MiR-148a suppressed the expression of stem cell markers and increased chemosensitivity, cell invasion, and cell migration. Tumor tissue WNT10b and beta-catenin signaling pathway. MiR-148a decreased angiogenesis and increased CRC cell apoptosis by downregulating HIF-1a/VEGF and Mcl-1, and serum miR-148a levels have prognostic or predictive value in patients with mCRC receiving bevacizumab. miR-154 miR-154, miR-323a, miR-369-3p, miR-377, miR-381, miR-382, miR-409, miR-410 MiR-377-3p expression levels in CRC samples (especially those from patients with stage III/IV CRC) were significantly lower than those in normal mucosa tissues. Overexpression of miR-377-3p enhanced the chemosensitivity of CRC cells by inhibiting Wnt/beta-catenin signaling by directly targeting ZEB2 and XIAP, which are positive regulators of Wnt/b-catenin signaling. Tumor tissue ZEB2 and XIAP MiR-382 functions as a tumor suppressor and chemosensitizer in CRC. miR-155 miR-155 Circulating expression levels of miR-20b, miR-29b, and miR-155 were significantly associated with progression-free survival (p < 0.05) and overall survival (p < 0.05). Serum No data MiR-155 induced radioresistance by targeting FOXO3a. FOXO3a. miR-193 miR-193a/b MiR-193b-5p enhanced chemosensitivity to 5-FU. Tumor tissue HMGA2/MAPK pathway CRC tissues and adjacent noncancerous tissues were obtained from 67 patients who had undergone surgery. Upregulation of miR-193-5p, particularly in combination with 5-FU and oxaliplatin, reduced the expression levels of CXCR4. A miR-193a-5p mimic suppressed CXCR4-induced CRC cell proliferation. CXCR4. miR-218 miR-218-1/2 MiR-218 enhanced 5-FU cytotoxicity by suppressing thymidylate synthase and MiR-218 promoted apoptosis, inhibited cell proliferation, and caused cell cycle arrest Tumor tissue thymidylate synthase; BIRC5 miR-330 miR-330 MiR-330 inhibited CRC cell proliferation and enhanced CRC cell chemosensitivity to 5-FU Tumor tissue Hexokinase 2 Thymidylate synthase miR-375 miR-375-3p MiR-375 enhanced CRC cell chemosensitivity to 5-FU by directly targeting YAP1 and SP1. Tumor tissue YAP1 and SP1 MiR-375 enhanced CRC cell chemosensitivity to 5-FU by targeting thymidylate synthase. Tumor tissue thymidylate synthase miR-488 miR-488 MiR-488 mimics transfected into CRC cell lines induced decreases in glucose uptake and increases in oxaliplatin/5-FU chemosensistivity. Serum PFKFB3 miR-1207 miR-1207-5p Upregulation of miR-1207-5p inhibited bevacizumab resistance in CRC cells. Tumor tissue ABCC1. miR-1287 miR-1287-5p Lower miR-1287-5p expression levels upregulate the mRNA expression of Y-box binding protein 1 (YBX1) and protein levels of YBX1, thereby inducing CRC cell proliferation and migration. Tumor tissue YBX1 The multifunctional YBX1 is overexpressed and phosphorylated in CRC and is associated with cetuximab resistance. miR-1915 miR-1915 Exosomal delivery of miR-1915-3p can improve the chemosensitivity of oxaliplatin by suppressing the epithelial-mesenchymal transition. Plasma PFKFB3 and USP2 cancers-15-01358-t005_Table 5 Table 5 Anti-oncomiRs that enhance radiosensitivity in CRC and mCRC. Family MiRs Verified Targets in CRC or Other Cancers Sample Source Target for miRNA Ref. miR-10 miR-10a/b, miR-99a/b, miR-100, miR-125a/b-1/b-2 MiR-125b was highly expressed both in tissues and serum obtained from nonresponders to CRT. Circulating miR-125b levels exhibited greater discriminatory power for treatment responses than did serum CEA levels. Serum No data miR-155 miR-155 Circulating miR-20b, miR-29b, and miR-155 levels were significantly associated with progression-free survival (p < 0.05) and overall survival (p < 0.05). MiR-155 induced radiation resistance. Serum No data FOXO3a miR-214 miR-214 MiR-214 promoted radiosensitivity by inhibiting ATG12-mediated autophagy in CRC. Tumor tissue ATG12 miR-296 miR-296-5p MiR-296-5p enhanced the radiosensitivity. Serum IGF1R; MSI1 cancers-15-01358-t006_Table 6 Table 6 MiRs involved in chemoradiosensitivity or chemoradioresistance in CRC and mCRC. Anti-OncomiRs That Enhance Chemoradiosensitivity Family MiRs Verified Targets in CRC or Other Cancers Sample Source Target for miRNA Ref. miR-148 miR-148a/b, miR-152 MiR-148a enhances the chemoradiosensitivity of patients with rectal cancer. Tumor tissue c-Met miR-34 miR-34a/b/c/d MiR-34a attenuates the chemoresistance of colon cancer to 5-FU by inhibiting E2F3 and SIRT1. The miR-34a mimic MRX34 is the first synthetic miRNA to undergo clinical trials. Serum E2F3; SIRT1 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000072

Despite the challenges the pandemic presented for university teaching, it opened up opportunities to set up and explore digital teaching formats like never before. This paper presents a case study of teaching introductory animal ethics in a digital format with flipped-classroom methods. The Interactive Literature Lecturing Format (ILLF) was designed along the following criteria: 1. Conformity with students' varying educational needs; 2. Consistent high level of interaction; 3. Maximum transparency in an application-oriented exam; 4. No further contribution to the workload of the teaching staff; 5. Flexibility regarding online or on-site conversions. Rather than provide the students with input in lecture sessions, the ILLF presents students with selected literature and a list of structured questions. This literature questionnaire serves as the main didactic element that guides the knowledge transfer, the structure of the sessions and the exam. This paper reviews the outcome of the redesigning process and the steps we took to implement it. To discuss the overall quality of the format from a student's perspective, the data from the systematically conducted students' evaluation (n = 65) are interpreted using quantitative and qualitative methods. Bringing these results together with the perspective of the teaching staff, the following question is discussed: did the ILLF meet these criteria? This case study explores the potential and limits of flipped-classroom methods for applied ethics teaching in a university setting. didactics of ethics animal ethics flipped classroom online teaching empirical evaluation This research received no external funding. pmc1. Introduction: COVID-19 as a Chance to Rethink Teaching Formats The pandemic presented numerous disruptions in many areas of life, including university teaching. Right from the start, university teaching around the world underwent severe restrictions, which led to a collective conversion from on-site events to digital formats. Despite the challenges this presented for teachers and students, it opened up opportunities to set up and explore digital teaching formats like never before. This paper presents a case study of an introductory animal ethics course that was significantly improved by reconstructing it in a digital format with flipped-classroom methods. The course in question, offered at the Institute of Philosophy at the University of Vienna, includes a lecture and reading components. It is taught annually and forms a part of the curriculum for philosophy students in the BA and MA Philosophy programs, as well as for students working through a philosophical extension curriculum. Thus, it is credited in many different modules, but it is not mandatory in any module. It is evaluated with 5 ECTS credit points in the European Credit Transfer and Accumulation System, in which 1 ECTS credit point is equal to 25 working hours. Thus, the course involves 125 working hours in total and is taught in German. As a lecture course, it does not require attendance throughout the semester, as there is only a written exam at the end of the semester for evaluation and three additional exams that take place throughout the following semester. The content is divided into two parts, the first one dealing with traditional theoretical approaches in the animal ethics debate and their critique, i.e., Peter Singer , Tom Regan and alternative approaches, such as those of Clare Palmer , Cora Diamond and Cheryl Abbate . The second part of the lecture focuses on fields of application of animal ethics, i.e., farm animals, laboratory animals and pets. When they complete the lecture course, students should meet the following three learning objectives:To reproduce central arguments in animal ethics and integrate their corresponding theoretical strands within the current animal ethics debate; To apply the theoretical approaches to practical examples, i.e., to be able to identify ethically relevant aspects in practical situations and interpret them by drawing from animal ethical theories; To critically reflect on animal ethics theories both in their practical application and by comparing them to other arguments in the present debate. After the first semester was spent merely switching a pre-pandemic lecture format to the virtual classroom, there was room for improvement. Multiple incidents of plagiarism in the final exam challenged the examination format. Transferring the exam from an on-site mode to an open-book, online mode jeopardized any effective control over whether or not the learning objectives were actually met. The exams revealed that it was no longer apparent whether students could reproduce and apply their actual knowledge or whether they were just skilled in sharing and rewriting existing texts. This presented not just an immediate need for change but an opportunity to rethink established teaching methods and structure the course's contents in a novel set-up, asking anew: which elements define good quality lecture formats, safeguard the learning objectives and can be transferred to an online setting? In answering these questions, the authors identified a list of five criteria that helped guide the design for this new lecturing format. Unfortunately, at the time of the redesign of this format, i.e., in the first months of the pandemic, the transition from ethics courses to online teaching had not yet been adequately addressed in the literature. Therefore, the authors have drawn on their own teaching experience and exchange with students to determine these criteria. The final list of five criteria has been separated into three criteria that qualify a good lecture from the students' perspective and two criteria that any viable format should meet from a teacher's perspective. From a student's perspective, what makes a lecture format worthwhile is, firstly, that the course should be tailored to the diverse needs of students. As the course in question is an elective and a lecture format does not demand mandatory attendance throughout the semester, the format should enable the students to decide when and how much they invest in the provided program. On the one hand, it should provide possibilities for increased engagement for students who are willing to dedicate more of their time. On the other hand, if students have only limited time resources, the materials provided should enable them to prepare independently and time efficiently for the exam. Secondly, a good lecture format should safeguard a high level of interaction between students and the teacher and allow for in-depth discussions. For those students who attend weekly sessions, there should be sufficient room for questions and discussion points. In order to safeguard this interaction, the format should further offer systematized opportunities for students to report on their experience with the literature so that the teacher can tailor the weekly input according to the students' needs. Lastly, the exam should be designed in a transparent way that challenges students without causing unnecessary stress. Safeguarding transparency here allows students to prepare for the exam in a target-oriented way and to avoid any guesswork. The exam should position students to demonstrate what they understood, not what they memorized. The following two requirements for a good teaching format are from the teacher's perspective. First, and most important, is that the new format must not permanently increase the teaching workload. This is a precondition to the requirements above, since many ways to meet student needs demand too much work from the teaching staff and are thus unlikely to be implemented. For example, giving individual feedback to voluntary weekly text submissions promises to be very helpful for students. However, correcting more than 20 submissions per week can easily add up to another full working day for teaching staff and, therefore, conflicts with other commitments. The more time-intensive any format is for the teacher, the less sustainable its implementation. Second, a good lecture format should provide sufficient flexibility for the teaching staff. It must be easily adjustable to online or on-site modes of teaching without compromising quality. In the following case, the course was designed expecting 40 to 60 students to attend the lectures and participate in the first examination at the end of the semester. Approximately 80 to 100 students were expected for the following three examinations that would take place throughout the next semester, thereby raising the total number of expected students from 120 to 160. The redesign, implementation and evaluation processes were carried out by a team consisting of one professor and an assistant. This paper reviews the outcome of the redesigning process, the steps to implement it and the results of the systematically conducted students' evaluation (n = 65). In the first step, the outcome of the design process, the Interactive Literature Lecturing Format (ILLF) is introduced and its implementation was elaborated on by the teaching staff. In the second step, the materials and methods of the evaluation survey are sketched to interpret the data in the third step. In the fourth step, the perspective of the teaching staff is brought together with the students' evaluation survey results, representing the students' perspective. Then the ILLF's ability to meet the criteria for a good lecturing format is discussed and potential future directions are delineated. A fifth step concludes the case study by explaining the added value of the ILLF as a didactic tool for ethics teaching. 2. Design and Structure: The Interactive Literature Lecturing Format (ILLF) The course design model that promised to meet all of the requirements is the Interactive Literature Lecturing Format (ILLF). In order to bring the criteria introduced above together in a feasible way, the ILLF draws from the flipped-classroom methodology: "The flipped classroom teaching model is colloquially defined as one in which the activities traditionally carried out by students outside class (e.g., practicing problem-solving) are moved into the classroom session, whereas what is traditionally completed in class (e.g., expository, information transmission teaching) is completed outside and prior to class." (Lag & Saele , p. 1; see also Galindo-Dominguez ) For an animal ethics lecture course, flipped-classroom methodology refrains from knowledge transfer consisting of one professor lecturing in front of the whole class throughout most of the lecture. While in a usual lecturing format, the arguments and central concepts of the literature are presented in the lecture by the teacher, in the ILLF, the students gain most of the relevant knowledge on their own by reading the selected literature. The activity that usually takes place outside of the lecture, i.e., developing understanding by putting theory into a practical context, is transferred to the weekly sessions. These sessions are not used to repeat the arguments from the literature, but rather to exclusively apply theoretical knowledge, providing opportunities to ask questions and have in-depth discussions. Therefore, attending the lecture is not beneficial for students who have not read the corresponding texts for the session. The central, flipped-classroom inspired, didactic element is a questionnaire containing a comprehensive list of semi-closed questions that guide readers through each of the texts provided in anticipation of the live discussion (see Appendix A for the full list of guiding questions in the first session of the lecture course). The questionnaire runs through all phases of the lecture course: It facilitates knowledge transfer by systematizing the reading process with guiding questions; The students' answers to the questions provide the basis for structuring the weekly sessions; The questions cover the exam's theoretical foundation and can, therefore, systematically guide exam preparation. The new format begins with an online introductory session that presents an overview of the topics and explains the new format to the students in detail. It includes instructions on how to use the material and the online learning platform (Moodle). Furthermore, it points to interactive tools, such as the discussion forum for students, and a tool that enables students to give feedback anonymously at any time. A detailed breakdown of the credit points into working hours made the requirements for the seminar as transparent as possible. It would take approximately 18 h (12 sessions of 1.5 h) of attendance or listening to the recordings of the sessions, 66 h for reading (11 weekly sessions of 6-h literature preparation), 22 additional hours to fill out the questionnaire (two hours per literature/session) and, lastly, 19 h for final exam preparations. This comes to a total estimated workload of 125 h required for 5 ECTS. 2.1. Knowledge Transfer: Guided and Independent Literature Work For the flipped-classroom methodology, the main challenge from a teacher's perspective is ensuring the correct understanding of the texts. However, by refraining from lecturing, the teacher also relinquishes control of how the arguments are encountered and thus how students interpret them. To enable a comprehensive understanding of the relevant positions, the teacher must rely on relevant secondary literature, which complements the primary texts. The primary text, e.g., Singer's All Animals Are Equal , introduces the original key terms and argumentation styles. The secondary literature for this text, e.g., Der Praferenz-Utlitarimus Singers by Grimm & Wild , explains the primary text, positioning it in the wider debate. The questionnaire functions as a third element to safeguard understanding by navigating through the primary reading alongside secondary literature. The questionnaire guides students by focusing on the central concepts and facilitating critical reflection on arguments and positions. For each session, there are five to eight guiding questions, such as, "According to Peter Singer, why are animals to be considered morally?", or "Explain the following central concepts in Singer's approach and their significance for his argument: Speciesism and interspecies equality; Universalizability; Vital vs. trivial interests". This form of independent knowledge transfer enables students to engage directly with texts according to their own time management. If some students have a higher level of prior knowledge, they can finish reading quickly. This stands in contrast to traditional lecturing, where students would have to sit through a lecture in which the teacher presents arguments they might have already understood. Furthermore, students can give detailed or brief answers as they deem appropriate. If some are not as familiar with philosophical texts, they can repeat central passages and express lingering unclarity through the three questions that complete each list of questions for any session, i.e., "Did the theoretical approach convince you? Why/Why not?", "Are there any aspects that are not yet clear to you?" and "Are there aspects of Singer's theory that you would like to discuss in the session?". These are key for the second function of the questionnaire--structuring the weekly sessions. 2.2. Weekly Sessions: Prepared In-Depth Discussions The answers to the literature questions could be uploaded to Moodle for the teacher to analyze the submissions before the weekly session, three hours at the latest before the session. This submission deadline provides a fixed timeslot that is sufficient for the teacher to adjust the sessions based on student input. The uploaded answers (on average, 17 submissions per week) provided the exclusive basis for structuring each session. This allows the teacher to prepare the sessions time efficiently and make them target-oriented, as there is no need for slides that introduce arguments in the first place. As the lecture format allows for non- or irregular attendance, any submissions throughout the semester were on a voluntary basis. Thus, submitting the answers to the questionnaire via Moodle is an offer from the teacher to the students to partake in designing the content of the weekly sessions. The weekly discussions take place via Zoom only and are recorded and uploaded to Moodle for students to rewatch. Within the weekly discussions, no exam-relevant material is introduced. However, in taking part in the discussions regularly, students also deepen their skills to apply theories to practical situations and to reflect on ethical theories critically, which is vital for the exam (see learning objective 2 and 3). The discussion is broadly structured into two parts. The first one focuses on resolving any confusion from the students' reading. When reading the students' submission to the questionnaire, the teacher checks whether there are recurring misunderstandings from the reading, e.g., connecting the idea of animals having dignity to Singer's approach. If there are, the teacher quotes them in slides for the presentation and explains what the misunderstanding consisted of. Furthermore, what students found unclear in their submissions is directly addressed, ensuring an overall understanding of the theoretical positions, e.g., "Does the interests of a being have to be consciously experienced in order to count morally?" The second part of the discussion consists of deepening and applying the theoretical knowledge. The students' comments regarding what they would like to discuss are scanned by the teacher. The most common comments from the submissions are addressed in the session, e.g., which beings we can safely ascribe sentience to and questioning the idea of self-conscious beings and non-self-conscious beings. In some sessions, it is possible to present real examples highlighting particular challenges for theoretical approaches. In the third session, Clare Palmer's approach to the distinction of wild and domesticated animals is discussed using the example of rewilding in a nature reservoir in the Netherlands, Oostvaardersplassen. As a rewilding project, animals are left to themselves, which leads to animal suffering due to limited nutritional resources and overpopulation, thereby motivating activist intervention . The reserve, in its function as a nature protection zone, is fenced, which hinders wild animal movement and foraging. Scarce winter resources were especially hard on the overpopulated wild horses. This illustrated a critique of the categorization of wildness based on whether or not animals are located in unimpacted environments. As there are barely any unimpacted zones for animals, according to this distinction alone, animals can barely ever be considered wild. To guide the discussion, the teacher encouraged participants to raise their hands and pose contributions via the microphone, as well as via chat. The chat kept the discussion going by allowing the teacher to pose a question and ask for quick and short answers in the chat. From the chat conversation, the teacher selected interesting short answers, asking students to elaborate on them. Furthermore, this enabled students to point out unclarities right away, which was helpful for the teacher as well, provided that there was sufficient time to filter through the questions in the chat. Overall, it was not the teacher who gave the input in the lectures, but the students themselves. Uploading the answers to the questionnaire regularly invited students to partake in the design and structure of the sessions. The teacher just selected and structured the input. This guaranteed a maximum level of interaction with the students, while maintaining a comparable level of working hours for the teaching staff compared to more traditional lecturing formats. This has been reflected in the quality of the discussions in the sessions via Zoom that the teaching staff experienced. Everyone attending was prepared for the discussions by having read the texts and students were generally very engaged. Since the lecturing did not entail content transfer, there was sufficient time to broaden the debate around animal ethics so that each session added further literature to the platform for interested students. For the teacher, the discussions were also more engaging due to the participants' knowledge. All of this resulted in a significant increase in the teacher's satisfaction with preparing and moderating the weekly sessions, even though the total amount of working hours put into the preparation did not decrease but remained roughly the same. However, the hours invested into preparation via the students' submissions were more purposeful due to the immediate interactions with the explicit students' demands in understanding the texts. 2.3. Exam: Transparent and Beyond Knowledge Reproduction The third and final function of the questionnaire was especially important for meeting the requirement to tailor the lecture to the diverse students' needs. It provided the theoretical foundation for the exam, i.e., no exam questions went beyond what was addressed by the questionnaire. If the questionnaire was filled out properly, students would not face topics beyond their knowledge in the exam. The questionnaire thus served as a guide not only through the literature, but also through any independent exam preparation. Since any lecture has to provide four exams in total, with no required attendance during the semester, it was common for students to study exclusively on the basis of the materials provided and prepare independently for the exam. The questionnaire facilitated this way of studying, as it allowed for consecutive preparation. Since it contained the condensed contents of the lecture, it was helpful in refreshing knowledge. Students could, for example, start at the beginning of the semester with active engagement in the course, pause it and then pick up studying at the end of the semester again without having wasted any time. Alternatively, students who regularly attend the sessions and submit answers to the weekly questionnaire may have to study less. The more hours one puts in during the semester, the less one has to prepare for the exam. To keep such a balance, the exam is designed in two parts, testing whether the learning objectives were met. The first part is the independently filled-out questionnaire that covers the first learning objective, i.e., to reproduce central arguments in the animal ethics debate. If the questionnaire is filled out properly, the reproductive knowledge requirement is fulfilled, because uploading a filled-out questionnaire on the exam date is a prerequisite to participate in the exam. The questionnaire is not evaluated in terms of quality, since this would increase the workload involving the evaluation drastically. Plagiarism software tests reveal whether or not the submissions were filled out individually. This is essential insofar as it prevents students from circulating filled-out questionnaires and using them as the sole material for preparation and re-uploading answers on the exam. As helpful as the questionnaire is, without this precaution, the ILLF is vulnerable insofar as it allows students to surpass any original texts in the exam preparation. The mandatory uploading of the filled-out questionnaire before the exam also reduces the number of participants who go into the exam heavily unprepared and thereby decreases the workload of evaluation for the teacher afterwards. The second part is an application-oriented, open-book, 2-h take-home exam. This part covers the second and third learning objectives, i.e., applying theories to practical examples and critically reflecting on them. For the second part of the exam, the students receive two relevant case scenarios in animal ethics to choose from, together with the exam questions via e-mail. They have to upload their answers to Moodle within two hours. As the reproductive basis has been covered by the first part of the exam, they are welcome to use any material for addressing the questions. Due to this division of the exam format, the aim of the take-home exam is for students to show that they understand the theoretical approaches and can thus apply them and reflect critically. From a student's perspective, the online format has the benefit of allowing to participate in the exam from any place. The two-hour limit minimizes the potential length of the answers, as it is expected that answers tend to become longer the more time is provided, which would increase the total evaluation hours of the teacher. Furthermore, two hours are sufficient for students to demonstrate an understanding of the lecture contents. The exam is designed in an application-oriented format, i.e., students have to analyze the animal ethics scenario with the theoretical approaches learned in the lecture. The scenarios are roughly a quarter of a page long and display challenging dilemmatic situations for a person who has to make a decision. For example: "Martina keeps five hens and a rooster in her backyard in a coop with an outdoor area. She and her family enjoy the fresh eggs the hens lay every day. One day she notices that one hen has started to hatch three eggs. However, the coop is clearly too small for a total of nine hens, and their quality of life would suffer from the limited freedom of movement. What should Martina do?" The exam questions then provide the interpretative frame for analyzing the case examples, for example: "Elaborate on the morally relevant aspects in this example against the background of the theories by Peter Singer and Tom Regan. In doing so, discuss possible answers to the question "What should Martina do?" and explain the central differences between the two approaches by illustrating them with features of the example." Overall, theoretical approaches are utilized to interpret the scenario and reflect on the theories, e.g., where do problems arise in their application? Thus, the online take-home exam provides the opportunity for students to apply and further explore their knowledge on a case-by-case basis rather than reproducing memorized knowledge. 2.4. Evaluation of the Exam In order to evaluate the exams, teaching staff checks whether both documents, i.e., the filled-out questionnaire and the answers to the exam questions, were submitted. Otherwise, the exam cannot be passed. The exams are then pre-evaluated by assisting staff before the teacher grades them. The pre-evaluation follows a template that is constructed for each exam individually. It breaks down the maximum points for each question into requirements for the answer. A good answer to the question above regarding Singer's approach should, for example, mention the concepts of speciesism, interspecies equality, universalizability and vital vs. trivial interests, illustrating it in the example provided. Furthermore, the pre-evaluation looks at how the question is answered: Is it correct? Did the participant apply the theories to the example sufficiently? Is sufficient justification provided? Lastly, the style is evaluated, i.e., is the answer clear and well structured? Pre-evaluating exams in this structure reduces the time invested by the teacher in grading to a minimum. Switching the examination method to an open-book and application-oriented format increases the satisfaction of pre-evaluating and grading the submissions significantly for the teaching staff. This is because the answer quality is higher, and it is thus more interesting to read the answers. Furthermore, this format allows for more diverse answers and approaches from students, which makes evaluating also more diverse. This offers students flexibility when taking the exam and the teaching staff when evaluating answers, as two very different approaches to one and the same question can be equally right. With these application-oriented, reflective questions, it is not about what the student determines to be the answer but how they argue for it. 3. Evaluation Survey: Materials and Method In order to gather a comprehensive view of the students' perspective on the ILLF, an evaluation form was sent out to the students after each examination date. The exam is the element which eventually reveals how the flipped-classroom knowledge transfer worked for students overall, including those students who never participated in the weekly sessions. Thus, it is crucial to evaluate the students' experiences not just throughout the semester but on the exam as well. That is why the evaluation targeted those students who were registered for an examination date after the corresponding exam. To gain targeted insights into the students' perspective on the ILLF and its elements, evaluation forms were specifically designed via the established online survey tool, Umfrageonline.com. Using this tool, the links to the evaluation forms were generated and the responses were collected. 3.1. Study Design and Measurements The links to the evaluation were sent by e-mail within 24 h after each examination date exclusively to the students who were registered for the respective examination. This was carried out using the platform of the central teaching organization platform of the University of Vienna (u:space), which allows e-mails to be sent to the registered student accounts as blind copies. After each of the four examination dates, two links were sent out, which enabled the evaluation of two separate groups of students with different evaluation forms. The active group consisted of those students who regularly participated in the lecture during the semester. The non-active group consisted of those students who independently prepared for the examination with the documents provided. The students were informed that participation in the evaluation is anonymous, on a voluntary basis and has no effect on the evaluation of the exams. The online survey was open for 30 days after sending out the link. After 30 days, the answers were downloaded as Microsoft Excel documents for data analysis. Despite sending out the evaluation forms after the examination dates, the response rate was sufficiently high to allow for conclusions on the quality of the format based on students' perspectives. A total of 132 students participated over the course of four examination dates, out of which 65 filled out the evaluation forms, resulting in a response rate of 49%. The evaluation after the examination with independently designed evaluation forms enabled a comprehensive evaluation tailored to the structure of the format. The evaluation forms included both closed and open questions. If not indicated otherwise, only single responses were possible. Closed questions with a numeric scale were integrated to establish comparability between individual assessments for those aspects that were most relevant for future targeted modifications of the format for both groups. This included the estimated amount of work compared to other lectures and the effort invested in exam preparation. Students could evaluate this from 1 to 5, 1 being too low and 5 being too high. The difficulty of the exam could be evaluated from 1 to 5, with 1 being too easy and 5 being too difficult. The amount of information about the format and the exam could be evaluated on a scale from 1 to 5, 1 being too little and 5 being too much. The overall format could be evaluated from 1 to 5, 1 being very good and 5 being very bad. Furthermore, the evaluation form for the active group asked students to assess the quality of the discussions in the sessions from 1 to 5, 1 being very good and 5 being very bad. The evaluation forms were supplemented by further closed questions with several answer options to understand how students used the flipped-classroom tool. Students from the active group were asked how regularly they prepared the literature and answered the guiding questions, choosing between six answers: prepared literature and guiding questions each week; prepared literature and guiding questions almost each week; prepared literature and/or guiding questions sometimes; prepared literature sometimes but never prepared guiding questions; prepared literature and/or guiding questions rarely; and never. Both groups were asked whether the literature questionnaire was perceived as helpful on the following scale, which allowed for multiple responses: yes, to work with the literature; yes, to systematically prepare for the exam; not really; no; and other (including the possibility to state a short open response). Students from the non-active group were further asked whether they felt sufficiently prepared by working through the material independently on the following scale: yes, very much; yes, sufficiently; no, too little; and no, not at all. Open-ended questions captured more closely the motivation of the students to register for the lecture course in the first place. Both groups were asked why they decided to register for this lecture. The non-active group was further asked why they chose not to attend the sessions regularly during the semester. These dimensions, however, were included only to define the target group more precisely for future semesters and do not reflect the quality of the ILLF. They are, therefore, not of further interest here. The final open question posed to both groups was intended to give students the opportunity to evaluate the lecture in their own words, i.e., what they particularly liked about the teaching format and where they see potential for improvement. 3.2. Quantitative Data Analysis For the quantitative analysis of the responses in the numeric and Likert scales, the data have been systematized with Microsoft Excel, and the overall responses of all evaluations have been divided into one document per group (active vs. non-active). Furthermore, one document has been created containing all the responses in total. The relevant mean values, their standard deviations, and percentages have been retrieved for each group using statistic operators provided by Excel. 3.3. Qualitative Data Analysis The responses to the open questions in the evaluation form (n = 53) have undergone a qualitative content analysis that systematizes the raw material into categories to reflect on and stress regularly mentioned points, (see the methodology of Mayring ). The first step consisted of scanning the text corpus and noting relevant points. The second step involved clustering the most frequently recurring relevant points into categories. These categories then served in a second analysis of the material. Thus, the categories were retrieved directly from the material, even though the exclusion of points targeted by the closed question could be considered a form of negative pre-selection of categories for this analysis. Consequently, the material was scanned again according to the inductively developed category system and coded according to correspondent keywords of the categories, e.g., sentences containing the keywords or phrases "questionnaire," "literature guiding questions" or "questions for literature" were coded as the category "questionnaire." Due to the size of this corpus, there was no need to develop further subcategories. 4. Results 4.1. Quantitative Results Of the overall participating students (n = 65), 21 (32.3%) reported to have been active throughout the semester, 19 (90.5%) of which registered for the first examination date. Thus, 44 (67.7%) students claimed to have studied for the exam independently from the lecture sessions. The data suggest that students were largely in favor of the ILLF, as the mean value of the assessment is 1.37 (+-0.63) on a scale from 1 to 5, 1 equaling "very good" (see Table 1). In fact, 20 (95.2%) of the active students and 42 (93.3%) of the non-active estimated the format to be at 1 or 2. This shows that the ILLF worked for both targeted student groups alike, with a slightly higher tendency of the non-active students to consider it very good. How the overall satisfaction is composed was inquired by questions focusing on the central didactic elements in different phases of the semester. The questionnaire was regarded as helpful for guiding through the literature by 12 (57.1%) of the active students and by 31 (68.9%) of the non-active students. Furthermore, it was reported to systematically help prepare for the exam by 17 (81.0%) of the active students and 38 (84.4%) of the non-active students. Only 1 (1.54%) person in total evaluated it to be not particularly helpful. This suggests that the students overall reported the questionnaire to work well as a didactic tool, but more so in terms of structuring the exam preparation than guiding through the literature. In addition to this, the active group flexibly adopted the questionnaire, as the answers regarding how regularly they prepared the literature for the sessions and fill out the questionnaire varied: fourteen persons (66.6%) stated that they prepared for the session every week or almost every week, while four (19.1%) sometimes prepared the literature or answered the questionnaire and three (14.3%) rarely or never prepared literature or filled out the questionnaire in advance of the weekly sessions. The active students evaluated the discussions on a scale from 1 to 5, 1 being "very good" and 5 being "very bad", as on average 1.71 (+-0.90). The workload, compared to other lecture formats, was generally estimated higher. On a scale from 1 to 5, 1 being too low and 5 being too high, the active group rated it at 3.76 (+-0.70) and the non-active group on 3.64 (+-0.66). However, only 6 (11.1%) students overall estimated the workload as generally too high and no one estimated it to be too low. The final phase of the lecture, i.e., the exam, was evaluated using three questions. Both groups estimated their exam preparation on a scale from 1 to 5, 1 being "very easy" and 5 being "very hard" as 2.40 (+-0.86) in total (active group 2.41 (+-0.74) and non-active group 2.41 (+-0.92)). Furthermore, 41 (93.2%) of the non-active participants claimed to have felt "very prepared" or "sufficiently prepared" for the exam by studying independently with the materials provided. Both groups evaluated the exam as adequately difficult on a scale from 1 to 5, 1 being "very easy" and 5 being "very difficult"; the active group rated it as 2.62 (+-0.67) and the non-active group as 2.64 (+-0.65). None of the participants found it very easy or very difficult, suggesting that the overall exam format worked for the students as well. 4.2. Qualitative Results The overall impression that the ILLF was received well by the students is solidified through the comments to the open question, which asks what they particularly liked about the seminar and where they see potential for improvement. Recurring issues pointed out by students fall into six categories: format, discussion, interaction with teaching staff, selection of literature, workload and exam. The following section will focus on points brought forward by the students regarding these categories, which go beyond what has been targeted by the quantitative results. 4.2.1. Format The impression from the quantitative evaluation that the format was received well by the students could be bolstered by analyzing the written comments. Students described the format as "great" and "very successful," especially pointing out that the questionnaire was "genius" or "incredibly good." Furthermore, they stated that "the flipped-classroom concept was implemented well" and that even "away from covid restriction [...] this format [is] fantastic." Participants from the non-active group also commented that they were "not feeling at a disadvantage compared to others who attended the lecture on a regular basis". 4.2.2. Discussion and Interaction with Teaching Staff The evaluation of the discussions, however, is more diverse. On the one hand, students pointed out how "interesting" and "extremely engaging" they found the discussions to be, that "time flew by" and, overall, the discussions were of "added value" for the lecture. Students mentioned that the structure of the sessions was flexible and that the teacher was competent in moderating and embedding single contributions into the larger animal ethics debate. Also, students from the non-active group reported rewatching the recorded discussions to deepen their knowledge, even stating that they regretted not having been able to participate in the discussions. On the other hand, students reported that the discussions were sometimes far off-topic from the literature. They felt that "the red thread was missing" and that the structure of the discussion was too "associative and free" or "excessive" in areas that were not relevant for exam preparation. Therefore, a few students reported missing reassurance as to whether they understood the central arguments correctly. They wished discussions were restricted to the sessions' literature and guiding questions. Furthermore, students pointed out that the interaction with the teacher was "pleasant," "motivating above average," "respectful" and "at eye level". Participants from the non-active group also said they appreciated the "maximum transparency" in the communication about the structure of the lecture, and especially the exam. 4.2.3. Selection of Literature and Workload Students reported that they found the selection of literature to be "good" and "interesting," but regularly pointed out that particularly texts in the English language added significantly to the workload and that they would have liked more translations. Another issue brought up regarding the literature directly connects to the next category; that the literature selection was too comprehensive and thus created a higher workload than other lecture formats. Furthermore, they reported that filling out the questionnaire was time-consuming, albeit students claimed that it turned out to be worth the effort, as it reduced the time for exam preparation drastically. Still, the overall impression remained that the workload was higher compared to other lectures. 4.2.4. Exam The format of the exam, however, was generally appreciated for its being "very pleasant," "very good because it was application-oriented" and the exam questions being "very exciting, because you had to deal with the material in a different way." The separation of the exam into a reproductive part (filled-out questionnaire) and the application part (take-home exam) was evaluated positively, as students did not need any further exam preparation when the questionnaire was filled out properly. 4.3. Limitations of the Survey The time for the evaluation survey was set 24 h after students took the exam, to have students evaluate the exam experience as well. Sending the evaluation forms this late, however, came at a price: Only those students who took the exam were targeted for the evaluation. We did not manage to include those students who refused to take the exam altogether, e.g., because the readings were too comprehensive, or they did not like the format in the first place. As the number of deregistration, which has been retrieved from the online administration system of the University of Vienna (u:space), is rather high compared to other semesters when the same lecture was offered in its more traditional format, the overall drop-out rate of this course seems to be relatively high. In the winter semesters of 2019 and 2020, 29% and 25% of the once-registered students for the four exam times deregistered again. In the winter semester of 2021, when the ILLF was adopted, the deregistration rate was 44%. Thus, 44% of the students who possibly intended to take the exam for the lecture eventually decided against taking the exam. It is worth noting here that there are no numbers available for when students deregistered after having registered; it is unclear whether it was shortly after registering, when reviewing the material and the requirements or when preparing for the exam had already started. Even though it would have been interesting to examine the reasons for drop-outs, this particular group of students was difficult to target. The interaction with them probably only consists of them looking at the online learning platform, the selection of literature and the requirements. If there were particular reasons for dropping out throughout the semester connected to the format, the anonymous feedback tool provided via Moodle could have been used. The question thus arises, even if one could target this group of students, on what grounds would their evaluation be based? Sending out an evaluation form at this late stage of the lecture course also gives rise to students' biases. Firstly, students who are outstandingly happy with the format are more likely to report this than those who found it merely average. Similarly, students who are very dissatisfied with the format or the exam are expected to show more initiative when it comes to evaluating. Since these biases tend to level with each other in the final findings, the results can still be regarded as reliable. Furthermore, it must be admitted here that the evaluation data from this semester alone only allow for a limited interpretation of the results. To make definite statements on the success of the redesign with the ILLF, a systematic comparison with prior semesters would have been necessary. As the redesign initially prompted the intensive student evaluation, there is no comprehensive set of evaluation data for prior semesters to allow for systematic comparability. Considered on its own, the data can only tell how the transformation to the ILLF and online teaching mode was experienced by teachers and students, or which specific elements have worked well and which have not. 5. Discussion 5.1. Does the ILLF Meet the Established Criteria for a Good Lecture Format? Designed amidst the restrictions of the pandemic, the ILLF significantly improved the animal ethics teaching beyond that. Both from a student's perspective as well as from a teacher's perspective, the implementation of a structured flipped-classroom format resulted in a high quality of interaction and proved to be viable for students with varying studying styles. Therefore, the ILLF met three initially posed requirements for a good lecture format for students, namely that it should be tailored to diverse students' needs, offer a high level of interaction with the teacher and be transparent in the way the exam is set up. Firstly, the ILLF effectively managed to target the two central student groups equally. Students who regularly attended the lecture throughout the semester reported being satisfied with the format as well as students who only prepared for the exam individually. It thus allows for maximum flexibility in the students' time management, which is crucial, especially for those students with work or other commitments. Furthermore, the possibility to submit answers to the literature guiding questions (i.e., questionnaire) each week enabled the teacher to interact with the students and prepare the sessions according to their interests. Therefore, each session is designed by the input of the students and not by the teacher's interests. This provided maximum room for application-oriented discussions that enabled students to grasp the bigger picture within the debate. Lastly, the two-part exam afforded students a level of transparency, as one part was the direct outcome of the exam preparation, i.e., the filled-out questionnaire. Moreover, the format of the exam provided another way for students to demonstrate their knowledge without the stress of memorizing since, due to its application orientation, it was an open-book examination. From a teacher's perspective, the requirements of a good lecture format could be met overall as well. The format allowed for sufficient flexibility in times when the teaching mode was subjected to quick changes. The ILLF was implemented as an online teaching format and it worked well as such. Nevertheless, it is possible to make the same adjustments to weekly on-site sessions. This would require only a few alterations, but it is yet unclear whether the quality of discussion remains equally high in an on-site mode, for central elements of structuring the discussion are missing, e.g., the teacher cannot use the chat easily to engage students in the discussion by asking for short answers. Furthermore, it can be expected that those students on the quiet side may be more hesitant to participate in discussions in a full classroom of more than forty persons. The format of the exam should also remain online, as it is an open-book format. This is because of the plagiarism software that is required to check the students' submissions for authenticity: hand-written exams cannot be easily uploaded for plagiarism checks. Even if students were on-site during the exam and later submit their answers digitally, there would be no reason for them to come on-site in the first place. Therefore, the exam structure only makes sense as an online format. If one wanted to provide open-book formats for students, there is no good reason to ask them to come on-site for the exam. On the other hand, a more immediate interaction between students and teachers in future semesters might support the learning objectives of this course. In this case, their skills regarding the application of theories and their critical reflection might benefit from an on-site transfer. The second requirement for a good lecture format from a teacher's perspective, i.e., keeping the workload minimal, was met. The time invested by the teacher throughout the semester in designing each lecture remained largely the same, even though it was much more motivating to react directly to the student's educational needs. Still, the effective time-saving will only become apparent in future semesters. Implementing the ILLF constituted a great deal of work: Selecting the literature, creating the questionnaire, setting up all relevant documents and transferring the information to the learning platform. However, this format is promising for future semesters--all it takes is a few clicks in the online learning platform to reinstall it for another semester. This is only possible because of the high level of interaction with the students, where each session is tailored according to student input. This level of interaction that is integrated into the framework makes it easy to react to current trends in the debate without having to alter the program at large. When it comes to the overall time invested by teaching personnel in correcting the exams, it is roughly the same as traditional lecturing formats. In order to receive the take-home exam, the students must have filled out the questionnaire beforehand and therefore have a solid knowledge basis to succeed in the exam. Making the upload the questionnaire a prerequisite part of the exam effectively reduces the number of those students who come underprepared. Nevertheless, it must be said that the experience of the teaching staff only allows for limited conclusions as the number of teaching staff involved here was limited (n = 2). 5.2. What Can Be Improved in the ILLF? Learning from Students' Input Overall, the results of the regular student evaluations showed that the ILLF did not only meet the predefined requirements, but revealed that not only the satisfaction of the teaching personnel was high. However, the evaluations can also be a tool to reflect on the format further. Two issues stand out from the written comment section of the evaluation, i.e., the discussion and the workload. The comments suggest that, for future semesters, it might be worthwhile to orient the weekly sessions more toward the students' input to provide more guidance when it comes to exam topics. The ILLF challenges students to work independently with literature, which allows for a flexible framework. This may be harder for some students. The question that arises here is whether one should tailor discussions to those students who tend to feel more lost because of the literature and thereby risk other students being underchallenged. Overall, it is advisable to tailor the discussion along the submitted answers to the guiding questions while allowing for extensive discussions for other students. Any insecurities regarding the understanding of the topics might be better dealt with outside of the weekly lectures, e.g., in student reading groups or regular tutorials with a student assistant. Regarding the workload, it makes sense to accommodate the students' wishes to a certain degree and slightly reduce the selection of mandatory readings. The question that arises here is what to align the expected workload with. Should one align the workload with what students expect to do in the framework of their university? Or should one use the credit points (ECTS) as a baseline to decide upon an expected workload? For future semesters, the authors will continue to use the ECTS as a measure to guide the expected workload, for a good lecture should be challenging to a certain degree. This is crucial, especially in undergraduate courses, where students must build the basic habits for efficient text work. Furthermore, providing current research literature from the animal ethics debate will remain a central part of the lecture. As such, it will often be in English, which introduces the debate's key terminology to the students. 5.3. Future Directions Redesigning the animal ethics lecture format was illuminating in many ways. What stands out most, however, is not directly connected with the quality of teaching. Evaluating the teaching format so regularly revealed an insight that can redefine what a lecture is about--two-thirds of students that take the exam study independently. Thus, when giving a lecture, two-thirds of the teacher's target audience is not in the room. The implementation of ILLF and all the material provided is, therefore, why so many students take the exam independently from weekly sessions. This clearly shows, however, that there was an interest in such formats among students. It is an interest also recognized in most curricula offering both mandatory attendance, such as seminars, and those without the need for attendance, such as lecture courses. One could argue further that, for lectures to conform to the format of non-mandatory attendance, the teaching staff has to provide sufficient materials for students to prepare for the exam independently. If a lecture does not provide sufficient material besides the weekly sessions, students are obligated to physically attend to take the exam. The implementation of ILLF has further demonstrated that knowledge transfer does not necessitate attendance. Students are able to learn independently if provided with well-structured material and sufficient room for interaction. Thus, in order to offer student-friendly formats, it is crucial to not only have the independent learners in mind, but to think of them as the majority of the target group and, respectively, realign priorities in lecture formats. This way, teachers can establish fair formats that respect students' varying time resources and thereby create flexible formats that make participating in worthwhile. This creates a paradoxical situation, as a third of the students that are regularly present produce the content for the other two-thirds. Even though the majority of students are "absent participants," one has to make the weekly sessions as attractive as possible for the ones that participate, so as to safeguard good discussions for others to rewatch. Thus, attending weekly sessions should provide added value for students through close interaction between students and teachers. If there is no room for discussion or no further explanations in the weekly sessions, a large part of the material for the non-active group of students is lost. Implementing the ILLF sheds a different light on the role of the teacher: the teaching person is no longer the primary knowledge mediator, as students have to extract the knowledge from the literature themselves. Rather, the teacher works as a guide or a facilitator for students to retrieve the information from selected literature. Even though this change of role promises to impact the learning outcomes of students more immediately, it imposes some limitations on potential applications of the format. Firstly, the teacher has to have sufficient expertise on a topic to have the competence to apply it in a flexible setting. The teaching staff must be able to embed students' criticism and questions regarding their theories into a larger theoretical framework regarding the animal ethics debate. Secondly, the teacher must be comfortable in that particular role and willing to take the risk of going along with the students' input. This includes preparing slides not to provide more information, but rather to entertain questions and discussions that may go beyond the provided literature. Lastly, the teacher must have good moderating skills to react to students and embed their input into a larger debate. Overall, this particular role might not suit everyone, but, as this case study has shown, there are good reasons to try becoming accustomed to flipping the classroom. Taking all of this into account, we regard any further application of the ILLF, be it in an online or on-site format, as desirable. This is especially true because transferring to an on-site mode promises to be insightful for future semesters. Only then can it be discerned whether ILLF actually works well as an online format or whether it has worked well despite being an online format. It is possible that the ILLF can be further improved upon in an on-site mode with all the benefits of more immediate interaction. 6. Conclusions This case study presented the implementation of an online teaching format that was developed by the authors as a reaction to the pandemic restrictions on university teaching. Taking this setting as the opportunity to explore digital formats, the authors drew from flipped-classroom methodology to design a novel lecture format, the Interactive Literature Lecturing Format (ILLF). The ILLF has been presented here as a structured didactic tool that introduces a novel format to recurring lecture courses with reasonable effort. It not only displays a high quality of the students' engagement with animal ethics due to the independent knowledge transfer and the consequently engaging discussions, but it is also viable and enjoyable for the teaching staff. The evaluation survey further shows that the students' satisfaction was very high throughout all phases of the lecture course, regardless of whether they were actively participating or whether they prepared independently for the exam. The ILLF restructures the lecture in a way that enables the teaching staff to reimplement it each semester without any alterations or compromises in the quality of teaching, as the high level of interaction with students is embedded within the structure of the ILLF. Thus, it is promising not only to remain with the ILLF for the animal ethics lecture discussed here, but to explore its further potential in other teaching areas, in other fields and in on-site teaching. However, when it comes to online-teaching, this case study has shown that it should not be regarded as a deficient or second-choice option per se. The implementation of the ILLF in an online mode shows that there are lecturing formats that can ensure high-quality teaching online. Thus, online teaching should be regarded independently from on-site teaching, as a didactic mode in its own right, that can obtain very good outcomes for both students and teachers, if the format actually fits the setting. Therefore, teachers should be encouraged to explore novel digital formats beyond any pandemic restrictions. Acknowledgments The authors would like to thank Svenja Springer for her support in writing the methods section and data analysis. Furthermore, they thank Dennis Papadopoulos, Erich Linder, Konstantin Deininger and the two anonymous reviewers for their helpful remarks on this case study. Author Contributions In the conversion process to ILLF, the second author, H.G. was the teaching professor, the first author, K.D. worked as the assistant; Original idea and didactic design of the ILLF, K.D.; Implementation, K.D. and H.G.; Teaching, H.G.; Organization of teaching and evaluation, K.D.; Conceptualization of Case Study, K.D. and H.G.; Methodology, K.D.; Software, K.D.; Investigation, K.D.; Writing--original draft, K.D.; Writing--review and editing, H.G. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement According to the statute of the Ethics Committee of the University of Vienna (SS2; re-release of the statute section "Ethikkommission", as per university gazette 2012-03-16, 18th piece, no 106), the Ethics Committee evaluates research projects on or with humans: These are investigation which may affect the physical or psychological integrity, the sphere of privacy, other subjective rights or predominant interests of research participants. None of the mentioned criteria apply to application number 00904, based on the submitted documents, therefore; hence, the Ethics Committee does not object to the investigation being implemented and conducted [Chair of Ethics Committee, 28 December 2022]. Informed Consent Statement The Data Protection Officer of the University of Vienna has audited the data analyses throughout several stages of this paper. It is confirmed that the storing, processing, and publication of the data has been carried out in anonymized form and thereby in compliance with GDPR (General Data Protection Regulation (EU) No 2016/679 of the European Parliament and Council). Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Appendix A The first session deals with Peter Singer's position. Primary Literature: Peter Singer, All Animals Are Equal (1976) . Secondary Literature: Herwig Grimm & Markus Wild, Grimm, Der Praferenzutilitarismus Singers (2016) , (pp. 57-78). Both texts are mandatory readings. Literature Guiding Questions, 1st Session According to Peter Singer, why are animals to be considered morally? Explain the following central concepts in Singer's approach and their significance for his argument: speciesism and interspecies equality; universalizability; and vital vs. trivial interests. In what way does Singer categorize life hierarchically? What is "the argument from marginal cases"? How does Singer weigh up different interests against each other? Did Peter Singer's approach convince you? Why/Why not? Are there any aspects that are not yet clear to you? Are there aspects of Singer's theory that you would like to discuss in the session? animals-13-00826-t001_Table 1 Table 1 Students' evaluation of the Interactive Literature Lecturing Format (ILLF): Comparison between active and non-active group. Active (n = 21) Non-Active (n = 44) Total (n = 65) Overall evaluation of the format Mean +- Std 1.48 +- 0.75 1.32 +- 0.56 1.37 +- 0.63 Workload compared to other lecture formats * Mean +- Std 3.76 +- 0.70 3.64 +- 0.66 3.69 +- 0.67 Questionnaire ** Count (%) Helpful for guiding text work 12 (57.1) 31 (68.9) 43 (65.2) Helpful for exam preparation Not particularly helpful 17 (81) 0 (0) 38 (84.4) 1 (2.3) 55 (83.3) 1 (1.5) Perceived difficulty of exam preparation Mean +- Std 2.38 +- 0.74 2.41 +- 0.92 2.40 +- 0.86 Perceived difficulty of exam Mean +- Std 2.62 +- 0.67 2.64 +- 0.65 2.63 +- 0.65 * Please note that there has been an inconsistency in the numeric scale on the evaluation forms in the two collectors for the first exam date. The numeric scale for the non-active group's evaluation form ranges from 1 to 7 instead of 1 to 5. Thus, the responses (n = 11) to this question in this collector have been excluded. ** Multiple answers were possible. 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PMC10000073

Different results have been reported concerning the relationship of the apparent diffusion coefficient (ADC) values and the status of methylation as the promoter gene for the enzyme methylguanine-DNA methyltransferase (MGMT) in patients with glioblastomas (GBs). The aim of this study was to investigate if there were correlations between the ADC values of the enhancing tumor and peritumoral areas of GBs and the MGMT methylation status. In this retrospective study, we included 42 patients with newly diagnosed unilocular GB with one MRI study prior to any treatment and histopathological data. After co-registration of ADC maps with T1-weighted sequences after contrast administration and dynamic susceptibility contrast (DSC) perfusion, we manually selected one region-of-interest (ROI) in the enhancing and perfused tumor and one ROI in the peritumoral white matter. Both ROIs were mirrored in the healthy hemisphere for normalization. In the peritumoral white matter, absolute and normalized ADC values were significantly higher in patients with MGMT-unmethylated tumors, as compared to patients with MGMT-methylated tumors (absolute values p = 0.002, normalized p = 0.0007). There were no significant differences in the enhancing tumor parts. The ADC values in the peritumoral region correlated with MGMT methylation status, confirmed by normalized ADC values. In contrast to other studies, we could not find a correlation between the ADC values or the normalized ADC values and the MGMT methylation status in the enhancing tumor parts. apparent diffusion coefficient glioblastoma MRI MGMT This research received no external funding. pmc1. Introduction Glioblastoma (GB) is the most common and most aggressive primary brain neoplasm. The prognosis of GB is very poor, which is often explained by the molecular heterogeneity of its genome, which leads to an unpredictable clinical course in treatment response . The most well-known gene alterations according to the recent World Health Organization (WHO) Classification of Tumors of Central Nervous System include, among others, the genes for the enzyme methylguanine-DNA methyltransferase (MGMT). Modifications of this gene have implications on treatment response and prognosis . Magnetic resonance imaging (MRI) is widely accepted as the modality of choice for the diagnosis and evaluation of treatment response . GB has an infiltrative pattern of growth and can expand into normal-appearing brain tissue, which is further than the conventional radiological margin. Computer tomography (CT) and standard MRI testing have underestimated the actual tumor extent . In order to achieve successful surgical resection in a best-case scenario, the greatest possible extent of the tumor must be resected without injuring nearby seemingly unaffected brain tissue . Diffusion weighted imaging (DWI), in combination with other sequences, is used for the assessment of brain tissue function and physiology. As a subset of DWI, apparent diffusion coefficient (ADC) maps/values represent Brownian motion in water molecules at a sub-voxel level . ADC maps and DWI are technically robust and can be obtained without administration of a contrast agent. Since the extracellular volume fraction is linked to water diffusion and is highly involved in tissue cellularity, tissue edema, and tissue necrosis, DWI and ADC maps are helpful in the initial assessment and post-treatment assessment of GBs . Furthermore, when evaluated against MRI results, ADC values offer a unique characteristic, in that they are numeric values (as compared to the Hounsfield units in CTs) that are easily measurable and can be monitored between different examinations. Studies have shown that low ADC values before treatment were correlated with high cellularity and the overall survival of glioma patients . Furthermore, mean ADC values were correlated with overall survival . In the difficult diagnostic dilemma of pseudo-progression vs. radiation-induced necrosis, ADC values have also been used for differentiation . The molecular profiles of tumors of the central nervous system (CNS) have increased in value in recent years, which has been reflected in the the most recent classifications of CNS tumors by the WHO, setting a greater emphasis on molecular profiling concerning tumor sub-types and management. Previous studies concerning MGMT status and ADC values have shown correlations with differing results . Normalization of ADC values is a process that aims to reduce variations by calculating the de facto ratio of tumor ADC values and normal-appearing white matter (NAWM) using the patient as their own control .The normalization of ADC values has also shown differing results, though, especially concerning progression-free survival . The aim of this study was to investigate if there were a correlation between ADC values (absolute and normalized) and the MGMT status in glioblastoma, with a focus on the enhancing tumor and the peritumoral region, identified in dynamic susceptibility (DSC) perfusion and T1-weighted sequences after contrast administration. 2. Materials and Methods This retrospective study was approved by the Ethical Committee of the Medical University of Innsbruck (AN 5100 325/4.19). Informed consent was obtained by all participants. 2.1. Patients The time period of recruitment was June of 2015 to March of 2018. Inclusion criteria were patients over 18 years of age, newly diagnosed GB with MRI scans before any therapy, biopsy, or surgery, and a post-surgical neuropathological diagnosis, including MGMT status. The time of survival after the initial MRI-study was retracted from the local patient archive. Exclusion criteria concerning image quality were based on general quality standards in MR-imaging, especially assessing for motion artifacts since they could affect the co-registration of the ROI. Only IDH wild-type glioblastomas were included in this retrospective study. Analysis was performed via immunochemistry in all 42 patients with further DNA-sequencing in 10 patients. Histopathological data were considered insufficient if there was no assessment for MGMT status (which was due to less representative tumor material) or the analysis was inconclusive (which was due to poor DNA-quality). 2.2. Image Acquisition and Analysis The MR images for each patient were acquired with a 3T MR imaging scanner (Siemens Healthcare, Verio, Germany) using identical acquisition protocols and parameters. The protocol included DWI and ADC maps (b-values were between 0 and 1000 s/mm2) in an axial plane, T2 turbo spin echo in an axial plane, susceptibility weighted imaging in an axial plane, T2-FLAIR in a coronal plane, T1-MPRAGE before and after contrast administration with multi-planar reconstructions in the axial, sagittal, and coronal planes and dynamic susceptibility contrast perfusion in an axial plane. The specific parameters for DWI are included in Table 1. The obtained MR images of the aforementioned protocol were digitally transferred from the picture-archiving and communication system (Infinitt; Infinitt, Seoul, Korea) to the post-processing program Olea (Olea Medical, La Ciotat, France). After co-registration via overlay in Olea between anatomical images (T1-MPRAGE after contrast) and cerebral blood volume (CBV) of DSC-perfusion, specific regions of interest (ROI) were defined as following: ROI 1 was defined in the center of the enhancing tumor with the highest visible area of perfusion, excluding necrotic components. ROI 3 was defined directly next to ROI 1, outside the enhancing margin of the tumor in the adjacent white matter. ROI 5 was defined 2 cm from the enhancing tumor core in normal-appearing white matter, and ROI 7 was defined as far away as possible from the enhancing tumor core, also in normal-appearing white matter. In order to minimize the heterogeneity of data according to different brain regions , we further defined ROI 2 (corresponding to ROI 1), ROI 4 (corresponding to ROI 3), ROI 6 (corresponding to ROI 5), and ROI 8 (corresponding to ROI) as the mirrored areas in the contralateral healthy hemisphere in order to calculate normalized ADC values. All manually drawn ROI were co-registered with the concordant ADC maps. After evaluation of the measured, mean ADC values in our ROI, four ratios (Q1, Q2, Q3, Q4) of ADC values were calculated. Q1 was equal to ROI 1 divided by ROI 2, Q2 was equal to ROI 3 divided by ROI 4, Q3 was equal to ROI 5 divided by ROI 6, and Q4 was equal to ROI 7 divided by ROI 8. The ratios corresponded to normalized ADC values. To minimize age-related change, special care was observed not to place ROIs in white matter affected by microvascular damage. We also ensured special care when assessing peritumoral edema in the contralateral hemisphere in order to only include ROIs in normal-appearing white matter that was not affected by peritumoral edema. This was achieved by also assessing T2 turbo spin echo sequences and DWI sequences. The selection of the ROIs, analysis, and evaluation of the ADC values were conducted by an experienced neuroradiologist. 2.3. Neuropathological Assessment All patients underwent either biopsy or resection following a baseline MRI. The tissue was sent to neuropathology for further diagnostics. The MGMT methylation status was assessed by pyrosequencing, using the Therascreen MGMT Pyro Kit (QIAGEN, Hilden, Germany). Tumors with a mean methylation percentage of more than 8% were considered to be MGMT methylated . 2.4. Statistical Analysis Statistical analysis was performed using R (R Core Team v. 3.6.1). The Shapiro-Wilk test and a one-sample Kolmogorov-Smirnov test were used to assess the normality of the data, which were presented with quantile-comparison plots and histograms. The data were normally distributed. Student's t-test was used for comparison between the groups. Total p-values < 0.05 were considered statistically significant. The results were presented using boxplots. For the significant areas, the receiver-operating characteristic (ROC) curves were created, and the area under the curve (AUC) was calculated. 3. Results 3.1. Descriptive Analysis A total of 63 patients were initially included in the study. Of those, 21 patients had to be excluded due to poor image quality, or insufficient or inconclusive histopathological data. A total of35 patients received surgical resections with histopathological confirmations after surgery. A single patient received a biopsy prior to surgical resection. Seven patients received biopsies with no surgical resection afterwards. After exclusion, 42 patients remained (11 female, 31 male; mean age 64.0 years +/- 14.2). A total of 19 patients (45.2%) had MGMT-methylated tumors (group 1), while in 23 patients, (54.8%) the tumors were MGMT unmethylated (group 2). The frequency and location of the GBs are included in Table 2. 3.2. MGMT Status We could show significantly (p = 0.002) higher ADC values in the peritumoral white matter (ROI 3) of the patients with a negative MGMT methylation status, as shown in Figure 2. The mean ADC values in the peritumoral white matter of patients with a negative MGMT methylation status were 1.21x10-3 mm2/s; however, in patients with a positive MGMT methylation status, the mean ADC values were 0.99x10-3 mm2/s. The difference of normalized peritumoral ADC values was even more pronounced (p = 0.0007), as shown in Figure 3. There were no significant differences in the ADC values of the enhancing tumor regions or the subsequently calculated normalized ADC values (Q1 and ROI 1) that concerned MGMT methylation status, nor the normal-appearing white matter 2 cm (ROI 5), nor even further away (ROI 7) from the enhancing tumor parts, nor the subsequently calculated normalized ADC values (Q3 and Q4). The mean ADC values in the enhancing tumor region of patients with a negative MGMT methylation status were 0.76x10-3 mm2/s, and in patients with a positive MGMT methylation status, it was 0.79x10-3 mm2/s. The ROC curves were generated for the peritumoral white matter regions (T2) and normalized values of the peritumoral white matter regions (Q2) . The AUC for the T2-region was 0.75, and for the Q2-region 0.79.The cut-off value for the T2-region was 1.055 with the sensitivity and specificity of 0.739 and 0.737, respectively, and cut-off value for the Q2 region was 1.305 with the sensitivity of 0.783 and specificity of 0.737. 3.3. Survival Analysis In this study, the group the patients with MGMT-methylated tumors did not show significantly increased survival rates, as compared to the patients with MGMT-unmethylated tumors. This was expected, as this group was part of a larger cohort with similar results, which was already published and discussed in detail in a different study . 4. Discussion Our results indicated that there were significant differences in the ADC values in patients with GB in their peritumoral white matter, correlating with their genetic profiles of their MGMT methylation status. In a study by Ellingson et al., a positive MGMT methylation status was linked to more pronounced peritumoral edema, though these findings were only based on visual impressions (T2 and FLAIR sequences) and not validated via absolute ADC values . Since ADC values are not solely influenced by edema but also by tissue necrosis and tissue cellularity, they may be superior in assessing peritumoral white matter, in our estimation. The center of the T1 enhancing tumor corresponded to the necrosis area, so we did not expect to find any reasonably explainable differences in these necrotic areas. The analysis of the ADC values of the more peripherally placed ROIs (ROI 5-8) in normal-appearing white matter also did not show any significant differences. This finding was expected, as well, since conventional MRI sequences cannot depict the real, microscopic invasion of normal-appearing white matter . However, peritumoral T2-hyperintense white matter in GB corresponded not only to the vasogenic edema found in other encountered brain-mass lesions, but it also contained significant amounts of tumor invasion areas . When combined with other predictors, this correlated with the likelihood of tumor recurrence after treatment . This was why we expected to find the most pronounced difference in exactly these areas with significant neoplastic presence and close proximity to the enhancing and perfused tumor area, but without visually appreciable necrosis. Choi et al. showed the prognostic value in assessing the ADC histogram analysis, but they found no correlation between ADC parameters and MGMT status. In their analysis, they assessed the extent of the complete conventional tumor but did not assess the peritumoral ADC values, nor did they assess or include tumoral perfusion characteristics, which was fundamentally different to our study design. In other studies, lower ADC values have been associated with more malignant tumors and tumors with higher cellularity . It has even been suggested by various authors that the radiological reports of low grade gliomas should include the location of the areas with the lowest ADC values. These low ADC value areas, along with other imaging indices and characteristics, could represent the most malignant parts of these tumors and should, therefore, be assessed in the surgical strategy concerning resection or biopsy. Because if the tissue sampling was localized in a part of the mass-like lesion with higher ADC values, final pathological diagnosis could under-grade the tumor, thus influencing therapy options. However, various studies have pointed out the influence of MGMT methylation status on the survival of GB patients . In this light, our results were somewhat unexpected since higher peritumoral ADC values in MGMT-unmethylated tumors suggested lower cellularity and lower peritumoral neoplastic infiltration than their MGMT-methylated counterparts.In this study, this finding could be explained by more pronounced apoptotic or necrotic activity in peritumoral areas of the MGMT-methylated tumors, as shown in studies with phosphorous MR-spectroscopy . Recent studies have shown different results concerning the usefulness of normalizing ADC values. In this study, normalization slightly increased the statistical significance in the peritumoral ADC values (ROI3 and ROI4). The other evaluated areas and ratios (Q1, Q3, Q4) did not show any significant differences in absolute and normalized values. Since only ADC values of ROI 3 (peritumoral white matter) showed differences that were highly statistically significant, this finding was expected. Normalization slightly increased the statistical significance but, ultimately, did not produce any new aspects of the defined ROIs, so the additional value may solely be in confirmation. In further studies concerning ADC values in the cerebrum, data confirmation may be important, and the normalization of ADC values is neither complex nor prone to mistakes, so it be a reasonable addition. MGMT methylation status is an important prognostic factor since the level of methylation of MGMT corresponds to the therapeutic effects of chemotherapeutic alkylating agents, such as temozolomide . Although many studies have been conducted concerning the correlation of visual parameters (tumor location and laterality, enhancement characteristics such as ring enhancement) and MGMT methylation status, there is still no broadly accepted consensus . Further advancements in radiological diagnostics could lead to a future where molecular profiles can be predicted on the basis of an initial MRI study, which could have vast implications for treatment options prior to any surgical intervention (biopsy or resection) and potentially for the surgical strategy, as well. Patients who are unfit for surgery or biopsy could significantly benefit from such advancements. Presently, there is still much uncertainty concerning the prognostic value of ADC values without clear results. Our results indicated that extending the scientific scope beyond conventionally accepted tumor boundaries could be an area of interest for evaluation, but further research is warranted. Since ADC maps are usually part of a routine cerebral MRI tumor protocol, there are large datasets available to evaluate. Enhanced analysis using radiomics could be very helpful for discovering further insights into the diagnostic value of ADC values in the diagnostic stage of GB. Ideally, these studies should include an evaluation of the connection of peritumoral ADC values and the epidermal growth factor receptor (EGFR), which has also been commonly reported as mutated in GB . A study in which we assess the peritumoral ADC values prior to any treatment and after treatment could also be of great interest, especially concerning possible alterations as a marker of the treatment response connected to the genetic profile. Limitations of the Study The evaluation of ADC values is a process prone to inaccuracy with low inter-rater reliability, as the values vary regarding the positioning of the ROI. Although we established a very strict model for positioning the ROI based on multiple sequences in order to link anatomical parameters with a high spatial resolution (T1-MPRAGE after contrast administration with 1mm slice thickness) to functional parameters (DSC perfusion and ADC Maps), it was not possible to exclude all bias due to the manual drawing. The slice length of 3 mm in ADC maps was rather small, but it was adequate for the purpose of interpreting diagnostic studies. However, the co-registration with T1-MPRAGE sequences after contrast administration and DSC perfusion maps could have led to further inaccuracy since ADC maps could be affected by a partial volume effect. This was a retrospective study with a rather small sample size (42 patients); in order to gain further insights, we recommend that our results be validated by a larger study, ideally in a prospective setting. 5. Conclusions The ADC values in the peritumoral region correlated with the MGMT methylation status, confirmed by normalized ADC values. In contrast to other studies, we could not find a correlation between the ADC values or the normalized ADC values and the MGMT methylation status in the enhancing tumor. However, with non-perfect sensitivity and specificity, these values could be a part of future prediction algorithms in order to estimate the MGMT status of glioblastomas in vivo. Acknowledgments The authors would like to thank Milovan Regodic for his useful comments regarding the statistical analysis. Author Contributions Conceptualization, V.K.L., M.G. and A.E.G.; methodology, V.K.L., M.G., J.K., C.F.F., M.N., A.M.B.-T., J.H., E.R.G., S.M. and A.E.G.; validation, V.K.L., M.G., E.R.G., S.M. and A.E.G.; formal analysis, V.K.L. and M.G.; investigation, V.K.L., M.G. and A.E.G.; resources, C.F.F., J.H., E.R.G. and A.E.G.; data curation, V.K.L.; writing--original draft preparation, V.K.L. and M.G.; writing--review and editing, V.K.L., M.G. and S.M.; visualization, V.K.L. and M.G.; supervision, A.E.G.; project administration, E.R.G. and A.E.G.; funding acquisition, E.R.G. and A.E.G. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethical Committee of the Medical University of Innsbruck (AN 5100 325/4.19). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The original data regarding this study can be made available upon request at the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Abbreviations The following abbreviations were used in this manuscript: ADC apparent diffusion coefficient GB glioblastoma MGMT methylguanine-DNA 2 methyltransferase MRI magnetic resonance imaging DSC dynamic susceptibility contrast ROI region-of-interest ROC receiver-operating characteristics AUC area under the curve Figure 1 Defined regions of interest in a representative case. Overlay images were acquired in Olea (c). From left to right: Overlay of DSC perfusion and ADC maps; T1 MPRAGE post-contrast administration; and overlay of T1 MPRAGE post-contrast administration and DSC perfusion. Figure 2 Absolute ADC values in peritumoral white matter (ROI 3) in patients with MGMT-unmethylated GB (MGMT -) and patients with MGMT-methylated GB (MGMT +). Figure 3 Normalized ADC values in peritumoral white matter (Q2) in patients with MGMT-unmethylated GB (MGMT -) and patients with MGMT-methylated GB (MGMT +). Figure 4 ROC curves for the peritumoral white matter regions (T2) and normalized values of the peritumoral white matter regions (Q2). cancers-15-01384-t001_Table 1 Table 1 Parameters for ADC Maps. Parameters for ADC Maps Echo time 95 ms Repetition time 7500 ms Matrix 256 x 256 Field of view 230 x 230 Time of acquisition 2 min 51 s Plane axial Slice thickness 3 mm cancers-15-01384-t002_Table 2 Table 2 Location of GBs and frequency. Location of GBs and Frequency Temporal left 11 Temporal right 4 Temporoparietal left 1 Temporoparietal right 1 Frontal left 4 Frontal right 6 Parietal left 5 Parietal right 3 Basal ganglia right 2 Occipital left 3 Temporo-Occipital left 1 Temporo-Occipital right 1 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000074

We aimed to investigate the effect of different levels of nutritional restriction on mammary gland development during the embryonic period by gradient nutritional restriction in pregnant female mice. We started the nutritional restriction of 60 female CD-1(ICR) mice from day 9 of gestation based on 100%, 90%, 80%, 70% and 60% of ad libitum intake. After delivery, the weight and body fat of the offspring and the mother were recorded (n = 12). Offspring mammary development and gene expression were explored by whole mount and qPCR. Mammary development patterns of in offspring were constructed using Sholl analysis, principal component analysis (PCA) and regression analysis. We found that: (1) Mild maternal nutritional restriction (90-70% of ad libitum intake) did not affect offspring weight, while body fat percentage was more sensitive to nutritional restriction (lower at 80% ad libitum feeding). (2) A precipitous drop in mammary development and altered developmental patterns occurred when nutritional restriction ranged from 80% to 70% of ad libitum intake. (3) Mild maternal nutritional restriction (90% of ad libitum intake) promoted mammary-development-related gene expression. In conclusion, our results suggest that mild maternal nutritional restriction during gestation contributes to increased embryonic mammary gland development. When maternal nutritional restriction reaches 70% of ad libitum intake, the mammary glands of the offspring show noticeable maldevelopment. Our results help provide a theoretical basis for the effect of maternal nutritional restriction during gestation on offspring mammary development and a reference for the amount of maternal nutritional restriction. mammary development nutritional restrictions pregnancy offspring Modern Agro-industry Technology Research System of ChinaCARS-36 This research was funded by the Modern Agro-industry Technology Research System of China, grant number CARS-36. pmc1. Introduction Nutritional challenges that occur during gestation, a critical period for embryonic growth and development, may lead to alterations in the physiological development and metabolism of the offspring after birth . The most possible nutritional challenges during gestation are undernutrition and overnutrition, which can affect the health of both the fetus and the maternal body . In particular, malnutrition during pregnancy, which still exists in underdeveloped regions, as a global problem, has important implications for the healthy development of the mother and the newborn . These nutritional damages can cause permanent adjustments in the embryonic physiological state and organ development by inducing genetic changes in the proliferation/differentiation pathways during embryonic development . Current research on nutritional restriction during pregnancy has focused on the placenta, brain and other organs that affect fetal survival , with little attention paid to fetal mammary gland development. The embryonic period, puberty and pregnancy are known as the three main stages of mammary gland development. The embryonic development of the mammary gland begins in many mammals at mid-gestation ; for mice, with a gestation period of 19-21 days, the mammary gland initiates development on day 10 of embryonic life . The embryonic mammary glands are formed by a bilaterally multilayered ectodermal stripe from the forelimb bud to the hindlimb bud on the ventral surface of the embryo, referred to as the mammary line . At day 11.5 of the mouse embryonic stage, these milk lines form five visible pairs of placebos. These placebos then become embedded in the mammary mesenchyme. At day 15-16 of the mouse embryonic stage, primary bud formation invades the secondary mammary mesenchyme and begins to develop a branching morphology . Before birth, the mammary gland consists of a small ductal tree with a dominant duct and 10-15 branches embedded in the nascent fat pad . The basic mammary duct system that forms at this time arises in the absence of hormonal input and remains essentially quiescent until puberty . This basic ductal system forms the framework from which the mammary glands develop further during puberty and pregnancy to form the mature mammary glands . If the development of the embryonic mammary glands is slowed at this period due to nutritional deficiencies and other factors, it directly affects the development of the mammary glands after birth and may even affect the amount of milk produced during lactation . Although the embryonic stage is the initiation of mammary gland development, nutritional regulation has remained less studied for this stage of mammary gland development. Since puberty is considered a critical window for nutritional regulation, most nutrition-related research has focused on this period . The impact of embryonic nutrition on fetal mammary gland development is often unnoticed. Although the mammary gland that develops during the embryonic period is considered a basic ductal system, it has the ability to produce milk, known as neonatal milk or switch's milk . This indicates that the mammary gland is already equipped with basic lactation functions after birth, and if there are problems with the development of the mammary gland during the embryonic period, these basic functions are affected. Terminal end buds, an important structure in the extension of the mammary ducts during puberty, form only at the tips of the elongated ducts which are based on branches generated during the embryonic period . Multipotent mammary stem cells (MASCs) from embryonic mammary gland formation are the source of MASCs and progenitor cells required for mammary duct development during puberty and alveolar luminal formation during pregnancy . These results all suggest that there is a connection between embryonic, pubertal and gestational mammary development, and that mammary gland damage caused by nutritional fluctuations received during the embryonic period further affect mammary gland development after birth. The embryonic stage is the period of initial mammary gland formation, when the mammary gland gradually begins to expand through proliferation and differentiation in multipotent MASCs . Many genes associated with mammary stem cells during the embryonic period have been shown to influence the future developmental fate of the mammary gland. The Axin2 gene was found to have the ability to allow cell regeneration in mammary gland transplantation assays, and the expression of the Axin2 gene during the embryonic period has been shown to be associated with future development in the ductal cell lineage . During this period, Wnt5a has also been shown to be required for normal development of the mammary ducts . In addition, MASCs marker genes such as Sox10, Procr, ELF5 and Aldh1a1 were identified by knockout studies and regulate key functions of mammary gland development . After birth, MASCs become lineage-restricted with some becoming progenitor cells and contributing to the development of the mammary gland base or lumen . Thus, nutritionally induced changes in embryonic mammary development may continue to affect mammary development in adulthood. The effect of altered nutrient levels on mammary stem cells has been demonstrated in previous studies with cells and adult mice . Maria Theresa E. Montales et al. found that angiotensin in food affects the number of mammary cell-like/progenitor cells . Omar M. Rahal et al. speculated that diet-regulated hormonal signaling could influence MASC self-renewal . Studies on the effects of nutritional restriction on mammary stem cells have had mixed results, with one study suggesting that nutritional restriction attenuates mammary stem cell viability and inhibits mammary gland development , while another study suggests that nutritional restriction induces the self-renewal of mammary stem cells . These results may be due to differences in the amount of nutrient limitation. Mild nutrient limitation mediates the restoration of stem cell self-renewal capacity through nutrient and energy-sensing pathways . When nutrient limitation exceeds the regulatory level of the cells, apoptosis and necrosis of stem cells can occur due to nutrient deficiency. Compared to nutritional treatment after birth, nutritional treatment for the embryonic period is more difficult and requires nutritional interventions for the maternal body. The most common approach in studies of fetal undernutrition is accomplished through food or caloric restriction of the mother during gestation . The mammalian placenta has evolved mechanisms that help buffer the fetus from short-term fluctuations in maternal diet and energy status . In order to avoid this buffering mechanism, most of the studied protocols reduce maternal nutritional intake to 50-60% of the normal amount, exerting a significant impact on fetal growth and development through high levels of food restriction . Moderate or low levels of food restriction may better mimic the clinical features of malnourished women, but few studies have investigated the effects of moderate food restriction during pregnancy on embryonic development. In addition to maternal nutritional interventions, the smaller size of the embryonic mammary gland presents challenges for the study of mammary gland development. The most visual method of viewing mammary gland development is the whole mount, a method of viewing a three-dimensional overview of the mammary gland, which provides a dense ductal epithelial structure within the complete mammary gland . The whole mount requires the complete mammary gland to be isolated from the skin of the mouse and spread out as naturally as possible, which is more challenging for embryonic and newborn mice. In earlier studies, the results of the whole-mount analysis were difficult to quantify and were only used as a display image in the studies . The complex ducts of the mammary gland in puberty can be evaluated in terms of the area covered and the denseness of the ducts observed visually. However, this unquantifiable observation is difficult to evaluate in the primary mammary gland, which has only 10-15 branches at birth. Jason P. Stanko et al. reported the use of Sholl analysis, an ImageJ plug-in for neuronal analysis, to quantify whole-mount results of the mammary gland . The Sholl analysis creates a series of concentric rings based on a custom center (origin of the mammary duct) and extends to the most distal portion of the branch (enclosing radius). The Sholl analysis plug-in calculates the number of intersections that occur on each ring and then returns a Sholl regression coefficient (k), which is a measure of the rate of decay of the epithelial branches. In Sholl analysis, the sum inters (N) is the number of intersections of multiple concentric circles centered on the primary ducts with the ducts, reflecting the complexity of the mammary gland. The sholl regression coefficient (k) is a measure of the distal mammary branch complexity, which is close to 0, indicating more complex and well-developed distal mammary branches. Branch density is calculated using the formula N/MEA. Sholl analysis provides a valid quantitative measure of mammary branch complexity and has become a reliable method for studying mammary gland development. Mammary gland development in embryonic mice can be evaluated through a combination of fine dissection and whole-mount and Sholl analysis. Different levels of maternal nutritional restriction may have different effects on embryonic mammary gland development due to different maternal nutritional buffering and stem cell responses to nutrition. To investigate this, we established a pattern of nutritional restriction on mammary gland development during embryonic period by setting 100%, 90%, 80%, 70% and 60% diet intakes for female mice during pregnancy. The objective of our study was to investigate the effect of maternal gradient nutritional restriction on mammary gland development in offspring and provide a reference for the amount of maternal nutritional restriction. 2. Materials and Methods 2.1. Animals and Experimental Design Sixty female 8-week-old CD-1(ICR) mice were provided by Vital River Laboratory Animal Technology Co., (Beijing, China) and mated with males of similar age. Each male mouse was put in a cage with 1 female mouse. Mating of mice was demonstrated by the presence of vaginal plugs. Female mice were individually housed after the discovery of the vaginal plugs and recorded as day 0 of gestation. All mice in our study were fed commercially available irradiated sterile growth and reproduction diets for experimental mice (SFS9112, Xietong Biotechnology, Yangzhou, China). To reduce the impact of nutritional restriction on early embryonic growth, it began on the ninth day of pregnancy. Pregnant mice were divided into five groups (n = 12): the 100% group was fed ad libitum (control group), and the 90%, 80%, 70% and 60% groups were fed 90%, 80%, 70% and 60% of the ad libitum food weight daily, respectively. The ad libitum group was mated one day earlier and their intake was used as the basis multiplied by 90%, 80%, 70% and 60% as the feed intake for the gradient nutrient restriction. The weight of the mice was recorded daily. The number of litters as well as the weight of the female mice and offspring were recorded on the day of delivery. 2.2. Body Fat Percentage Assay On the day of delivery, whole body image and body fat percentage were evaluated in vivo using dual-energy X-ray absorptiometry (DEXA) on an InAlyzer (Medikors Co., Seongnam, Republic of Korea). Female mice and female offspring were anesthetized using isoflurane (RWD, Shenzhen, China) and placed on a scanner bed and operated according to the instructions. After in vivo imaging, female offspring mice were euthanized using CO2. 2.3. Collection and Preservation of Mammary Glands The mammary glands were removed immediately after euthanasia, the #4 inguinal mammary glands were placed on slides and immersed in Carnoy's solution for whole mount, and the other mammary glands were stored at -80 degC for real-time quantitative polymerase chain reaction (qPCR). 2.4. Mammary Whole Mount The mammary glands were fixed in Carnoy's solution (60% absolute ethanol, 30% chloroform, 10% glacial acetic acid) for 4 h and then placed sequentially in ethanol at 100%, 70%, 50% and 10% concentrations for 15 min each. After soaking in deionized water for 5 min, the mammary glands were stained using carmine alum solution (1 g carmine alum, 2.5 g aluminum potassium sulfate in 500 mL dH2O) for 4 h. The stained mammary glands were soaked for 5 min using distilled water, then sequentially soaked in 70%, 95% and 100% alcohol, each concentration for 15 min. The mammary glands were placed in xylene for 12 h for transparency and then sealed with neutral resin. Whole-mount slices of mammary glands were sectioned for image acquisition using an upright microscope (Nikion, Japan). 2.5. RNA Extraction and qPCR RNA from offspring mammary glands was extracted using RNA-easy Isolation Reagent (R701-01, Vazyme Biotech, Nanjing, China) according to the instructions. RNA quality was evaluated by 1% agarose gel electrophoresis, while the purity of the total RNA was determined by NanoDrop 2000 (NanoDrop, ThermoFisher Science, Waltham, MA, USA). The genomic DNA was removed from each RNA sample and reverse-transcribed into cDNA using an Evo M-MLV Mix Kit (Accurate Biology, AG11728, Hunan, China). Then qPCR was performed using a SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, AG11701, Hunan, China) with a LightCycler 96 Instrument (Roche, Basel, Switzerland). The reaction program was set to pre-denaturation at 95 degC for 30 s, followed by denaturation at 95 degC for 5 s and extension at 60 degC for 30 s, for a total of 40 cycles, with each reaction repeated 3 times. The primer sequences are shown in Table 1. The amplification efficiency and the specificity of the amplified products of each primer pair were verified using standard curves and melting curves, respectively. The mRNA expression of each sample was normalized relative to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Relative gene expression levels of each target gene were analyzed using the 2-DDct method. 2.6. Statistical Analysis We performed Sholl analysis on mammary whole-mount results according to the method described in a previous study . Mammary gland whole-mount analysis was performed using ImageJ 2.1 software, and the Sholl analysis plugin 4.0.1 for ImageJ was used for Sholl analysis. The distance from the primary ducts to the most distal end of the mammary epithelium (enclosing radius) and the mammary epithelial area (MEA) were measured using ImageJ. The Sholl analysis was performed with the primary duct as the center, the enclosing radius as the ending radius and a radius step size of 0.02 mm. Since the mammary ducts in newborn mice are less developed and farther away from the mammary lymph nodes, the area occupied by the mammary lymph nodes was not calculated in Branch density. Body weight, body fat, litter size, Sholl analysis results and gene expression were analyzed using one-way ANOVA in the ad libitum feeding, 90%, 80%, 70% and 60% groups. One-way ANOVA was performed using IBM spss 25 (Armork, NY, USA), with Sidak correction for multiple testing. Body weight, body fat, litter size and gene expression data were presented as the mean +- the standard deviation (SD). Principal component analysis (PCA) was performed on enclosing radius, MEA, sum inters and k from the results of Sholl analysis in all groups. PCA was performed using the FactoMineR and factoextra packages in R4.2.1, and PCA biplot figures were generated. The enclosing radius of each group in the Sholl analysis results were regressed against MEA, sum inters and k. Linear regression analysis of the mammary Sholl analysis was performed using simple linear regression in GraphPad Prism software 9.1.0 (San Diego, CA, USA). 3. Results After nutrient restriction management, a significant difference in body weight was observed in mice from day ten of pregnancy, and the difference persisted until the end of gestation . After parturition, the adult female mice showed a significant decrease in body weight compared to the control group (p < 0.05), except for the 90% group . However, there was no significant difference in body fat percentage in adult female mice after parturition . When the nutritional intake was only 60% of the normal intake, a significant decrease in litter size was observed compared to the control group , while the individual offspring weight was significantly lower than that of the other groups . The body fat percentage of the offspring was significantly higher in the control group than in the 80%, 70% and 60% groups . The mammary whole-mount images are shown in Figure 3A. For the enclosing radius, a significant increase was observed in the control group compared to the 70% and 60% groups (p < 0.05), while a significant increase was observed in the 90% group compared to the 60% group (p < 0.05; Table 2). MEA did not differ in the control, 90% and 80% groups (p < 0.05), while it was significantly lower in the 70% and 60% groups than in the former three groups (p < 0.05; Table 2). Sum inters were significantly higher in the control, 90% and 80% groups than in the other two groups (p < 0.05; Table 2). The k of 70% and 60% were significantly higher than the other three groups (p < 0.05; Table 2). Branching density was not significantly different among the groups (p > 0.05; Table 2). Consistent with the results in Table 2, an identifiable change in mammary gland development was observed from the 80% group to the 70% group in Figure 3B. Figure 3C shows the number of intersections of each concentric circle with the mammary ducts in the Sholl analysis. The control group had the longest duct extension distance. At a radius of 0.5 mm, the control, 90% and 80% groups reached the highest number of intersections with a similar peak, all higher than the 70% and 60% groups. In order to further investigate the reasons for the dramatic decline in offspring mammary development from the 80% group to the 70% group, we performed a PCA of the mammary whole-mount results (enclosing radius, MEA, sum inters and k). After dimensionality reduction, the data points in the control, 90% and 80% groups were nearer to each other, forming visible distance differences with the 70% and 60% groups, indicating that a massive reduction in mammary gland development in the offspring occurs when the maternal nutritional limit is reduced from 80% to 70%. The PCA bipartite plot shows the scores and loadings of the first two components (dim1 and dim2), revealing the projection of the observed indicators on a space with dim1 and dim2 as axes. In our study, the indicators of mammary gland development were explained by 79.1% of dim1 and 10.2% of dim2, respectively. The variable with the highest weight in the first principal component is the enclosing radius, indicating that the main reason for the difference in distance from the 80% to the 70% group in the mammary glands was the change in enclosing radius. A positive correlation between enclosing radius and MEA and sum inters and a negative correlation with k are presented in the PCA biplot. To analyze the effect of the enclosing radius, which has the highest weight in PCA, on the pattern of mammary gland development in the offspring, we performed a regression analysis of the whole-mount results . In the regression analysis of the enclosing radius with MEA, the 90% and 60% groups had larger slopes compared to the control group, while the 80% and 70% groups had smaller slopes. In the regression analysis of the enclosing radius versus sum inters, as maternal nutritional restriction increased, the slope first increased in the 90% group, then gradually decreased in the 80% and 70% groups and then showed an increase in the 60% group. In the regression analysis of the enclosing radius versus k, the slope of each group is less than the control group, with the 60% group having the lowest slope. We analyzed the expression of development-related genes (Sox10, Axin2, Elf5, Lgr5, Wnt5a, Aldh1a1, Procr), mammary basal cell marker genes (K5), mammary luminal cell marker genes (K18), estrogen (ERa,ERb) and progesterone receptor (PR) genes in the mammary glands by one-way ANOVA followed by a Sidak multiple-comparison test. In the 90% group, Sox10 expression was significantly higher than in the other four groups (p < 0.05), and Elf5 was significantly higher than in the control and 60% groups (p < 0.05). Sox10 was significantly lower in the control group than in the 90%, 70% and 60% groups (p < 0.05), and Axin2 was significantly higher in the control group than in the 60% group (p < 0.05). Aldh1a1 was significantly higher in the 80% group than in the 60% group (p < 0.05). The expression of K5 was significantly higher in the control group than in the 80%, 70% and 60% groups (p < 0.05). In the 60% group, ER1 was significantly lower than in the 90% group (p < 0.05) and ER2 was significantly lower than in the control group (p < 0.05). The expression of other genes did not differ significantly among the groups (p > 0.05). 4. Discussion Nutritional deficiencies during gestation cause irreversible effects in fetal organs , but nutritional deficiency research on embryonic mammary gland development remains vacant. The impairment of mammary gland development at this phase may directly lead to delayed fetal mammary gland development in adulthood . The small size of the mammary gland, which is difficult to observe, and the buffering through the placenta, which reduces the impact of nutritional fluctuations in the embryo, present challenges for the study of mammary gland development during this period. We performed a quantitative study of the mammary glands using whole mount combined with Sholl analysis and further analyzed the developmental pattern of the mammary gland by PCA and regression analysis. Through maternal gradient nutrient limitation, we established a pattern of offspring mammary gland development and revealed stem cell-related gene expression through a gradient reduction in maternal nutrient intake from 100% to 60% during gestation. The main findings of the study were: (1) Mild maternal nutritional restriction (90-70% of ad libitum intake) did not affect offspring weight, while body fat percentage was more sensitive to nutritional restriction (lower at 80% ad libitum feeding). (2) A precipitous drop in mammary development and altered developmental patterns occurred when nutritional restriction ranged from 80% to 70% of ad libitum intake. (3) Mild maternal nutritional restriction (90% of ad libitum intake) promoted mammary development-related gene expression. Inadequate nutrition during pregnancy can have an impact on maternal and fetal health, most notably in the form of weight loss . In our study, differences in body weight of female mice emerged from the tenth day of gestation after nutritional restriction. After delivery, maternal mice in the 80% group lost significant body weight, while body fat percentage was not affected. For offspring, body fat percentage decreased first when nutritional intake was 80% of ad libitum, and weight loss occurred when it was 60%. Our result is similar to a previous study, which found no significant change in offspring birth weight during gestation for a maternal restriction to 75% ad libitum feeding . A reduction in offspring body weight occurs when nutritional restriction reaches 60% or less of the ad libitum intake . It seems that embryonic body fat percentage is more susceptible than body weight when faced with nutritional constraints. Mammary ducts and epithelium need to be embedded in the mammary stroma for growth, which is composed of homogeneous adipose tissue . In studies on obesity, there is a strong association between mammary fat pads and obesity . Although mammary fat pad and body fat have not been studied in studies on nutritional restriction, the possibility exists that a decrease in whole body fat percentage may affect mammary fat pad development. To assess mammary gland development, we performed a Sholl analysis on the mammary glands of offspring with different levels of nutritional restriction. Based on the Sholl analysis reported in the previous study , we innovatively performed PCA analysis and regression analysis on the results of the Sholl analysis to explore the developmental pattern of mammary glands. We found a dramatic decrease in mammary gland development when nutritional restriction was dropped from 80% to 70% of ad libitum intake. In contrast, there was no significant difference in the effect of normal feeding versus 90% and 80% of ad libitum feeding on mammary gland development. We hypothesize that the dramatic delay in mammary gland development may be due to the buffering of embryonic nutrients by the placenta as a "nutrient sensor" . For maternal nutritional restriction to 90% and 80% of normal intake, the buffering mechanism in the maternal body mitigates the effect of nutrition on fetal mammary development, and as the intake decreases to 70%, the maternal buffering limit is exceeded, resulting in delayed mammary development. Inconsistently with our results, the nutritional intake of sows being restricted to 70% of ad libitum intake had no effect on the weight of mammary parenchyma, fat content, protein content and DNA content of the offspring . The weight, DNA content and other methods used in their study to evaluate the mammary glands do not provide a complete view of the development of the mammary ducts compared to the whole mount. In addition, the species may also be responsible for the discrepancy between their results and our findings. From the results of the PCA analysis, we determined that the variable with the greatest weight is the enclosing radius. This suggests that the dramatic decrease in mammary development from the 80% to the 70% group was mainly caused by changes in the enclosing radius. To analyze the developmental pattern of the mammary glands, we performed a regression analysis of the enclosing radius with MEA, sum inters and k. Our study found a positive regression relationship between the enclosing radius and MEA and sum inters and an inverse regression relationship with k. This suggests that as the distance of the terminal duct from the primary duct increases, the mammary gland will cover a larger area with more complex branching, while the terminal decay will be slower. Similar to our results, the similar trend of the mammary longitudinal extension distance with the mammary epithelial area was found in a previous study . We found that compared to controls, mild nutritional restriction (90% of ad libitum intake) had larger regression coefficients in regression analyses with MEA and sum inters and smaller regression coefficients with k. This suggests that offspring with mild maternal nutritional restriction have better potential for mammary gland development. When nutrition was restricted to 80% of ad libitum feeding, the regression coefficients of the enclosing radius and MEA reflected reduced mammary epithelial area growth potential, despite no difference in mammary gland developmental indicators compared to the control group. Interestingly, the regression coefficients of MEA, sum inters and k all showed greater absolute values when the nutritional restriction was 60% of the ad libitum intake. At this level of nutritional restriction, the enclosing radius already showed a significant shortening and, therefore, mammary gland development slowed down. Sox10, Axin2 and Elf5 have been shown to function as key genes in embryonic mammary gland development. Sox10 is expressed in fetal mammary gland stem cells during embryonic mammary gland development and plays a central role in mammary gland development . Axin2, a target gene of the Wnt/b-catenin pathway, has been used as a marker of functional stem cells in the mammary gland in a lineage-tracing approach . Elf5 is required for the proliferation and differentiation of mammary epithelial cells in embryonic mouse mammary glands . We found that mild nutritional restriction (90% of ad libitum intake) increased the gene expression of Sox10 and Elf5, suggesting a positive effect on mammary gland development. In a previous study on dietary control, alternate-day nutritional restriction was proven to increase the activity of tissue-specific stem cells and had positive implications for life extension . Combined with our regression analysis of whole-mount results, our results suggest that mild maternal nutritional restriction does not impair offspring mammary development and may even increase offspring mammary growth potential by increasing the expression of stem cell-related genes. In addition, 60% of ad libitum feeding reduced Axin2 expression, suggesting that high levels of nutritional restriction inhibit mammary stem cell development and mammary gland development. Consistent with our results, in a study of high levels of maternal gestational nutritional restriction (50% of ad libitum feeding), the ability to differentiate neural progenitor cells was decreased . These results suggest that stem cell activity in the embryonic mammary gland is related to the level of maternal nutritional restriction, with mild nutritional restriction contributing to stem cell-associated gene expression and high nutritional restriction inhibiting them. K5 is a known marker gene in the myoepithelial/basal layer of the mammary gland . Our study shows that a decrease in K5 gene expression occurs in the basal lamina of the mammary glands when nutrition is restricted to 80% of ad libitum feeding. Combined with regression analysis, our results showed that the expansion potential of mammary basal and mammary gland area was affected by maternal nutritional restriction up to 80% of ad libitum feeding, despite no significant difference in the results of whole-mount analysis. Embryonic mammary gland development is considered to be hormone-nondependent, and previous studies have demonstrated that embryonic mammary glands are able to develop in mice lacking estrogen (ER-a and ER-b) and progesterone receptors . After birth, especially during puberty, the mammary glands are stimulated by these hormones to develop rapidly. Estrogen is required for the branching of the mammary ducts during puberty, and estrogen and progesterone are required for lobuloalveolar development during pregnancy. In our study, ER-b receptor expression appeared to be reduced when nutritional restriction reached 60% of the ad libitum intake. This suggests that high levels of maternal nutritional restriction may affect the development of offspring mammary estrogen receptors whose impairment may have further effects on mammary development during puberty. 5. Conclusions In conclusion, our results suggest that mild maternal nutritional restriction (90% of ad libitum intake) during gestation contributes to increased embryonic mammary gland development. When nutritional restriction ranges from 80% to 70% of ad libitum intake, mammary gland development decreases dramatically, and changes in developmental patterns occur. Author Contributions Conceptualization, X.D. and Z.W.; Methodology, X.D. and Z.W.; Software, X.D. and X.L.; Validation, Z.W.; Formal Analysis, Z.H. and Z.W.; Investigation, X.D. and Q.H.; Resources, Z.W.; Data Curation, X.D. and Y.W.; Writing--Original Draft Preparation, X.D.; Writing--Review and Editing, Z.W.; Visualization, X.D.; Supervision, Z.W.; Project Administration, Z.W.; Funding Acquisition, Z.W. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All animal procedures used in this study were approved by the Institutional Animal Care and Use Committee of Shandong Agricultural University (Number: SDAUA-2021-021). Informed Consent Statement Not applicable. Data Availability Statement If requested, the authors guarantee that the data supporting the results reported in this article may be provided. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Body weight and body fat of gestational nutritional restriction female mice. (A) Body weight of female mice during gestation. (B) Postpartum weight of female mice. (C) Body fat percentage and X-ray images of female mice after parturition. Identical letters are not significant difference (p > 0.05), while different letters indicate significant difference (p < 0.05), determined by one-way ANOVA followed by a Sidak multiple-comparison test. Bar charts represent mean, error bars represent SD. Different colors represent different groups. Asterisk stands for p < 0.05 and no asterisk means no significant difference. Figure 2 The number, body weight, body fat and X-ray images of maternally nutritionally restricted offspring. (A) Litter size in different nutrient-restricted groups. (B) Birth weight of offspring mice in different nutritionally restricted groups. (C) Body fat percentage of offspring mice in different nutrient restriction groups. (D) X-ray images of offspring mice in different nutritionally restricted groups. Identical letters are not significant difference (p > 0.05), while different letters indicate significant difference (p < 0.05), determined by one-way ANOVA followed by a Sidak multiple-comparison test. Bar charts represent mean, error bars represent SD. Different colors represent different groups. Figure 3 Whole-mount and Sholl analysis of #4 inguinal mammary glands from maternally restricted offspring. (A) Whole-mount image of mammary glands in different nutrient-restricted groups. (B) Bubble plot of mammary gland Sholl analysis. (C) Linear Sholl plots of mammary glands. (D) Principal component analysis (PCA) biplot of mammary gland Sholl results. Figure 4 Regression analysis of mammary whole-mount results from maternally restricted offspring. Regression analysis between (A) mammary enclosing radius and mammary epithelial area (MEA), (B) mammary enclosing radius and sum inters and (C) mammary enclosing radius and Sholl regression coefficient (k). The dots represent individual measured values, the black lines represent linear regressions, and the gray areas represent their 95% confidence intervals. Figure 5 Mammary gland proliferation- and hormone-related gene expression in offspring. Identical letters or no letters are not significant difference (p > 0.05), while different letters indicate significant difference (p < 0.05) determined by one-way ANOVA followed by a Sidak multiple-comparison test. Bar charts represent mean, error bars represent SD. Sox10: SRY (sex-determining region Y)-box 10. Axin2: Axis inhibition protein 2. Elf5: E74-like factor 5. Aldh1a1: aldehyde dehydrogenase family 1. Wnt5a: wingless-type MMTV integration site family, member 5A. Lgr5: leucine-rich repeat-containing G-protein-coupled receptor 5. Procr: protein C receptor. ERa: estrogen receptor 1 (alpha). ERb: estrogen receptor 2 (beta). Pr: progesterone receptor. K18: keratin 18. K5: keratin 5. animals-13-00946-t001_Table 1 Table 1 Nucleotide sequences of real-time PCR primers. Gene 1 Accession Number Primer Sequence, 5'-3' Sox10 NM_011437.1 F: CTGAGCTCAGCAAGACACTAG R: GTTGGTACTTGTAGTCCGGATG Axin2 NM_015732.4 F: AGCCTAAAGGTCTTATGTGGCTA R: ACCTACGTGATAAGGATTGACT Elf5 NM_001145813.1 F: GAGCATCAGACAGCCTGTGA R: CCATTCCAGGATGCCACAGT Aldh1a1 NM_001361503.1 F: CAGTGAGCGGCAAGAAA R: GGAGAGCCAATCTGGAAAG Wnt5a NM_001256224.2 F: GAATCCCATTTGCAACCCCTCACC R: GCTCCTCGTGTACATTTTCTGCCC Lgr5 NM_010195.2 F: GCTCAACTCTCTCTGTTTCCTCA R: GGTGAGGTTTAGCAAAGAGGAGA Procr NM_011171.2 F: CTCTCTGGGAAAACTCCTGACA R: CAGGGAGCAGCTAACAGTGA ERa NM_001302531.1 F: TGTGTCCAGCTACAAACCAATG R: CATCATGCCCACTTCGTAACA ERb NM_010157.3 F: TGTGTGTGAAGGCCATGATT R: TCTTCGAAATCACCCAGACC Pr NM_008829.2 F: GGTCCCCCTTGCTTGCA R: CAGGACCGAGGAAAAAGCAG K18 NM_010664.2 F: CTGGGGTGGCTCTGTGGGGT R: GGCTTCCAGACCTTGGACTTCCTCT K5 NM_027011.3 F: GTACCAGACCAAGTATGAGGAGC R: CCTCTGGATCATTCGGTTCATCT Gapdh NM_001289726.2 F: TGTGTCCGTCGTGGATCTGA R: TTGCTGTTGAAGTCGCAGGAG 1 Sox10: SRY (sex-determining region Y)-box 10. Axin2: Axis inhibition protein 2. Elf5: E74-like factor 5. Aldh1a1: aldehyde dehydrogenase family 1. Wnt5a: wingless-type MMTV integration site family, member 5A. Lgr5: leucine-rich repeat-containing G-protein-coupled receptor 5. Procr: protein C receptor. ERa: estrogen receptor 1 (alpha). ERb: estrogen receptor 2 (beta). Pr: progesterone receptor. K18: keratin 18. K5: keratin 5. Gapdh: glyceraldehyde-3-phosphate dehydrogenase. animals-13-00946-t002_Table 2 Table 2 Sholl analysis of mammary grands from maternally restricted offspring. 100% 90% 80% 70% 60% SEM p-Value Enclosing radius (mm) 1.062 a 1.021 ab 0.985 ab 0.851 bc 0.776 c 0.022 <0.01 MEA (mm2) 1 1.033 a 1.010 a 0.937 a 0.615 b 0.622 b 0.036 <0.01 Sum inters (n) 357.07 a 337.44 a 317.97 a 213.57 b 215.33 b 8.80 <0.01 k 2 6.071 a 6.199 a 6.539 a 8.232 b 8.122 b 0.179 <0.01 Branching density (N/mm2) 369.417 367.253 343.607 364.029 349.753 6.147 0.425 The different letters indicate statistically significant differences (p < 0.05). 1 MEA: mammary epithelial area. 2 k: Sholl regression coefficient. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000075

Objective: To review somatic genetic changes in nephrogenic rests (NR), which are considered to be precursor lesions of Wilms tumors (WT). Methods: This systematic review is written according to the PRISMA statement. PubMed and EMBASE were systematically searched for articles in the English language studying somatic genetic changes in NR between 1990 and 2022. Results: Twenty-three studies were included in this review, describing 221 NR of which 119 were pairs of NR and WT. Single gene studies showed mutations in WT1 and WTX, but not CTNNB1 to occur in both NR and WT. Studies investigating chromosomal changes showed loss of heterozygosity of 11p13 and 11p15 to occur in both NR and WT, but loss of 7p and 16q occurred in WT only. Methylome-based studies found differential methylation patterns between NR, WT, and normal kidney (NK). Conclusions: Over a 30-year time frame, few studies have addressed genetic changes in NR, likely hampered by technical and practical limitations. A limited number of genes and chromosomal regions have been implicated in the early pathogenesis of WT, exemplified by their occurrence in NR, including WT1, WTX, and genes located at 11p15. Further studies of NR and corresponding WT are urgently needed. nephrogenic rests nephroblastomatosis Wilms tumors genetic changes epigenetic alterations This research received no external funding. pmc1. Introduction Nephrogenic rests (NR) are foci of aberrant embryonal tissue in the kidney, which are still present after 36 weeks of gestation . They represent non-obligate precursor lesions of WT and may regress over time or progress to WT due to as yet unknown molecular factors . The presence of multifocal or diffuse NR is referred to as nephroblastomatosis . NR have been described as either perilobar NR (PLNR) or intralobular NR (ILNR), based on the topographic site in the kidney. Both types can also occur in the same kidney as well as in both kidneys of the same individual. PLNR are located at the periphery of the renal cortex and have been shown to consist mainly of blastemal or immature epithelial components . ILNR are located within the lobes and are mostly composed of a combination of cystic epithelial elements surrounded by moderately cellular stromal elements . In addition, heterologous components, including fat, may be present. ILNR are considered to arise prior to PLNR, and are usually not as well demarcated as PLNR . Histologically, according to Beckwith's criteria , NR can be subdivided into different histological subtypes such as dormant rests (especially blastemal cells with minimal proliferation), sclerosing rests (minimal blastemal components and presence of stromal maturation), hyperplastic rests (proliferative signs manifested by blastemal and epithelial cells and increased size), neoplastic rests (usually a discrete spherical nodule), and obsolete rests ((sclerotic) stromal and epithelial components, and minimal blastemal). Any histological subtype of NR has been shown to be able to transition to another NR subtype . In infants, microscopic NR are identified in about 1% of autopsies, the majority of which will either remain stable or go into regression . The occurrence of NR is partly determined by ethnic and demographic factors . In White American children, 20% reveal WT-associated PLNR compared to 7% in Asian American children, and 2% in Japanese children. ILNR, on the other hand, are more common in Asian American (33%) and Japanese children (25%) relative to the White American children (17%) . As ILNR seem to arise from abnormalities earlier in development, they also occur at a younger age in children, namely at a median age of 23 months, whereas PLNR are usually not discovered until a median age of 36 months . PLNR occur mainly in females and ILNR are more often seen in males . The age at diagnosis also differs between ethnic groups. NR are generally diagnosed earlier in Asian American patients (median age at diagnosis 31 months) than in White American patients (median age at diagnosis 39 months) . NR have been described to lead to the development of WT in a subset of cases . WT are the most commonly found malignant renal tumor in children . WT are morphologically heterogeneous embryonic tumors, including epithelial, blastemal, stromal, and sometimes rhabdomyomatous elements . Tumors can be classified in different histological types, which currently grossly determines risk stratification after neoadjuvant chemotherapy treatment . The distinction between NR, mainly PLNR, and WT can be extremely challenging, both radiologically and pathologically, especially if a biopsy is submitted for evaluation. In fact, no single radiological or histological criterion, including size and shape of the lesion, can distinguish NR from WT and a 25% misdiagnosis rate has been reported for radiological assessment . PLNR are associated with epithelial, blastemal, or mixed type WT and stromal or heterologous elements are limited or absent. In contrast, there is an association with ILNR and stromal type WT and heterologous components are frequent . Most WT occur sporadically and are unilateral, but they can also be familial and bilateral . According to the International Society of Paediatric Oncology Renal Tumour Study Group (SIOP-RTSG), kidneys with unilateral WT contain NR in 40% of the cases (25% PLNR, 9% ILNR, 5% both PLNR and ILNR and 1% nephroblastomatosis). Bilateral WT are significantly more frequently associated with NR, as NR have been reported in 94% of the stage V cases . Bilateral WT occur significantly more often in syndromes in the context of which WT recurrently occur . It is beyond the scope of this review to discuss these syndromes, except for their association with NR. Examples are Denys-Drash and WAGR syndrome, which are associated with the presence of ILNR. Patients with these syndromes have a risk of about 30% and 95%, respectively, to develop WT . Patients with these syndromes have a risk of up to 95% and 50%, respectively, to develop WT . PLNR occur more frequently in patients with overgrowth syndromes such as Beckwith-Wiedemann syndrome (BWS) . Some of the abovementioned syndromes have a known genetic driver, such as the occurrence of germline WT1 mutations in Denys-Drash and WAGR syndrome . In addition, numerous recurrent somatic genetic abnormalities, both gene mutations and copy number variations (CNV) at specific chromosomal locations, as well as hypermethylated regions have been found in non-syndromic, sporadic WTs. These include mutations in WT1, CTNNB1, MYCN, TP53, AMER1, FBXW7, GPC3, MLLT1, DIS3L2, DICER1, DROSHA, DGCR8, SIX1 and SIX2, SMARCA4, ARID1A and chromosomal aberrations such as gain of chromosome arm 1q, and loss of 16q and 1p . Although knowledge on tumor-driving changes in WT is increasing, the genetic changes underpinning the development of NR have not been extensively studied. Notably, most NR regress and only some develop into WT . Identifying molecular changes underpinning the transformation of NR to WT may aid the understanding of WT pathogenesis and guide the development of targeted therapies. Therefore, we performed a systematic review of the literature in which molecular analysis was completed in NR or in a combination of NR and WT. This systematic review presents the current knowledge of somatic molecular changes in NR. 2. Materials and Methods 2.1. Search Strategy and Eligibility Criteria This review was written according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement and the protocol was not registered. PubMed, EMBASE and Cochrane were systematically searched for all the available literature on molecular changes in NR in the English language, published from 1990 to 2022. The full search strategy is provided in Table S1. Articles were included if somatic molecular changes in NR were studied, including case reports in which specific analysis for NR was performed. Studies without molecular analysis in NR, non-English literature, animal studies or germline mutation analyses were excluded. Three authors (T.B., A.M., R.K.) assessed all articles independently based on the in- and exclusion criteria. After a comparison and the consensus of the authors on potentially relevant articles, the remaining publications were screened for eligibility criteria, based on their full text. Controversies were resolved by consensus. 2.2. Quality Assessment The "Standards for Reporting Diagnostic Accuracy 2015" (STARD 2015) checklist was used to assess the quality of the included studies individually and is provided in Supplementary Table S2a. The included case reports were assessed using the case report guidelines (CARE checklist 2013), the checklist is presented in Supplementary Table S2b . The included studies were further assessed with the Oxford Centre for Evidence-Based Medicine Levels of Evidence Classification rubric , independently by the three authors (T.B., A.M., R.K.), for methodologic quality. In case of disagreement, it was solved by reaching consensus. 2.3. Data Extraction From the included articles, the number of NR, WT, WT-NR pairs analyzed, mean age at time of diagnosis of WT, sex, histological type of WT and NR and whether a WT was bilateral or hereditary, were obtained. Furthermore, from each study, the genes or chromosomal regions investigated with associated molecular techniques were extracted. 3. Results 3.1. Search Strategy and Eligibility Criteria Our search in databases PubMed, EMBASE, and Cochrane generated a total of 203 articles. Seventy-three were duplicate articles that were found with more than one search engine and were therefore removed. The 130 remaining articles were screened for title and abstract after which 98 articles were excluded because of the in 2.1 mentioned criteria . We performed full-text screening in 32 potentially relevant studies, critically examining the in- and exclusion criteria. Consequently, 14 more articles were excluded, of which five were abstracts only and nine others were excluded since they did not meet the inclusion criteria. The remaining 18 articles were included in this systematic review. There were five additional articles identified by forward and backward snowballing, resulting in a total of 23 articles. In Figure 2, a flowchart of the search and selection process is presented based on the PRISMA scheme. 3.2. Quality Assessment Quality assessment was completed for all 23 included articles. An overview of the quality assessment of 18 included articles, using the STARD checklist, is provided in Supplementary Table S3a (including the STARD Checklist in Table S2a). The remaining five case reports, using the CARE checklist, are presented in Supplementary Table S3b. All included articles could be classified as Oxford level 2 to 4, as shown in Table 1. 3.3. Characteristics of the Included Studies Patient characteristics of the 23 included studies are presented in Table 1. In those studies, 221 NR were analyzed, of which 53 were ILNR, 136 were PLNR, 11 were nephroblastomatosis and 21 were not further specified. A total of 119 WT-NR pairs were analyzed with different methods including methylation analysis, sequencing analysis, LOH analysis, expression analysis and in situ hybridization. There were 28 WT-NR pairs reported with concomitant bilateral WT, whereas this was not explicitly reported in the other cases. Sex and age were described in only a few studies. In the eight studies describing sexual gender, there were 42 females and 30 males. The age at diagnosis for WT was variable ranging from 11 months to 144 months and for NR from 10 months to 192 months. 3.4. Chromosomal Changes Table 2 presents the somatic changes that were found in NR, including the gene or chromosomal region that was involved and the detection method that was used. We identified seven studies in which LOH analysis was used. LOH of 11p15 was found in 13/64 investigated NR (20%), and the same loss was confirmed in the corresponding WT (n = 13) . Three studies demonstrated LOH of 11p13, which revealed that this was present in 4/28 WT-NR pairs (14%) all representing ILNR. There was a single WT-PLNR pair that showed LOH of 11p13 in the WT only . Hoban et al. described a case in which the LOH of chromosome 11 (both 11p13 and 11p15) was observed, where in fact all other maternal chromosomal loci were lost in the NR and WT in a Beckwith-Wiedemann patient . Three studies involved LOH of 16q showing loss in 13/53 WTs (25%). In two studies, loss of 16q was not found in their associated NR , but Austruy et al. detected loss of 16q in one case of nephroblastomatosis . LOH of 7p was investigated in two pairs of NR-WT, but LOH of 7p was only present in WT, not in the corresponding NR . Furthermore, genome-wide comparative genome hybridization (CGH) was performed in two other studies. Vuononvirta et al. investigated 50 PLNR and divided them into three subgroups. The first group showed no copy number changes at all (44%). The second group contained eight cases in which whole chromosome changes were observed (16%). The remaining group, involving 20 cases, presented multiple partial chromosomal gains or losses (40%). In 76%, the NR contained part of the copy number changes seen in the corresponding WT . Steenman et al. also performed whole genome analysis by CGH. This revealed losses of 1p, 7p, 4q and gains of 1q and 12q in nephroblastomatosis and their associated WT. Loss of 11p was found in nephroblastomatosis only. Changes only present in WT were loss of 9p and gain of 8, 10q and 18. Loss of 16q was detected in one case of WT and in one case of nephroblastomatosis adjacent to WT . MdZin et al. found loss of chromosome 22 and showed that the frequency of chromosome 22 loss depended on NR morphology. Dormant, involuted and sclerosing PLNR presented monosomy 22 in 30%, whereas the hyperplastic and adenomatous PLNR showed monosomy 22 in 50%, increasing to a rate of 60-80% in WT . 3.5. Structural Aberrations in Candidate Gene Studies While LOH analysis allows for detecting larger chromosomal regions without exactly identifying the responsible gene(s), candidate gene analysis by targeted sequencing might highlight abnormalities in putative oncogenes and tumor suppressor genes. Seven studies investigated single nucleotide changes in NR compared to WT. Four studies found mutations in the WT1 gene and showed that these can be present in both NR and WT . Using Sanger sequencing analysis, Fukuzawa et al. showed that CTNNB1 mutations occurred only in WT (n = 6) and not in paired NR (n = 6) . Another gene that may play a role in WT tumorigenesis, WTX, presented mutations in both NR (n = 1) and WT (n = 4) . A KRAS gene mutation was found in one case of WT and adjacent NR in the setting of mosaicism . In this same patient Slack et. al. also described a somatic FBXW7 mutation in two WT but not in another WT nodule or the associated NR. Two studies reported an absence of mutations of specific candidate genes. In the first study by Wegert et al., EGFR-internal tandem duplications (ITD) and BRAF-internal deletions (ID) were investigated, involving 208 WTs and 12 NR, but no changes in any of these genes were found . In the second study, where PTEN was investigated, no abnormalities were found in the WT and NR included in the study by Grill et al. . 3.6. Epigenetic Studies Epigenetic changes were described in ten studies, and six of them focused on DNA methylation changes. The different platforms used for methylation profiling in these studies included bisulfate sequencing , combined bisulfate and restriction analysis (COBRA) , pyrosequencing , and Illumina BeadChip microarray . Charlton et al. , using the latter technique, performed a longitudinal study to find differences in DNA methylation to compare NR to NK as well as to WT. For this purpose, differentially methylated regions (DMR) were compared and shown to differ between WT, NR and NK . Hypermethylation was seen in 55% of 629 differentially methylated regions (DMR) in NR as compared to NK . When NR were compared to WT, in paired analysis, two subgroups of WT could be distinguished. In one group, NR and WT grossly harbored the same epigenetic profiles. Yet, the other group of WT showed hypervariability in the methylation profiles in comparison to NR, suggesting that there is a shift in methylation during the development from NK to NR and/or WT . Vuononvirta et al. used pyrosequencing to analyze H19. Hypermethylation of this gene is associated with LOI of IGF-2, which was found in 23/33 (70%) cases and hypermethylation of H19 in 37/40 (93%) of the PLNR cases. Brown et al. analyzed H19 in relation to LOI at 11p13 and 11p15. They found H19 hypermethylation in two WT-NR pairs using COBRA . LOI at 11p13 and 11p15 was found in both WT-NR pairs. LOI of 11p13 leads to the decreased methylation of WT1 antisense regulatory region (ARR), which results in LOI of the noncoding antisense RNA WT1-AS and the alternate coding WT1 transcript AWT1. LOI of 11p15 is responsible for IGF-2 overexpression, another imprinted gene on chromosome 11 . Hypomethylation of WT1 ARR in WT was also studied by Hancock et al. in two WT-NR pairs using bisulfate sequencing, revealing the lowest methylation levels in WT, the highest in NK, and NR showing methylation percentages between fetal kidney (FK) and NK. They also looked at the expression of AWT1 and WT1-AS, and found biallelic expression of AWT1 and WT1-AS in NR and WT, where monoallelic expression was found in the NK . Cui et al. studied RNA expression of the H19 gene in association with IGF-2 expression, using in situ hybridization (ISH) in WT, NR and associated renal medulla. H19 was not expressed in WT and NR, but was present in normal renal medulla . Yun et al. also investigated IGF-2 by in situ hybridization and Northern blotting in NR and WT. Both studies displayed comparable patterns in NR and WT, but there was a variable and heterogeneous level of expression. IGF-2 expression was frequently associated with blastema . LOI of IGF-2 was studied by Ravenel et al. and was present in a WT-NR pair with two PLNR . Coorens et al. used Illumina BeadChip microarray to demonstrate the presence of H19 hypermethylation in NK with clonal expansions (58%), while this hypermethylation was not found in NK without clones. One WT-NR pair was included, in which the WT and NR emerged from a similar ancestral clone at different time points, which is indicative of an association between clones, NR and WT all showing H19 hypermethylation . Finally, Chilukamarri et al. investigated the GLIPR1/RTVP-1 gene. Hypomethylation of this gene was shown in 21 out of 24 WTs. There were two associated NR analyzed, which also showed hypomethylation of this gene . 4. Discussion We have presented an overview of all molecular studies on nephrogenic rests between 1990 and 2022. As a result of this long-time frame, a wide range of techniques were used to examine chromosomal regions, copy number variations, individual genes and epigenetic changes in NR. A total of 23 studies were found showing loss of chromosomal arms 11p13 and 11p15, 1p, 4q and 11p, and gains in 1q, 7q and 12q, as well as mutations in WT1, WTX and KRAS to occur in both NR and WT, suggesting these are early events (Table 3). Mutations in CTNNB1 and FBXW7 and LOH of 16q and 7p are only present in WT, but not the associated NR, therefore likely representing late events (Table 3). Furthermore, differential methylation levels display a relationship between WT, NR, and NK. Summary of early molecular events that occur in both NR and WT and late molecular events that occur in WT only. Late events are therefore likely not involved in the progression from NR to WT. Little is known about the molecular pathogenesis of WT, including the mechanisms that affect transition from NR to WT, although NR have been recognized as precursor lesions . Studying molecular changes in pairs of NR and WT might shed light on the timing of such events. If the same alteration occurs in NR and the associated WT, this can be considered as an early event. Indeed, LOI and LOH at 11p13 and 11p15 were found in both NR and WT, representing the chromosomal regions where the WT1 and the IGF2 genes are located, respectively . No such gene correlations are known for the other chromosomal arms that were recurrently lost (1p, 4q, 7p and 11p), or gained (1q, 7q and 12q) . MdZin et al. found an increasing frequency of monosomy of chromosome 22 from sclerotic/dormant PLNR to hyperplastic/adenomatous PLNR and then finally toward WT, suggesting that loss of chromosome 22 is an early event and that tumor suppressor genes on this chromosome might be involved in WT tumorigenesis . Mutations in WT1 and WTX can also be considered early events, as they were found in both NR and WT. WT1 was first investigated in WT in 1991 , and WTX was first described in association with WT in 2007 . Genetic changes that are found in WT only, and not in NR, are likely to be late events and therefore not involved in the transition from NR to WT. For instance, LOH of 16q appears to be a late event . However, in one case, loss of 16q was also found in the associated nephroblastomatosis . Loss of 7p appears to occur later in development as well , although Steenman et al. found LOH of 7p in a case of nephroblastomatosis . Furthermore, CTNNB1 mutations, which were first described in WT in 1999 , are considered late events in Wilms tumorigenesis . No PTEN, EGFR and BRAF mutations or rearrangements were found in both WT and NR, implying that these genes do not seem to contribute to the transition of NR to WT . It should be noted that, due to the long time interval, there was large variation between studies with regard to the number and type of polymorphic markers used for LOH or LOI analyses, which may have had an effect on the detection of molecular abnormalities. Likewise, for the candidate gene studies, not all genes have been investigated completely or information on the extent of screening is lacking. In one study, all exons of CTNNB1 were sequenced , while Park et al. examined exons 2-10 for detecting mutations in WT1. In studies describing WT1, two studies used primers for only two exons to detect mutations in WT1, not mentioning if all other exons of WT1 were sequenced . In one other study that examined both CTNNB1 and WTX, it was not reported which exons were sequenced . Furthermore, many of the genes that have been shown to be involved in WT tumorigenesis, such as SIX1, SIX2, and DROSHA, have not been systematically analyzed in NR . Thus, comprehensive analyses should be performed using WES or WGS to examine all genes and to prevent important genes from not being detected. Regarding epigenetic changes, there appears to be a correlation between the methylation and expression patterns of NR as compared to WT. The similar or increased methylation of WT and NR with respect to NK suggests that this might play a role in tumorigenesis . There was increased methylation of H19 in WT and NR, and also in the clonal expansions of NK, which suggests a transformation from NK to WT in the levels of methylation of H19. It is remarkable that the WT-NR pair included in this study arose from the same clone and that they are therefore phylogenetically related . Together this may indicate that the clonal beds of NK are "primed" to become a WT. LOI at 11p15 and 11p13 and LOI of IGF-2 were detected in WT and NR, and thus seem to be early events . The presence of LOI of 11p13 correlates with the decreased methylation of WT1 ARR, suggesting that imprinting defects at 11p13 may be involved in tumorigenesis . H19 and IGF-2 are two parentally imprinted genes on 11p15, with opposite regulatory mechanisms: if H19 is silenced, IGF-2 on the other hand is upregulated . This explains why there was expression of IGF-2 in both NR and WT, while H19 expression was not detected . Together with ISH studies showing variable IGF-2 expression patterns in NR and WT, these data suggest a role for IGF-2 as an early driver of WT development . Likewise, hypomethylation of GLIPR1/RTVP1 was found in WT and NR, suggesting that this specific change might also contribute to WT development . As previously described, NR are present in approximately 40% of unilateral WT and in almost all cases of bilateral WTs . Nephroblastomatosis is more frequently present in bilateral WT . This suggests that not every WT might arise from NR. Conversely, not every NR develops into a WT either. WT and NR both develop from the same embryonic tissue, and both morphologically reflect embryonal renal tissue. Normally, nephrogenesis stops at 34 weeks of gestation and any remaining nephrogenic tissue is considered NR. However, little is known about the underlying factors that lead to such persistence. ILNR are frequent in tumors with stromal histology that are typically associated with aberrations in WT1 on chromosome locus 11p13. PLNR occur more often in blastemal and/or epithelial type WT and are associated with alterations in IGF-2 on 11p15 . These associations have been confirmed in several included studies where a distinction was made between PLNR and ILNR. WT1 mutations and LOH of 11p13 are found in ILNR , and LOI of IGF-2 in PLNR . However, LOH of 11p15 can also be found in ILNR . Interestingly, there is a known association between WT1 and CTNNB1 mutations in WT . However, it is notable that CTNNB1 mutations occur as late events and are only present in WT , while WT1 mutations can occur in NR and WT . Even though NR are precursors of WT, there are only few studies which focus on and describe the molecular pathogenesis of NR. In this review, all studies between 1990 and 2020 which analyzed molecular changes in NR and used molecular techniques were included, resulting in only 23 relevant articles, i.e., less than 1 per year over the investigated time frame. We chose 1990 as a starting point , as it coincides with the first histopathologic description of NR and with the wider applicability of molecular methods in biomedical research, but even until 2000 few molecular studies were performed. In addition, most techniques required frozen lesional tissue, while frequently only formalin-fixed paraffin embedded (FFPE) material was available, limiting the possibilities of molecular analyses, at least in previous decades. Finally, it is virtually impossible to distinguish NR macroscopically, apart from patients with nephroblastomatosis, requiring expert microscopic detection on FFPE material with its inherent limitations. Throughout this review, we have applied the histological definition of NR and nephroblastomatosis over the radiological definition, which is not 100% superimposable with the histological term of NR. The supplied histological material was used as a reference point. The diagnosis of NR and WT can only be made after histological confirmation. On CT imaging, NR and nephroblastomatosis may show distinct features from WT, mainly with regard to the shape and size of the lesion . However, up to 25% of the NR and WT are still misdiagnosed, with half of nodules radiologically diagnosed as WT, turning out be NR after histological confirmation . It is outside the scope of this review to further discuss the potential a newer imaging technology, such as diffusion-weighted imaging (DWI) MRI for the above distinction. As presented in Table 1, the number of WT and NR examined varied greatly from study to study. Some genes have been studied on less than five tumors. Therefore, firm conclusions cannot be drawn from these studies. To do so in the future, larger-scale studies of WT and corresponding NR would be needed to assign certain genes a significant role in the development of NR to WT. In addition, mutations such as DROSHA and SIX1 and SIX2, which are known driver mutations in WT, have not been examined in NR. These genes, as can be seen in Figure 3, have been discovered from 2010 onwards. Many studies described in this review were completed at a time when these candidate genes were not yet identified and have therefore not been investigated in NR. Until 2000, mRNA in situ hybridization (ISH), sequencing analysis, LOH analysis and CGH analysis were used to analyze NR . From 2006 methylation studies emerged in which the difference in methylation patterns between NR and WT was investigated . Several methods that were used in studies presented in this review may currently be considered outdated with the advent of new molecular methods, including whole exome and whole genome sequencing, RNA sequencing and also spatial transcriptomics, which allows tissue context while analyzing gene expression profiles. These techniques may allow faster progress and seem particularly relevant for NR analysis, which still relies on morphological identification. It is important to consider tests for the early detection of genetic, epigenetic and somatic changes in NR that may become clinically relevant to the patient before NR develop into a WT. At the moment, not all genetic predispositions have been explored. Therefore, all children with WT or nephroblastomatosis should be referred to a clinical geneticist and advised to undergo whole exome or whole genome sequencing, as there is a high risk (up to 33%) of genetic predisposition, as was recently shown in a Dutch cohort . This certainly applies to children with bilateral WT, in whom an 80% chance of genetic predisposition has recently been revealed . 5. Conclusions In conclusion, over the past 30 years, several studies have looked at NR with or without associated WT. As a result, much has become known about the genetic changes in NR. LOH of 11p13 and 11p15, expression of IGF-2 and mutations in WT1 and WTX appear to play a role in the early tumorigenesis of WT. LOH of 16q and 7p and mutations in CTNNB1 seem to occur later in development. Methylation patterns of NR in comparison to WT appear to be similar. Due to rapid advances in (genome-wide) molecular techniques, the increased possibilities to use FFPE material and the availability of histologically confirmed frozen material, genetic changes in NR and corresponding WT may be investigated in larger series and might unravel early steps in the progression of WT tumorigenesis. Supplementary Materials The following supporting information can be downloaded at: Table S1: Search strategy PubMed and EMBASE, Table S2a: "Standards for Reporting Diagnostic Accuracy 2015" (STARD 2015) checklist, Table S2b: Case report guidelines (CARE checklist 2013), Table S3a: Quality assessment of 18 out of 23 included articles based on the STARD 2015 checklist, Table S3b: Quality assessment of 5 out of 23 included articles based on the CARE 2013 checklist. Click here for additional data file. Author Contributions Conceptualization, T.B., J.D., A.M.C.M.-G. and R.R.d.K.; methodology, T.B., J.D., A.M.C.M.-G. and R.R.d.K.; writing--original draft preparation, T.B., A.M.C.M.-G. and R.R.d.K.; writing--review and editing, T.B., J.D., A.M.C.M.-G., M.M.v.d.H.-E. and R.R.d.K. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement No new data were created in this study. Data sharing is not applicable to this article. Conflicts of Interest The authors declare no conflict of interest. Figure 1 (A) Hematoxylin and eosin-stained slide with an overview of a hyperplastic perilobar nephrogenic rest, composed of epithelial structures without cytonuclear atypia of the composing cells. Magnification 2x. (B) Hematoxylin and eosin-stained slide with overview of an intralobar nephrogenic rest, composed of cystic structures with some intervening stroma. Both types of rests are surrounded by normal renal parenchyma. Magnification 2x. (C) Hematoxylin and eosin-stained section of a 9 mm lesion of the left kidney of 2-year-old male patient for which nephron-sparing surgery was performed. The patient underwent pre-operative chemotherapy according to the UMBRELLA protocol and also had a lesion of the contralateral kidney that was simultaneously removed. The image shows fibrous tissue of the kidney capsule at the top. Below there are glandular structures compatible with epithelial elements of a perilobar nephrogenic rest, as well as a blastemal component with limited nuclear atypia. Magnification 10x. (D) Hematoxylin and eosin-stained section of the same lesion as (C). This lesion was eventually classified as compatible with a perilobar nephrogenic rest after the careful review of multiple expert pathologists. Please note the bland nuclear morphology of the epithelium and blastema in this close-up image. Magnification 20x. Figure 2 Flowchart of the search and selection process. Figure 3 History of identified aberrations in WT and introduction of molecular methods. The above timeline shows selected WT-associated genes and there first description in relation to WT. The lower part indicates the year of the first description of the selected molecular methods. LOH, loss of heterozygosity; CGH, comparative genomic hybridization; RNA, ribonucleic acid; WES, whole exome sequencing; WGS, whole genome sequencing. cancers-15-01363-t001_Table 1 Table 1 Characteristics of reviewed NR cases. Study Oxford Level NR (n) NR Type WT (n) WT Type WT-NR Pairs Bilateral WT Sex Age at Diagnosis Chang et al., 2021 * 4 Multiple 1 PLNR NBL 1 - epithelial type - 1 - Yes - M F 21 months 36 months Slack et al., 2021 4 Multiple PLNR 1 epithelial type 1 Yes M 21 months Coorens et al., 2019 4 1 ND 23 ND 1 No ND ND Wegert et al., 2018 2 12 ND 208 ND ND ND ND ND Charlton et al., 2015 2 22 17 PLNR, 5 ILNR 36 ND 20 ND ND ND MdZin et al., 2011 4 9 1 9 PLNR 1 PLNR 2 1 blastemal type mixed type 1 1 Yes No M M 42 months 48 months Fukuzawa et al., 2010 4 4 4 ILNR 4 3 stromal type, 1 blastemal type 4 ND 3F, 1M ND Grill et al., 2010 2 26 18 PLNR, 8 ILNR 22 14 mixed type, 2 stromal type, 4 blastemal type and 1 regressive type, 1 not known 22 ND ND ND Vuononvirta et al., 2008 2 50 50 PLNR 25 ND 25 17/50 NR ND ND Brown et al., 2008 ** 4 Multiple PLNR 51 ND 2 2; 2 WT-NR pairs ND ND Chilukamarri et al., 2007 4 2 ND 24 5 stromal type, 1 epithelial type, 4 blastemal type, 8 mixed type, 6 not known 2 5; 2 WT-NR pairs 8M, 16F (WT-NR pairs 2F) WT 36 ^ months Hancock et al., 2007 2 Multiple ND 2 mixed type 2 2 WT-NR pairs ND ND Fukuzawa et al., 2006 4 3 3 ILNR 2 stromal type 2 ND ND ND Ravenel et al., 2001 4 2 2 PLNR 60 ND 1 ND ND ND Powlesland et al., 1999 2 2 PLNR 7 3 mixed type, 1 stromal type, 3 blastemal type 2 2, 1 WT-NR pair 4 M, 3F (WT-NR pair M) WT 43 months ^ Charles et al., 1998 2 42 22 PLNR, 17 ILNR, 3 both types 139 ND ND 9; 2 WT-NR pairs ND NR 49 months ^ PLNR 54 ^, ILNR 44 ^ Cui et al., 1997 2 8 7 PLNR, 1 ILNR 14 ND 7 ND ND ND Steenman et al., 1997 2 7 7 NBL 46 ND 6 2, no WT-NR pairs ND ND Austruy et al., 1995 2 2 NBL 28 ND 2 3, no WT-NR pairs ND ND Hoban et al., 1995 4 Multiple PLNR, ILNR 1 ND 1 ND F 11 months Park et al., 1993 4 Multiple 1 ILNR, multiple PLNR 19 blastemal type 2 ND 2F (WT-NR pair) 11 months and 48-84/144 months Yun et al., 1993 2 15 13 ILNR, 2 PLNR 31 6 blastemal type, 5 stromal dominant, 20 mixed type 15 ND 14M, 17F (WT-NR pairs 8M, 7F) WT 43 months ^ Pritchard-Jones et al., 1991 2 Multiple NBL and not known 32 18 mixed type, 4 epithelial type and 10 not known ND ND ND ND NR, nephrogenic rests; WT, Wilms tumors; n, population size expressed as number of NR or WT; ND, no (complete/clear) data; PLNR, perilobar nephrogenic rests; ILNR, intralobular nephrogenic rests; NBL, nephroblastomatosis; ^, mean; *, one case already reported by Slack et al.; **, two cases already reported by Hancock et al. cancers-15-01363-t002_Table 2 Table 2 Genetic changes in NR. Study Gene/Chromosomal Region Method Results Conclusion Chang et al., 2021 KRAS, FBXW7 NGS/WGS Association between nephroblastomatosis and KRAS. Possible association between KRAS and bilateral WT and between mosaic KRAS and NBL. Slack et al., 2021 KRAS, FBXW7 NGS Mosaic KRAS has similar frequencies in WT and adjacent NR. Bilateral WT, but not two adjacent NR, contained the FBXW7 mutation. Similar KRAS allele frequencies in WT and NR. FBXW7 mutation seems to be a late event in WT tumorigenesis Coorens et al., 2019 Genome wide WGS, WES Methylation analysis Clonal nephrogenesis in 14/23 (61%) WTs (4/4 bilateral). WT and NR from same patient arose at different times from the same ancestral clone. H19 hypermethylation in 7/12 NK with clonal nephrogenesis but not in NK without clonal nephrogenesis. There is an association between VAF of embryonal clonal expansions, H19 hypermethylation and development of WT. Wegert et al., 2018 EGFR, BRAF WGS No mutations in both NR (n = 12) and WT (n = 208) No EGFR, BRAF mutations in NR and WT Charlton et al., 2015 Genome wide Comprehensive methylome analysis NR vs. NK: 629 DMR, 55% showed hypermethylation. NR vs. WT: 2 subgroups WT, one group showed the same epigenetics as NR and one group presented increased methylation variability. Methylation profiles vary significantly between NK, NRs and WTs and alterations in the methylome lead to NR formation and transformation to WTs. MdZin et al., 2011 Chromosome 22 FISH and microsatellite analysis Dormant, involuted and sclerosing NR displayed monosomy 22 in 30%, hyperplastic and adenomatous NR in 50%, and 60-80% in nuclei of WT. More common loss of chromosome 22 in the development of PLNR (from dormant to hyperplastic) to WT. Fukuzawa et al., 2010 WTX, CTNNB1 Sequencing analysis, MLPA/microsatellite analysis CTNNB1 mutation: n = 0 in NR, n = 4 in WT. WTX mutation: n = 1 in NR, n = 4 in WT. WTX can occur as an early event, or in later stages of development, CTNNB1 is a late event. Grill et al., 2010 PTEN LOH-analysis, sequencing analysis None of the WT and none of the ILNR/PLNR showed LOH. PTEN does not play a role in tumorigenesis. Downregulation does not cause WNT-pathway activation. Vuononvirta et al., 2008 Genome wide aCGH LOH-analysis Methylation analysis PLNR 3 groups: no copy number changes (44%); single, whole chromosome changes (16%); multiple gains or losses (40%). In 76% NR changes correspond to WT. 11p15 LOH in 10/39 (26%), in NR and tumor (n = 9). H19 hypermethylation in 37/40 (93%) PLNR. PLNR are non-obligate precursors of WT. Brown et al., 2008 LOI 11p13/15, LOH 16q, 7p LOH-analysis Methylation analysis LOI 11p13 and 11p15; LOH 7p/16q not in NR, but in WT. H19 DMR analysis increased in NR and WT (n = 2). Reduced methylation of WT1 ARR in NR and WT. LOI at 11p13 and 11p15 are early events in NR, and occur prior to LOH at 7p or 16q. H19 DMR methylation was increased in NR and WT. Chilukamarri et al., 2007 GLIPR1/RTVP-1 Methylation analysis Hypomethylation WT (21/24) and NR (n = 2) Hypomethylation of GLIPR1/RTVP1 may play a role in WT tumorigenesis. Hancock et al., 2007 WT1 Methylation and expression analysis WT showed hypomethylation of WT1 ARR on both alleles. WT1 methylation differs between FK, NR and WT. Imprinting defects at 11p13 contribute to WT tumorigenesis. Fukuzawa et al., 2006 CTNNB1, WT1 11p13 Sequencing analysis LOH-analysis No CTNNB1 mutations in NR, n = 2 in WT. WT1 mutation: n = 3 in NR and n = 2 in associated WT. 11p13 LOH in ILNR and tumor (n = 1) Mutations in the CTNNB1 occur in the later stages of WT tumorigenesis. Mutations of WT1 are early events in ILNR. Ravenel et al., 2001 IGF-2 Expression analysis LOI of IGF-2 in 2 PLNR and associated WT. LOI of IGF-2 seems to be an early event in the development of WT. Powlesland et al., 1999 7p LOH-analysis LOH of 7p in 7/77 WT, one associated NR has no LOH of 7p. LOH of 7p seems to be a late event in WT tumorigenesis. Charles et al., 1998 WT1 11p15, 11p13, 16q Sequencing analysis LOH-analysis Two pairs of ILNR and WT showed WT1 mutation. LOH at 11p15 in 3/25 (12%, all ILNR). LOH at 11p13 in 3/26 (12%, 2 in ILNR, in case with PLNR only in tumor). Loss of 16q in 4/23, only in tumors. LOH at 11p13 and 11p15 is seen in ILNR and WT. PLNR showed no LOH 11p events occur early in WT development. Genetic changes at 16q are a late event. Cui et al., 1997 H19, IGF-2 ISH IGF-2 expression present in NR, WT and kidney medulla. H19 is not expressed in NR and WT in contrast to renal medulla. Pattern of IGF-2 expression differs in NR and WT. Association between expression of IGF-2 and H19, but H19 inactivation also without effect on IGF-2 expression status. Loss of H19 expression possibly involved in blastemal overgrowth. Steenman et al., 1997 Genome wide CGH Losses in 1p, 4q, 7p and gains in 7q, 1q and 12q can occur in both tumor and NBL, even as LOH at 1p and 11p13; loss of 11 only in NBL; loss of 9p, 16q and gain of 8, 10q and 18 are only seen in WT. Two specific 1p regions involved in WT etiology. Austruy et al., 1995 16q LOH analysis LOH of 16q in 7/28 WT and in one out of two associated NBL. LOH of 16q can also occur as an early event Wilms tumorigenesis. Hoban et al., 1995 Genome wide LOCH analysis LOCH present on chromosome 11 (11p13/p15), but also on all other chromosomes. Loss of all maternal loci, including #11, suggests to be an early genetic event. Park et al., 1993 WT1 Sequencing analysis WT1 mutations in both cases in NR and WT (n = 2), both somatic. WT1 inactivation seems to be an early genetic event. Yun et al., 1993 IGF-2 mRNA ISH IGF-2 hybridization patterns of NR equal to WTs. IGF-2 expression in NR variable. IGF-2 transcripts more frequent in tumors with blastema. Occasional NR also displayed different IGF-2 expression, suggesting NR could be precursor lesions of WT. Pritchard-Jones et al., 1991 WT1 mRNA ISH NBL have high levels of expression, similar to WT. WT1 contributes to WT tumorigenesis. NGS, next generation sequencing; WGS, whole genome sequencing; WES, whole exome sequencing; NK, normal kidney; VAF, variable allele frequency; DMR, differently methylated regions; FISH, fluorescent in situ hybridization; MLPA, multiplex ligation dependent probe amplification; LOH, loss of heterozygosity; aCGH, array comparative genomic hybridization; LOI, loss of imprinting; WT1 ARR, WT1 antisense regulatory region; FK, foetal kidney; ISH, in situ hybridization. CGH, comparative genomic hybridization; LOCH, loss of constitutional heterozygosity. cancers-15-01363-t003_Table 3 Table 3 Early and late events. Early Events Late Events LOI and LOH of 11p13 LOI and LOH of 11p15 Loss of 1p, 4q and, 11p Gains in 1q, 7q and 12q Loss of chromosome 22 Mutations in WT1, WTX and KRAS LOH of 16q LOH of 7p Mutations in CTNNB1 and FBXW7 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000076

Non-Hodgkin Lymphoma (NHL) represents a severe complication and the main cause of morbidity in patients with primary Sjogren's syndrome (pSS). This study aimed to assess the role of textural analysis (TA) in revealing lymphoma-associated imaging parameters in the parotid gland (PG) parenchyma of patients with pSS. This retrospective study included a total of 36 patients (54.93 +- 13.34 years old; 91.6% females) diagnosed with pSS according to the American College of Rheumatology and the European League Against Rheumatism criteria (24 subjects with pSS and no lymphomatous proliferation; 12 subjects with pSS and NHL development in the PG, confirmed by the histopathological analysis). All subjects underwent MR scanning between January 2018 and October 2022. The coronal STIR PROPELLER sequence was employed to segment PG and perform TA using the MaZda5 software. A total of 65 PGs underwent segmentation and texture feature extraction (48 PGs were included in the pSS control group, and 17 PGs were included in the pSS NHL group). Following parameter reduction techniques, univariate analysis, multivariate regression, and receiver operating characteristics (ROC) analysis, the following TA parameters proved to be independently associated with NHL development in pSS: CH4S6_Sum_Variance and CV4S6_Inverse_Difference_Moment, with an area under ROC of 0.800 and 0.875, respectively. The radiomic model (resulting by combining the two previously independent TA features), presented 94.12% sensitivity and 85.42% specificity in differentiating between the two studied groups, reaching the highest area under ROC of 0.931 for the chosen cutoff value of 1.556. This study suggests the potential role of radiomics in revealing new imaging biomarkers that might serve as useful predictors for lymphoma development in patients with pSS. Further research on multicentric cohorts is warranted to confirm the obtained results and the added benefit of TA in risk stratification for patients with pSS. radiomics textural analysis lymphoma primary Sjogren's syndrome MRI Increasing the Performance of Scientific Research, Supporting Excellence in Medical Research and Innovation, PROGRES40PFE This work was granted by project PDI-PFE-CDI 2021, entitled Increasing the Performance of Scientific Research, Supporting Excellence in Medical Research and Innovation, PROGRES, no. 40PFE/30 December 2021. pmc1. Introduction Primary Sjogren's syndrome (pSS) represents an inflammatory autoimmune disease, commonly affecting the salivary and lacrimal glands, resulting in the progressive destruction of the glandular parenchyma, which is replaced by fat and fibrous tissue. Consequently, patients with pSS suffer from exocrine dysfunction and sicca syndrome . The hallmark of the pathological process in patients with pSS is the accumulation of mucosa-associated lymphoid tissue (MALT) due to chronic inflammation . This tissue represents a substrate for non-Hodgkin lymphoma (NHL) development in this group of patients (especially the NHL-MALT histologic subtype). It is estimated that patients with pSS have an increased risk of developing lymphoma, up to 40-fold higher than in the general population . Therefore, predicting the pSS outcome both at disease onset and during follow-up is of paramount importance. Clinical and biological predictors of lymphoma in pSS have been assessed, the two major ones being persistent salivary gland swelling and cryoglobulinemic vasculitis. Composite indexes/scores have also been proposed as valuable tools in predicting lymphoma . Several imaging features proved to be associated with lymphoma development in pSS, such as the severity of main salivary gland (MSG) parenchymal destruction based on the ultrasonographic aspect , a parotid gland (PG) stiffness value > 11.5 kPA assessed using 2D shear-wave elastography , and an increased diffusion restriction with low apparent diffusion coefficient . However, despite the obtained results, biopsy and histopathological analysis remain mandatory for lymphoma diagnosis . Recently, radiomics has emerged as a rapidly evolving post-processing imaging technique that can extract high-dimensional quantitative data from radiological images and aims to surpass the limits of the subjective observational imaging assessment . Textural analysis (TA) features proved to be correlated to tissue heterogeneity at a cellular level and, therefore, might represent novel biomarkers that could improve diagnostic accuracy and facilitate the decision-making process for clinicians, especially considering the current age of targeted treatment and patient-customized medicine . In the field of salivary gland imaging, the role of radiomics was mainly assessed in the following two clinical settings: oncology and radiation-induced xerostomia, while few studies focused on inflammatory pathologies . Radiomic studies with a specific focus on pSS are scarce. One study proposed a radiomic-based evaluation of the pSS scoring system using MSG ultrasound images and identified the best-performing classifier (multilayer perceptron) among the considered artificial intelligence algorithms . Additionally, the efficiency of deep learning algorithms was tested for the automated PG segmentation on ultrasound images . Regarding the role of radiomics in pSS using MRI, one study revealed textural features in the lacrimal gland's parenchyma that were able to distinguish between patients with pSS and healthy controls . Another MRI radiomic study found imaging biomarkers that could stage disease activity in pSS. The histogram derived from the PG segmentation on the ADC map provided quantitative parameters that reflected different tissue characteristics between pSS patients and healthy volunteers . Although MRI is a useful technique in the local staging of malignant PG tumors and pSS-associated lymphomas of salivary and lacrimal glands , to the best of our knowledge, no radiomic study regarding lymphomatous proliferation in pSS has been performed so far. NHL represents the main complication and cause of morbidity in pSS patients, and the gold standard diagnostic method remains biopsy, which is an invasive procedure with a nonnegligible risk of sampling error . Although pSS-associated lymphomas often present an indolent evolution, these malignancies still present the risk of dissemination to other mucosal sites or organs . Therefore, the aim of this radiomic study was to identify alternative, non-invasive MRI textural features in the PG parenchyma associated with NHL development in patients with pSS that might serve as prognostic biomarkers, complement biopsy, and provide new directions in assessing the disease. 2. Materials and Methods This study was performed according to the Declaration of Helsinki and received approval from the Ethical Committee of the "Iuliu Hatieganu" University of Medicine and Pharmacy Cluj-Napoca (registration number: 43; date: 11 February 2022). Due to the study's retrospective design, all participants' informed consent was waived. 2.1. Patients and Standard Reference This retrospective nonrandomized study was conducted on patients with previously documented pSS who underwent head and neck MRI examination between January 2018 and October 2022 to assess MSG. The inclusion criteria were the fulfillment of the American College of Rheumatology and the European League Against Rheumatism (ACR/EULAR) diagnostic criteria (2016) for pSS and age older than 18 years. One rheumatologist with 5 years of clinical experience evaluated all patients. The Schirmer's test and the unstimulated whole salivary flow (UWSF) were assessed for all subjects, and the EULAR Sjogren's Syndrome disease activity index (ESSDAI) was computed . Biological analysis (anti-Ro/La autoantibodies and rheumatoid factor (RF)) was performed for each patient. The exclusion criteria were secondary Sjogren's syndrome (in patients with systemic lupus erythematosus, rheumatoid arthritis, progressive systemic sclerosis, or mixed connective tissue disease), sialolithiasis, previous neck radiation, history of hepatic virus C infection and patients with sicca syndrome that did not fulfill the pSS criteria. After applying the inclusion and exclusion criteria, a cohort of 36 consecutive patients was constituted. For the statistical analysis of the clinical and biological features, subjects were divided into two groups: one included 12 patients with pSS and NHL at the time of the MRI examination (pSS NHL group), and another control group of 24 pSS patients without lymphomatous transformation (pSS control group). The gold standard for pSS patients with NHL was the histopathological result obtained after core needle biopsy or surgery. All samples were analyzed in the same institution. All subjects in the pSS NHL group were confirmed with NHL-MALT subtype and on MRI presented solitary solid nodules or masses identified due to the increased diffusion restriction and extremely low apparent diffusion coefficients (<0.650). Patients in the control group were followed-up at 6 months and 1 year after the MRI exam. They did not present any clinical or imaging changes (assessed with the ultrasonography of MSG) suggesting lymphomatous transformation compared to the initial evaluation. For the radiomic analysis, a total of 65 PGs were included (representing the training dataset). PGs were divided into two groups: 48 PGs were included in the pSS control group (both PGs of one patient underwent segmentation and feature extraction), while 17 PGs were included in the pSS NHL group (seven patients with unilateral involvement, and five patients with bilateral involvement, respectively). 2.2. Image Acquisition All patients underwent head and neck MRI exams on a 1.5 Tesla scanner (SIGNATM Explorer, General Electric) using an eight-channel high-resolution head coil. The MRI protocol comprised the following standard sequences: axial and coronal FSE T1-WI and FSE T2-WI, coronal STIR PROPELLER (short tau inversion recovery with periodically rotated overlapping parallel lines with enhanced reconstruction), coronal SE T1-WI with fat saturation obtained after intravenous administration of gadolinium chelate, diffusion-weighted imaging with the ADC map, and heavily three-dimensional T2-WI (MRI sialography) covering all MSG. For the TA, the coronal STIR PROPELLER sequence was used, acquired employing the following specifications: field of view 256 x 256 mm; slice thickness 3 mm; slice gap 0.3 mm; echo time 60 ms; repetition time 3500 ms; inversion time 1900 ms; bandwidth 437.1 Hz/pixel; acquisition time 4 min. 2.3. Texture Analysis Protocol and Statistical Analysis 2.3.1. Image Preprocessing and Segmentation One head and neck specialized radiologist reviewed each MRI examination on a dedicated workstation (General Electric, Advantage workstation, 4.7 edition) and identified the best artifact-free slice for assessing PG on the STIR PROPELLER sequence. After anonymization, the selected images were retrieved in a DICOM format and imported into an open-source texture analysis software, MaZda 5 (Institute of Electronics, Technical University of Lodz, Lodz, Poland) . To decrease image variations in brightness and contrast that might impair the natural texture of PG, the first step in the MaZda program was to apply a grey-level normalization technique to all images. The mean (m) and standard deviation (s) of grey levels of voxels inside the ROIs were computed, and all outlier voxels (beyond m +- 3s) were consequently removed. A preprocessing wavelet filter was applied, using high and low bandpass filters. A total of 65 PGs divided into two groups (48 PGs in the pSS control group and 17 in the pSS NHL group) underwent segmentation and feature extraction. The segmentation process for the pSS control group implied incorporating each PG into a 2D region of interest (ROI) using a semiautomatic algorithm. Firstly, a seed was placed inside the PG parenchyma, and the ROI was automatically traced following the PG contour using gradient and geometrical coordinates. When necessary, manual corrections were further applied. An example of PG segmentation is shown in Figure 1. The segmentation was performed for both PGs of one subject. The segmentation process for the pSS NHL group was performed first by automatically tracing a 2D ROI that comprised the focal lesion corresponding to NHL using gradient and geometrical coordinates. Then, the PG parenchyma outside the lesion was manually delineated using a nonoverlapping 2D ROI at a 2 mm distance from the first 2D ROI . The 2D ROIs represented the regions in which the radiomic features were calculated. 2.3.2. Feature Extraction From each segmented ROI, MaZda software automatically extracted a total of 275 parameters belonging to six texture classes (Absolute gradient, Histogram, Co-occurrence Matrix, Run Length Matrix, Auto-regressive Model, and Wavelet transformation). Details regarding the extracted radiomic features and the computation settings of each class are shown in Table 1. 2.3.3. Feature Selection and Statistical Analysis From the total of 275 extracted features, the MaZda program allowed the selection of the most discriminative features through several preset reduction techniques. As a first step, the probability of classification error and average correlation coefficients (POE + ACC) reduction technique was applied , and a set of 10 features was generated. This algorithm, available within the MaZda program, selects features with the highest discriminative ability while being poorly correlated, thus making them suitable for building prediction models. To assess the stability and reproducibility of the selected TA feature set after computing the POE + ACC reduction technique, 30 PGs (randomly chosen from both studied groups) underwent re-segmentation 1 month apart from the initial procedure. The same radiologist redefined another ROI and a second round of feature extraction was performed. Then, the intraobserver reproducibility of the radiomic features was assessed using the intraclass correlation coefficient (ICC). Radiomic parameters that presented an ICC higher than 0.850 were regarded as stable, and their corresponding absolute values from the initial segmentation were considered suitable for the subsequent statistical analysis. A univariate analysis test (Mann-Whitney U) was further performed to assess which features were best suited to discriminate between the pSS control group and the pSS-NHL group. The statistically significant level was set at a p-value lower than 0.05. All texture parameters that showed univariate analysis results above this threshold were excluded from further processing. The receiver operating characteristic (ROC) analysis was performed, with the calculation of the area under the curve (AUC) for parameters that demonstrated statistically significant results in the univariate analysis (p < 0.05). Optimal cutoff values were chosen using a common optimization step that maximized the Youden index. Sensitivity (Se), specificity (Sp), positive likelihood ratio (+LR), and negative likelihood ratio (-LR), with their corresponding 95% confidence intervals (CI), were computed from the same data without further adjustments. Parameters that showed statistically significant results in the univariate and ROC analysis were included in a multiple regression using the "enter" input model. The resulting features independently associated with lymphoma development in patients with pSS were used to generate a radiomic model, computed using the regression coefficients. An overview of the radiomic workflow used in this study is offered in Figure 3. This step-by-step feature selection method was used in previous texture analysis studies , and the resulting parameters demonstrated good discriminative ability. The statistical analysis regarding the clinical and biological features implied assessing the differences between the means or medians using the independent-samples T test or Mann-Whitney U test, as necessary. The exact Fisher test was used to evaluate the association between categorical variables. The statistical analysis was performed using the commercially available dedicated software, MedCalc version 14.8.1 (MedCalc Software, Mariakerke, Belgium). 3. Results A total of 36 patients diagnosed with pSS referred to our imaging department during the study period (mean age 54.93 +- 13.34; age range 29-83) were included in this study (Table 2). Statistically significant risk factors for NHL development in our cohort of patients with pSS were the ESSDAI score value and the positive presence of the rheumatoid factor. Subjects in the pSS NHL group presented a higher disease activity than the pSS control group, using an ESSDAI score cutoff value of 5 (p < 0.001). The overall ESSDAI score values were higher in the pSS NHL group (p < 0.001). Disease duration did not influence the lymphomatous transformation (p > 0.05). The rheumatoid factor was present in all patients in the pSS NHL group and only 62.5% of the subjects in the pSS control group (p = 0.016). For the textural analysis, 48 PGs from the pSS control group and 17 PGs from the pSS NHL group underwent segmentation, and a total of 275 radiomic features were extracted. Following the POE+ACC reduction technique, 10 unique texture features with the highest discriminatory values between the two studied groups were selected. Seven of the 10 previously selected texture features showed statistically significant results in the univariate analysis (Table 3). All selected parameters presented high ICC values (>=0.850). One variation of Sum Variance, Run Length NonUniformity, and Sum Average were excluded from further analysis, as the p-value exceeded 0.05. The receiver operating characteristics (ROC) analysis was further performed. Three parameters (CH4S6SumVarnc, CV4S6InvDfMom, and Perc1) presented 88.24% sensitivity in differentiating between the two groups of patients, with specificities of 64.58%, 77.08%, and 62.50%, respectively. The highest area under the curve (AUC) was reached by CV4S6InvDfMom (0.875). The extended ROC analysis results are presented in Table 4. The seven texture features that showed statistically significant results in the univariate analysis, high ICC values, and ROC analysis were included in the multivariate regression. The multivariate analysis showed a coefficient of determination (R2) of 0.5524, an adjusted R2 of 0.4975, a multiple correlation coefficient of 0.7433, and a residual standard deviation of 0.3140 (Table 5). Four parameters proved to be independent predictors for NHL development in patients with pSS (CH4S6SumVarnc, Perc90, Mean, CV4S6InvDfMom). A radiomic model (RM) was generated, including two independent parameters (CH4S6SumVarnc and CV4S6InvDfMom) revealed in the multivariate analysis. Two parameters (Mean, Perc90) were excluded from the model due to the high variance inflation factor (VIF), indicating multicollinearity. RM was computed using the regression coefficients (RM = -27.7065 + 0.00417CH4S6SumVarnc + 3.3534CV4S6InvDfMom). At the cutoff value >= 1.556, the RM was associated with NHL development with high sensitivity and specificity (94.12% and 85.42%, respectively), presenting an AUC of 0.931. (Table 6). The areas under the ROC curve of the independent features associated with lymphoma development in pSS and the resulting radiomic model are depicted in Figure 4. 4. Discussion The exocrine glands of patients with pSS are characterized by an increased content of mucosa-associated lymphoid tissue, which consequently increases the risk of developing lymphoma . The ability to forecast the pSS outcome on disease onset and during follow-ups is still limited despite years of research. Significant clinical and biological predictors of lymphoma in pSS proved to be persistent enlargement of the MSG (defined as lasting at least 2 months) and mixed cryoglobulinemia . Currently, there are no studies to certify the role of any imaging technique as a validated predictive tool in pSS. Therefore, discovering noninvasive imaging biomarkers that might be associated with lymphoma development in pSS represents a crucial step for further clinical and applied research. MRI represents a valuable imaging technique in pSS diagnosis, allowing both the anatomical assessment of MSG by using T1- and fat-suppressed T2-weighted images and the functional evaluation with MRI sialography, based on the spontaneously increased signal of stagnant fluids on heavily T2-weighted sequence and simultaneously signal suppression of the adjacent tissue . The association of more than 5% fat areas with diminished intact parenchyma replaced by areas with increased signal in the MSG on T2-weighted images with fat saturation, together with an increased number of foci of salivary duct ectasia (>=6), reached a 96.4% sensitivity and 100% specificity for pSS diagnosis . MRI sialography of PG has outstanding diagnostic performance in pSS (sensitivity and specificity ranging between 83-96% and 83-100%) . MRI also proves helpful in parotid lymphoma diagnostic guidance and local staging . According to the algorithm proposed by Jousse-Joulin et al. , the association of solid and cystic lesions with very low apparent diffusion coefficient values requires a biopsy, as there is high lymphoma suspicion. Recently, radiomics proved to be a promising tool in oncological imaging, especially in diagnosing cancer, evaluating the response to therapy, or predicting prognosis . Therefore, the present radiomic study aimed to assess the role of TA in discovering imaging biomarkers in the PG of patients with pSS that are associated with lymphoma development and could potentially become surrogates of the histopathological results. Using fat saturation techniques, the PG parenchymal architecture was outlined by suppressing additional signals originating from interlobular fat structures . Therefore, the STIR sequence was selected to perform PG segmentation and TA feature extraction. The STIR technique provided a more uniform fat suppression than the fat-saturated FSE technique, especially for cervical MR imaging, where the air-filled structures and complex anatomy might generate susceptibility effects and cause magnetic field inhomogeneity . Moreover, the PROPELLER technique was utilized to reduce motion artifacts, improve image quality and obtain a more homogenous set of images . In this study, two independent texture analysis features were found to be associated with lymphoproliferation in pSS : CH4S6SumVarnc and CV4S6InvDfMom, each presenting a good AUC (>0.750). The combination radiomic model proved to perform better than the individual parameters, reaching an AUC of 0.931. Sum variance (CH4S6SumVarnc) and inverse difference moment (CV4S6InvDfMom) were obtained from the co-occurrence matrix. Within a given ROI, sum variance measures the deflection extent of the sum of grey-level intensity distribution from the mean grey-level value . Therefore, sum variance is a parameter that reflects heterogeneity by emphasizing the deviation of neighboring grey levels from the mean in the co-occurrence matrix . In our study, sum variance presented higher values in the pSS NHL group, reflecting more inhomogeneous parenchyma of the PG in comparison to the pSS control group (234.60 vs. 199.24, p < 0.001). The inverse difference is an indicator of homogeneity . A wide range in levels of grey-level co-occurrences is less quantified and consequently lowers the overall feature's value. In other words, the maximum value for this feature is obtained if there is no difference in the grey levels. The inverse difference moment is conceptually similar to the inverse difference feature. However, it gives less weight to items further away from the diagonal and is also linked to homogeneity . In the pSS NHL group, CV4S6InvDfMom proved to be lower than in the pSS control group (0.10 vs. 0.18, p < 0.0001); therefore, a large grey level variation equivalent to an increased parenchymal inhomogeneity of PG favors NHL development. Our results show that a high PG parenchymal heterogeneity quantified by TA features is associated with NHL development. This observation agrees with other studies that proved that severe parenchymal MSG destruction, with a consequently increased inhomogeneity assessed on ultrasonography, could be a risk factor for progression to MALT lymphoma . To the best of our knowledge, this is the first study to assess the role of radiomics in depicting features associated with lymphoma development in the PG of patients with pSS, and the obtained preliminary results are promising. However, this study has several important limitations that need to be addressed. Firstly, TA was performed in a relatively small sample of PG (n = 65) that represented the training dataset. Consequently, the small training dataset might lead to unintentional overfitting, which would hinder the generalization of the radiomic model. To address this issue, a validation dataset is warranted . Generally, approximately 70% of the acquired dataset is used for training, and the remaining samples are used to evaluate the classifier's performance on another validation dataset . We were unable to split the acquired data into a training and validation dataset, due to the limited number of observations in the pSS-NHL group (n = 17, 26% of all observations). Although pSS is one of the most common autoimmune disorders, its prevalence in the general population is still relatively low (0.06% worldwide in the general population) . This fact, together with the monocentric aspect of this study, has unfortunately contributed to a limited number of patients with pSS referred to our imaging center, and an even lower number of pSS patients that developed lymphoma. Therefore, the discovered radiomic features associated with lymphoma development in the parotid glands of patients with pSS could not be tested on a separate validation set in this study. This would have significantly increased the reliability of the obtained results and counteracted any potential overfitting. More extensive multicentric studies (which would also provide external datasets) are required to collect a sufficient number of pSS subjects that could be appropriately used for training and validation groups in AI algorithms based on TA. Moreover, important limitations in MRI radiomic studies are related to the presence of confounding factors, some related to differences in image acquisition , scanner , or vendor differences. Variations in image acquisition settings such as technical parameters (matrix size, time of repetition, time of echo, signal-to-noise ratio,) or voxel size (slice thickness, pixel size) may lead to pictures of varying quality, which may impact the performance of the radiomic signatures and limits generalization . MRI radiomic studies also present great potential in identifying predictive biomarkers in several head-neck pathology studies. However, due to the high variability in methodology, collective and accurate data assessment is limited . Radiomics reporting guidelines, including Radiomics Quality Score (RQS) or the Image Biomarker Standardization Initiative (IBSI) , proposed different approaches to conduct reproducible and generalizable radiomics studies. However, there is still a lack of consensus on how to control and reduce the effect of potential confounding factors. RQS stresses the importance of reporting exact details of the used imaging protocol, but no reliable strategies for reducing the confounding effects have been provided. Conversely, the IBSI guideline emphasizes having a pre-processing standardized algorithm for feature extraction and focuses less on limiting the confounding factors. Some studies have assessed radiomic feature robustness by using test-retest repeated scans or multiple MRI scans . The means to control confounding factors in our study were by using a standardized MRI protocol, with fixed technical parameters for all patients with pSS that were examined in our department, and by performing image processing and computation before feature extraction. Although we strongly acknowledge the importance of feature robustness assessment that impacts model generalization , due to the retrospective nature of this study, additional experiments could not be performed, and therefore, we could not test the effect of confounding parameters. This represents an important limitation of the present study but represents an important objective for future prospective radiomic studies in our department. Another limitation of the study is that the radiomic features were extracted from a 2D ROI segmentation. However, in daily clinical practice, a 3D segmentation of the PG, which has an irregular shape, might be harder to adopt, given the longer time required for segmentation and possible increased operator variability. Moreover, differences between PGs with lymphoma and the contralateral PGs without lymphoma in the pSS NHL group could not be tested, given the low number of subjects with unilateral PG lymphomatous proliferation. Finally, since all patients in the pSS NHL group were diagnosed with MALT lymphoma, one cannot draw generalizations about other NHL subtypes. 5. Conclusions This study suggests that radiomic analysis of the parotid gland's parenchyma performed on MR images has the potential to reveal new imaging biomarkers that reflect tissue heterogeneity associated with lymphoma development in patients with pSS. However, the results obtained in this study must be confirmed in larger prospective studies, using ideally multicentric cohorts, to validate the role of textural analysis in the risk stratification of patients with pSS. Author Contributions Conceptualization, D.D.M., P.A.S. and L.M.L.; methodology, D.D.M., L.M.L. and G.M.R.; validation, L.M.L., D.F. and S.M.D.; formal analysis, D.D.M. and P.A.S.; investigation, D.D.M., P.A.S., G.M.R., M.B. and C.C.; data curation, M.B. and C.C.; writing--original draft preparation, D.D.M.; writing--review and editing, S.M.D. and G.M.R.; visualization, C.C. and L.M.L.; supervision, L.M.L., D.F. and G.M.R.; project administration, S.M.D. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted following the Declaration of Helsinki and approved by the Ethics Committee of "Iuliu Hatieganu" University of Medicine and Pharmacy Cluj-Napoca (DEP43/11 February 2022). Informed Consent Statement Informed consent was waived, given the study's retrospective nature. Data Availability Statement The data are available only by request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 (A) MRI coronal STIR PROPELLER sequence of a 57-year-old female patient diagnosed with primary Sjogren's syndrome. (B) The region of interest (ROI) covering almost the entire parotid gland was semiautomatically delineated by the software following geometry and gradient coordinates (green circle). (C) The final ROI after the manual correction was performed (green area). Figure 2 (A) MRI coronal STIR PROPELLER sequence of a 45-year-old female patient diagnosed with primary Sjogren's syndrome and non-Hodgkin lymphoma, MALT subtype in the right parotid gland. (B) The software automatically traced the region of interest (ROI) covering the focal lesion corresponding to lymphoma using geometric and gradient coordinates (red area). (C) A second ROI was manually delineated (green area), covering the parotid gland's parenchyma surrounding the focal lesion (this 2D segmentation was used for the textural analysis). Figure 3 Radiomics workflow diagram. ROI = region of interest; STIR PROPELLER = short tau inversion recovery with periodically rotated overlapping parallel lines with enhanced reconstruction; PG = parotid gland; pSS = primary Sjogren's syndrome; NHL = non-Hodgkin lymphoma; POE + ACC = probability of classification error and average correlation coefficients; ICC = Intraclass Correlation Coefficient, SumVarnc = Sum Variance; InvDfMom = Inverse Difference Moment. Figure 4 Receiver operating characteristics (ROC) curve of the independent parotid glands' texture features (CH4S6SumVarnc, CV4S5InvDfMom) and the resulting radiomic model, associated with lymphoma development in patients with primary Sjogren's syndrome (orange dotted line--ROC curve; thin blue lines--ROC Confidence Interval). Figure 5 Texture maps presenting the distribution of the independent features (CH4S6SumVarnc, CV4S6InvDfMom) associated with NHL development in PG of patients with pSS on the selected MRI coronal STIR PROPELLER image. (A-C) images of a patient with pSS and without lymphomatous proliferation in the parotid gland; (D-F) images of a patient with pSS and NHL development in the right parotid gland. cancers-15-01380-t001_Table 1 Table 1 The extracted radiomics features and the computation settings of each category. Category Feature Computation Variation Number of Features Histogram Mean, Kurtosis, Percentile 01/10/50/90/99% Skewness, Variance - - 9 Co-occurrence matrix Angular second moment, Contrast, Correlation, Difference entropy, Difference Variance, Entropy, Inverse difference moment, Sum average, Sum entropy, Sum of squares, Sum variance 6 bits/pixel Computed 20 times for distance values from 1 to 5 220 Run length matrix Fraction of image in runs, Grey level nonuniformity, Long run emphasis, Run length nonuniformity, Short run emphasis 6 bits/pixel Computed four times for horizontal, vertical, 45deg, and 135deg directions 20 Gradient Kurtosis, Mean, Percentage of pixels with nonzero gradient, Skewness, Variance 4 bits/pixel - 5 Autoregressive model Sigma, Teta 1-4 - - 5 Wavelet Wavelet energy with high- and low-pass filters 8 bits/pixel 4 scales 16 cancers-15-01380-t002_Table 2 Table 2 Patient clinicobiological features. Feature All Patients (n = 36) pSS Control Group (n = 24) pSS NHL Group (n = 12) p Age (years) 54.93 +- 13.34 58.79 +- 12.44 46.92 +- 12.50 0.013 Gender (female) 33 (91.6) 23 (95.8) 10 (83.3) 0.207 BMI (kg/m2) 26.11 +- 4.39 25.23 +- 3.98 27.39 +- 5.01 0.130 Disease duration (months) 34 29 37 0.416 Disease duration 0.349 <5 years 24 (66.7) 21 (87.5) 3 (25) >=5 years 12 (33.3) 3 (12.5) 9 (75) ESSDAI score 2 0 13 <0.001 Disease activity <0.001 Low (ESSDAI < 5) 22 (61.1) 20 (83.3) 2 (16.6) Moderate-high (ESSDAI >= 5) 14 (38.9) 4 (16.7) 10 (83.4) Positive Schirmer's test 33 (91.6) 21 (87.5) 12 (100) 0.522 UWSF (mL) 1.24 +- 0.34 1.23 +- 0.34 1.25 +- 0.28 0.861 Anti-Ro/La autoantibodies 32 (88.9) 20 (83.3) 12 (100) 0.139 Rheumatoid factor 27 (75) 15 (62.5) 12 (100) 0.016 The results are expressed as mean +- standard deviation, median and [interquartile range], or percentage (%), n = number of patients, BMI = body mass index, ESSDAI = EULAR Sjogren's syndrome disease activity index; UWSF = unstimulated whole salivary flow. cancers-15-01380-t003_Table 3 Table 3 Texture features associated with lymphoma development in patients with pSS after the POE + ACC (probability of classification error and average correlation coefficients) reduction technique and the univariate analysis results. Texture Parameters PG pSS Control Group (n = 48) PG pSS NHL Group (n = 17) p ICC Median IQR Median IQR CH4S6SumVarnc 199.24 175.39-221.77 234.60 214.35-257.19 0.0004 0.956 WavEnHL_s-4 270.38 186.15-370.19 497.10 283.74-727.07 0.0094 0.910 Perc90 33,280.50 33,186.00-33,354.50 33,512.00 33,359.75-33,563.50 0.0001 0.922 Mean 33,147.03 33,101.45-33,229.68 33,377.18 33,230.06-33,389.89 0.0001 0.933 CV4S6InvDfMom 0.18 0.16-0.22 0.10 0.10-0.13 <0.0001 0.924 CH3S6Correlat 0.14 0.04-0.25 0.405 0.17-0.49 0.0027 0.901 CN1S6SumVarnc 349.22 327.72-369.30 360.04 320.52-388.26 0.3547 0.897 RNS6RLNonUni 1371.17 1104.56-1720.21 1603.50 1272.27-2083.16 0.2383 0.905 Perc1 32,990.50 32,942.00-33,050.00 33,109.00 33,028.25-33,157.25 0.0005 0.911 CV5S6SumAverg 65.00 64.50-65.29 62.86 59.58 to 65.09 0.4737 0.934 IQR = interquartile range; p = statistical significance level; ICC = intraclass correlation coefficient; SumVarnc = Sum Variance; WavEnHL = Wavelet Energy High-Level; Perc = percentile; InvDfMom = inverse difference moment; Correlat =correlation; RLNonUni = Run Length NonUniformity; SumAverg = Sum Average. cancers-15-01380-t004_Table 4 Table 4 The receiver operating characteristic analysis results for texture parameters associated with the lymphomatous transformation of the parotid gland's parenchyma in patients with primary Sjogren's syndrome. Parameter Cutoff AUC Se (%) Sp (%) +LR -LR Youden Index p CH4S6SumVarnc >207.62 0.800 88.24 (63.6-98.5) 64.58 (49.5-77.8) 2.49 (1.64-3.79) 0.18 (0.04-0.68) 0.528 <0.0001 WavEnHL_s-4 >388.84 0.713 58.82 (32.9-81.6) 81.25 (67.4-91.1) 3.14 (1.54-6.39) 0.51 (0.28-0.91) 0.400 0.0021 Perc90 >33,363 0.816 76.47 (50.1-93.2) 79.17 (65.0-89.5) 3.67 (1.99-6.76) 0.30 (0.12-0.71) 0.554 <0.0001 Mean >33,233.87 0.821 76.47 (50.1-93.2) 81.25 (67.4-91.1) 4.08 (2.14-7.78) 0.29 (0.12-0.69) 0.577 <0.0001 CV4S6InvDfMom <0.145 0.875 88.24 (63.6-98.5) 77.08 (62.7-88.0) 3.85 (2.23-6.65) 0.15 (0.04-0.57) 0.653 <0.0001 CH3S6Correlat >0.321 0.746 52.94 (27.8-77.0) 89.58 (77.3-96.5) 5.08 (1.98-13.05) 0.53 (0.31-0.88) 0.425 0.0008 Perc1 >33,006 0.787 88.24 (63.6-98.5) 62.50 (47.4-76.0) 2.35 (1.57-3.53) 0.19 (0.05-0.70) 0.507 <0.0001 The 95% confidence interval values are shown in parentheses. AUC = area under curve; Se = sensitivity; Sp = specificity; +LR = positive likelihood ratio; -NR = negative likelihood ratio; p = statistical significance level; SumVarnc = Sum Variance; WavEnHL = Wavelet Energy High-Level; Perc = Percentile; InvDfMom = Inverse difference moment; Correlat = correlation. cancers-15-01380-t005_Table 5 Table 5 Multivariate analysis results revealing the texture features independently linked to lymphoma development in patients with primary Sjogren's syndrome. Independent Variables Coefficient Std. Error p VIF (Constant) -27.7065 CH4S6SumVarnc 0.00417 0.001495 0.0072 3.284 WavEnHL_s-4 0.00009 0.0001514 0.5478 1.303 Perc90 -0.00242 0.0006944 0.001 15.771 Mean 0.00423 0.001474 0.0058 27.424 CV4S6InvDfMom 3.3534 0.8530 0.0002 1.529 CH3S6Correlat 0.0356 0.3662 0.9229 3.776 Perc1 -0.001 0.001147 0.3841 7.217 R2 0.5524 R2-adjusted 0.4975 MCC 0.7433 RSD 0.3140 Std. Error = standard error; p = statistical significance level; VIF = Variance Inflation Factor; R2 = coefficient of determination; R2-adjusted = coefficient of determination adjusted for the number of independent variables in the regression model; MCC = multiple correlation coefficient; RDS = residual standard deviation; SumVarnc = Sum Variance; WavEnHL = Wavelet Energy High-Level; Perc = Percentile; InvDfMom = Inverse difference moment; Correlat = correlation. cancers-15-01380-t006_Table 6 Table 6 The receiver operating characteristic analysis for the radiomic model predictive of the lymphomatous transformation of the parotid gland's parenchyma in patients with primary Sjogren's syndrome. Parameter Cutoff AUC Se (%) Sp (%) Youden Index p Radiomic Model >=1.556 0.931 94.12 (71.3-99.9) 85.42 (72.2-93.9) 0.795 <0.0001 The 95% confidence interval values are shown in parentheses. AUC = area under curve; Se = sensitivity; Sp = specificity; p = statistical significance level. 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PMC10000077

Apicomplexan parasites such as Toxoplasma gondii, Neospora caninum, and Besnoitia besnoiti are widely recognized as causes of production diseases in ruminants. This study aimed to investigate the serological occurrence of T. gondii, N. caninum, and B. besnoiti in cattle and goats from smallholder farms in Selangor, Malaysia. A cross-sectional study was conducted on 19 farms by collecting 404 bovine (n = 225) and caprine (n = 179) serum samples, which were then essayed for T. gondii, N. caninum, and B. besnoiti antibodies using commercially available ELISA test kits. Farm data and animal characteristics were documented, and the data were analyzed using descriptive statistics and logistic regression models. The seroprevalence of T. gondii at animal and farm levels in cattle was 5.3% (95% CI 1.2-7.4%) and 36.8% (95% CI 22.4-58.0%), respectively. Animal-level seropositivity for N. caninum was 2.7% (95% CI 0.4-4.2%) and 5.7% for B. besnoiti (95% CI 1.3-9.4%) with corresponding farm-level seropositivity of 21.0% and 31.5%, respectively. For the goat samples, a high animal- (69.8%; 95% CI 34.1-82.0%) and farm-level (92.3%) seropositivity was recorded for T. gondii, but was relatively lower for N. caninum antibodies, at 3.9% (95% CI 1.5-6.2%) and 38.4% (5/13). The factors associated with T. gondii seropositivity were older animals (above 12 months) (OR = 5.3; 95% CI 1.7-16.6), semi-intensive farms (OR = 2.2; 95% CI 1.3-6.2), the presence of either dogs or cats (OR = 3.6; 95% CI 1.1-12.3), a large herd size (>100 animals) (OR = 3.7; 95% CI 1.4-10.0), and a single source of replacement animals (OR = 3.9; 95% CI 1.6-9.6). These findings are vital in developing effective control measures against these parasites in ruminant farms in Selangor, Malaysia. More national epidemiological research is required to elucidate the spatial distribution of these infections and their potential impact on Malaysia's livestock industry. seroprevalence Toxoplasma gondii Besnoiti besnoiti Neospora caninum cattle goats Malaysia Universiti Putra MalaysiaGP-IPM/2016/9509800 This research was funded by Universiti Putra Malaysia grant (GP-IPM/2016/9509800). pmc1. Introduction Apicomplexa protozoan parasites, Toxoplasma gondii, Neospora caninum, and Besnoitia besnoiti, have a common heterogeneous life cycle and contribute significantly to the reproductive failures and low efficiency in ruminant farms globally . Toxoplasma gondii infects all warm-blooded animals and causes toxoplasmosis, a cosmopolitan zoonotic disease that is of public health significance in humans, especially in immunocompromised individuals and unborn fetuses. Human toxoplasmosis causes several health issues including fever, dizziness, arthralgia, fever, myalgia, and lymphadenopathy, whereas abortions, blindness, and encephalitis may occur in severe conditions . Several routes of T. gondii infection exist in humans, with the most common route being via the consumption of food products of animal origin . Small ruminants' meat contains a high number of tissue cysts, which is linked to grazing in environments contaminated with oocysts . Hence, a major source of infection stems from poor food hygiene practices and/or undercooking of meat, especially in countries where the consumption of sheep and goat meat is part of the culinary tradition . High rates of T. gondii infection in small and large ruminants results in severe economic losses and imposes significant zoonotic health hazards among susceptible populations . The wide availability of the definitive (domestic cats and other wild felids) and intermediate (mammals and birds) hosts contribute to the high infection rates of T. gondii and variation in the prevalence in different countries . Neospora caninum is also a major concern in large and small ruminants. The protozoan parasite is widely recognized as a major abortifacient pathogen in cattle but has also been shown to cause abortion in goats and sheep . Additionally, the pathogen is common in these ruminant species with varying seroprevalence values, which are strongly associated with breeding conditions . Previous studies have shown that extensive management systems, herd size, and pasture access are common risk factors for neosporosis in small ruminants . Bovine besnoitiosis contributes to economic losses in affected farms due to skin lesions, weight loss, prolonged recovery, complete or transient sterility in males, and reduced milk production . Typical cases of bovine besnoitiosis transit from acute to chronic stages with varying pathogenicity and abortion might occur in infected cows . Overall, the clinical impact and epidemiology of B. besnoiti on ruminants are not fully understood. Nevertheless, their capacity to induce abortion and general disorders in the domestic intermediate host have been documented . For instance, bovine besnoitiosis was recognized as an emerging disease in European countries following the reported cases in Germany , Spain , and Italy . Overall, several researchers concluded that limited information is available on the epidemiology, prevalence, and clinical evidence of besnoitiosis infections in ruminants . The rearing of cattle and goats are key activity by Malaysian smallholder farmers, thus reflecting the importance of ruminants in income generation for several livelihoods in the country. A recent study conducted in Selangor, Malaysia, highlighted the importance of T. gondii infection in meat samples collected from wet markets and abattoirs in the region . Meanwhile, a seroprevalence survey has also documented the potential definitive hosts of the parasite in Selangor . On the other hand, Kyaw et al. reported the first seropositivity of Neospora in goats and sheep in Malaysia. The study was undertaken by sampling 472 animals from 10 districts of Kelantan, and the resulting prevalence of Neospora antibodies was 1.1%. Other local studies by Cheah and Rahman et al. focused on calf and cattle, respectively. Thus, epidemiological data about T. gondii and Neospora, especially in the ruminant population, remain scarce in Malaysia. Presently, there is a dearth of information regarding the exposure of ruminant farms to N. caninum and B. besnoiti in the country. Seroprevalence and risk factors information of the aforementioned protozoan parasites is important in developing and implementing future public health programs to control their infections in Malaysian ruminant farms. Hence, this study aimed to determine the serological occurrence of T. gondii, N. caninum, and B. besnoiti in cattle and goats from smallholder farms in Selangor, Malaysia. The secondary goal was to identify the factors associated with the seropositivity of each parasite in the study location. 2. Materials and Methods 2.1. Study Design, Study Area, and Sampling Technique A cross-sectional study was conducted involving ruminant farms in Klang Valley, Selangor, Malaysia. As of 2018, there were 122 registered cattle and goat farms with the Department of Veterinary Services (DVS), Selangor. The farmers were contacted either via phone contacts or email addresses and informed about the purpose of the study while seeking their oral consent to participate. Farmers willing to participate were briefed about the inclusion criteria, which entailed the production of either cattle or goats for commercial or subsistence purposes, herd size > 10 animals, location within any district in Selangor, and being free from any reportable diseases as at the proposed time of farm visit. The required sample size was calculated using the EpiTools website ) (accessed on 4 August 2019) by assuming an expected seroprevalence of 35.5% from a previous study conducted in Malaysia by Chandrawathani et al. at a confidence level (CI) of 95%, precision level of 5%, and a target population of ruminants (cattle, goat, and sheep) in Selangor as 40,000 animals . The minimum required sample size based on the aforementioned parameters was 350. 2.2. Collection of Farm and Animal Data A well-structured datasheet was used to record vital on-farm data such as herd size, farm location, management systems, pest control, presence of stray animals (cats, dogs, wild animals, etc.), feeding regimen, water source, and the source of animals. Meanwhile, animal characteristics such as sex, age, breed, and identification number were recorded. All the information was provided by the farm owner or available farm staff during the researcher's visit. 2.3. Blood Sampling and Serology All the animals were restrained effectively for blood sampling via the jugular vein. The samples were transported immediately to the laboratory for further analysis. Of the 22 farms that were visited in this study, a total of 404 serum samples were collected, comprising 225 from cattle and 179 from goats. The samples were centrifuged at 5000 rpm to obtain the sera, transferred into 1.5 mL microcentrifuge tubes, and stored frozen at -80 degC prior to essay for T. gondii, N. caninum, and B. besnoiti antibodies. This study was approved by the Institutional Animal Care and Use Committee University Putra Malaysia (IACUC) (Approval code: U066/2018). 2.3.1. ELISA Testing for Specific T. gondii Antibodies All sera collected were tested using a commercially available ELISA test kit (ID Screen(r) Toxoplasmosis Indirect Multi-Species Test Kit, ID.vet, France). This indirect assay utilizes T. gondii P30 antigen with a multi-species peroxidase (HRP) conjugate and detects antibodies against T. gondii antibodies in samples from cats, dogs, swine, and ruminants and plates were read at 450 nm. For interpretation of the results, the sample to positive (S/P) percentage was calculated as: S/P % = (ODsample - ODNC)/(ODPC - ODNC) x 100. Any sample with an S/P % less than or equal to 40% was considered negative, a value greater or equal to 50% was considered positive, and between 40-50% was considered doubtful. According to the manufacturer, this cut-off has more than 99% specificity and 97% sensitivity. The test was considered valid if the mean value of the positive control OD (ODPC) is greater than 0.350 (ODPC > 0.350) and the ratio of the mean OD values of the positive and negative controls (ODPC and ODNC) is greater than 3 (ODPC/ODNC > 3). 2.3.2. ELISA Testing for Specific N. caninum Antibodies All samples were also analyzed for the presence of N. caninum antibodies using a commercial competitive ELISA kit (cELISA- VMRD, VMRD, Pullman, DC, USA). According to the manufacturer's instructions and cut-off recommendations, serum samples were considered positive when the inhibition percentage was greater than 30%. Farms were considered positive when their true seroprevalence was greater than 0 as described by von Blumroder et al. . 2.3.3. ELISA Testing for Specific B. besnoiti Antibodies Only cattle sera were tested for B. besnoiti using a commercially available ELISA test kit (ID Screen(r) Besnoitia Indirect, IDvet, France). The procedure was based on the manufacturer's instructions. Briefly, the test kit was specific for cattle only and coated with B. besnoiti purified antigen. The conjugate used was anti-ruminant IgG-HRP conjugate and the assay was read using a spectrophotometer with a wavelength of 450 nm. This assay has a 97.2% sensitivity and 100% specificity and the result was interpreted and calculated using the formula below. S/P % = (Net OD sample/Net OD positive control) x 100 Sample to Positive (S/P) values of less than or equal to 25% were considered negative, whereas those greater than or equal to 30% were considered positive. Meanwhile, S/P values less than 30% but greater than 25% were considered doubtful and recorded as negative in this study. 2.4. Data Analysis All farm and animal data were manually transferred from the datasheet to the Microsoft Excel spreadsheet (NY, United States). Upon completing the sampling, the data were sorted and checked for any entry errors before transferring to SPSS Version 23 (IBM, Armonk, NY: IBM Corp, the United States) for statistical analysis. Descriptive statistics were employed to summarize the farm and animal data, thereby presenting the farm and animal characteristics. Continuous variables were assessed for data normality using the level of kurtosis and skewness. The data were then presented either as means (+-SD) or medians (+-IQR) depending on the normality test results. All categorical data were presented in frequencies and percentages. Seropositivity was computed based on the number of seropositive animals to the total number of animals under investigation for each apicomplexan protozoan. A mixed-effects logistic regression model was only built separately to determine the factors associated with seropositivity for T. gondii in cattle and goat populations, as the seroprevalence of N. caninum and B. besnoiti was low to accommodate risk factor analysis. Seropositivity to T. gondii at animal levels was considered the dependent variable and the farm was introduced into the model as a random factor. All other independent variables with complete data records were introduced into the model in a backward conditional method. Factors such as water source and feeding regimen were not considered since a significant number of farms failed to provide the information. Variables were removed from the model upon assessing the Akaike information criterion (AIC) before obtaining the final model fit. All biologically plausible pair-wise interactions were evaluated. 3. Results 3.1. Descriptive Analysis Table 1 shows the characteristics of the study population and the corresponding seroprevalence towards each apicomplexan. Overall, a higher proportion (42.8%) of the sampled animals were between 13 and 24 months old and female (89.0%). Most of the animals (82.1%) were reared under an intensive management system and not exposed to pest control (79.4%). Other farms and animal characteristics such as herd size, presence of stray animals, source of animals, type of production, and water source are presented in Table 1. 3.2. Seroprevalence at Animal and Farm Levels As shown in Table 2, the individual seroprevalence of T. gondii in cattle was low (5.3%; 12/225), while seven farms (7/19; 36.8%) were seropositive for the parasite. Likewise, a low number of cattle tested positive for N. caninum (2.7%; 6/225) and B. besnoiti (5.7%; 13/225) with a corresponding number of seropositive farms of four (21.0%) and six (31.5%), respectively. Only a single farm (Farm 5; F5) was seropositive for T. gondii and N. caninum involving only one animal. Meanwhile, two farms (F5 and F13) were seropositive for N. caninum and B. besnoiti, involving three animals. Interestingly, Farm 5 was also seropositive for the three apicomplexan parasites: T. gondii, N. caninum, and B. besnoiti. Table 3 depicts the seroprevalence of T. gondii and N. caninum in the goats sampled from 13 farms. High seropositivity was recorded for T. gondii at 69.8% (125/179) at the animal level, with a corresponding farm-level seroprevalence of 92.3%, as only one farm (F8) had no seropositive animal. Meanwhile, the seroprevalence for N. caninum antibodies was low at 3.9% (7/179) but five of the farms (38.4%; 5/13) had at least one seropositive animal. All the farms that were seropositive for N. caninum had at least one animal seropositive for T. gondii. 3.3. Factors Associated with Seropositivity for T. gondii, N. caninum, and B. besnoiti Table 4 presents the final mixed-effects logistic regression model for the factors associated with the seroprevalence of T. gondii in goats. The factors associated with T. gondii seropositivity included age, management system, presence of stray animals, animal source, and herd size (Table 5). Older goats (above 12 months) were more likely to be seropositive (OR = 5.26; 95% CI 1.72-16.66) compared to younger ones (less than 6 months). Likewise, there were higher odds of seropositivity in semi-intensively kept goats (OR = 2.17; 95% CI 1.34-6.21) relative to those raised intensively, as well as in farms with the presence of either dogs or cats (OR = 3.63; 95% CI 1.1-12.3) relative to those without such animals. Farms with fewer animals (less than 50) were less likely to be seropositive (OR = 0.27; 95% CI 0.10-0.72) compared to those with more than 100 animals. Farms using a single source or own animals for replacement had higher seroprevalence (OR = 3.92; 95% CI 1.61-9.69) compared to farms with multiple or external sources. For cattle, only the management system was significantly associated with seropositivity for T. gondii antibodies (Table 5). Cattle raised under the intensive system were less likely to be seropositive (OR = 0.20; 95% CI 0.06-0.69) compared to those raised under semi-intensive systems. Meanwhile, the presence of stray animals (i.e., dogs, cats, wild animals) and pest control were not significant in the final models. Given the low seroprevalence of N. caninum and B. besnoiti in both cattle and goats, risk factor analysis was not conducted. 4. Discussion This study was the first attempt to investigate the seroprevalence of the three most common apicomplexan protozoa, T. gondii, N. caninum, and B. besnoiti, in bovine and caprine livestock in Malaysia. The results revealed a low exposure of cattle farms to T. gondii as both the animal-level seroprevalence was 5.3% while only 7 of the 19 farms had at least one seropositive cattle. This result aligns with a previous conducted in the same study area in Malaysia where 9.0% of sampled cattle were seropositive for T. gondii antibodies . In comparison to studies conducted elsewhere, the estimate from the cattle population in this study differs from pooled seroprevalence of 28.5% in Asia, 18.8% in Africa, 21.9% in Europe, 22.2% in America, and 1.36% in Australia/Oceania, as reviewed by Shariatzadeh et al. . Direct comparisons between studies conducted in different countries might not be feasible due to variations in study designs, the serological tests employed, cut-off values used, geographic locations, and farm practices. These events might explain the discrepancies in seropositivity rates reported in different studies. Nevertheless, the majority of seroprevalence studies employed a cross-sectional design. In this study, a relatively higher seroprevalence of T. gondii was detected in goats (69.8%), with 12 of the 13 farms having at least one seropositive animal. These findings are similar to previous reports in goat and sheep samples collected from wet markets in Selangor in which 69.0% and 35.0% were seropositive for T. gondii . Likewise, a high prevalence of T. gondii antibodies was reported in goat populations from Egypt (62.0%), Italy (63.3%), Iran (48.0%), and Pakistan (42.8%) , while relatively lower estimates were found in Myanmar (11.4%) . Factors contributing to the varying exposure to the parasite, the serological assay and cut-off values used, and contamination at farm levels might explain the various seroprevalences detected in the aforementioned studies. The similarity in the seroprevalence found in the present study and earlier research conducted on wet market samples suggest consistent levels of exposure to T. gondii and environmental contamination by the parasite in various ruminant farms in Selangor. Nevertheless, given that the present study included both young and adult goats, the seroprevalence of T. gondii is alarming since the previous study involved adult animals slaughtered at abattoirs, which are presumably at a higher risk of infection . Thus, the high seroprevalence of T. gondii detected in this study implies a significant public health risk. This study supports previous research findings that small ruminants (goats and sheep) are more susceptible to T. gondii infection compared to large ruminants such as cattle and buffaloes . Accordingly, 69.3% of sampled goats were seropositive compared to 5.3% of sampled cattle from various farms in Selangor. The lower infection rate in the latter might be due to greater resistance and a stronger immune system compared to that obtainable in goats . Besnoitia besnoiti and N. caninum have become important pathogens in cattle and goat farms, causing severe economic losses in endemic farms worldwide. A low number of cattle were seropositive for N. caninum (2.7%; 6/225) and B. besnoiti (5.7%; 13/225) in this study. Most studies conducted elsewhere have reported varying seroprevalences for B. besnoiti in cattle populations, including 2.7% in Turkey and Australia , as was found in the present study, and are comparatively lower than the 22.0% reported in Greece and 44.1% in Italy . Although there is no report of bovine besnoitiosis in Malaysia, evidence of the spread of the disease was documented in other nations such as Germany, Italy, and Portugal following subsequent outbreak reports (reviewed by Jacquiet et al. . In the current study, four farms had a history of importation of cattle mainly from Australia and Thailand. Some studies reported that the importation of cattle from endemic countries such as France and Spain increased the risk of bovine besnoitiosis . For instance, an outbreak of besnoitiosis occurred in a beef cattle herd in Bavaria while positive animals were discovered in Italy following the importation of cattle from France . These findings indicated that cattle from endemic regions were able to cause transmission of the disease to a naive region. One reason for the geographical expansion of the besnoitiosis is the trade between countries. Presently, Australia is one of the main countries from where Malaysian farmers source their cattle . Though Malaysia does not share direct animal trade with reported endemic countries, seropositive findings among cattle farms in Australia were linked to trade with other European nations. Previous studies conducted in Australia also reflected a higher presence of antibodies against B. besnoiti in cattle sampled from different parts of the country . Hence, the importation of cattle from endemic regions into Malaysia could be a risk factor and requires further investigation. B. besnoiti are transmitted either by direct contact with oocysts or by mechanical transmission via vectors. Fly control is important as flies play a role as the vector for the parasite. In the current study, six farms practiced the burning of coconut husks to produce smoke as their method of controlling the number of flies, especially in the morning. The transmission of B. besnoiti from cattle to cattle might occur through mechanical transmission by blood-sucking flies, and B. besnoiti DNA had been detected in stable flies after ingestion of blood from the infected cattle . In Malaysia, biting flies such as stable and Tabanidae flies have been reported . Though further investigation is required, farmers must be made aware of the importance of hygiene and fly control on their farms. The overall seroprevalence of N. caninum in the present study (2.7%; 6/225) is slightly higher than the report (0.32%) by Kyaw et al. among sheep and goats in Musang district in Kelantan, Malaysia, but is similar to the earlier study conducted by the same group of researchers, which was 1.1% . These seroprevalence estimates are generally lower than the pooled estimate of 6.2% recorded by Reichel et al. in a detailed systematic review and meta-analysis. We also found that the prevalence of N. caninum antibodies in goats was 3.9%, which is slightly higher than the 1.1% recorded locally , but lower than the seroprevalence of 26.4% reported in Brazil and other Asian countries . The low prevalence coincides with reports from Iran (6.2%) and the Czech Republic (6.0%) . Nevertheless, exposure to N. caninum should be considered a vital outcome as the parasite has been associated with deleterious effects on the feto-maternal structures and inhibitory action against post-parturient macrophage activities, which are required for normal fetal membrane release . The presence of co-infection with any of the three protozoan parasites was also investigated in this study. Resultantly, only one animal was seropositive for T. gondii and N. caninum among the sampled cattle, and three were positive for N. caninum and B. besnoiti. These findings depict a low level of co-infection with any of the three parasites. In contrast, a study conducted on water buffaloes, which are considered large ruminants and the same category as cattle, revealed a 15.3% co-infection for both T. gondii and N. caninum . Although the seroprevalence of co-infection of T. gondii and N. caninum was very low in goats (>2.0%), 5 of the 13 farms had at least one seropositive animal for both parasites. These findings suggest that the risk factors for both infections might be common on seropositive farms. Bartova et al. employed the same cELISA for detecting anti-N. caninum antibodies and also found that all goats testing positive for N. caninum were also positive for T. gondii. The present study used an ELISA technique for the detection of both parasites, which is considered to have higher specificity and sensitivity compared to other serological methods . Cross-reactions between T. gondii and N. caninum have been demonstrated to occur when IFAT was used given that a high concentration of fluorescent antibodies against apical organelles antigens of numerous apicomplexan parasites is common . Notably, the majority of goats seropositive for T. gondii and N. caninum were adult animals. These results reflect that most of the seropositive goats were probably chronically infected by both parasites. Given the public health significance and the risk of zoonotic transmission of both parasites, preventive measures to mitigate the exposure of ruminants to T. gondii and N. Caninum are pertinent. Apart from the seroprevalence results, risk factor analysis is essential to identify and implement effective control programs against the three protozoan parasites investigated in this study. For the sampled cattle, the odds of having a T. gondii-seropositive animal were higher in semi-intensive compared to intensive farms. In Malaysian ruminant farms, semi-intensive management systems provide opportunities for livestock to graze externally in contrast to intensive systems where the animals are confined. Such practices heighten the exposure to oocysts, which are widely distributed in the environment, thereby increasing the risk of T. gondii infection . For the goat population in this study, higher seroprevalence to T. gondii was observed among older animals (>12 months), semi-intensively managed farms, as well as those with larger herd sizes, presence of cats or dogs, and using single-source/own animals as replacement stock. Recent seroprevalence studies in goats have also demonstrated positive associations between seropositivity for T. gondii antibodies and increasing age , the rearing system used, and the presence of dogs and cats . The association between increasing age and a higher T. gondii infection suggests sustained exposure to the parasite and the situation could become exacerbated due to a weakening immune system. Such animals are more likely to be exposed to these infections from various sources since they have lived longer. Therefore, older goats are less resistant to T. gondii infection as a result of lower defense mechanisms associated with aging . On the other hand, cats are the definitive host for T. gondii, and a single infected cat sheds millions of oocysts which allows them to rapidly contaminate a wide area. Moreover, in most of the visited farms in this study, cats and dogs had access to water troughs and animal feeds that were kept outdoors. These events might heighten the seropositivity for goats included from the various farms. A study conducted in Klang Valley, Malaysia by Tan detected that 10.5% of cats sampled shed T. gondii DNA in their feces while 5.5% were seropositive against T. gondii antibodies, indicating a significant threat to farming, as well as representing a public health hazard. As also reported in a recent review , the presence of cats on farms and their movements in housing or feed storage areas increase the risk of T. gondii infection. The significant role of cats in the maintenance and transmission of caprine toxoplasmosis has been demonstrated in previous research . Hence, animal sheds or farms should be subjected to good hygienic practices. The limitations in this study are attributed to the study design, diagnostic method used, and study location. Given that this research entailed a cross-sectional design, the findings only reflect snap-shot information of exposure levels to the three apicomplexan parasites investigated among the studied population. Causal relationships could not be obtained as only associations between potential risk factors and seropositivity to T. gondii antibodies were analyzed. The prevalence of N. caninum and B. besnoiti was too low to accommodate risk factor analysis, hence future studies might consider enrolling a larger sample size to facilitate such analysis. The indirect diagnosis of the three protozoans by ELISA tests may impact some level of cross-reactivity . Notwithstanding, the three commercial ELISA kits employed in this study were specific for multiple species of small and large ruminants, with high sensitivity and specificity values as reported in previous research . Since only ruminant farms located in Selangor were considered in the present study, similar investigations are required to elucidate the information in other states in Malaysia. 5. Conclusions In conclusion, this study suggests that toxoplasmosis is highly prevalent among the goat population in farms located in Selangor, Malaysia, whereas N. caninum infections were relatively low. Overall, sampled cattle from the studied area demonstrated an overall low exposure to the three protozoan parasites. The semi-intensive management of cattle farms was identified as the main risk factor for bovine toxoplasmosis, whereas multiple factors such as older animals, a larger herd size, the presence of cats or dogs, and using single source/own animals as replacement stock were associated with a higher prevalence of T. gondii in goats. Given the vital role of these parasites in causing reproduction disorders, controlling the infections is pertinent for economic, animal welfare, and public health reasons. These findings are vital for veterinarians, animal, and human health bodies to strategize effective control measures against these parasites in Malaysia. More national epidemiological research is needed to succinctly elucidate the spatial distribution of these infections and estimate the potential impact on Malaysia's livestock industry. Acknowledgments The authors appreciate the support from Md Nazim Razali Kanini, Mohd Jefri Norsidin and the staff of the Department of Exotic and Large animal for helping throughout the study. Author Contributions Conceptualization, A.S.M., S.A.H. and S.S.S.-H.; Data curation, M.B.S.; Formal analysis, Funding acquisition, N.A.A.A. and S.S.S.-H.; Investigation, A.S.M., S.A.H., S.Z.R., Z.Z., N.A.A.A., R.M., N.A.H. and S.S.S.-H.; Methodology, A.S.M., S.A.H., S.Z.R., J.K. and S.S.S.-H.; Project administration, M.B.S., A.S.M., N.A.A.A., R.M., N.A.H., J.K. and S.S.S.-H.; Resources, S.S.A., N.K., Z.Z., R.M., N.A.H., J.K. and S.S.S.-H.; Supervision, S.Z.R., Z.Z. and S.S.S.-H.; Writing--original draft, M.B.S. and S.S.S.-H.; Writing-review & editing, M.B.S. and S.S.S.-H. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by the Institutional Animal Care and Use Committee University Putra Malaysia (IACUC) (Approval code: U066/2018). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement Data will be made available upon request. Conflicts of Interest The authors declare no conflict of interest. animals-13-00948-t001_Table 1 Table 1 Farm and animal characteristics of the sampled animals from ruminant farms in Selangor, Malaysia. Cattle (n = 225) Goats (n = 179) Overall (n = 404) Variables Frequency Percentage Frequency Percentage Frequency Percentage Age (months) 1-12 44 19.6 49 27.4 93 23.0 13-24 83 36.9 90 50.3 173 42.8 Above 24 98 43.6 40 22.3 138 34.1 Sex Male 16 7.1 27 15.1 43 10.6 Female 209 92.9 152 84.9 361 89.4 Management Intensive 193 85.8 139 77.7 332 82.1 Semi-intensive 32 14.2 40 22.3 72 17.9 Pest control Yes 47 20.9 36 20.1 83 20.5 No 178 79.1 143 79.9 321 79.5 Presence of dogs, cats, and wild animals Yes 131 58.2 137 76.9 268 66.3 No 94 41.8 42 23.5 136 33.7 Animal source Single/internal 124 55.1 84 46.9 208 51.4 Multiple/external 101 44.9 95 53.1 196 48.6 Herd size Less than 50 77 34.2 45 25.1 122 30.1 50-99 30 13.3 54 30.2 84 20.7 100 and above 118 52.4 80 44.7 198 49.0 Main production Meat 24 10.7 127 70.9 151 37.4 Milk 126 56.0 30 16.8 156 38.6 NA 75 23.3 22 12.3 97 24.0 Water source Tap water 147 65.3 145 81.0 292 72.3 Tap water + underground 6 2.7 10 5.6 16 3.9 Underground 9 4.0 8 4.5 17 4.2 Public source 55 24.4 16 8.9 71 17.5 Well 8 3.6 - 8 1.9 NA = not available. animals-13-00948-t002_Table 2 Table 2 Seroprevalence of T. gondii, N. caninum, and B. besnoiti antibodies in cattle (n = 225) sampled from 19 farms in Selangor, Malaysia. Farms Label Number of Sampled Animals Seropositivity for T. gondii (%) Seropositivity for N. caninum (%) Seropositivity for B. besnoiti (%) F1 10 0 (0.0) 1 (10.0) 0 (0.0) F2 5 1 (20.0) 0 (0.0) 1 (20.0) F3 2 0 (0.0) 0 (0.0) 1 (50.0) F4 9 0 (0.0) 0 (0.0) 5 (55.6) F5 9 4 (44.4) 1 (11.1) 1 (11.1) F6 4 0 (0.) 0 (0.0) 0 (0.0) F7 5 0 (0.0) 0 (0.0) 0 (0.0) F8 8 1 (12.5) 0 (0.0) 0 (0.0) F9 9 0 (0.0) 0 (0.0) 0 (0.0) F10 8 0 (0.0) 0 (0.0) 1 (12.5) F11 16 0 (0.0) 1 (6.3) 0 (0.0) F12 37 0 (0.0) 3 (8.1) 4 (10.8) F13 13 0 (0.0) 0 (0.0) 0 (0.0) F14 12 1 (8.3) 0 (0.0) 0 (0.0) F15 2 1 (50.0) 0 (0.0) 0 (0.0) F16 10 0 (0.0) 0 (0.0) 0 (0.0) F17 30 1 (3.3) 0 (0.0) 0 (0.0) F18 30 3 (10.0) 0 (0.0) 0 (0.0) F19 6 0 (0.0) 0 (0.0) 0 (0.0) Total 225 12 (5.3) 6 (2.7) 13 (5.7) animals-13-00948-t003_Table 3 Table 3 Seroprevalence of T. gondii, N. caninum, and B. besnoiti antibodies in goats (n = 179) sampled from 13 farms in Selangor, Malaysia. Farms Label Number of Sampled Animals Seropositivity for T. gondii (%) Seropositivity for N. caninum (%) F1 20 5 (20.0) 2 (10.0) F2 30 14 (30.0) 0 (0.0) F3 10 10 (100.0) 0 (0.0) F4 15 13 (86.7) 0 (0.0) F5 6 6 (100.0) 1 (16.7) F6 10 9 (90.0) 1 (10.0) F7 11 10 (90.9) 2 (18.2) F8 5 0 (0.0) 0 (0.0) F9 8 6 (75.0) 1 (12.5) F10 18 15 (83.3) 0 (0.0) F11 24 22 (91.7) 0 (0.0) F12 16 13 (81.3) 0 (0.0) F13 6 2 (33.3) 0 (0.0) Overall 179 125 (69.8) 7 (3.9) animals-13-00948-t004_Table 4 Table 4 Final mixed-effects logistic regression model of the factors associated with the prevalence of T. gondii antibodies in goats from 13 farms in Selangor, Malaysia. Variables B S.E. Wald df OR 95% CI p-Value Age (months) 8.51 2 0.014 Above 12 -1.63 0.56 8.43 1 5.26 1.7-16.66 0.004 7-12 -1.01 0.56 3.23 1 2.77 0.91-8.33 0.072 Less than 6 Ref Management Intensive -0.76 0.49 2.43 1 2.17 1.34-6.21 0.12 Semi-intensive Ref Presence of dogs, cats, and wild animals Yes 1.29 0.62 4.28 1 3.63 1.1-12.3 0.03 No Ref Animal source Single/internal 1.37 0.45 9.09 1 3.92 1.61-9.69 0.00 Multiple/external Ref Herd size 6.84 2 0.00 100 and above -1.30 0.50 6.83 1 3.70 1.38-10.0 0.00 50-99 -0.50 0.49 1.05 1 1.67 0.63-4.34 0.30 Less than 50 Ref Note: df = degree of freedom, CI = confidence interval, OR = odds ratio, ref = reference group. animals-13-00948-t005_Table 5 Table 5 Final mixed-effects logistic regression model of the factors associated with the prevalence of T. gondii antibodies in cattle from 16 farms in Selangor, Malaysia. Variables B S.E. Wald df OR 95% CI p-Value Management Intensive -1.59 0.62 6.59 1 0.20 0.06-0.68 0.01 Semi-intensive Ref Presence of dogs, cats, and wild animals Yes 1.60 1.12 2.04 1 4.99 0.55-45.35 0.15 No Ref Pest control Yes -2.00 1.39 2.07 1 0.13 0.009-2.06 0.15 No Ref Note: df = degree of freedom, CI = confidence interval, OR = odds ratio, ref = reference group. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000078

The aim of this study was to evaluate the effect of a phytomelatonin-rich diet, including by-products from the food industry, on ram sperm quality and seminal plasma composition. Melatonin content in several by-products before and after in vitro ruminal and abomasal digestion was determined by HPLC-ESI-MS/MS. Finally, 20% of a mix of grape pulp with pomegranate and tomato pomaces was included in the rams' diet, constituting the phytomelatonin-rich diet. Feeding the rams with this diet resulted in an increase in seminal plasma melatonin levels compared with the control group (commercial diet) in the third month of the study. In addition, percentages higher than those in the control group of morphologically normal viable spermatozoa with a low content of reactive oxygen species were observed from the second month onwards. However, the antioxidant effect does not seem to be exerted through the modulation of the antioxidant enzymes since the analysis of the activities of catalase, glutathione reductase and glutathione peroxidase in seminal plasma revealed no significant differences between the two experimental groups. In conclusion, this study reveals, for the first time, that a phytomelatonin-rich diet can improve seminal characteristics in rams. circular economy diet melatonin phytomelatonin ram seminal quality Ministerio de Economia Industria y CompetitividadAGL-2017-83799-R Gobierno de AragonDGA A07 20R Diputacion General de Aragon (DGA)inisterio de Ciencia e Innovacion (MICINN)IJC2019-041482-I This study was funded by the Ministerio de Economia Industria y Competitividad (AGL-2017-83799-R) and the Gobierno de Aragon (DGA A07 20R). V.P.-D has a predoctoral contract from the Diputacion General de Aragon (DGA). G.A-R has a Juan de la Cierva-Incorporacion postdoctoral grant (IJC2019-041482-I) from the Ministerio de Ciencia e Innovacion (MICINN). pmc1. Introduction Livestock farming systems in the Mediterranean regions of the southern European Union countries, such as sheep husbandry, are important given that they are linked with the use of semi-natural and natural areas, and involve well-adapted autochthonous breeds , which is the case for the Rasa Aragonesa breed . However, these systems are currently threatened by economic, institutional, environmental and social factors . Furthermore, one of the limiting factors is reproductive seasonality, which is regulated by nocturnal melatonin secretion in the pineal gland. Melatonin is also synthesized in the male reproductive tract and is present in seminal plasma , having direct effects on ram sperm functionality. Experiments conducted with melatonin added in vitro to ovine sperm samples have demonstrated that this hormone. modulates sperm capacitation, decreases oxidative stress and apoptosis markers . Regarding the in vivo effects of melatonin on small ruminants, studies in the 1980s were performed by orally administering melatonin . This hormone, absorbed onto food pellets or added in saline solution, resulted in sustained elevated blood levels of melatonin for at least 7 h in ewes . Moreover, Arendt et al. reported that daily oral administration allowed advancing the reproductive season in ewes although the efficacy of this treatment decreased if the administration was reduced to three times a week . Most of these experiments were performed on ewes, but few studies examined the effects of the oral administration of melatonin on rams. Among these, the work performed on Suffolk rams by Kusakari and Ohara revealed that melatonin feeding could increase the reproductive activity of these rams during periods of seasonal regression . Since the 1990s, with the development of melatonin subcutaneous implants, the use of these devices has displaced studies with oral melatonin, as they are more practical . In this case, our group and others demonstrated that implants had beneficial effects on reproductive traits, sperm motility and fertilization parameters, the seminal plasma hormonal profile and the activity of some antioxidant enzymes in rams . There is abundant research into the effects of melatonin implants on the semen of other ruminants . However, in order to adapt sheep production to the new demands of consumers, who are increasingly concerned about organic, hormone-free production, it would be of interest to replace the synthetic melatonin present in implants with other natural sources of melatonin such as phytomelatonin, which is the melatonin present in plants and which can be administered with the diet. Plants contain phytomelatonin in highly variable concentrations . The highest levels have been found in seeds such as mustard (Brassica nigra and Brassica hirta, 129 and 189 ng/g of dry matter, respectively), goji (Lycium barbarum, 103 ng/g), fenugreek (Trigonella foenum-graecum, 43 ng/g), almond (Prunus amygdalus, 39 ng/g), sunflower (Helianthus annuuss, 29 ng/g), fennel (Foeniculum vulgare, 28 mg/g) and alfalfa (Medicago sativum, 16 ng/g) , and also in fruits, such as tomato (Solanum lycopersicum, 2-114 ng/g, depending on the variety, the fraction and the method of analysis), cherry (Prunus avium, 8-120 ng/g) and grapes (Vitis vinifera, 5-96 ng/g) . Modulating blood melatonin levels in mammals through the intake of these products has become a strategy of great interest . In fact, several studies have shown that the consumption of certain melatonin-rich vegetables, seeds and plant products increases the levels of this hormone in blood . Given that melatonin readily crosses physiological barriers such as the blood-testis barrier , we hypothesized that a phytomelatonin-rich diet could have a beneficial effect on ram semen. In the present study, by-products from food industry derivatives were included in the diet in order to achieve circular economy objectives. Specifically, our aims were to evaluate the effect of the phytomelatonin-rich diet on melatonin concentration and the activity of the antioxidant enzymes in seminal plasma, and on sperm quality of rams. 2. Materials and Methods Unless otherwise stated, all reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.1. Determination of Melatonin Content in Vegetables and Residues after In Vitro Digestion The melatonin content in several agri-food by-products was analyzed to choose which could potentially be used as a supplement in the animals' diet. The analyzed products were by-products of the juice (pomegranate pomace and peels), the canning (tomato pomace), the brewing (brewer's spent grain, malt sprouts and spent yeast) and the wine (grape pulp and grape seeds) industries. Another product with reported high melatonin content, sunflower meal , was included as a control. Melatonin was determined following the protocol published by Rebollo-Hernanz et al., 2019 . Briefly, by-products were homogenized until turning them into flour. Melatonin was extracted by treating the flours with MeOH under continuous stirring at 4 degC (16 h in darkness). After centrifugation, the supernatants were filtered under vacuum and dried using N2. The residues were resuspended in Milli-Q water and melatonin was isolated using solid-phase extraction (SPE, cartridge C-18, Waters) and measured by HPLC-ESI-MS/MS triple quadrupole. Melatonin content was expressed as ng g-1 sample. From each sample, triplicate extractions were made and each one was injected twice into the column. Melatonin content in the feed residues remaining after in vitro studies or ruminal and abomasal digestion was estimated by the same protocol. 2.2. Analysis of Chemical Composition of the By-Products The by-products selected on the basis of their melatonin content were: grape pulp, tomato pomace, pomegranate peels and pomegranate pomace, to which sunflower meal was added as control. The AOAC (Association of Official Analytical Chemists) methods were used for the analysis of dry matter (DM, method 934.01), organic matter (OM, method 942.05), crude protein (CP, method 976.05) and ether extract (EE, method 2003.05) content. Concentration of neutral detergent fibre (NDFom) was analyzed as described by Mertens in an Ankom 200 Fibre Analyser (Ankom Technology, New York, NY, USA), using a-amylase and sodium sulphite, with the results being expressed exclusive of residual ashes. The acid detergent fibre (ADF, method 973.18) and acid detergent lignin (ADL) were determined as described by AOAC and Robertson and Van Soest , respectively. 2.3. Analysis of In Vitro Digestibility of the By-Products The four selected by-products and the sunflower meal were ground (1 mm particle size), analyzed for chemical composition and incubated in vitro in a closed batch system. Rumen fluid was obtained from four adult, rumen-cannulated ewes. Extraction procedures were approved by the Ethics Committee for Animal Experimentation of the University of Zaragoza (protocol PI48/20). The care and management of animals followed the Spanish Policy for Animal Protection RD 53/2013, which complies with EU Directive 2010/63 on the protection of animals used for experimental and other scientific purposes. On each incubation run, the rumen contents (approximately 300 mL of each animal) were sampled before feeding, filtered through a cheesecloth, mixed, and immediately transferred to the lab for incubation. Four in vitro closed batch incubation series were carried out. Incubations were run at 39 degC in a water bath for 24 h under anaerobic conditions following Theodorou et al. with modifications . Five gas bottles were filled with 800 mg of sample sealed in nylon bags and then 80 mL of incubation medium including rumen inoculum (0.2 of total incubation volume) were added. Three bottles without substrate were also included as blanks. Pressure produced on the bottles was measured with a HD8804 manometer fitted to a TP804 pressure gauge (DELTA OHM, Caselle di Selvazzano, Italy). Readings were corrected for the atmospheric pressure and converted to volume (mL) using a pre-established linear regression (n = 103, R2 = 0.996) and expressed per unit of incubated organic matter (OM). At the end of the 24 h incubation, 40 mL of the liquid phase and the solid residue were collected from one bottle per treatment and immediately frozen until analysis of the melatonin content. The solid residues of the remaining three bottles were washed with tap water and dried (60 degC, 48 h) to determine ruminal digestibility. Thereafter, dry residues pooled by treatment for each incubation run were digested with HCl-pepsin to estimate abomasal digestibility. The melatonin concentration in the residues after ruminal and abomasal digestion was analyzed as described in Section 2.1. 2.4. Animals and Diets Sixteen 2-year-old Rasa Aragonesa rams were randomly assigned into two groups: eight rams were fed with 500 g of a commercial diet (Nutrifeed ovejas 800 MS(r), Agroveco, Spain) and the other eight were fed with a 500 g phytomelatonin-rich diet per day for five months (from February to July, non-breeding season). The phytomelatonin-rich diet consisted of a mixture of various sources of phytomelatonin (20%), such as pomegranate and tomato pomaces and grape pulp (all of them derivatives of the agri-food industry), which was added to the commercial diet (80%). Diets were formulated to contain the same amount of protein, fat and fibre (Section 2.2). All animals were fed straw ad libitum. All rams were housed at the Experimental Farm of the University of Zaragoza (Zaragoza, Spain) and all experimental procedures were accomplished as described for the Project License PI39/17 approved by the Ethics Committee for Animal Experiments, University of Zaragoza (Spain), in accordance with the Directive 2010763/UE of the European Parliament on the of animals used for scientific purposes. 2.5. Semen Collection and Seminal Plasma Extraction Ejaculates from each ram were obtained by artificial vagina prior to the beginning of the experiment (month 0, February) and every fifteen days for 5 months (month 5, July), so two complete semen analyses from all animals of each group were performed every month. After semen collection, sperm motility, morphology, membrane integrity, intracellular levels of reactive oxygen species (ROS) and phosphatidylserine (PS) inversion were assessed. Seminal plasma was obtained by centrifugation at 14,000x g for 10 min at 4 degC. The supernatant was collected and centrifuged again under the same conditions, and the recovered seminal plasma was stored at -20 degC until the analysis of the melatonin concentration and the activity of antioxidant enzymes (glutathione reductase, glutathione peroxidase and catalase). 2.6. Sperm Motility Analysis A computer-assisted sperm analysis system (CASA) was used for analyzing sperm motility (ISAS v. 1.04, Proiser S.L., Valencia, Spain). For this assessment, a dilution of each semen sample was made in a medium with the following composition: 0.25 M sucrose, 100 mM EGTA, 0.5 mM sodium phosphate, 50 mM glucose, 100 mM HEPES and 20 mM KOH. A drop of 8 mL of each diluted sample (3 x 107 cells/mL), was placed between a pre-warmed slide and a coverslip and maintained at 37 degC in a heated slide holder during analysis. Spermatozoa were recorded using a video camera (Basler A312f, Basler Vision Components, Exton, PA, USA) mounted on a microscope (Nikon Eclipse 50i, Nikon Instruments Int, Tokyo, Japan) equipped with a 10x negative-phase contrast lens. The recording was performed at 25 frames/s and 25 consecutive digitalized images were taken for a single field. Five fields of each drop were recorded, and percentages of total motile and progressive motile spermatozoa in all samples were evaluated. 2.7. Sperm Morphological Study The sperm morphology was evaluated by eosin-nigrosine staining . A volume of 10 mL of each dye was added to 20 mL of each sample (4 x 107 cells/mL). After mixing, a drop of the stained sample (20 mL) was placed on a slide and spread with the aid of another. The smears were air-dried and observed by bright field microscopy using a Nikon Eclipse E-400 microscope (Kanagawa, Yokohama, Japan). At least 200 spermatozoa were analyzed at 1000x magnification, and the percentage of normal morphology cells was evaluated, considering abnormal those cells that showed primary (detached head) and secondary (bent tail, coiled tail or proximal or distal droplet) abnormalities . 2.8. Flow Cytometry Analyses All flow cytometry measurements were performed using a Beckman Coulter FC 500 flow cytometer (Beckman Coulter Inc., Fullerton, CA, USA) equipped with CXP software, two lasers of excitation (argon-ion laser, 488 nm; and solid-state laser, 633 nm) and five filters of absorbance (FL1-525, FL2-575, FL3-610, FL4-675 and FL5-755; +-5 nm each bandpass filter). A flow rate stabilized at 200-300 cells/s was used, and a minimum of 20,000 events were recorded in all experiments. The sperm population was gated for further analysis on the basis of its specific forward (FS) and side scatter (SS) properties and other non-sperm events were excluded. 2.8.1. Evaluation of Sperm Membrane Integrity To determine cell membrane integrity (viability), a modification of the procedure described by Harrison and Vickers was followed. Samples (500 mL; 5 x 106 cells/mL) were fixed with 3 mL formaldehyde (0.5% (v/v) in water) and stained with 3 mL of 10 mM carboxyfluorescein diacetate (CFDA) and 3 mL of 7.3 mM propidium iodide (PI). After incubation (15 min, 37 degC in darkness), samples were analyzed by flow cytometry. The monitored parameters were FS log, SS log, FL1 log (CFDA) and FL4 log (PI). 2.8.2. Intracellular Content of ROS ROS levels were assessed by using 2',7'-dichlorohydrofluorecein-diacetate (H2DCFDA), which is freely permeable across cell membranes and converted into non-permeable and non-fluorescent 2',7'-dichlorodihydrofluorescein (H2DCF) by intracellular esterases. The H2DCF is oxidized by H2O2 to dichlorofluorescein (DCF), which emits fluorescence at 530 nm in response to 488 nm excitation . This probe was combined with PI to exclude the nonviable population from the analysis . For the assessment, sperm samples (final concentration 5 x 106 cells/mL) were stained with 5 mL of 10 mM H2DCFDA, and 3 mL of 1.5 mM PI. After 15 min of incubation (37 degC in darkness), samples were fixed with 5 mL formaldehyde (0.5% (v/v) in water) and analyzed. The monitored parameters were FS log, SS log, FL1 log (H2DCFDA) and FL4 log (PI). 2.8.3. Detection of Membrane Phosphatidylserine Translocation Annexin V is a protein with a high affinity for phosphatidylserine (PS); hence, it can be used as a sensitive probe to detect PS exposure upon the cell membrane . For the analysis, aliquots of 50 mL of sperm samples were diluted (final concentration 4 x 106 cells/mL) with 250 mL of 1X binding buffer (provided in the commercial kit; Binding Buffer Apoptosis Detection Kit, Life Technologies, Carlsbad, CA, USA) and stained with 3 mL of 1.5 mM PI and 2 mL FITC-Annexin V (conjugate also provided in the kit). After 15 min of incubation (37 degC in darkness), samples were assessed by flow cytometry. The monitored parameters were FS log, SS log, FL1 log (Annexin-V) and FL4 log (PI). 2.9. Melatonin Evaluation in Seminal Plasma Melatonin concentration in ram seminal plasma was measured using a commercial competitive immunoassay (Direct saliva melatonin ELISA kit, Buhlmann Laboratories AG, Schonenbuch, Switzerland) as previously described . Absorbance was measured at 450 nm on a microtiter plate reader (SPECTROstar Nano, BMG Labtech, Ortenberg, Germany). 2.10. Antioxidant Enzyme Activity Assays in Seminal Plasma 2.10.1. Glutathione Reductase (GRD, EC.1.6.4.2) All measurements were performed as previously described and all samples were loaded in duplicate. The reaction mixture contained 501.38 mM sodium phosphate buffer at pH 7.2; 0.5 mM EDTA; 85 mM NADPH+ + H+ and 0.8 mM GSSG. 5 mL of seminal plasma were added to complete a final volume of 200 mL. The enzymatic activity was evaluated for 3 min at 340 nm with a microtiter plate reader (SPECTROstar Nano, BMG Labtech, Ortenberg, Germany). 2.10.2. Glutathione Peroxidase (GPx, EC.1.11.1.9) Measurements were performed as previously described based on a modification of the procedure described by Plagia and Valentine . The reaction mixture contained 501.38 mM sodium phosphate buffer at pH 7.2; 0.5 mM EDTA; 85 mM NADPH+ + H+, 54 mUI GRD, 2 mM GSH and 1.2 mM t-BuO2H. A total of 6 mL of seminal plasma was added to complete a final volume of 200 mL. The absorbance change at 340 nm was monitored for 3 min with the microtiter plate reader (SPECTROstar Nano, BMG Labtech, Ortenberg, Germany). 2.10.3. Catalase (CAT, EC. 1.11.1.6) For catalase evaluation, the reaction mixture contained 62.5 mM sodium phosphate buffer at a pH 7, 200 mM H2O2 and 4 mL of seminal plasma to complete a final volume of 200 mL. The absorbance change at 240 nm was monitored for 120 s with the microtiter plate reader (SPECTROstar Nano, BMG Labtech, Ortenberg, Germany). For these measurements, a quartz microplate was used. 2.11. Statistical Analyses Results of in vitro digestion were analyzed statistically by ANOVA with the Statistix 10 package (Analytical Software. Statistix 10 for Windows; Analytical Software: Tallahassee, FL, USA, 2010), considering the incubation run as a block. Treatment differences among means with p < 0.05 were accepted as representing statistically significant differences. When significant, differences were contrasted by the Tukey t-test. The chemical compositions of the diets (control vs. phytomelatonin-rich) were compared by the Mann-Whitney test. Data are shown as mean +- S.E.M. (standard error of the mean). The effects of the diet and time on sperm motility, morphology, viability, intracellular levels of ROS, phosphatidylserine translocation, melatonin concentration and the activities of antioxidant enzymes in seminal plasma were analyzed using the mixed-model ANOVA and Fisher's LSD as a post hoc test. Statistical analyses were performed with SPSS Statistics v.26 (IBM Analytic, Armonk, NY, USA) and GraphPad Prism v.8 (La Jolla, CA, USA). 3. Results 3.1. Melatonin Content in Vegetables According to the HPLC-ESI-MS/MS analysis, the by-products containing the highest levels of phytomelatonin were pomegranate pomace and peels, tomato pomace and grape pulp (Table 1). We therefore selected these by-products for subsequent analysis, together with the sunflower meal. 3.2. Melatonin Content after In Vitro Digestion and Diet Composition The chemical composition of the selected by-products is summarized in Table S1 (Supplementary material). After incubation of these by-products in ruminal liquid and the treatment of residues with HCl-pepsin, the melatonin content in the obtained fractions was measured, and the results are shown in Table 2. The in vitro ruminal and abomasal substrate digestibility and rumen in vitro fermentation pattern of these four by-products and also the sunflower meal are summarized in Table S2 and Figure S1 (Supplementary Material), respectively. Based on these results, for the in vivo experiment, we selected pomegranate and tomato pomaces and grape pulp mixed in equal proportions. We opted for the pomegranate pomace instead of the pomegranate peels since the rumen fermentation was better , as well as the acceptability by the animals. The mixture accounted for 20% of the daily ration ingested by the treated animals. The chemical analysis of the phytomelatonin-rich and commercial (control) diets (Table S3, Supplementary Material) revealed that the differences in CP, EE and fibre were not significant (p < 0.05). 3.3. Effects of Phytomelatonin-Rich Diets on Seminal Plasma 3.3.1. Effect on Melatonin Levels in Seminal Plasma Melatonin levels in seminal plasma from rams fed with the phytomelatonin-rich diet showed an increase from month 2, which was statistically significant (p < 0.05) in month 3 when compared with rams from the control group. Afterwards, melatonin levels tended to decrease (month 4) and then rise (month 5) both in rams fed with phytomelatonin and rams fed with the commercial diet . 3.3.2. Effect on Antioxidant Enzymes Activity in Seminal Plasma There were no statistical differences in the activity of catalase, glutathione reductase and glutathione peroxidase between the two experimental groups throughout the duration of the study . 3.4. Effects of Phytomelatonin-Rich Diets on Sperm Quality 3.4.1. Effect on Sperm Motility For total motility, there was a significant effect of the diet (p < 0.01). Percentages of total motile sperm cells in samples from animals fed with the phytomelatonin diet tend to be higher than in the control group samples , and the post hoc test showed that this increment was statistically significant (p < 0.01) just in the first month after diet administration. However, there was no significant effect of diet on progressive motility. 3.4.2. Effect on Sperm Morphology Significant effects of the administered diet were observed on the percentages of morphologically normal spermatozoa (p < 0.0001). After month 2, rams fed with the phytomelatonin-rich diet showed higher percentages of morphologically normal cells than the control group until the end of the experiment , with the maximum differences reached at months 2 and 3 (p < 0.001). 3.4.3. Effect on Sperm Viability, ROS Levels and PS Translocation Regarding the percentages of viable, viable with low levels of ROS and viable with no PS translocation spermatozoa, the phytomelatonin-rich diet had a significant effect on sperm viability (p < 0.001), and on the content of ROS (p = 0.01). From the second month of the study, sperm viability was significantly higher in rams fed with the phytomelatonin-rich diet than in the control group (p < 0.01). These differences were also found until the end of the study, especially in the third and the fifth months . A similar pattern was observed when analyzing the production of ROS, since rams fed with the phytomelatonin-rich diet had significantly higher percentages of viable sperm with low ROS levels than the control group from month 2, this being more marked at month 3 . Nevertheless, this effect was not observed when studying the phosphatidylserine inversion, as there were no statistical differences in the percentages of viable spermatozoa without PS translocation between the two groups during the experiment . 4. Discussion Results of the present experiment have revealed that rams fed with a phytomelatonin-rich diet experience an increase in the content of melatonin in their seminal plasma, improving sperm viability and morphology, and protecting sperm cells against oxidative damage. In previous studies, the beneficial effects of melatonin subcutaneous implants on testis size, ejaculate volume, sperm concentration, motility and morphology, among others, have been reported . Likewise, melatonin implants increased melatonin levels in seminal plasma during the non-reproductive season . However, as consumers are increasingly aware of animal welfare and organic production, it is of interest to adapt animal production to new consumer demands. Replacing the synthetic melatonin used in subcutaneous implants with other natural sources of melatonin, such as phytomelatonin (melatonin from plants), could be a good option, provided that it does not adversely affect the nutritive feed value. In addition, obtaining these sources from by-products of the agri-food industry is consistent with the objectives of the circular economy. In accordance with the existing bibliography and having regard to the availability of by-products from the food industries in our region, we selected by-products from the wine, beer, juice and canning industries. Analysis of these products by HPLC-ESI-MS/MS revealed that pomegranate pomace and peels, grape pulp and tomato pomace had the highest melatonin content. After evaluating the chemical composition, the in vitro digestibility and the melatonin remaining after in vitro ruminal and abomasal digestion, we selected pomegranate pomace, grape pulp and tomato pomace as the ingredients for a phytomelatonin-rich diet. Pomegranate peels showed higher melatonin content after in vitro digestion than pomegranate pomace but a worse rumen fermentation pattern, digestibility, and acceptability by the animals. Sunflower meal could also have been a good alternative, but in this study it was included only as a control since the work focused only on by-products. Feeding the rams with a phytomelatonin-rich diet containing a 20% proportion of a mix of by-products (tomato pomace, pomegranate pomace and grape pulp) resulted in an increase in seminal plasma melatonin levels compared with the control group in the third month of the study. However, the use of melatonin subcutaneous implants led to an earlier, higher increase in seminal plasma melatonin levels than that resulting from administering the phytomelatonin-rich diet . This could be explained because the quantity of melatonin that can be administered through subcutaneous implants is much higher than the quantity found in the agri-food by-products used in the diet (18 mg in melatonin subcutaneous implants vs. 23.76 +- 1.37, 45.94 +- 4.19 and 35.81 +- 0.40 ng/g for tomato pomace, grape pulp and pomegranate pomace, respectively). Regarding the possible effects of the diet on sperm quality, parameters including sperm motility, morphology, membrane integrity, intracellular ROS levels and PS translocation were evaluated. We observed that total motility tends to be higher in semen collected from animals fed with the phytomelatonin-rich diet than in the control group samples, although this increase was only significant in the first month after administering the diet. This finding is in agreement with results previously described for Black Racka rams treated with subcutaneous melatonin implants during the non-breeding season, since treated animals from this breed showed better total sperm motility rates than the control group . However, feeding a phytomelatonin-rich diet had no significant effect on progressive motility, contrary to what was described when melatonin implants were used in rams of the same or other breeds . The phytomelatonin-rich diet led to higher percentages of morphologically normal and viable spermatozoa than in the control group from the second month to the end of the experiment. Beneficial effects on morphology were also reported in some studies using melatonin implants but not others . In the case of viability, no changes were reported after the use of melatonin implants in rams , but beneficial effects were described in other species, including bull , buck and buffalo . In ovine, improvement in sperm viability was observed when melatonin was added directly to spermatozoa in vitro . However, in this work, we describe, for the first time, how melatonin supplemented through diet can enhance the percentage of morphologically normal and membrane-intact sperm cells. A similar pattern occurred with the percentage of live sperm cells with low content of reactive oxygen species (ROS). It has been demonstrated that in vitro incubation of sperm samples with melatonin can reduce oxidative stress damage caused by ROS in human and boar spermatozoa . Recently, our research group has documented that melatonin, when added to sperm samples in capacitating conditions, can reduce ROS levels and partially prevent sperm capacitation . However, in the present study, no significant differences in the percentages of viable spermatozoa without inversion of phosphatidylserine were observed between the samples from the two groups. Although in vitro melatonin has an antiapoptotic effect on sperm from different species , including ovine , its oral administration through the assayed diet does not seem to influence this parameter in rams. According to the data presented, we can infer that a diet rich in phytomelatonin can protect sperm cells against oxidative damage by decreasing intracellular ROS levels. This antioxidant effect could be exerted directly or indirectly by influencing the activities of antioxidant enzymes . In the present study, the analysis of the activities of catalase (CAT), glutathione reductase (GRD) and glutathione peroxidase (GPX) in seminal plasma revealed no significant differences between the two experimental groups. Although melatonin implants modulate the activity of GPX and GRD, but not CAT, in rams and bucks , the tested phytomelatonin-rich diet seems not to modify the activities of these antioxidant enzymes, probably because the amount of exogenous melatonin provided with the diet is much smaller than that produced by implants . As no changes in the activities of the aforementioned antioxidant enzymes were detected, we hypothesized that phytomelatonin could modulate the levels of reactive oxygen species directly in the spermatozoa, either by crossing the sperm plasma membrane or by binding to its specific membrane receptors. Nevertheless, the molecular mechanisms by which the phytomelatonin-rich diet exerts this antioxidant action on spermatozoa remain unknown. Furthermore, we cannot overlook the fact that other antioxidants present in the by-products used in the diet may contribute synergistically with phytomelatonin to produce this effect. For instance, polyphenols and anthocyanins present in pomegranate have been shown to have powerful antioxidant activity , as well as lycopene, ascorbic acid, vitamin E and flavonoids present in tomato , or catechins, flavonols, benzoic acid and cinnamic acid present in grape pulp . 5. Conclusions This study reveals, for the first time, that a phytomelatonin-rich diet increases melatonin levels in seminal plasma, improves sperm viability and morphology, and protects sperm cells against oxidative damage by decreasing intracellular ROS levels; all these effects occurring, at the latest, three months after the beginning of the feeding. Acknowledgments The authors would like to acknowledge the use of the Servicio General de Apoyo a la Investigacion-SAI (Universidad de Zaragoza); special thanks to G. Gonzalo for his help in the diet preparation and the feeding of the animals. Thanks to ANGRA for helping to obtain the rams. Thanks to Gustavo Maria Levrino (Universidad de Zaragoza) and Carlos Perez Calvo (SAEQSA) for advice on using by-products and providing contacts. Thanks to Bodegas Tempore, Conservas Vega del Ebro S.A., Fabrica de cerveza La Zaragozana S.A. and Pomegranate juice manufacturer Vitalgrana for providing the by-products used in this study. Supplementary Materials The following supporting information can be downloaded at: Table S1: Chemical composition (g/kg dry matter) of the selected by-products; Table S2: In vitro ruminal and abomasal substrate digestibility (g/kg) of the selected by-products; Table S3: Chemical composition (g/kg dry matter) of the control and phytomelatonin-rich diets Figure S1: Rumen in vitro fermentation pattern (mL gas/g OM) of selected by-products Click here for additional data file. Author Contributions Conceptualization, A.C. and R.P.-P.; methodology, A.C., M.F., J.A.A. and R.P.-P.; validation, J.A.A., A.C., M.F., M.A.M.-C. and R.P.-P.; formal analysis, V.P.-D., M.C.-S., M.F., Y.A., M.A.M.-C. and A.C.; investigation, M.C.-S., V.P.-D., G.A.-R. and A.C.; resources, M.F., M.A.M.-C. and R.P.-P.; data curation, V.P.-D. and A.C.; writing--original draft preparation, V.P.-D.; writing--review and editing, J.A.A., A.C., M.F., M.A.M.-C. and R.P.-P.; visualization, V.P.-D., A.C., M.F. and R.P.-P.; supervision, R.P.-P. and A.C.; project administration, R.P.-P.; funding acquisition, R.P.-P. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by the University of Zaragoza Ethics Committee for Animal Experiments (Project Licenses PI39/17 and PI48/20). Care and management of animals followed the Spanish Policy for Animal Protection RD 53/2013, which complies with EU Directive 2010/63 on the protection of animals used for experimental and other scientific purposes. Data Availability Statement The datasets generated for this study can be found in the figshare repository doi: 10.6084/m9.figshare.22193245. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Figure 1 Monthly variations of melatonin levels in seminal plasma of animals fed with a phytomelatonin-rich (*) or commercial (control, #) diet from month 0 (before diet administration) to month 5. Values are shown as mean +- S.E.M., n = 16. * p < 0.05 indicates significant differences between both experimental groups. Figure 2 Monthly variations of catalase (CAT), (a), glutathione reductase (GRD), (b) and glutathione peroxidase (GPX), (c) activities in seminal plasma of animals fed with a phytomelatonin-rich (*) or commercial (control, #) diet from month 0 (before diet administration) to month 5. Values are shown as mean +- S.E.M., n = 16. Figure 3 Percentage of total motile (top) and progressive (bottom) spermatozoa in semen samples from animals fed with a phytomelatonin-rich (*) or commercial (control, #) diet from month 0 (before diet administration) to month 5. Values are shown as mean +- S.E.M., n = 16. ** p < 0.01 indicates significant differences between the two experimental groups. Figure 4 Percentage of sperm cells with normal morphology in samples from animals fed with a phytomelatonin-rich (*) or commercial (control, #) diet from month 0 (before diet administration) to month 5. Values are shown as mean +- S.E.M., n = 16. ** p < 0.01 and *** p < 0.01 indicate significant differences between both experimental groups. Figure 5 Percentage of viable (a), viable with low ROS levels (b) and viable without phosphatidylserine (PS) translocation (c) spermatozoa in samples from animals fed with a phytomelatonin-rich (*) or commercial (control, #) diet from month 0 (before diet administration) to month 5. Values are shown as mean +- S.E.M., n = 16. * p < 0.05, ** p < 0.01 and *** p < 0.01 indicate significant differences between the two experimental groups. animals-13-00905-t001_Table 1 Table 1 Melatonin content in agri-food by-products analyzed by HPLC-ESI-MS. Sample Melatonin (ng/g) Pomegranate pomace 35.81 +- 0.40 Pomegranate peels 127.03 +- 8.73 Tomato pomace 23.76 +- 1.37 Brewer's spent grains 3.01 +- 0.15 Malt sprouts 3.33 +- 0.04 Spent yeast 5.03 +- 0.11 Grape pulp 45.94 +- 4.19 Grape seeds 9.26 +- 0.52 Sunflower meal 41.55 +- 5.16 animals-13-00905-t002_Table 2 Table 2 Melatonin content in by-products after in vitro digestion. Sample Melatonin Content (ng/g) * After Ruminal Incubation After HCl-Pepsin Treatment Liquid Fraction Solid Fraction Pomegranate pomace 0.53 +- 0.04 n.d. 0.93 +- 0.58 Pomegranate peels 1.56 +- 0.47 0.62 +- 0.14 n.d. Tomato pomace 2.69 +- 0.69 0.32 +-0.02 0.54 +- 0.11 Grape pulp 1.58 +- 0.19 0.34 +- 0.12 n.d. Sunflower meal 1.22 +- 0.15 0.58 +- 0.06 0.32 +- 0.08 * Melatonin content expressed on dry matter of residue (mean +- SEM) (n = 4 corresponding to four incubation bottles). n.d.: not detected. 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PMC10000079

Surgical excision of solid tumors is required for local control of neoplasms. However, surgical trauma can stimulate the release of proangiogenic growth factors, suppressing cell-mediated immunity and favoring the development of micrometastases and progression of residual disease. The present study aimed to evaluate the intensity of the metabolic response to trauma induced via unilateral mastectomy in bitches with mammary neoplasia, the consequences of its joint performance with ovariohysterectomy, and their respective effects on the organic response. Two groups of animals were evaluated in seven perioperative moments, namely, unilateral mastectomy (G1) and unilateral mastectomy associated with ovariohysterectomy (G2). Thirty-two female dogs were selected, ten clinically healthy, and twenty-two diagnosed with mammary neoplasia. Surgical trauma reduced serum concentrations of albumin and interleukin-2 but increased blood levels of glucose and interleukin-6 in the postoperative of G1 and G2 patients. Moreover, serum cortisol levels increased after unilateral mastectomy associated with ovariohysterectomy. Our findings allowed us to conclude that unilateral mastectomy induces significant metabolic alterations in female dogs with mammary neoplasms and its joint performance with ovariohysterectomy increases the organic response to trauma. oncology surgical oncology surgical trauma mammary tumor dogs Sao Paulo Research FoundationFAPESP 2015/25400-7 FAPESP 2016/21474-9 National Research Council --CNPq306055/2011-2 This research was financially supported by the Sao Paulo Research Foundation (FAPESP 2015/25400-7; FAPESP 2016/21474-9) and National Research Council --CNPq (306055/2011-2). pmc1. Introduction Mammary neoplasms are commonly diagnosed in female dogs, and surgery is the main therapeutic modality for local control of the disease. Several surgical techniques have been used to treat mammary neoplasms in dogs. The most appropriate method is selected based on size, location, and number of tumors, as well as the occurrence of invasion into adjacent tissues . Unilateral mastectomy (UM) enables the removal of macroscopically visible lesions and occult tumors, reducing the risk of new lesions due to the excision of all neoplasia-affected mammary chain tissue . However, removing more tissue than enough to obtain tumor-free surgical margins has proved to be unnecessary, as radical techniques do not promote increased patient survival . Tumor recurrences and metastases are the main failures when treating mammary neoplasms, especially in dogs diagnosed with high-grade or advanced-stage malignant tumors. Thus, adjuvant therapies are required to maximize disease-free time . Sorenmo, Shofer, and Goldschmidt (2000) found that performing ovariohysterectomy (OH) concomitantly with UM increases the survival of female dogs with mammary neoplasia. Kristiansen et al. (2016) showed that OH can be beneficial in dogs diagnosed with grade-2 mammary carcinomas and positive tumor immunostaining for oestrogen receptors. Furthermore, metabolic response to trauma (MRT) is a set of neuroendocrine and inflammatory changes after injury aimed at re-establishing homeostasis . The main metabolic alterations associated with trauma are an increase in the secretion of pituitary hormones, activation of the sympathetic nervous system, and release of pro-inflammatory cytokines . The intensity of the organic response depends on the magnitude, nature, and duration of the stimuli. Medium and large surgeries can trigger such a large response that it causes a deterioration of the host's regulatory processes, contributing to postoperative morbidity and mortality . Attenuation of metabolic response to surgical trauma significantly reduces incidence of postoperative complications . Studies in human patients with cancer have shown that adequate analgesia in the perioperative period reduces susceptibility to tumor recurrences and metastases, as pain induces the suppression of cellular immunity . Protocols with inhalational anesthesia combined with analgesia with opioids follow a worse prognosis in cancer patients when compared to protocols using locoregional anesthetic blocks . Studies on humans and laboratory animals have highlighted that opioid analgesics, including morphine and fentanyl, enhance the surgery-induced suppression of cellular immunity. However, studies with tramadol have shown high analgesic potency associated with reduced postoperative immunosuppression . In veterinary medicine, there are no studies on the modulation of metabolic responses to surgical trauma in cancer patients. When considering cancer patients, surgical trauma-derived alterations may also promote neoplastic development through stimuli of micrometastases . Additionally, surgical manipulation of tumors can induce the release of tumor cells into the bloodstream, favoring the spread of neoplasms . However, studies on neuroendocrine and stress responses induced via oncological surgeries are scarce in veterinary medicine. In this sense, understanding metabolic responses to surgical trauma in cancer patients allows for selecting anesthetic protocols, surgical techniques, and therapeutic measures that reduce perioperative morbidity, risks of local recurrences, and metastases. Based on the above, our study aimed to evaluate the intensity of metabolic responses associated with surgical trauma induced via unilateral mastectomy in female dogs with mammary neoplasia and verify whether concomitant ovariohysterectomy increases the organic response to trauma. 2. Materials and Methods 2.1. Animal Selection Thirty-two female dogs were selected regardless of breed and age, ten clinically healthy, and twenty-two diagnosed with mammary neoplasia. The patients came from a university hospital in the city of Curitiba, Parana State (Brazil). The study was approved by the Committee on Ethics in Animal Use (CEUA) of the Evangelical College of Parana (Protocol ndeg 001333/2013). Anamnesis, physical examination, laboratory tests (complete blood count; blood glucose concentration; and serum levels of alanine aminotransferase, alkaline phosphatase, urea, and creatinine), and diagnostic imaging tests (abdominal ultrasound and chest X-ray in the right and left ventrodorsal and laterolateral projections) were performed to assess the clinical condition of the patients with mammary cancer and define tumour staging (World Health Organization; Table 1). In patients with multiple tumours, we chose to analyse those with the largest diameter for clinical staging. Caption: N0 (no metastatic involvement in lymph nodes), N1 (metastatic involvement in lymph nodes), M0 (no distant metastasis), M1 (distant metastasis present), T (tumor size). Animals that showed no clinical manifestations of systemic diseases, nor changes in physical examination or in laboratory and imaging tests, were considered clinically healthy. Moreover, we excluded females weighing less than 7 kg; pregnant; with other neoplasms; with detectable distant metastases (stage V); carriers of systemic diseases that interfere with blood glucose and serum concentrations of albumin, cortisol, and cytokines; and those which had been medicated with anti-inflammatories or subjected to other surgical procedures within 90 days prior to the study. Animals were initially split into two groups: a control group with healthy females (n = 10) and a mammary neoplasm group (n = 22). Then, mammary neoplasm patients were randomly distributed into two subgroups as a function of the surgical procedure performed. Eleven females that underwent unilateral mastectomy (UM) were included in group 1, and eleven females that underwent UM associated with ovariohysterectomy (OH) were included in group 2. In females diagnosed with tumours in both mammary chains, the chain with the largest tumours was resected. 2.2. Procedure and Parameter Evaluation Patients in the mammary neoplasm group were evaluated in the immediate preoperative period before preanesthetic medication (M0), at the end of the surgery (M1), three hours after the end of the surgery (M2), six hours after the end of the surgery (M3), twenty-fours hours after the end of the surgery (M4), seventy-two hours after surgery (M5), and ten days after surgery (M6). Patients in the control group were evaluated only at M0 and were not subjected to any surgical procedure. At all times of evaluation, we measured heart rate (HR); respiratory rate (RR); systolic blood pressure (SBP); body temperature; and blood levels of glucose, lactate, fibrinogen, albumin, cortisol, IL-2, IL-6, and TNF-a. The clinical parameters were evaluated before the blood collections to minimize the effect of stress on outcomes. SBP was measured using a non-invasive blood pressure device (portable vascular Doppler DV 610V, MedMega, Franca/SP, Brazil). Before the surgery, animals underwent a twelve-hour food fast and eight-hour water fast. After measuring clinical parameters and blood collections for laboratory evaluations (M0), acepromazine (0.03 mg/kg) and morphine (0.5 mg/kg) were administered via intramuscular as preanesthetic medication. After 20 min, the surgical site was shaved, and the cephalic vein was catheterized for fluid therapy, infusing 10 mL/kg/hour of lactated Ringer's solution. Anaesthetic induction was performed with 5 mg/kg propofol intravenously, followed by orotracheal intubation and maintenance with isoflurane diluted in 100% oxygen, using a universal vaporizer. Before the start of the surgery, 5 mg/kg intravenous fentanyl citrate bolus was given, followed by an infusion rate of 0.5 mg/kg/min for intraoperative analgesia. During anesthesia, a multi-parameter monitor, a DX-2010, Dixtal(r) (Biomedica e Tecnologia, Brasil) was used. After anaesthetic induction, the patients were positioned in dorsal decubitus for subsequent surgical site antisepsis. In group 2 patients, OH was performed through a median retro-umbilical celiotomy, preceding the UM technique. Then, UM was conducted via elliptical skin incision surrounding the cranial thoracic, caudal thoracic, cranial abdominal, caudal abdominal, and inguinal mammary glands, considering a 2-cm margin of safety to tumours, performing en bloc resection of the mammary chain and inguinal lymph nodes. After the procedure, clinical parameters were measured and blood collected (M1), and then the surgical wound was cleaned and dressed. In the immediate postoperative period, all animals were medicated with 3 mg/kg tramadol hydrochloride intravenously, remaining under observation until full recovery from anaesthesia. They were maintained on intravenous fluid therapy with lactated Ringer's solution and warmed with a blanket and room heater. After measuring clinical parameters and collecting blood samples for M2 and M3, the animals were released, and a follow-up was scheduled for the morning after the procedure (M4). All dogs were medicated in the postoperative period with 5 mg/kg enrofloxacin orally every twelve hours for ten days, 25 mg/kg dipyrone orally every eight hours for three days, and 3 mg/kg tramadol hydrochloride orally every eight hours for five days. Nonsteroidal anti-inflammatory drugs were not administered in the perioperative period to minimize interference with serum levels of cytokines and acute phase proteins. The patients returned for surgical dressing removal three days after surgery (M5), and then for suture removal in the presence of proper surgical wound sealing after ten days (M6). At all times of evaluation, the patients were evaluated for the presence of hematoma, oedema, or subcutaneous emphysema around the wound, oedema of the pelvic limbs, and the presence of suture dehiscence. To minimize the influence of the operative technique and circadian rhythm on the serum levels of cortisol, all surgical procedures were performed by the same surgeon and always in the morning. Samples of tumour and inguinal lymph nodes were stored for histopathological evaluation, following the classification method reported by Cassali et al. (2013) . 2.3. Statistical Analysis For statistical analysis, the data collected were initially subjected to the Anderson-Darling residual normality test and presented as mean +- standard deviations for parametric distribution (Gaussian), or median and minimum and maximum values for non-parametric distribution. The Gaussian distribution parameters at each evaluation time were compared with M0 via an analysis of variance (ANOVA) and post-Dunnett's test, and then a direct comparison between two groups was performed using the Student's t test. For the non-Gaussian distribution parameters, comparisons between each evaluation moment and M0 were performed using the Friedman's test and then Dunn's test, and the comparison between the two groups was performed using the Mann-Whitney test. When needed, the data were subjected to logarithmic transformation to fit normal distribution of residuals, establish homogeneity of variances, or reduce asymmetry. Pearson's test was used to determine correlations for parametric data, and Spearman's test for non-parametric data. All statistical analyses were performed using the GraphPad Prism19 software, at a 5% significance level. 3. Results 3.1. Animal Characteristics and Clinical Presentation of Tumors and Postoperative Evolution Healthy animals (control group) were aged between five and eleven years (6.9 +- 2.28) and weighed between 7.0 and 44 kg (13.18 +- 11.17). Females with mammary neoplasia subjected to UM (Group 1) were between six and fourteen years old (9.4 +- 2.84) and had 8.75 and 35.10 kg (18.92 +- 11.34). Finally, females subjected to UM and OH (Group 2) were between five and fourteen years old (9.4 +- 2.70) and weighed 7.10 and 34.70 kg (17.80 +- 8.31). No significant differences were observed for ages and body weights of patients in the different groups. As for breed, five female dogs (50%) in the control group were crossbreeds and the remainder comprised the breeds Pug (n = 2; 20%), Scottish Terrier (n = 1; 10%), Cane Corso (n = 1; 10%), and Schnauzer (n = 1; 10%). Ten females (45.5%) with mammary neoplasia were crossbreeds and the remainder were Pit Bull (n = 5; 22.7%), Poodle (n = 2, 9.1%), Cocker Spaniel (n = 2; 9.1%), Dachshund (n = 2; 9.1%), and Rottweiler (n = 1; 4.5%). At the time of diagnosis, among patients with mammary neoplasia, twenty (95.2%) had more than one tumor, eight (38.1%) had tumors only in the right mammary chain, three (14.3%) only in the left mammary chain, and ten (47.6%) in both mammary chains. Thoracic, abdominal, and inguinal mammary glands were involved in mammary tumors in 11 (52.4%), 19 (90.5%), and 18 (85.7%) female dogs, respectively. Sixteen animals (72.7%) had at least one tumor larger than three centimeters in diameter. Lastly, eight patients (38.1%) showed ulcerated tumors. Of the 22 patients who had mammary tumors, 8/22 (36%) had only malignant tumors; 11/22 (50%) had malignant and benign tumors; and 3/22 (14%) had only benign tumors. Regarding mammary tumor clinical staging, six patients (27.3%) presented stage I tumors, four (18.2%) stage II, ten (45.4%) stage III, and two (9.1%) stage IV . No deaths were recorded in the perioperative period of patients undergoing UM, with or without OH. The main surgical wound-related complications were: hematoma (n = 11; 50%), pelvic limb oedema (n = 5; 22.7%), seroma (n = 4; 18.2%), subcutaneous emphysema (n= 4; 18.2%), and partial suture dehiscence (n = 2, 9.0%). In all animals, skin sutures could be removed on the tenth postoperative day due to the adequate sealing of surgical wound edges. 3.2. Histopathological Features of Tumors Histopathological evaluation of mammary neoplasms revealed malignancy in 86% of patients. Malignant tumors were classified as: carcinoma in situ (n = 11; 31.4%), carcinoma in mixed tumors (n = 11; 31.4%), squamous cell carcinoma (n = 4; 13.6%), papillary carcinoma (n = 3; 8.6%), tubular carcinoma (n = 2; 5.7%), anaplastic carcinoma (n = 2; 5.7%), solid carcinoma (n = 1; 2, 9%), and mammary osteosarcoma (n = 1; 2.9%). As for tumor malignancy grade, 70.6% of carcinomas were classified as grade one, 23.5% as grade two, and 5.9% as grade three. Two animals had metastases in inguinal lymph nodes at the time of surgery, which were diagnosed as grade three anaplastic carcinoma and grade two tubular carcinoma, respectively. Benign or non-cancerous neoplasms were classified as: benign mixed tumor (n = 10; 42.1%), atypical ductal hyperplasia (n = 7; 31.6%), adenoma (n = 5; 21.1%), and ductal papilloma (n = 1; 5.2%). 3.3. Clinical and Laboratory Parameters Table 2 and Table 3 respectively detail the clinical and laboratory outcomes in healthy and diseased female dogs at M0. Higher blood levels of glucose, lactate, fibrinogen, and IL-6 were found in patients with mammary tumours (p < 0.05). Figure 2 graphically represents the results of clinical parameters evaluated at different times in the perioperative period of female dogs with mammary neoplasia subjected to UM, associated or not with OH. In group 1 patients, HR was significantly lower at M1, M2, and M3 than at M0 (p = 0.0121). However, the values remained within the physiological limits for canine species. In group 1 and 2 patients, RR and SBP were significantly low at M1 (p < 0.05). Four animals (36.4%) in group 1 and nine (81.4%) in group 2 had bradypnea at the end of the surgery. Group 2 patients showed a higher reduction in RR compared to group 1 patients at M1 (p = 0.001). As for SBP, none of the patients assessed at M0 had hypotension. Animals from both groups showed hypothermia at M1, M2, and M3, which was more pronounced at the end of the surgery. The association of OH with UM had no influence on the intensity of body temperature reduction. Figure 3 graphically represents the blood levels of glucose, lactate, fibrinogen, albumin, and cortisol measured at different times during the perioperative period. Both group 1 and 2 patients had glycaemia increases significantly higher at M1, M2, M3, and M4 when compared to that at M0 (p < 0.05). Seven animals (63.6%) of group 1 and six (54.5%) of group 2 had hyperglycaemia after surgery. At M6, glycaemic levels returned to values similar to those recorded preoperatively. No changes were observed in blood lactate levels at all evaluation times for both groups (p > 0.05). However, a significant increase in plasma fibrinogen was recorded at M5 for female dogs under UM associated with OH (p = 0.0238), with hyperfibrinogenaemia being evidenced in six animals (54.5%). Serum albumin concentrations were significantly lower at M1, M3, and M4 compared to that at M0 in group 2 patients (p = 0.0100). At M2, four animals (36.4%) had serum levels below 2 g/dL. Group 1 patients showed reduced concentrations of albumin only at M1 when compared to M0 (p = 0.0300). Despite the lack of significant difference between groups 1 and 2, hypoalbuminemia was evidenced at all times during the postoperative period for animals subjected to UM associated with OH. Although an increase in serum cortisol concentrations was observed at M1, M2, and M3 in group 2 patients, a significant difference was found only at M1 compared to M0 (p = 0.0492). Figure 4 graphically represents the serum levels of IL-2, IL-6, and TNF-a of female dogs with mammary neoplasms at different times during the perioperative period. Serum concentrations of IL-2 significantly decreased at M5 for group 1 patients (p = 0.0465), and at M3, M4, and M5 for group 2 (p = 0.0187). Serum concentrations of IL-6 increased significantly at M3 for group 1 patients (p = 0.0035), and at M2 and M3 for group 2 patients (p = 0.0035). At M4, they returned to preoperative levels in both groups. As for TNF-a blood levels, no significant changes were observed at all evaluation times for groups 1 (p = 0.1105) and 2 (p = 0.4555). Furthermore, no correlations were observed between the evaluated parameters and clinical stage of tumors, or even the duration of surgeries in patients with mammary neoplasia. We found a positive correlation between blood levels of fibrinogen and serum concentrations of IL-6 (r = 0.256; p < 0.05), but a negative correlation between serum concentrations of albumin and IL-6 (r = -0.298; p < 0.01) in patients undergoing UM and OH jointly. Positive correlations were also observed among serum concentrations of IL-6, IL-2, and TNF-a in group 1 and group 2 patients . 4. Discussion Surgical resection is the therapy of choice for mammary tumors, as it provides the greatest chance of cure . However, studies have suggested that surgical trauma can facilitate the progression of minimal residual disease and development of metastases, mainly due to the increased production of angiogenic growth factors and suppression of cellular immunity . Several studies in human medicine have investigated the occurrence and intensity of neuroendocrine and immunological changes in the perioperative period of patients undergoing oncological surgeries . However, in veterinary medicine, most studies have been limited to cortisol changes in dogs subjected to ovariohysterectomy, laparoscopic, or orthopaedic surgeries . In this sense, our study enables for understanding the metabolic response to trauma induced via unilateral mastectomy in female dogs with mammary neoplasia, evaluating neuroendocrine and inflammatory parameters. Higher blood glucose and lactate concentrations were found in females with mammary neoplasia than in healthy females. Increased blood glucose often results from hepatic neoglucogenesis due to high glucose uptake by tumor cells, producing lactate, even under adequate tissue perfusion. Recent studies have hypothesized that lactate may act as a signaling molecule for tumor angiogenesis, thus facilitating the progression of neoplasms . Inflammation has been correlated with tumor invasion and poor prognosis in women with mammary neoplasia. Besides acting in the immune and inflammatory responses, cytokines play major roles in tumor initiation, growth, and metastasis . In our study, female dogs with mammary cancer had higher serum levels of IL-6 than healthy ones. In this context, Ravishankaran and Karunanithi (2011) observed a correlation between IL-6 concentrations and tumor staging in women with breast carcinoma, with higher levels in patients with detectable metastases. Conversely, we found no correlation between serum IL-6 concentrations and tumor staging. Plasma fibrinogen concentrations were also higher in females with mammary neoplasia. It may be because IL-6 stimulates protein production in the acute phase . We observed hyperfibrinogenaemia in 22.7% of patients with mammary cancer in the preoperative period. Studies on men and women diagnosed with solid tumors have revealed that preoperative hyperfibrinogenaemia is associated with tumor progression, thus having a negative correlation with survival time . Andreasen et al. (2012) found that animals with detectable metastases had significantly higher plasma concentrations of fibrinogen. This could not be verified in our study due to the small number of patients with metastases in lymph nodes and the exclusion of females with distant metastases. The intensity of metabolic responses to surgical trauma can be influenced by different factors, including the extent of the surgery, surgical technique used, surgery length, and anesthetic protocol . UM is considered a highly invasive surgery due to incision and tissue resection extents, thus inducing moderate to severe pain . According to Krikri et al. (2013) , the intensity of abdominal surgery-related trauma is determined by the size of incisions in the skin, subcutaneous tissue, aponeuroses, and parietal peritoneum, in addition to the handling of viscera in the abdominal cavity. In OH, ovarian pedicle traction stands for the moment of greatest nociceptive stimulus . Due to the greater tissue trauma and nociceptive stimulus induced by performing UM and OH concomitantly, our goal was to compare MRT parameters of female dogs undergoing UM and those under UM associated with OH. The mean time of UM associated with OH was significantly longer than that of UM alone; however, no correlation was observed between surgical time and MRT. MRT begins preoperatively when the sympathetic nervous system (SNS) is stimulated by fear and anxiety associated with fasting, hospital stay, and handling for preanesthetic medication. Moreover, anesthetic induction and orotracheal intubation also stimulate release of catecholamines . In the hypodynamic phase, which begins immediately after tissue trauma, hypotension, hypothermia, and tachycardia may occur as a result of the reduced circulating blood volume . We noted reductions in blood pressure, bradypnea, and hypothermia at the end of both surgical procedures, but such changes might be related to the anaesthetic protocol. Acepromazine, which is used in preanesthetic medication (PAM), produces relevant effects on the cardiovascular system due to myocardial depression and the blockade of peripheral a-1 adrenergic receptors . Volatile anesthetics cause a dose-dependent reduction in blood pressure, which is related to a drop in systolic volume or peripheral vascular resistance . In dogs anesthetized with isoflurane, acepromazine tends to reduce mean arterial pressure by 24%, lasting between two and three hours. It, thus, justifies the drop in systolic blood pressure at the end of the surgery in both groups evaluated. Heart rate is not significantly altered after PAM with acepromazine and inhalational anesthesia with isoflurane , as we observed in group 2 patients. Propofol is a sedative and hypnotic used for anaesthetic induction, with a rapid onset of action that lasts only 20 min. It may transiently reduce blood pressure and myocardial contractility, in addition to inducing apnoea . Therefore, the effects observed at M1 for both groups may not be a consequence of its use, since its action is shorter than surgery lengths. Significant reduction in RR at the end of the surgery in both groups may be due to administration of morphine as a preanesthetic medication, continuous infusion fentanyl in the transoperative period, and isoflurane for anesthesia maintenance. Morphine is an opioid analgesic with a high affinity for m receptors, which has as main effects: sedation, analgesia, and respiratory depression. Fentanyl is a m-agonist opioid analgesic with high potency, but it can cause bradycardia and respiratory depression . Inhalation anesthesia with isoflurane causes respiratory depression associated with central (medullary depression) and peripheral (intercostal muscle dysfunction) mechanisms, in addition to a depression of the physiological ventilatory response to hypoventilation . We noticed that RR decrease was more severe in females under UM and OH in association, which might have been due to the greater respiratory depression induced by a longer exposure to isoflurane . Body temperature reduction in both groups after surgery may be associated with loss of heat via convection due to wide trichotomy, evaporation via humidifying solutions on the body surface during antisepsis, exposure of tissues and body cavities in the intraoperative period, and hypometabolism associated with the hypodynamic phase of trauma. Moreover, hypothermia may have resulted from an impaired thermoregulation due to the use of acepromazine in preanesthetic medication and trans- and post-operative opioid analgesia, in addition to a low heat production associated with the depression of metabolism via inhalational anesthesia . Mild-to-moderate hypothermia causes changes in hemodynamics, ventilation, and oxygenation. However, severe cases can lead to metabolic acidosis, arrhythmias, electrolyte disturbances, blood hyperviscosity, hyperglycaemia, and resistance to insulin action . Despite the use of a thermal blanket during surgery, seven patients (31.8%) had body temperatures below 35.5 degC at the end of UM, associated or not with OH, requiring additional artificial heating. In 63.6% of patients, it returned to a physiological standard six hours after surgeries, normalizing in all patients twenty-four hours after surgeries. Therefore, maintaining normothermia is essential to reducing the extent of MRT . In the hyperdynamic phase of trauma, there is an intense mobilization of energy and protein substrates with tachycardia, tachypnoea, and fever secondary to activation of the SNS, as well as an increased secretion of pituitary hormones and production of proinflammatory cytokines . In our study, plasma lactate concentrations did not increase at any evaluation times, possibly due to adequate tissue perfusion during the trans- and post-operative periods ; however, they remained above the reference range for dogs at M2, M3, M4, M5, and M6 in group 1 patients, and at M2, M3, and M4 in group 2 patients. Such an outcome may be a result of the greater energy demand in the hyperdynamic phase of MRT, resulting in anaerobic glycolysis . Floriano et al. (2010) reported that lactate production may decrease in the intraoperative and immediate postoperative periods due to a reduction in the peripheral vascular resistance induced via isoflurane, with consequent oxygen delivery. Although some studies have reported that high lactate levels in surgical patients indicate higher postoperative morbidity and mortality , others have shown that this substrate is a crucial source of energy, in addition to helping in the tissue repair process by stimulating angiogenesis and collagen deposition . Increased serum concentrations of catabolic hormones after trauma, including cortisol, glucagon, and catecholamines, induce hepatic gluconeogenesis and resistance to the peripheral action of insulin, with consequent hyperglycemia , of which the intensity reflects the severity of injuries . In our study, glycemia increased at M1, M2, M3, and M4 in patients undergoing UM, with or without OH; overall, at the end of the surgery, hyperglycemia was recorded in 63.6% of group 1 patients and 54.5% of group 2 patients. A proper glycemic control in the perioperative period is known to reduce mortality rates and postoperative infection . Serum cortisol levels have been widely used to assess pain or postoperative stress in humans and animals . Our study showed increased serum cortisol levels after UM associated with OH, but not in patients who underwent UM exclusively. Fox et al. (1994) reported that increased cortisol in the perioperative period is related to the severity of surgical trauma and is more pronounced in abdominal and thoracic surgeries than in surgical procedures on the body surface. Moreover, a neuroendocrine response to surgical trauma is mediated by nervous impulses from the traumatized area, which can be minimized via locoregional anesthesia or total intravenous anesthesia with opioid analgesia . Inhalational anesthesia does not block the neuroendocrine response to surgical trauma . However, in our study, the administration of morphine in preanesthetic medication, associated with continuous infusion of fentanyl during surgery, may have reduced it in the early postoperative period . Moreover, serum cortisol decreases associated with an absence of tachycardia and tachypnoea in the following evaluations suggest an adequate nociceptive control, although patients' behavior was not assessed. In this regard, Teixeira et al. (2013) pointed out that tramadol administration at 3 mg/kg every eight hours in the postoperative of female dogs under UM, as performed in our study, can control pain properly. Zografos et al. (2009) found increased blood glucose and serum concentrations of cortisol and growth hormone in women undergoing excisional biopsy of a mammary tumor under local anaesthesia, both during surgery and at the end of the surgical procedure. In another study with women undergoing mastectomy, serum glucose and cortisol levels four hours after the end of the surgery were higher in patients receiving opioid analgesia than in those subjected to paravertebral anaesthetic block . Still, Horta et al. (2015) did not observe neuroendocrine changes before anaesthetic induction or after orotracheal intubation in dogs undergoing regional mastectomy and UM; however, two hours after the end of the surgeries, there were significant increases in blood glucose and serum cortisol concentrations in patients undergoing UM . Alterations in serum levels of cytokines and acute-phase proteins reflect the presence and intensity of an inflammatory process after trauma, helping the prognosis of patients undergoing different surgical interventions . Under physiological conditions, concentrations of cytokines are extremely low and may even be undetectable. Changes in plasma cytokine levels may occur within two to four hours after surgery, while in the production of acute-phase proteins these changes occur after twelve to twenty-four hours and may persist for several days, depending on the extent of the trauma . Therefore, we observed an increase in the plasma concentration of fibrinogen three days after surgical trauma in females of group 2, possibly due to a greater inflammatory response associated with trauma induced via OH in association with UM. Fibrinogen is not routinely evaluated as an acute-phase protein in dogs and cats due to its late and low-intensity response, as observed in our study . However, in cancer patients, hyperfibrinogenemia may accompany blood hypercoagulability and progression of the neoplastic disease, shortening survival time for patients . We found a reduction in serum albumin levels after surgery in both groups; however, patients under UM associated with OH had hypoalbuminemia throughout the postoperative period. Reductions in circulating levels of albumin in surgical patients may be due to low hepatic production induced by the acute-phase reaction, blood dilution secondary to intraoperative intravenous fluid therapy, and loss of proteins to the interstitial space . Hypoalbuminemia has been considered a risk factor in surgical patients, following a poor prognosis; the main postoperative complications associated with it include oedema, delayed surgical wound healing, and infection . In our study, plasma fibrinogen concentrations and serum IL-6 levels displayed a positive correlation, while serum albumin levels and serum IL-6 levels exhibited a negative correlation; therefore, an acute-phase reaction occurred in the postoperative period of UM, with or without OH. IL-6 is the main mediator of an inflammatory response secondary to surgical trauma, and its serum concentrations are related to the extent of the tissue injured and postoperative complications . For these reasons, several studies have been aimed at evaluating the serum levels of IL-6 and acute-phase proteins in human patients undergoing oncological surgeries . Here, we evidenced an increase in serum IL-6 levels at M2 and M3 in group 2 patients and at M2 in group 1 patients, with no difference between groups. Okamura et al. (2015) described a positive correlation between serum IL-6 levels and systemic inflammatory response syndrome (SIRS) duration in the postoperative period of patients with esophageal tumors undergoing esophagectomy. In turn, IL-2 stimulates the production of T lymphocytes and immunoglobulins, acting on antitumour immunity . Therefore, the reduction in IL-2 circulating levels observed at M5 in patients under UM, and at M4 and M5 for dogs undergoing UM and OH, can lead to the suppression of cellular immunity, facilitating tumour recurrences and metastases . The main cause of reduced serum IL-2 levels after trauma is an increase in prostaglandin E2 concentrations . In our study, serum levels of TNF-a did not change at the different times of the postoperative period in both evaluated groups, possibly due to its half-life being less than 20 min . Horta et al. (2015) showed that postoperative complications are common in female dogs undergoing mastectomy and more often after radical surgical techniques. We found hematomas in 50% of animals, with pelvic limb oedema, seroma, subcutaneous emphysema, and partial suture dehiscence at lower incidences. Hematomas result from transoperative injuries to the complex vascular system of the mammary glands, while pelvic limb oedema may be related to lymphatic drainage damage after surgical removal of the inguinal lymph nodes. In turn, subcutaneous emphysema and seroma formation stem from the dead space formed after resection of the mammary chain. Finally, suture dehiscence occurs in the presence of seroma, infection, and ischemic necrosis at the edges of the surgical wound . We observed a low incidence of seroma and subcutaneous emphysema, which can be attributed to the reduction in the dead space achieved through the use of movable sutures during the transoperative period and application of a compressive bandage in the first three postoperative days. Vitug and Newman (2007) indicated that the main postoperative complications associated with mastectomy in women include: seroma formation, hematomas, surgical wound infection, chronic incisional pain, and venous thromboembolism, with surgeries considered of low morbidity, as observed in our study. Different studies have compared the benefits of radical surgical procedures for the removal of mammary neoplasms with the conservative excision of tumours . Researchers have concluded that radical surgical techniques do not promote longer survival times than conservative techniques since tumour excision promotes surgical margins free of neoplastic cells . Evaluating 99 female dogs with isolated mammary tumors, Stratmann et al. (2008) showed that 58% of animals undergoing regional mastectomy developed a new tumor in the remaining ipsilateral mammary gland, requiring a new surgical intervention. These authors emphasized that UM enables removing macroscopically visible lesions and occult tumors, reducing the risk of new lesions due to the resection of all mammary tissue in the chain affected by the neoplasm. However, Horta et al. (2015) reported that, although unilateral mastectomy can prevent the development of new mammary tumors, it should not be performed prophylactically, as it is an invasive surgical technique that results in surgical stress and postoperative complications. Although some studies have revealed that performing OH concomitantly with mammary tumor resection can bring benefits to some females with mammary neoplasms , most traumatic surgical interventions may lead to aggravated organic responses in the postoperative period. We found that unilateral mastectomy induces significant metabolic changes in bitches with mammary neoplasia, and these changes are intensified with concomitant ovariohysterectomy. Thus, additional studies on the modulation of the MRT in female dogs with mammary neoplasia are needed, aiming to minimize the incidence of postoperative complications, such as tumor recurrences and metastases associated with postoperative immunosuppression. 5. Conclusions An analysis of the outcomes allows us to conclude that female dogs with mammary neoplasia have metabolic changes in the preoperative period possible due to tumor development and progression, including increased blood concentrations of lactate, glucose, fibrinogen, and interleukin-6. We may also conclude that, even with general inhalational anesthesia associated with perioperative opioid-based analgesia, unilateral total mastectomy induces significant metabolic alterations in female dogs with mammary neoplasia, which may be enhanced when in concomitance with ovariohysterectomy. Thus, this fact must be included among the numerous criteria considered in decision making for surgical intervention. Acknowledgments We thank Sao Paulo Research Foundation (FAPESP) for supporting this study. Author Contributions Conceptualization, S.M.R. and A.B.d.N.; methodology, S.M.R., T.F. and A.B.d.N.; investigation, S.M.R., F.N.d.P., B.F.F., T.F., C.B.M. and A.B.d.N.; writing--original draft preparation, S.M.R., F.N.d.P., B.F.F. and A.B.d.N.; writing--review and editing, S.M.R., F.N.d.P., B.F.F., T.F., C.B.M. and A.B.d.N. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study was approved by the Institutional Ethics Committee on Animal Use (CEUA) of the Evangelical College of Parana (protocol ndeg: 001333/2013), approved on 15 March 2016. All owners signed an informed consent allowing the use of the obtained samples in research. Informed Consent Statement All owners signed an informed consent allowing the use of the obtained samples in research. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Graphical representation of the clinical staging (stages I, II, III or IV) of mammary neoplasms diagnosed in female dogs subjected to unilateral mastectomy (Group 1, n = 11) or unilateral mastectomy and ovariohysterectomy (Group 2, n = 11). Paulista State Universisty/Jaboticabal (2016). Figure 2 Graphic representation of means (heart rate and systolic blood pressure) and medians (respiratory rate and temperature) of clinical parameters of female dogs with mammary neoplasia subjected to unilateral mastectomy (group 1; n = 11) or unilateral mastectomy and ovariohysterectomy (group 2; n = 11), evaluated at different times of the perioperative period. G1: group 1. G2: group 2. *: significantly different in relation to M0 (group 1). deg: significantly different in relation to M0 (group 2). #: significant difference between groups 1 and 2 (p = 0.001). Paulista State Universisty/Jaboticabal (2016), Brazil. Figure 3 Graphical representation of mean (lactate and fibrinogen) and median (glucose, albumin, and cortisol) values of laboratory parameters of canine females with mammary neoplasia subjected to unilateral mastectomy (group 1; n = 11) or unilateral mastectomy and ovariohysterectomy (group 2); n = 11) at different times in the perioperative period. G1: group 1. G2: group 2. *: significantly different in relation to M0 (group 1). deg: significantly different in relation to M0 (group 2). Paulista State Universisty/Jaboticabal (2016), Brazil. Figure 4 Graphical representation of median values of serum concentrations of IL-2, IL-6, and TNF-a in canine females with mammary neoplasia subjected to unilateral mastectomy (group 1; n = 11) or unilateral mastectomy and ovariohysterectomy (group 2; n =11) at different times in the perioperative period. G1: group 1. G2: group 2. *: significantly different in relation to M0 (group 1). deg: significantly different in relation to M0 (group 2). Paulista State Universisty/Jaboticabal (2016), Brazil. Figure 5 Graphical representation of the Spearman correlation between serum concentrations of IL-6 and IL-2, IL-6, and TNF-a in canine females with mammary neoplasia subjected to unilateral mastectomy (group 1; n = 11) or unilateral mastectomy and ovaryhysterectomy (group 2; n = 11) at different times in the perioperative period. Paulista State Universisty/Jaboticabal (2016), Brazil. Figure 6 Graphical representation of the Spearman correlation between serum IL-2 and TNF-a concentrations in canine females with mammary neoplasms subjected to unilateral mastectomy (group 1; n = 11) or unilateral mastectomy and ovariohysterectomy (group 2; n = 11) at different times in the perioperative period. Paulista State Universisty/Jaboticabal (2016), Brazil. animals-13-00926-t001_Table 1 Table 1 Clinical stage of mammary neoplasms in dogs. Stage Tumor Size Lymph Nodes Distant Metastases I T1: <3 cm N0 M0 II T2: 3-5 cm N0 M0 III T3: >5 cm N0 M0 IV Any T N1 M0 V Any T N0 or N1 M1 animals-13-00926-t002_Table 2 Table 2 Clinical parameters [mean +- standard deviation or median (minimum-maximum)] of healthy females (control group) and females with mammary neoplasia evaluated at M0. Paulista State Universisty/Jaboticabal (2016), Brazil. Parameter Control Group (n = 10) Mammary Neoplasm Group (n = 22) p-Value Normal Values (Adult Dog) HR (bpm) 132 +- 27.51 121 +- 23.15 p = 0.2301 60-120 RR (mpm) 43 (24-80) 32 (12-90) p = 0.1652 18-36 SBP (mmHg) 148 +- 32.59 158 +- 26.80 p = 0.3917 80-120 T (degC) 148 +- 32.59 38.7 (38.1-39.7) p = 0.4258 37.5-39.2 HR: heart rate; RR: respiratory rate; SBP: systolic blood pressure; T: body temperature. animals-13-00926-t003_Table 3 Table 3 Laboratory parameters [mean +- standard deviation or median (minimum-maximum)] of healthy females (control group) and females with mammary neoplasia evaluated at M0. Paulista State Universisty/Jaboticabal (2016), Brazil. Parameter Control Group (n = 10) Mammary Neoplasm Group (n = 22) p-Value Normal Values (Adult Dog) Glucose (mg/dL) 85 (76-98) 98 (73-204) b p = 0.0483 65-118 Lactate (mmol/L) 2.18 +- 0.56 3.3 +- 0.94 a p = 0.0017 1.20-3.10 Albumin (g/dL) 2.95 (2.50-3.60) 2.93 (1.51-3.66) p = 0.7565 2.6-3.3 Fibrinogen (mg/dL) 0.2 (0.2-0.2) 0.3 (0.2-1.0) b p = 0.0109 2.0-4.0 Cortisol (ng/mL) 4.00 (1.08-79.11) 8.08 (2.97-57.13) - X CortisolLog 0.59 (0.03-1.90) 0.90 (0.47-1.75) p = 0.4385 X IL-2 (pg/mL) 1.15 (1.02-759) 22.64 (0.40-24.134) - X IL-2Log 0.06 (0.01-2.88) 1.35 (0.01-4.38) p = 0.0996 X IL-6 (pg/mL) 4.06 (1.18-469) 63.25 (3.13-20.182) - X IL-6Log 0.60 (0.07-2.67) 1.80 (0.58-4.30) b p = 0.0416 X TNF-a (pg/mL) 2.43 (1.06-216) 4.85 (1.19-1.900) - X TNF-aLog 0.38 (0.02-2.33) 0.68 (0.07-3.23) p = 0.1725 X a significant difference (p < 0.05) in relation to the control group using Student's t test; b significant difference (p < 0.05) in relation to the control group using the Mann-Whitney test. 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PMC10000080

This experiment investigated the effects of lactic acid bacteria and cellulase on the fermentation quality, in vitro digestibility, and aerobic stability of Flammulina velutipes spent mushroom substrate silage (F-silage) and Pleurotus eryngii spent mushroom substrate silage (P-silage). Silage treatments included groups without any additives (control), with lactic acid bacteria (L), with cellulase (E), and with lactic acid bacteria and cellulase (M). Data analysis was performed using independent sample t-test and analysis of variance. After 45 days of ensiling, the pH in F-silage and P-silage from the L, E, and M groups were lower than those in the control group (p < 0.05). The pH, acetic acid (AA), and propionic acid (PA) levels in P-silage were lower than those in F-silage, and the LA content in P-silage was higher than that in F-silage (p < 0.05). Compared with the control, the E treatment increased in vitro neutral detergent fibre digestibility (IVNDFD) and in vitro acid detergent fibre digestibility (IVADFD) in F-silage and P-silage (p < 0.05). The aerobic stability of F-silage inoculated with L increased (p < 0.05) by 24 h compared to the control. The aerobic stability of P-silage inoculated with M increased (p < 0.05) by 6 h compared to the control. The improvement in fermentation quality and aerobic stability is extremely large in terms of applying M in F-silage and P-silage. The E is effective in improving the in vitro digestibility of P-silage. The research results provide a theoretical basis for the production of high-quality spent mushroom substrate fermented feed. additives aerobic stability fermentation quality in vitro digestibility spent mushroom substrate Jilin Scientific and Technological Development Program of China20210101375JC Sanya Yazhouwan Science and Technology City AdministrationSYNO-2021-13 This research was funded by the Jilin Scientific and Technological Development Program of China, Grant/Award Number: No.20210101375JC. Sanya Yazhouwan Science and Technology City Administration, Grant/Award Number: No. SYNO-2021-13. pmc1. Introduction With the increase in feed prices, the beef cattle-breeding industry is facing great production cost pressures. Rising feed costs also herald a large emphasis on underutilized and inexpensive agricultural byproducts. Agricultural byproducts can be composted to form natural organic fertilizers or used as soil conditioners to improve soil structure, but the utilization rate of agricultural byproducts is not high. The optimal process is to prepare agricultural byproducts into animal feed, thereby enhancing the sustainability of feed resources and alleviating the shortage of roughage resources in production . Spent mushroom substrate (SMS), which usually uses agricultural byproducts, including corncob, wheat bran, rice straw, and residues, such as raw materials for edible fungi cultivation, is the base residue of edible fungi culture medium after harvesting . Edible fungi not only absorb nutrients in the culture medium to maintain their growth but also degrade certain fibrous substances in the culture medium through cellulose complex enzymes and peroxidase. There are many edible fungal mycelial residues in SMS that are complexes composed of amino acids, organic acids, and other nutrients produced by the enzymatic conversion of cellulose and biologically active substances . The agricultural byproducts of high fibre crops, such as straw, corncob, cottonseed shell, sawdust, and bran, can provide more abundant feed sources and large amounts of energy for ruminants. For example, most SMS raw materials are agricultural byproducts of high fibre crops, which have low nutritional value, poor palatability, low digestibility and absorption, and low feeding value. However, the nutritional value and palatability of SMS raw materials can be improved through microbial fermentation, thus improving the production performance of ruminants. Approximately 39.34 million tons of edible fungi were produced in China in 2019, accounting for more than 85% of the global total (China Edible Fungi Association 2020). Under normal conditions, 1 kg of edible fungi will produce 5 kg of SMS . According to this ratio, 197 million tons of fresh SMS could be produced in China in 2019. In China, the production of edible fungi is mainly divided into two production modes: family small-scale and factory standardization. Due to factors such as scattered production areas, complex culture medium composition, and unstable yield and quality, most of the SMS produced by family small-scale are treated by in situ disposal or nonfuel incineration, which has caused serious environmental pollution problems. Although the SMS produced by the factory standardization has stable nutrients and high feed value, it is mainly used in power plants to generate electricity, and only a small amount is used for beef cattle fattening. Therefore, to protect the environment and promote the sustainable development of recycling-based animal husbandry, it is necessary to develop effective SMS feed preparation and storage technology. The moisture content in SMS is generally between 50% and 75%, which is not easy to store. Ensiling is an important roughage processing method that can not only improve the palatability of feed but also prolong the storage time of high-water feed and maintain the original nutrient level . Biotransformation in the process of ensiling can delay the spoilage of SMS and can convert it into animal feed resources with high economic benefits. Lactic acid bacteria preparation is effective in improving the fermentation quality of cassava residue silage . During ensiling, the metabolites produced by lactic acid bacteria can inhibit the growth of Clostridium, moulds, and other harmful bacteria. The water-soluble carbohydrate content in SMS is low. Therefore, cellulase can be used to promote the growth of lactic acid bacteria. Cellulase promotes the decomposition of the plant cell wall, degrades plant fibres into monosaccharides, and provides a sufficient fermentation substrate for lactic acid bacteria growth . At present, due to the different physicochemical properties of various agricultural byproducts, there is no unified conclusion on the effects of using microbial additives in agricultural byproducts . According to the previous research results of soybean residue corn straw mixed silage , we speculate that the combined application of lactic acid bacteria and cellulase can increase the lactic acid content of SMS silage, delay the deterioration of SMS silage, and further improve the fermentation quality, in vitro digestibility, and aerobic stability of SMS silage. Therefore, to provide a theoretical basis for the production of high-quality SMS fermented feed, the effects of lactic acid bacteria and cellulase on the fermentation quality, in vitro digestibility, and aerobic stability in the industrially produced SMS silage were investigated in this experiment. 2. Materials and Methods 2.1. Silage Production and Treatments The experiment was conducted at the Animal Nutrition and Feed Science Experimental Base of Jilin University, Changchun City, Jilin Province, China (43.88deg N, 125.35deg E, above sea level 300 m). The Flammulina velutipes spent mushroom substrate (F-SMS) and Pleurotus eryngii spent mushroom substrate (P-SMS) used in this experiment were the base residue of edible fungi culture medium after harvesting. The raw material composition of the two edible fungal cultivation media is shown in Table 1. The experimental treatments included (1) no additive (control); (2) lactic acid bacteria (L, strain number of the L. plantarum: Lactobacillus plantarum LP1, provided by SNOW BRAND SEED Co., Ltd., Chikusou-1) applied at 50 mg kg-1 FW, and the actual number of lactic acid bacteria was 4.7 x 107 colony forming units (CFUs) g-1 FW; (3) cellulase (E, utilizing Trichoderma viride and Acremonium cellulolyticus Y-94) applied at 50 mg kg-1 FW, and the actual enzymatic activity was 4.2 x 10-3 U g-1 FW; and (4) a mixed preparation of lactic acid bacteria and cellulase (M). The amount of lactic acid bacteria and cellulase added was 50 mg kg-1 FW. The actual number of lactic acid bacteria was 4.7 x 107 CFUs g-1 FW, and the actual enzymatic activity was 4.2 x 10-3 U g-1 FW. After the fresh SMS was treated with additives, it was placed into a 5 L polyethene plastic bucket with an inner lid (five 5-L-polyethylene-plastic-bucket drums per treatment). The packing densities (mean +- standard deviation) of F-SMS and P-SMS were 550.1 +- 10.0 kg/m3 FW and 575.0 +- 9.2 kg/m3 FW, respectively. The fermentation barrels were sealed, weighed, and stored at ambient temperatures (21-25 degC) for 45 days to prepare F. velutipes spent mushroom substrate silage (F-silage) and P. eryngii spent mushroom substrate silage (P-silage). Five replicates per treatment were used for the in vitro digestibility analysis. Three replicates were used for the fermentation quality analysis and the other replicates were mixed and used for the aerobic stability test. 2.2. Chemical Composition Analysis The silage samples were dried in a forced-air oven at 60 degC for 72 h and then ground and sieved through a 1 mm sieve for chemical analysis. Dry matter (DM), organic matter (OM), ether extract (EE), ash, crude fibre (CF), and crude protein (CP) contents were determined using the method from the Association of Official Analytical Chemists . Neutral detergent fibre (NDF), acid detergent fibre (ADF), and acid detergent lignin (ADL) contents were determined using the procedure reported by Van Soest et al.--the NDF and ADF were determined sequentially using neutral detergent and acid detergent, respectively . Buffering capacity (BC) was measured by the method of Playne and McDonald . Calcium (Ca) and total phosphorus (P) were determined by the potassium permanganate method and the ammonium molybdate spectrophotometric method, respectively . Water-soluble carbohydrate (WSC) was determined by the sulfuric acid-anthrone colorimetric method . Gross energy (GE) was determined using an oxygen bomb calorimeter . The digestible energy (DE), metabolizable energy (ME), net energy for maintenance (NEm), net energy for weight gain (NEg), and net energy for lactating cows (NEl) were calculated according to the method of Gao et al. . The following formula were used for the calculations. DE = GE x [70.19 - 1.364 x (ADF - 29.83) + 0.104 x CP + 0.149 x EE + 0.022 x NDF - 0.244 x ash]/100(1) ME = DE x [86.38 - (9.9 x CF + 19.6 x CP)/(100 - ash)]/100(2) NEm = ME x (0.287 x ME/GE + 0.554)(3) NEg = ME x (0.78 x ME/GE + 0.006)(4) NEl = ME x [0.6 + 0.24 x (ME/GE - 0.57)](5) Notes: (1) The units of GE, DE, ME, NEm, NEg, and NEl are MJ/kg DM. (2) The unit of CF, CP, EE, NDF, ADF, and ash is g/kg of DM. Formula (1) was derived from 347 experimental data in different countries. The CP content in the feed was 90-350 g/kg, with an average content of 175 g/kg. The CF content in the feed was 150-430 g/kg, with an average content of 256 g/kg. In addition, Formulas (2)-(5) were derived based on Formula (1). 2.3. Fermentation Quality and Microbiological Analyses A total of 20 g of sample was diluted with 180 mL of distilled water; then, the solution was stirred until homogenous and stored in the refrigerator at 4 degC for 24 h. Thereafter, the mixture was filtered through quantitative filter paper . The pH was determined by a pH meter (PHSJ-4F, Yidian Scientific Instrument Co., Ltd., Shanghai, China). Ammonia nitrogen (AN) was determined by steam distillation . Lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA) were determined by high-performance liquid chromatography (Column: Shodex RS Pak KC-811, Showa Denko KK, Kawasaki, Japan; Detector: DAD, 210 nm, SPD-20A, Shimadzu Co., Ltd., Kyoto, Japan; mobile phase: 3 mmol/L HClO4; flow rate: 1 mL/min; detection wavelength: 210 nm; temperature: 50 degC; injection volume: 10 mL). The fresh sample (20 g) was evenly mixed with 180 mL of sterile physiological saline (0.85% NaCl), shaken for 30 min on a shaker, and then sequentially diluted in sterile physiological saline according to a 10-1-10-6 gradient. The three best dilutions (10-3-10-5) for determining the bacterial colony count were selected. Finally, 100 ml dilutions of different concentrations were spread evenly on the agar medium described below. Lactic acid bacteria were counted on De Man, Rogosa, and Sharp agar medium (Budweiser Technology Co., Ltd., Shanghai, China) and incubated at 37 degC for 48 h under anaerobic conditions. Aerobic bacteria were counted on nutrient agar medium (Hope Biotechnology Co., Ltd., Qingdao, China) and incubated at 37 degC for 48 h. Yeast and moulds were counted on potato dextrose agar medium (Budweiser Technology Co., Ltd., Shanghai, China) and incubated at 28 degC for 48 h. The number of microorganisms was counted on plates yielding 20-200 CFUs. All microbiological data were converted to log10 CFUs g-1, and the results are reported as fresh weight . 2.4. Aerobic Stability Analysis Aerobic stability was determined based on the time required for the silage temperature to be 2 degC higher than the ambient temperature . The 1 kg sample participating in the aerobic stability test was put into a clean plastic bucket with a volume of 1 L, and the fermented silage was exposed to aerobic exposure at 23.5 degC +- 1 degC for 14 days. A thermometer was placed in the centre of the silage, and the temperature was recorded every 6 h. 2.5. In Vitro Degradability Analysis This study strictly followed the Chinese Laboratory Animal Welfare Ethical Review Guidelines and was approved by the Laboratory Animal Welfare Ethics Committee of Jilin University (Number of permit: SY202009600). A 0.5 g sample was taken and put into a filter bag (ANKOM F57; aperture 25 um; ANKOM Technology Corporation; Macedon, NY, USA), which was heat-sealed and placed in a 130 mL serum bottle. In the presence of CO2, rumen fluid (collected from four sheep fed alfalfa and corn silage) was filtered through four layers of cheesecloth and mixed in equal volumes. The mixed filtrate was mixed with McDougall's artificial saliva at a ratio of 1:4 (v/v). A 60 mL mixture was placed into a 130 mL serum bottle, rinsed with CO2, and incubated at 39 degC for 72 h in a water bath. After 72 h of incubation, the filter bag was removed from the serum bottles and rinsed with cold distilled water. The filter bag was dried in a forced air drying oven at 100 degC for 24 h, and the residue was weighed to determine the in vitro dry matter digestibility (IVDMD) . In vitro neutral detergent fibre digestibility (IVNDFD) and in vitro acid detergent fibre digestibility (IVADFD) were determined, respectively, by analysing the content of residual NDF and ADF in the residue . Hemicellulose (HC) = NDF-ADF (the NDF and ADF run sequentially on the same original sample). IVDMD, IVNDFD, IVADFD, and in vitro hemicellulose digestibility (IVHCD) are calculated as follows: (respective weights of DM, NDF, ADF and HC before digestion--respective weights of DM, NDF, ADF and HC remaining)/(respective weights of DM, NDF, ADF and HC before digestion). 2.6. Statistical Analyses All data were analysed by SPSS statistical software (version 26; International Business Machines Corporation; Armonk, NY, USA). An independent sample t-test was used to analyse the significant differences between F-silage and P-silage for each item. One-way analysis of variance (ANOVA) was used to examine the significant differences between the mean values of the fermentation quality, chemical composition, energy, aerobic stability, and in vitro digestibility of the silage. Based on the results from the significance test, Tukey's method was used for multiple comparisons. Lactic acid bacteria (L) and cellulase (E) were used as fixed factors, and a two-way analysis of variance was used to evaluate the main effects of each factor and interactions on fermentation quality, chemical composition, energy, and in vitro digestibility. 3. Results The chemical composition, BC, microbial population, and energy of F-SMS and P-SMS before ensiling are shown in Table 2. The DM content of P-SMS was 12 g kg-1 FW greater than that of F-SMS and the WSC content was 1.4 times higher than that of F-SMS. However, the BC value of F-SMS was 1.8 times higher than that of P-SMS. The epiphytic numbers of lactic acid bacteria in F-SMS and P-SMS were 4.68 log10 CFUs g-1 FW and 4.30 log10 CFUs g-1 FW, respectively. Except for NDF and Ca, there were significant differences between F-SMS and P-SMS in terms of the other indicators (p = 0.000-0.001). 3.1. Fermentation Quality of Spent Mushroom Substrate Silage The fermentation quality of F-silage and P-silage is shown in Table 3. The pH, AA, and PA in P-silage were lower than those in F-silage (p = 0.000-0.041), and the LA content was higher than that in F-silage (p = 0.000-0.020). The L and E treatments influenced pH in F-silage (p = 0.002-0.005). The L treatment and the interaction (L x E) influenced LA content in F-silage (p = 0.002) and pH in P-silage (p = 0.000-0.001). The E treatment influenced pH in P-silage (p = 0.000). The L treatment influenced ammonia nitrogen/total nitrogen (AN/TN) ratio in P-silage (p = 0.008). The pH in F-silage and P-silage from the L, E, and M groups was lower than those in the control group (p < 0.05). The LA content in F-silage from the L, E, and M groups was higher than that in the control group (p < 0.05). The AN/TN in F-silage from the M group was lower than that in the control group (p < 0.05). The AN/TN in P-silage from the L group was lower than that in the control group (p < 0.05). 3.2. Chemical Compositions of Spent Mushroom Substrate Silage The chemical compositions of F-silage and P-silage are shown in Table 4. The DM, ADF, and ADL contents in F-silage were lower than those in P-silage (p = 0.000-0.040), and the CP content was higher than that in P-silage (p = 0.001-0.009). The E treatment influenced ADL content in P-silage (p = 0.001). The interaction (L x E) influenced DM and NDF contents in P-silage (p = 0.002-0.007). The DM content in P-silage from the L, E, and M groups were higher than that in the control group (p < 0.05). The ADF content of P-silage treated with L was lower than that in the control (p < 0.05). The CP and NDF contents in P-silage from the M group was higher than those in the control group (p < 0.05). Compared with the control, the E and M treatments produced lower ADL contents in P-silage (p < 0.05). 3.3. Energy and In Vitro Digestibility of Spent Mushroom Substrate Silage The energy and in vitro digestibility of F-silage and P-silage are shown in Table 5 and Table 6. The DE, ME, NEm, NEI, and NEg in F-silage were higher than those in P-silage (p = 0.000-0.011), and the IVDMD, IVADFD, and IVNDFD were higher than those in P-silage (p = 0.000-0.017). The L treatment and the interaction (L x E) influenced IVHCD in F-silage (p = 0.002-0.007). The E treatment and the interaction (L x E) influenced IVNDFD in F-silage (p = 0.000-0.020). The E treatment influenced IVADFD in F-silage (p = 0.000). The L treatment influenced IVHCD in P-silage (p = 0.012). Compared with the control, the E and M treatments produced higher IVNDFD and IVADFD in F-silage (p < 0.05). The L treatment resulted in higher IVDMD of P-silage (p < 0.05), while the L treatment produced a lower IVHCD of F-silage (p < 0.05). The E treatment increased IVNDFD and IVADFD in F-silage and P-silage (p < 0.05). Compared with the control, the L and E treatments produced higher IVHCD in P-silage (p < 0.05). 3.4. Aerobic Stability of Spent Mushroom Substrate Silage The changes in temperature of F-silage and P-silage aerobic exposure are shown in Figure 1 and Figure 2. The temperature of all treatments in F-silage increased within 24-96 h, but the temperature increase range was small . Compared with F-silage, the temperature fluctuation in P-silage was more obvious . After 72 h, the temperature of the L and E treatments began to increase evidently. After 144 h, the temperature in the L treatment began to decrease. After 120 h, the temperature in the control and M treatment groups began to obviously increase. The aerobic stability of F-silage inoculated with L and M increased (p < 0.05) by 24 h compared to the control; additionally, inoculation with E increased (p < 0.05) the aerobic stability of F-silage by 18 h . The aerobic stability of P-silage inoculated with M increased (p < 0.05) by 6 h compared to the control . The aerobic stability of P-silage inoculated with L reduced (p < 0.05) by 42 h compared to the control; additionally, inoculation with E reduced (p < 0.05) the aerobic stability of P-silage by 48 h . 4. Discussion In the introduction, we speculated that the combined application of lactic acid bacteria and cellulase is able to increase the lactic acid content of SMS silage, delay the deterioration of SMS silage, and further improve the fermentation quality, in vitro digestibility, and aerobic stability of SMS silage. From Table 3, F-silage and P-silage obtained good fermentation, and the fermentation quality of M treatment group was the best, which was consistent with our previous speculation. From Table 6, for F-silage, the IVDMD, IVNDFD, and IVADFD of the M treatment group was the highest. However, the M treatment group did not affect the in vitro digestibility of P-silage. From Figure 3 and Figure 4, in F-silage and P-silage, the aerobic stability of the M treatment group was the best, which was consistent with our previous speculation. 4.1. Effects of Additives on Fermentation Quality In general, high-quality silage has a pH of 3.8-4.2, at which point the activity of harmful bacteria in the silage is attenuated . In this study, the pH of the L, E and M treatment groups of P-silage was 3.93-3.96, the LA content was higher, and the AN/TN ratio was lower, indicating that P-silage obtained good fermentation. The fermentation effect of F-silage was lower than that of P-silage, as reflected in the high pH (4.2-4.4) and BA (5.0 g kg-1 DM-11.2 g kg-1 DM), despite the higher LA content than well-preserved silages (30 g kg -1 DM) . In the presence of a sufficient LA content, the high water content and BC value (282 mEq kg-1 DM) of the F-SMS feedstock were also important factors inducing low-quality fermentation . High pH may also affect the activity of the proteolytic Clostridium and Enterobacter bacteria, causing secondary fermentation . The number of lactic acid bacteria on the feed surface must be at least 5 log10 CFUs g-1 FW for the silage to be fermented with good quality. The number of lactic acid bacteria in the two SMS was measured to be < 5 log10 CFUs g-1 FW before silage, and thus a lactic acid bacteria preparation was needed to improve silage fermentation. Studies have shown that adding L. plantarum during ensiling effectively improves the fermentation quality of P. ostreatus spent mushroom substrate silage . The L. plantarum reduced the pH and BA content of Leymus chinensis silage, while increasing the LA content . Compared with the control group, the addition of L. plantarum accelerated the accumulation of LA, decreased the pH and AN/TN ratio, and improved the fermentation quality of forage or alfalfa silage, which was consistent with the results of this experiment in F-silage . Structural polysaccharides in plant cell walls are decomposed into monosaccharides by cellulase, which provide sufficient substrates (fermentable sugars) for lactic acid bacteria fermentation, thereby improving fermentation quality . Zhao et al. showed that adding cellulase reduced the pH and AN/TN of bean dregs and corn stover mixed silage, and improved the fermentation quality of silage, consistent with the experimental results obtained for P-silage. Compared to the control group, the mixed addition of L. plantarum and cellulase significantly reduced the pH of corn stover silage, and the pH of the mixed addition treatment group was minimal, which was consistent with the results of this experiment . The AN produced by Clostridium was also an important indicator for evaluating the quality of silage fermentation, reflecting the degradation level of proteins and amino acids during ensiling . The AN/TN ratio of F-silage and P-silage in each additive treatment group was lower than the control treatment. The explanation was that a substantial decrease in pH inhibits plant enzymes and aerobic microorganisms, reducing protein degradation . The concentration of AA in both the L treatment and M treatment groups of P-silage was lower than that in the control treatment group. This result may be due to the higher fermentation efficiency of silage due to the exogenous addition of lactic acid bacteria, while the fermentation efficiency of the lactic acid bacteria attached to the surface of the feed material was lower and the pH decreased more slowly . This hypothesis was supported by the experiment reported by Silva et al. , who found that the AA concentration of alfalfa silage inoculated with lactic acid bacteria was lower than that of the control treatment group because the lactic acid bacteria attached to the feed surface in the control treatment group were less efficient in inducing rapid acidification in the early fermentation stage. This finding was in stark contrast to the results reported by Filya that the AA concentration of low dry matter maize silage inoculated with heterofermentative lactic acid bacteria (Lactobacillus brucei) was significantly higher than that of control and L. plantarum treatments. This difference may be attributed to the difference in AA concentration caused by the different fermentation types mediated by lactic acid bacteria. The metabolism of homofermentative lactic acid bacteria produces only LA, while the metabolism of heterofermentative lactic acid bacteria produces higher contents of AA and various metabolites. The significant difference in the AA content of F-silage and P-silage in this experiment might also be attributed to the different types of lactic acid bacteria populations that epigenetically grow on the surface of feed ingredients. The M treatment of P-silage increased the LA content by 3.3-6.8% and decreased the pH by 0.02-0.28 units compared with the control and L and E treatments. The reason for this was that L. plantarum and cellulase were added at the same time, and the two exerted a synergistic effect. Cellulase promotes the degradation of fibrous substances, provides an additional fermentation substrate for lactic acid bacteria, and helps lactic acid bacteria dominate the microbial community in silage such that the fermentation moves towards homogenous fermentation . Therefore, in this study, the addition of M was suggested to improve the quality of P-silage. 4.2. Effects of Additives on Chemical Composition and In Vitro Digestibility Compared with the two types of SMS raw materials, the content of OM did not change significantly in the silage of the two types of SMS in this experiment, and the CP content was increased, indicating that the two types of SMS better preserve their nutritional components. This increase may be due to the rapid inhibition of proteolysis or a concentration effect caused by organic carbon loss during ensiling . High-quality silage fermentation reduces protein hydrolysis in non-protein nitrogen, ammonia nitrogen, and other non-protein compounds and generally retains the nutrients of raw silage materials . The CP content is also an important indicator for evaluating the nutritional value of silage. The higher the CP content, the better the feed value. Compared with the control treatment, better protein preservation was observed in alfalfa and corn silage inoculated with homofermentative lactic acid bacteria, indicating that the addition of lactic acid bacteria may reduce the loss of nutrients to a certain extent, consistent with the test results for the L treatment of P-silage . Compared with the control treatment, E or M treatment decreased the NDF, ADF, and ADL contents in F-silage; increased the CP content; and improved the feed value of F-silage. The explanation for this result was that cellulase decomposed the fibre and other components of F-silage plant cell wall, fully releasing the cell contents, increasing the available WSC content for lactic acid bacteria, and reducing the consumption of CP . The AN concentration is one of the key indicators to determine the degree of protein hydrolysis in silage. The extensive hydrolysis of proteins will affect nitrogen utilization by ruminants. The smaller AN/TN ratio also proved the good effect of E and M treatment on preserving proteins from another aspect. Dehghani et al. showed that adding cellulase directly hydrolysed the fibrous material in silage, which was the most effective at reducing the NDF content of corn stover silage, consistent with the results of our experiment. The contents of NDF, ADF, and ADL in the L treatment group of P-silage were lower than in the control group, and this result was surprising because lactic acid bacteria do not directly decompose plant fibres and lignin . Some microorganisms might degrade fibre and lignin during the silage process, which may explain the decrease in fibre and lignin contents in silage. The chemical composition of the silage determines the nutritional value of the livestock. Dry matter loss in silage is associated with the depletion of soluble carbohydrate and organic acid components . In P-silage, the DM content in L, E, and M treatment groups was significantly higher than that in the control group, indicating that the DM loss of P-silage was reduced after the additive was applied, and the P-silage nutrients were better preserved because of the higher WSC content in the P-SMS raw material. Therefore, during the fermentation process of lactic acid bacteria, WSC, a fermentation substrate, was metabolized into organic acids, thereby reducing the loss of nutrients . The study showed that about 2% of the DM content were consumed through the action of the additive . After 45 days of ensiling, the DM content of both silages was lower than the DM content of the raw material. In general, the energy distribution potential of the feed was mainly assessed by determining the IVDMD in ruminant fluids of ruminants . The IVDMD and IVNDFD of F-silage were higher than those of P-silage. This difference was related to the difference in carbohydrate types between the two SMS feedstocks (Table 1). The IVDMD of F-silage in the E and M treatment groups were slightly higher than that in the control treatment group. A potential explanation for this result was the enzymatic saccharification of exogenously added cellulase, where sugars and proteins were enzymatically generated to glycoproteins, increasing the rumen-fermentable fraction of silage . The experimental results reported by Rinne et al. support this result. The enzymatic saccharification reaction of cellulase contributes to lactic acid fermentation, thereby increasing the in vitro OM digestibility of the silage. The additive treatment also increased the digestible and metabolizable energy of F-silage. Bala at al. added an enzyme preparation containing cellulase to the diet of goats and found that IVDMD, IVNDFD, and IVADFD were increased, indicating that cellulase effectively improved the digestibility of cellulose feed in the rumen. The reason for this might be that cellulase could destroy glycosidic bonds and disintegrate the cell wall structure of silage, thus reducing the resistance of the cell wall to rumen microorganisms and improving the in vitro digestibility of silage, which was consistent with the results of F-silage. The L treatment increased the IVDMD of P-silage compared to the control treatment, due to the effect of lactic acid bacteria on reducing dry matter loss during silage fermentation. Lactic acid bacteria improved the fermentation quality of P-silage, inhibited the digestion and hydrolysis of protein, and subsequently increased IVDMD. The addition of lactic acid bacteria to vegetable residue silage, such as cabbage and lettuce, increases the IVDMD of the silage, which is consistent with the aforementioned experimental results . The fermentation effect of F-silage was lower than that of P-silage, as reflected in the high pH (4.2-4.4) and BA (5.0 g kg-1 DM-11.2 g kg-1 DM). If F-silage treated with additive is introduced into a sheep's diet, it affects the food intake of the sheep and will very likely to lead to weight loss due to poor feed and fermentation quality. The barley silage ration inoculated with cellulase (0.75 mL/kg) improved milk production efficiency, milk fat-corrected milk yield, and the feed digestibility of dairy cows . Therefore, if the E-treated P-silage is introduced into a sheep's diet, it may maximize the use of agricultural byproduct, promote the balance of the animal's diet, and improve the production performance of the sheep. 4.3. Effect of Additives on Aerobic Stability The aerobic spoilage of fermented feed is mainly caused by the proliferation of harmful microorganisms, such as yeast and mould . After the silage is opened, the anaerobic environment is destroyed, and the yeast becomes active, which breaks down lactic acid and protein, releases carbon dioxide and ammonia nitrogen, and causes a loss of nutrients. Due to the depletion of LA, the pH of the silage will increase, causing the rapid proliferation of moulds that spoil the silage. AA and PA, acting as inhibitors of aerobic microorganisms, improve the aerobic stability of silage, which increases exponentially with the concentration of AA . In F-silage, L, E, and M treatments all contained high AA concentrations and the aerobic stability increased at 24 h, 18 h, and 24 h, respectively . Compared with F-silage, P-silage contained very low levels of AA and PA, which was more likely to cause aerobic spoilage. If the silage maintains a high aerobic stability, it will greatly preserve the nutrition of the silage and reduce the accumulation of mycotoxins . The LA and WSC are substrates for the growth and reproduction of aerobic microorganisms, such as yeast and mould . Silage additives such as L. plantarum or cellulases also reduced the aerobic stability of silage . For example, the addition of L. plantarum did not inhibit the reproductive growth of yeast, resulting in the instability of maize and alfalfa silage during aerobic exposure , consistent with the results from the P-silage test. The potential explanation was that the L treatment had a higher LA content and produced fewer short-chain fatty acids that inhibited the growth and reproduction of yeasts and moulds on P-silage. In addition, the addition of L. brucei reduced the number of harmful bacteria in sunflower and sorghum silage and prolonged the spoilage time of the silage, which was in sharp contrast to the results of this experiment . At the same time, the addition of E decreased the aerobic stability of P-silage and caused the silage to spoil earlier. The reason was that the rapid accumulation of a fermentation substrate promoted the reproduction and growth of yeasts and moulds, resulting in the secondary fermentation of P-silage . The P-SMS raw material contained a higher WSC content, which provided a fermentation substrate for the growth of undesired bacteria and may be responsible for the reduced aerobic stability of P-silage treated with L and E . The AA and PA possessed strong antifungal activities and inhibited the growth and reproduction of yeast and mould . The aerobic stability of the M treatment group was the best among F-silage and P-silage at 96 h and 138 h, respectively. Therefore, the M treatment was more stable during aerobic exposure and improved the aerobic stability of F-silage and P-silage. 5. Conclusions Overall, the combination of application of lactic acid bacteria and cellulase is an effective strategy for improving the fermentation quality and aerobic stability of F-silage and P-silage. Cellulase can be used as an additive to improve the in vitro digestibility of P-silage. However, in vivo studies are needed to validate our findings and examine the silage quality and effect of cellulase-treated P-silage on beef cattle fattening before recommending it to farmers. Author Contributions Conceptualization, Q.Y. and P.W.; methodology, Q.Y.; M.Y.; Z.S.; H.L. and H.T.; software, Q.Y.; T.Z.; M.Y.; Z.S. and H.T.; validation, P.W.; H.L.; M.Y. and T.Z.; formal analysis, Q.Y.; P.W.; Z.S. and T.Z; investigation, M.Y. and H.L.; resources, H.T.; data curation, Q.Y.; P.W. and M.Y; writing--original draft preparation, Q.Y. and P.W.; writing--review and editing, Q.Y.; P.W. and T.Z; visualization, P.W.; supervision, P.W.; project administration, P.W.; funding acquisition, P.W. and H.L. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study strictly followed the Chinese Laboratory Animal Welfare Ethical Review Guidelines and was approved by the Laboratory Animal Welfare Ethics Committee of Jilin University (Number of permit: SY202009600). Informed Consent Statement Not applicable, as this research did not involve any humans. Data Availability Statement The authors are solely responsible for the completeness of all data and the accuracy of data analysis in this study. Complete data in this study are available from the corresponding authors upon reasonable request. Conflicts of Interest The authors declare that there are no conflict of interest in this study. Figure 1 Effect of application of additives on the temperature change of Flammulina velutipes spent mushroom substrate silages after exposure to aerobic conditions. L, lactic acid bacteria; E, cellulase; M, the mixture of lactic acid bacteria and cellulase; AT, ambient temperature. Figure 2 Effect of application of additives on the temperature change of Pleurotus eryngii spent mushroom substrate silages after exposure to aerobic conditions. L, lactic acid bacteria; E, cellulase; M, the mixture of lactic acid bacteria and cellulase; AT, ambient temperature. Figure 3 Time required for Flammulina velutipes spent mushroom substrate silages temperature to exceed room temperature by 2 degC after exposure to air (one-way ANOVA: F = 117.7, p = 0.000). L, lactic acid bacteria; E, cellulase; M, the mixture of lactic acid bacteria and cellulase. a-c Means with different superscripts differed significantly (p < 0.05). Figure 4 Time required for Pleurotus eryngii spent mushroom substrate silages temperature to exceed room temperature by 2 degC after exposure to air (one-way ANOVA: F = 545.5, p = 0.000). L, lactic acid bacteria; E, cellulase; M, the mixture of lactic acid bacteria and cellulase. a-d Means with different superscripts differed significantly (p < 0.05). animals-13-00920-t001_Table 1 Table 1 Percentage of raw material in spent mushroom substrates. Flammulina velutipes Pleurotus eryngii Corncob meal (%) 37 Corncob meal (%) 23 Rice bran (%) 38 Corn straw powder (%) 18 Sawdust (%) 8 Sawdust (%) 39 Soybean hulls (%) 5 Cottonseed hull (%) 5 Soybean meal (%) 6 Brewer's grain (%) 5 Corn meal (%) 12 Lime (%) 1 Lime (%) 1 Calcium carbonate (%) 1 Calcium carbonate (%) 1 Total (%) 100 Total (%) 100 animals-13-00920-t002_Table 2 Table 2 Characteristics of spent mushroom substrates. Spent Mushroom Substrate + Item ++ F-SMS P-SMS SEM p-Value Chemical composition and buffering capacity Dry matter (g kg-1 FW) 492 504 0.351 0.000 Organic matter (g kg-1 DM) 872 903 0.677 0.000 Crude protein (g kg-1 DM) 90.4 80.7 0.664 0.000 Neutral detergent fibre (g kg-1 DM) 729 727 4.112 0.684 Acid detergent fibre (g kg-1 DM) 428 497 4.141 0.000 Acid detergent lignin (g kg-1 DM) 111 176 3.164 0.000 Water-soluble carbohydrate (g kg-1 DM) 24.4 34.2 0.120 0.000 Calcium (g kg-1 DM) 17.0 16.5 0.896 0.609 Phosphorus (g kg-1 DM) 7.31 1.82 0.247 0.000 Buffering capacity (mEq kg-1 DM) 282 157 12.209 0.001 Energy GE (MJ kg-1 DM) 19.2 18.8 0.034 0.000 DE (MJ kg-1 DM) 10.3 7.69 0.090 0.000 ME (MJ kg-1 DM) 8.30 6.18 0.071 0.000 NEm (MJ kg-1 DM) 5.59 4.00 0.059 0.000 NEl (MJ kg-1 DM) 4.69 3.35 0.047 0.000 NEg (MJ kg-1 DM) 2.82 1.63 0.051 0.000 Microbial counts Lactic acid bacteria (log10 cfu g-1 FW) 4.68 4.30 0.032 0.000 Yeasts (log10 cfu g-1 FW) 3.10 4.20 0.040 0.000 Moulds (log10 cfu g-1 FW) 0.21 2.33 0.072 0.000 SEM, standard error of the mean; + F-SMS, spent mushroom substrate from Flammulina velutipes; P-SMS, spent mushroom substrate from Pleurotus eryngii; ++ FW, fresh weight; DM, dry matter; GE, gross energy; DE, digestible energy; ME, metabolizable energy; NEm, net energy for maintenance; NEl, net energy for lactating cow; NEg, net energy for weight gain; cfu, colony-forming units. animals-13-00920-t003_Table 3 Table 3 Fermentation quality of spent mushroom substrate silages prepared with lactic acid bacteria and cellulase. Item ++ S-Silage SS Additives + SEM p-Value Significance of Main Effects and Interactions Control L E M L E L x E pH value F-silage 4.40 b 4.26 a 4.24 a 4.20 a 0.033 0.002 0.005 0.002 0.072 P-silage 4.21 b 3.96 a 3.95 a 3.93 a 0.031 0.000 0.000 0.000 0.001 SEM 0.041 0.027 0.032 0.025 p-value 0.041 0.000 0.001 0.000 LA (g kg-1 DM) F-silage 56.9 a 77.2 b 67.4 b 67.9 b 0.318 0.002 0.002 0.802 0.002 P-silage 118 121 122 126 0.889 0.839 0.596 0.488 0.947 SEM 0.624 0.198 0.861 0.784 p-value 0.001 0.000 0.020 0.002 AA (g kg-1 DM) F-silage 144 151 141 151 1.554 0.887 0.462 0.925 0.886 P-silage 2.72 0.90 0.94 1.04 0.149 0.583 0.443 0.460 0.393 SEM 1.004 0.273 1.635 1.059 p-value 0.000 0.000 0.013 0.000 PA (g kg-1 DM) F-silage 19.5 20.6 18.3 20.4 0.295 0.850 0.456 0.757 0.793 P-silage 3.44 4.50 5.91 4.70 0.112 0.249 0.903 0.123 0.191 SEM 0.134 0.101 0.382 0.158 p-value 0.000 0.000 0.032 0.001 BA (g kg-1 DM) F-silage 5.01 5.00 11.2 9.76 0.317 0.184 0.757 0.040 0.757 P-silage 4.33 4.17 5.52 4.18 0.121 0.638 0.398 0.505 0.505 SEM 0.110 0.062 0.346 0.308 p-value 0.577 0.250 0.177 0.210 AN (g kg-1 TN) F-silage 12.8 b 10.9 ab 10.4 ab 9.40 a 0.085 0.023 0.048 0.011 0.490 P-silage 11.6 b 8.06 a 9.84 ab 9.92 ab 0.068 0.007 0.008 0.894 0.006 SEM 0.107 0.103 0.028 0.029 p-value 0.324 0.050 0.130 0.140 Different lowercase letters (a, b) in the same line indicate that there is a significant difference between different additive treatment groups under the same spent mushroom substrate (p < 0.05); the p-value in the same column represents the p-value of the independent sample t-test; SEM, standard error of the mean; + L, lactic acid bacteria; E, cellulase; M, the mixture of lactic acid bacteria and cellulase; ++ DM, dry matter; LA, lactic acid; AA, acetic acid; PA, propionic acid; BA, butyric acid; AN, ammonia nitrogen; TN, total nitrogen; SS S, spent mushroom substrate; F, Flammulina velutipes spent mushroom substrate; P, Pleurotus eryngii spent mushroom substrate. animals-13-00920-t004_Table 4 Table 4 Chemical composition of spent mushroom substrate silages prepared with lactic acid bacteria and cellulase. Item ++ S-Silage SS Additives + SEM p-Value Significance of Main Effects and Interactions Control L E M L E L x E DM (g kg-1 FW) F-silage 414 414 416 412 0.312 0.705 0.398 0.965 0.446 P-silage 457 a 462 b 462 b 460 b 0.141 0.015 0.084 0.134 0.007 SEM 0.322 0.122 0.324 0.100 p-value 0.000 0.000 0.000 0.000 OM (g kg-1 DM) F-silage 885 887 880 876 1.184 0.803 0.891 0.382 0.740 P-silage 899 898 897 899 0.130 0.234 0.218 0.469 0.128 SEM 1.476 0.358 0.595 0.423 p-value 0.457 0.033 0.054 0.006 CP (g kg-1 DM) F-silage 115 113 120 119 0.555 0.612 0.737 0.219 0.928 P-silage 88.2 a 88.8 ab 92.9 ab 95.3 b 0.216 0.032 0.369 0.006 0.564 SEM 0.381 0.510 0.500 0.230 p-value 0.002 0.009 0.006 0.001 NDF (g kg-1 DM) F-silage 690 694 668 662 1.765 0.262 0.910 0.063 0.689 P-silage 682 ab 650 a 684 bc 717 c 1.035 0.002 0.944 0.002 0.002 SEM 0.213 0.855 2.295 1.529 p-value 0.022 0.007 0.527 0.022 ADF (g kg-1 DM) F-silage 402 406 393 389 0.886 0.260 0.965 0.067 0.566 P-silage 464 b 445 a 465 b 478 b 0.469 0.001 0.472 0.001 0.001 SEM 0.281 0.461 1.134 0.656 p-value 0.000 0.001 0.003 0.000 ADL (g kg-1 DM) F-silage 135 ab 150 b 95.3 a 101 a 1.372 0.011 0.327 0.002 0.658 P-silage 188 c 183 bc 159 ab 156 a 0.819 0.009 0.562 0.001 0.828 SEM 1.760 0.783 0.930 0.727 p-value 0.040 0.013 0.002 0.002 Different lowercase letters (a, b and c) in the same line indicate that there is a significant difference between different additive treatment groups under the same spent mushroom substrate (p < 0.05); the p-value in the same column represents the p-value of the independent sample t-test; SEM, standard error of the mean; + L, lactic acid bacteria; E, cellulase; M, the mixture of lactic acid bacteria and cellulase; ++ FW, fresh weight; DM, dry matter; OM, organic matter; CP, crude protein; NDF, neutral detergent fibre; ADF, acid detergent fibre; ADL, acid detergent lignin; SS S, spent mushroom substrate; F, Flammulina velutipes spent mushroom substrate; P, Pleurotus eryngii spent mushroom substrate. animals-13-00920-t005_Table 5 Table 5 Energy of spent mushroom substrate silages prepared with lactic acid bacteria and cellulase. Item ++ S-Silage SS Additives + SEM p-Value Significance of Main Effects and Interactions Control L E M L E L x E GE (MJ kg-1 DM) F-silage 19.3 19.3 19.3 19.1 0.251 0.813 0.468 0.607 0.778 P-silage 18.9 ab 18.7 a 18.9 ab 19.0 b 0.058 0.034 0.937 0.025 0.032 SEM 0.343 0.094 0.060 0.048 p-value 0.288 0.006 0.002 0.040 DE (MJ kg-1 DM) F-silage 11.1 10.9 11.3 11.2 0.316 0.664 0.698 0.272 0.773 P-silage 7.89 7.75 7.74 7.79 0.059 0.115 0.254 0.254 0.054 SEM 0.297 0.063 0.310 0.134 p-value 0.007 0.000 0.000 0.001 ME (MJ kg-1 DM) F-silage 8.82 8.69 8.95 8.98 0.262 0.668 0.787 0.279 0.671 P-silage 6.27 6.19 6.17 6.20 0.046 0.225 0.380 0.216 0.137 SEM 0.247 0.039 0.262 0.100 p-value 0.008 0.000 0.008 0.001 NEm (MJ kg-1 DM) F-silage 6.04 5.94 6.15 6.19 0.205 0.625 0.807 0.249 0.642 P-silage 4.07 4.01 4.00 4.01 0.037 0.277 0.461 0.193 0.193 SEM 0.180 0.031 0.215 0.083 p-value 0.007 0.000 0.009 0.001 NEl (MJ kg-1 DM) F-silage 5.05 4.96 5.14 5.17 0.171 0.624 0.810 0.250 0.633 P-silage 3.40 3.36 3.35 3.36 0.029 0.298 0.446 0.236 0.188 SEM 0.150 0.024 0.180 0.069 p-value 0.007 0.000 0.009 0.001 NEg (MJ kg-1 DM) F-silage 3.19 3.11 3.30 3.35 0.165 0.503 0.890 0.175 0.583 P-silage 1.66 1.63 1.61 1.62 0.026 0.265 0.356 0.129 0.356 SEM 0.120 0.028 0.190 0.066 p-value 0.005 0.000 0.011 0.001 Different lowercase letters (a, b) in the same line indicate that there is a significant difference between different additive treatment groups under the same spent mushroom substrate (p < 0.05); the p-value in the same column represents the p-value of the independent sample t-test; SEM, standard error of the mean; + L, lactic acid bacteria; E, cellulase; M, the mixture of lactic acid bacteria and cellulase; ++ DM: dry matter; GE, gross energy; DE, digestible energy; ME, metabolizable energy; NEm, net energy for maintenance; NEl, net energy for lactating cow; NEg, net energy for weight gain; SS S, spent mushroom substrate; F, Flammulina velutipes spent mushroom substrate; P, Pleurotus eryngii spent mushroom substrate. animals-13-00920-t006_Table 6 Table 6 In vitro digestibility of spent mushroom substrate silages prepared with lactic acid bacteria and cellulase. Item ++ S-Silage SS Additives + SEM p-Value Significance of Main Effects and Interactions Control L E M L E L x E IVDMD (g kg-1 DM) F-silage 516 ab 512 a 522 ab 526 b 4.383 0.034 0.968 0.066 0.568 P-silage 471 a 487 b 468 a 461 a 3.856 0.000 0.478 0.001 0.001 SEM 0.218 0.358 0.870 0.504 p-value 0.000 0.001 0.003 0.000 IVNDFD (g kg-1 DM) F-silage 445 a 439 a 483 b 495 c 0.437 0.000 0.345 0.000 0.020 P-silage 409 a 416 ab 430 b 405 a 0.500 0.006 0.040 0.225 0.002 SEM 0.375 0.469 0.657 0.301 p-value 0.001 0.008 0.001 0.000 IVADFD(g kg-1 DM) F-silage 353 a 361 a 431 b 452 c 0.479 0.000 0.003 0.000 0.085 P-silage 339 a 341 a 355 b 329 a 0.465 0.004 0.006 0.560 0.003 SEM 0.352 0.340 0.556 0.587 p-value 0.017 0.004 0.000 0.000 IVHCD(g kg-1 DM) F-silage 573 b 549 a 560 a 557 a 0.418 0.003 0.002 0.405 0.007 P-silage 560 a 576 b 589 c 556 a 0.382 0.000 0.012 0.138 0.000 SEM 0.196 0.430 0.538 0.359 p-value 0.003 0.003 0.006 0.755 Different lowercase letters (a, b, and c) in the same line indicate that there is a significant difference between different additive treatment groups under the same spent mushroom substrate (p < 0.05); the p-value in the same column represents the p-value of the independent sample t-test; SEM, standard error of the mean; + L, lactic acid bacteria; E, cellulase; M, the mixture of lactic acid bacteria and cellulase; ++ IVDMD, in vitro dry matter digestibility; IVNDFD, in vitro neutral detergent fibre digestibility; IVADFD, in vitro acid detergent fibre digestibility; IVHCD, in vitro hemicellulose digestibility; SS S, spent mushroom substrate; F, Flammulina velutipes spent mushroom substrate; P, Pleurotus eryngii spent mushroom substrate. 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PMC10000081

Recent advances in animal nutrition have indicated that bovine colostrum (BC), due to its content of macronutrients, micronutrients and bioactive compounds, is an excellent health supplement. To the best of our knowledge, no studies on the effect of BC on antioxidant status have been performed in rabbits. This study aimed to investigate the effect of two BC concentrations on antioxidant status and gene expression of antioxidant enzymes in some tissues of rabbits. Thirty New Zealand White male rabbits were randomly divided into three experimental diets, containing 0% (CON), 2.5%, and 5% of BC (BC-2.5 and BC-5, respectively). The activity of antioxidant enzymes in plasma (catalase: CAT; glutathione peroxidase: GPx; superoxide dismutase: SOD), and the enzymes' gene expression in the liver and longissimus dorsi muscle, were determined. Results showed no significant differences, neither in plasma nor in tissues. A significant tissue-related effect has been observed regarding the mRNA levels of SOD and GPx, which were higher in the LD (p = 0.022) and liver (p = 0.001), respectively. Further studies, considering modifications of the length and dosage of dietary BC supplementation, are required to update the current state of knowledge in rabbits, as well as to fully understand the potential value of BC for possible application in farming use. rabbit antioxidant status bovine colostrum gene expression UNIMI Research Support PlanThis research was funded by UNIMI Research Support Plan--Research Funds of the Department of Veterinary Medicine and Animal Sciences--Call Line 2--Project entitled "Effects of colostrum supplementation on reproductive and productive performance, immunity and meat quality of rabbit". pmc1. Introduction Colostrum, also known as first milk, is produced by the mammary gland immediately following parturition. Bovine colostrum (BC), due to its role in immunity, growth and antimicrobial factors, has been used for centuries as a traditional or complementary therapy for a wide variety of disorders and in veterinary practice. Its role for a newborn is not only to provide nutrition for the first few days of life, but also to provide protection against infections while the immune system is still developing . Several studies support its beneficial activity for the treatment of medical conditions in children and adults, particularly gastrointestinal disorders . Its use is not limited to humans; in fact, its role as an effective nutraceutical for the enhancement of the immune function in several animal species, both farm and pet, has been documented . Colostrum is also rich in enzymatic (lactoperoxidase, catalase, superoxide dismutase, glutathione peroxidase) as well as non-enzymatic (vitamins E, A, C, lactoferrin and selenium) antioxidants, which confer it antioxidant activity. The evaluation of the potential antioxidant effects of BC dietary supplementation in rabbit species could be relevant, due to the high proportion of poly-unsaturated fatty acids (PUFAs) that rabbit meat contains . For the rabbit meat industry, Italy represents the third-largest producer within the European Union, after Spain and France. Although Italy is among the top five producers of rabbit meat in the world, its consumption has decreased over the past five years in the Mediterranean region . Rabbits are sensitive to many bacterial infections, such as respiratory and intestinal diseases, which cause a high mortality of the animals, thus weighing heavily on the productivity of farms . These pathologies have multifactorial etiologies, connected to diet, hygiene, environmental and climatic factors, and stress. Oxidative stress occurs when the production of potentially destructive reactive oxygen species (ROS) exceeds the body's natural antioxidant defense . Oxidative stress can be measured directly, by detecting free radical production, or indirectly, by detecting the antioxidant defenses of the organism. Since oxidative stress is involved in several pathological conditions in farm animals, the evaluation of oxidative status is a good indicator of the health status of animals. Antioxidant enzyme activities are generally used for the evaluation of oxidative stress status in animals . Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) enzymes represent the primary components of the enzymatic antioxidant defense system against oxidative stress. Dietary antioxidant supplementation is considered a promising way for mitigate oxidative stress. For this reason, the additional supply of antioxidant substances is able to contribute to the direct and indirect extinction of free radicals, complementing the activities of primary antioxidant enzymes . Currently, great attention is paid to natural products with antioxidant effects, of which BC is a valuable example. To the best of our knowledge, no information about the effects of BC on blood antioxidant status and antioxidant gene expression in rabbits is available. Based on the positive antioxidant activity of BC described in the literature in piglets and calves , we hypothesized that a similar effect could also be exerted in rabbits. The study was planned to evaluate the effect of two increasing levels of dietary BC on plasma antioxidant enzymes' activity and the expression of primary antioxidant enzymes in the liver and longissimus dorsi muscle of rabbits. 2. Materials and Methods 2.1. Animals Diets and Samples Collection The experimental protocol was conducted at the Department of Agricultural, Food, and Environmental Science of the University of Perugia's experimental farm, in compliance with the guidelines of the Legislative Decree No. 146, implementing Directive 98/58/EC on the protection of animals bred for farming purposes. A total of 30 post-weaning New Zealand White male rabbits (37 days old), with an average body weight of 550 +- 24 g, were randomly assigned to three experimental diets: a control group (CON) fed with a pelletted commercial standard diet, CON supplemented with 2.5% of liquid bovine colostrum (BC-2.5), and CON supplemented with 5.0% of liquid bovine colostrum (BC-5) (Table 1). Rabbits were fed ad libitum and had free access to water. The experimental colostrum, of high quality (>23% Brix; ), was collected from multiparous Holstein-Friesian cows during the first milking, and immediately stored in sterile containers at -20degC until the start of the experiment. All rabbits were kept under the same management, and the same hygienic and environmental conditions; their clinical condition was monitered throughout the trial. Thirty-seven days after the beginning of the feeding trial, the animals were slaughtered (body weight of 1977 +- 26 g), and fasting blood samples were collected by jugular vein puncture using vacutainers. The blood was then centrifuged at 3000x g at 4 degC for 15 min to separate the plasma; the plasma aliquots were stored at -80 degC until subsequent analyses. The carcasses were left for 24 h at 2.5 +- 0.5 degC, and the LD muscle (left side) and liver (a portion of the right lobe) were taken from each animal, for a total of 60 samples. Samples were stored in RNAlaterTM (ThermoFisher Scientific, Waltham, MA, USA) soon after the sampling procedure, and maintained at -80degC until their subsequent use in RNA extraction. 2.2. Total Phenol Content and Antioxidant Activity of Bovine Colostrum and Experimental Diets 2.2.1. Sample Treatment All samples were extracted using 70% (v/v) ethanol/double-distilled water. The BC sample was extracted at a ratio of 1 mL in 2 mL 70% EtOH, while finely ground samples of control diet, and control diet supplemented with 2.5% and 5% of BC, were extracted at a ratio of 2 g in 10 mL of 70% EtOH (CON, BC-2.5, and BC-5, respectively). All samples were shaken for 1 h in the dark, and were centrifuged at 13,000 rpm for 15 min at 4 degC. Recovered supernatants were stored at -20 degC until analyses. 2.2.2. Total Phenol Content The Folin-Ciocalteu method was used to determine total phenol contents (TPs) . Different concentrations of gallic acid (from 2 to 10 mg/mL) were used as a standard to construct the calibration curve, and values were expressed as milligrams of gallic acid equivalents: (GAE)/mL for BC, and GAE/g for CON, BC-2.5, and BC-5 samples. 2.2.3. Antioxidant Activities (DPPH, ABTS, ORAC Assays) The antioxidative properties of BC and experimental diets were measured in terms of the Trolox equivalent antioxidant capacity (TEAC), using DPPH, ABTS and ORAC assays. The DPPH assay was carried out as reported in Saltarelli et al. , with minor modifications. Briefly, 850 mL of 100 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH) ethanol solution was added to 150 mL of sample at various concentrations. Samples were rested for 20 min in the dark at room temperature, and then the absorbance, decreasing at 517 nm, was detected (UV Beckman Spectrophotometer CA, USA); 70% (v/v) ethanol/double-distilled water was used as a blank. The DPPH radical-scavenging activity (DSA) of the sample was calculated as follows: DSA (%) = [(blank Abs - sample Abs)/ blank Abs] x 100. For the ABTS assay , 10 mL of the samples and 1 mL of 2,2,-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) ethanolic solution were left for 5 min in the dark at room temperature, and then the absorbance was detected at 734 nm (UV Beckman spectrophotometer CA, USA); 70% (v/v) ethanol/double-distilled water was used as blank. The ABTS radical-scavenging activity (ASA) was reported as follows: ASA (%) = [(blank Abs - sample Abs)/ blank Abs] x 100. The oxygen radical absorbance capacity (ORAC) was performed as in De Bellis et al. , detecting fluorescence until total extinction (485 nm ex. and 520 nm em.) on a Fluostar Optima plate reader (BMG Labtech, Offenburg, Germany). All antioxidant assays were compared to the Trolox antioxidant capacity, and, for this reason, data are reported as the micromoles of Trolox equivalents (TE)/mL, or TE/mg, of the samples. 2.3. Antioxidant Enzymes Activity Determination The activity of catalase (CAT), glutathione peroxide (GPx) and superoxide dismutase (SOD) enzymes was tested in plasma samples using commercial kits supplied by Cayman Chemical (Ann Arbor, MI, USA), according to the instructions provided by the manufacturer. CAT activity in plasma was measured with the Catalase Assay Kit (ref. No.: 707002, Cayman Chemicals, Ann Arbor, MI, USA), which utilizes the peroxidase function of CAT for the determination of enzyme activity. In the presence of an optimal H2O2 concentration, the CAT enzyme reacts with methanol, producing formaldehyde, which is measured colorimetrically with the chromogen 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald). This chromogen specifically forms a bicyclic heterocycle with aldehydes, which changes from colorless to purple upon oxidation. One unit is defined as the amount of CAT enzyme that forms 1.0 nmol of formaldehyde per minute at 25degC (according to the manufacturer's protocol). GPx activity in plasma was measured with a Glutathione Peroxide Assay Kit (ref. No.: 703102, Cayman Chemicals, Ann Arbor, MI, USA), which measures GPx activity indirectly by a coupled reaction with glutathione reductase. The reduction of hydroperoxide by GPx produces oxidized glutathione, which is recycled to its reduced state by glutathione reductase and NADPH. The latter is oxidized to NADP+, decreasing the absorbance at 340 nm. One unit of GPx is defined as the amount of enzyme that oxidizes 1.0 nmol of NADPH to NADP+ per 1 min at 25 degC (according to ref. No.: 703102, Cayman Chemicals, USA Kit). The assay of SOD activity was performed using the commercial kit "Superoxide Dismutase Assay Kit" (ref. No.: 706002, Cayman Chemical, Ann Arbor, MI, USA), which utilizes a tetrazolium salt for the detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. The amount of SOD enzyme required to exhibit a 50% dismutation of the superoxide radical represents one unit of SOD (according to the manufacturer's protocol). 2.4. RNA Extraction and cDNA Synthesis Total RNA was isolated from liver and LD samples using a commercial kit (ReliaPrepTM RNA Tissue Miniprep System; Promega Corporation, USA), following the manufacturer instructions, and was eluted in 30 mL of RNase-free water. The purity and concentration of each RNA sample was measured using a NanoDrop ND-1000 spectrophotometer (ThermoFisher Scientific, Massachusetts, USA). One microgram of each RNA sample was treated with DNase enzyme to remove any genomic DNA contamination using DNase I, RNase-free kit (ThermoFisher Scientific, Waltham, MA, USA). Synthesis of cDNA was performed from 500 ng of total RNA using the GoScriptTM Reverse Transcription Mix (Promega Corporation, Madison, WI, USA). For each sample, a no-reverse transcriptase (no-RT) control was used to verify the complete DNA removal. The cDNA samples were stored at -20degC until quantitative real-time PCR analyses. 2.5. Real Time RT-qPCR The gene expression of specific target genes was determined: catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD); glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB) were used as reference genes for real-time quantitative PCR, which was used to compare the expression levels of the three experimental groups (CON, BC-2.5 and BC-5). The primers were designed using the tool Primer-BLAST ), and were supplied by Eurofins Genomics (Eurofins Genomics, Ebersberg, Germany). Table 2 reports the primer sequences, annealing temperatures (Tm) and amplicon size of each fragment. cDNA samples were used as templates for the gene expression analysis, which was performed using the thermocycler BioRad iQ5 Real-Time PCR (Bio-Rad, CA, USA), and Universal SYBR(r) Green Supermix (Bio-Rad, CA, USA) was used as a fluorescent molecule. Each reaction was performed as follows: 500 ng of cDNA, 10 mL of Universal SYBR(r) Green Supermix, 150 nM of each forward and reverse primer pair (GAPDH, ACTB, and GPx), 250 nM of CAT and SOD genes, and nuclease-free water to reach a final volume of 20 mL. The thermal profiles were as follows: 95 degC for 3 min, 40 cycles of 95 degC for 15 sec, 59 degC (GAPDH, ACTB, GPx, CAT) or 58 degC (SOD) for 30 sec; a melting profile was included after the last amplification cycle to exclude the presence of non-specific amplifications. All data were normalized to the reference genes GAPDH and ACTB, and the relative gene expression of each target gene was calculated using the 2-DDCt method. 2.6. Statistical Analyses Statistical analyses were performed using SPSS 26.0 (SPSS Inc., Chicago, IL, USA). Values are expressed as means +- SD. The distribution of all data was firstly explored for their agreement with normal distribution using the Kolmogorov-Smirnov goodness-of fit test. Where data were not normally distributed, they were log-transformed, and retested for normality before analysis. The effect of treatment and tissue was analyzed by ANOVA. For all analyses, differences were considered significant at p < 0.05. 3. Results 3.1. Total Phenol Content and Antioxidant Activity of Bovine Colostrum and Experimental Diets The total phenol content (TPC) of BC was equal to 0.084 +- 0.005 mg GAE/mL, while DPPH, ABTS and ORAC assays gave results equal to 0.34 +- 0.05, 2.00 +- 0.13 and 3.92 +- 0.19 mmol/TE, respectively. Table 3 reports the TPC and antioxidant activities found in the three experimental diets (CON, BC-2.5 and BC-5). 3.2. Antioxidant Enzymes Activity in Plasma Antioxidant enzyme activity data are listed in Table 4. No significant differences in plasma CAT, GPx and SOD activities were found among dietary treatments (p > 0.05) (Table 4). 3.3. Antioxidant Enzymes Gene Expression The results of relative mRNA expression levels in liver and LD muscle are presented in Table 5. No significant differences were observed in terms of mRNA expression between CAT, GPx and SOD in the liver and LD muscle of the rabbits among the three groups . A significant tissue-related effect has been observed in terms of the mRNA levels. Indeed, mRNA levels were significantly higher in the LD than the liver for SOD (p = 0.022) and GPx (p = 0.001) enzymes . 4. Discussion Bovine colostrum (BC) has been widely studied for its antioxidant activity. It is rich in essential nutrients, such as proteins, fats, vitamins and minerals. Additionally, it contains high levels of bioactive compounds, including oligosaccharides, immunoglobulins, lactoferrin, and lysozyme , which contribute to passive immunity, antimicrobial protection, and to the development of the gastrointestinal system in the early life of calves . It is recognized to provide beneficial nutrients essential for postnatal growth . Due to the lack of knowledge about the potential of this product, the amount of BC produced in excess in dairy farms is usually discarded. Nowadays, researchers have investigated BC's potential as a product with health benefits, although the utilization of its potential needs to be more effective among the producers. Firstly, BC for human use has been utilized as a functional ingredient in several food categories, such as cheeses, yoghurts, ice cream, and beverages. There is also evidence that the biologically active proteins present in BC exert not only antibacterial and antiviral activity, but also improve peristalsis and regulate the work of the digestive tract . The positive effects of BC on the human body have also been described during antibiotic therapy; its administration at the beginning of therapy reduced diarrheal episodes and increased bacterial susceptibility to the antibiotic, thus reducing the drug dose. The use of BC is not confined to humans, but its supplementation as a nutraceutical for both production and companion animals of all ages has been documented. Concerning farm animals, the supplementation of BC in post-weaning piglets improved the GPx activity by 19% and decreased the production of MDA in blood, thus providing supplementary protection against oxidative stress . The dietary supplementation with 0.1% of BC for 40 weeks improved the function of the gut-associated lymphatic tissue of dogs, as higher fecal IgA levels and increased plasmatic responses against canine distemper virus vaccination were observed . The higher antioxidant capacity of colostrum than mature milk depends on presence of a higher level of anti-stress vitamins, such as vitamins A (retinol), E (tocopherol), and C, which are present at higher levels in colostrum than in normal milk . In the current study, antioxidant activities were evaluated by three different methods to perform a comparative overview. The most interesting results came from the ORAC assay, evidencing that BC inclusion in the supplement diet provides better antioxidant activity compared to CON samples. The highest values of Trolox equivalent indicate the sample's good ability to protect against the formation of peroxyl radicals, and, therefore, indicate a potentially high antioxidant activity. The antioxidant capacity of BC found herein agrees with the data present in the literature on bovine milk , being slightly higher than that reported in human milk ; the potential antioxidant activity of diets, as measured by ORAC, increases in the feed according to BC inclusion, confirming the appropriate incorporation of the BC inclusion in the complete diet. Antioxidants, which are divided in two classes, protect organisms from oxidative damage by free radicals . The enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) belong to the first category, while the second one is found in exogenous sources, such as dietary supplements . Enzymes such as SOD, CAT and GPx convert free radicals into safer compounds; briefly, SOD reduces superoxide to hydrogen peroxide, and CAT and GPx reduce hydrogen peroxide, via the superoxide dismutase reaction, to water. Bovine colostrum has been reported to improve serum SOD and GPx activities, while depressing MDA production in piglets . Likewise, the supplementation of BC to Holstein calves has determined an improvement in the antioxidant status, by increasing the SOD activity . In the present study, contrary to our expectations, we found that BC dietary supplementation had no effect on the plasma levels of CAT, GPx and SOD in the rabbits. Nevertheless, a significant tissue-related effect in mRNA levels of SOD and GPx has been found. To the best of our knowledge, the mechanisms by which BC could interact with the antioxidant enzymes are not clear. Up to now, the lack of literature data on antioxidant enzyme activity in rabbits supplemented with BC makes comparisons and considerations difficult. A numerical increase of GPx concentrations in the plasma of BC-2.5 group in comparison with the control animals has been observed; the result could be due to an initial activation of the antioxidant response. The activity of the two other main enzymes involved in the antioxidant defense mechanism, SOD and CAT, remained unchanged. We speculate that the weak activity of antioxidant enzymes is due to a sparing effect of dietary antioxidants. Alia et al. reported that, in presence of high concentrations of exogenous antioxidants in the circulatory system, a low demand for enzymatic antioxidant function is required. Mokhtarzadeh et al. found a significant increase (p < 0.01) in SOD serum concentrations in Japanese quails supplemented with BC (2% and 4%) for six weeks. In our study, dietary supplementation of BC failed to affect CAT, GPx and SOD gene expression levels in the liver and LD muscle of treated rabbits. We speculate that the similar content of phenolic compounds found in all the three experimental diets somehow affected the response of gene expression; the increase in enzyme activity and gene expression found in sheep likely resulted from a combined effect of the n-3 PUFA and the phenolic compounds in the administered perilla seeds. In the present study, all three diets showed similar polyphenol content, according to the responses of the relevant enzymes. In the organism, dietary compounds must pass several barriers during their metabolic path before being adsorbed. Furthermore, the various biotransformation processes they undergo reduce their bioavailability and accumulation in tissues . In rabbits, the cecum is the site of microbial fermentation, which permits the release of nutrients from ingested food, and the fermentation products are absorbed directly through the wall of the cecum or re-ingested as cecotrophs. The efficiency of the rabbit's digestion depends, primarily, on the production and ingestion of cecotrophes . Another possible explanation for the lack of alteration in antioxidant enzymes' gene expression in tissues could be due to the biotransformation processes that antioxidant compounds present in BC undergo, limiting their bioavailability. Under normal physiological conditions, the oxidation and antioxidant defense systems are in dynamic equilibrium. Our understanding of how cells maintain the capacity to respond to shifts in antioxidant challenges associated with shifting metabolic demands of healthy animals is incomplete, and remains a crucial issue of growth biology. However, the difference between chronic and oxidative stress has been investigated well , and it is known that oxidative stress promotes changes in metabolic balance and the efficiency of nutrient use . In the present study, no oxidative stress was induced. A hypothesis associated with growing animals is that an increased redox state (generation of more reactive oxygen species than can be reduced by enzymatic and non-enzymatic antioxidant systems) may result in sub-optimal growth . Growth performances of all three groups were in accordance with those of rabbits in trading conditions, with no significant effect (p > 0.05) of the dietary treatment on the final body weight (2.06 vs. 1.93 vs. 1.94 kg in CON, BC-2.5 and BC-5 respectively); moreover, in healthy animals, reactive oxygen species' production is counterbalanced by antioxidant defenses, and an imbalance between their generation and inactivation leads to oxidative stress . We speculated that no activation of antioxidant system enzyme has been found, since animals were not subjected to any oxidative stress challenges, and the rabbits remained healthy (absence of clinical signs, absence of lesions, shiny coat) throughout the study. Important variations in enzyme activity between organs or tissues are usual. For example, variations among SOD and/or CAT levels have been reported in rats by comparing brain, heart, kidney, liver, lung and spleen samples , in primates by comparing liver, brain and heart samples , and also in rabbit, comparing the liver, brain and testis . In the present work a significant tissue-related effect in mRNA level of SOD and GPx, has been found. The GPx was widely expressed in both tissues examined, but the highest expression was found in the liver, according to the fact that this organ is known to be a rich source of GPx . This antioxidant enzyme normally decreases and detoxifies the hydrogen peroxide and organic hydroperoxides; its mRNA expression is particularly sensitive to any changes in ROS accumulation . GPx activity being higher in the liver than the muscle agrees with a study conducted on rats by Zhang et al. , who found the following order of tissue expression: liver > kidney > heart > lung > brain = muscle. As a potential explanation, we speculate that, due to different oxygen consumption rates, different tissues express distinct antioxidant enzyme activities when subjected to the same treatment . 5. Conclusions This study was set out to determine whether increased levels of BC would alter the antioxidant response in rabbits. The results provided information for the first time, on the effect of BC on the antioxidant status of different tissues in rabbits. The antioxidant activity in BC seems to be confirmed. Anyway, the dietary inclusion of BC into diet produced an unchanged antioxidant defense system in rabbits. Based on this study's finding, the use of BC at concentrations of 2.5% and 5% did not influence the antioxidant status of the rabbits, since muscular and hepatic expression of the antioxidant enzymes did undergo significant changes. No detrimental or positive alterations have been also observed in terms of the concentrations of antioxidant enzymes in plasma. Certainly, a study considering some modifications of BC use could be performed, in order to better understand the effect of this potentially promising nutraceutical. Increasing doses, length of supplementation or addition with other factors that act synergistically with BC could be investigated. Author Contributions Conceptualization, M.C., G.B., G.P. and VS; formal analysis, V.S., G.P. and R.D.B.; investigation, G.B., D.V., M.C., V.S., G.P., C.M.B., G.C. and S.A.; data curation, V.S., G.P. and R.D.B.; writing--original draft preparation, V.S., G.P. and R.D.B.; writing--review and editing, V.S., M.C., S.A., G.C., D.V., A.D.G., S.C.M., F.R., C.M.B., R.D.B., G.B. and G.P.; visualization, V.S., M.C., S.A., G.C., D.V., A.D.G., S.C.M., F.R., C.M.B., R.D.B., G.B. and G.P.; supervision, G.P.; project administration, M.C. and G.B; funding acquisition, G.B. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethic Committee Name: Universita degli Studi di Milano, Approval Code: OPBA_42_2021, 7 May 2021. Informed Consent Statement Not applicable. Data Availability Statement The data presented in the current study are available from the corresponding authors on reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 mRNA expression levels of antioxidant enzymes CAT, GPx and SOD in the liver (a) and LD muscle (b) of rabbits fed diets supplemented with bovine colostrum. The data are reported as means +- SD. CON: group without supplemented diet; BC-2.5: group supplemented with 2.5% of bovine colostrum; BC-5: group supplemented with 5.0% of bovine colostrum; CAT: catalase; GPx: glutathione peroxidase; SOD: superoxide dismutase; SD: standard deviation. Figure 2 mRNA expression level of SOD and GPx in the liver and LD muscle of rabbits. GPx: glutathione peroxidase; SOD: superoxide dismutase; a,b Means within a row with different letters are significantly different at p < 0.05. animals-13-00850-t001_Table 1 Table 1 Ingredients (percentage wet weight) of control (CON) and experimental diets of post-weaning rabbits. Ingredients Basal Diet Experimental Diets CON BC-2.5 BC-5 Dehydrated alfalfa meal 32.40 32.40 32.40 Barley 22.40 19.90 17.40 Wheat bran 20.50 20.50 20.50 Sunflower meal 8.00 8.00 8.00 Soybean meal 4.00 4.00 4.00 Cane molasses 3.00 3.00 3.00 Carob pods 2.70 2.70 2.70 Wheat meal 2.50 2.50 2.50 Calcium Carbonate 2.00 2.00 2.00 Vitamin-mineral premix 1 1.60 1.60 1.60 Soybean oil 0.50 0.50 0.50 Sodium chloride 0.40 0.40 0.40 Bovine colostrum - 2.50 5.00 Chemical composition 2 Dry matter 92.34 91.71 91.69 Crude protein 14.82 14.76 15.23 Ether extract 2.79 2.95 3.02 Ash 7.04 7.23 7.62 NDF 40.00 36.81 35.79 ADF 27.04 24.92 24.31 ADL 12.02 10.03 9.11 CON: group without supplemented diet; BC-2.5: CON supplemented with 2.5% of bovine colostrum; BC-5: CON supplemented with 5.0% of bovine colostrum; NDF: neutral detergent fiber; ADF: acid detergent fiber; ADL: acid detergent lignin. 1 Contains, per kg of feed: retinyl acetate, 4800 IU; vitamin D3, 1800 IU; calcium-D pantothenate, 6.6 mg; choline chloride, 300 mg; ferrous sulfate monohydrate, 37.5 mg; manganous sulphate monohydrate, 54.0 mg; copper sulphate pentahydrate, 6.0 mg; zinc oxide, 36.0 mg; potassium iodide, 0.66 mg; sodium selenite, 0.12 mg; butylhydroxytoluene, 30.0 mg; butylhydroxyanisole, 30.0 mg.2 Analyzed. animals-13-00850-t002_Table 2 Table 2 Primers used for quantitative real-time PCR analysis of gene expression in the liver and L. dorsi muscle of this study. Gene Forward Primer Reverse Primer Tm F (degC) Tm R (degC) Amplicon Size (bp) GAPDH GTCAAGGCTGAGAACGGGAA TCTCCATGGTGGTGAAGACG 59.4 59.4 142 ACTB ACATGGAGAAGATCTGGCAC GCGTGTTGAACGTCTCGAAC 57.3 59.4 147 SOD TCGGGAGATATGTCCGTC GACACCACAGGCCAAACG 56.0 58.2 126 CAT GCTGAGATTGAACAGTTGGC GGTGAGTATCGGGATAGGAG 57.3 59.4 109 GPx CAGTTTGGGCATCAGGAGAAC GCATGAAGTTGGGCTCGAAC 59.8 59.4 94 GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ACTB: actin beta; SOD: superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase. animals-13-00850-t003_Table 3 Table 3 Total phenol contents and antioxidant activities of experimental diets, as measured using different methods (values are given as a mean +- SD of three independent measurements). CON BC-2.5 BC-5 p-Value TPC (mg GAE/g) 3.85 +- 0.15 4.09 +- 0.27 3.84 +- 0.08 ns DPPH (mmol TE/g) 11.13 +- 0.20 11.14 +- 0.19 11.16 +- 0.17 ns ABTS (mmol TE/g) 36.42 +- 1.84 34.88 +- 0.23 36.12 +- 0.42 ns ORAC (mmol TE/g) 113.00 +- 3.8 136.3 +- 4.5 150.70 +- 5.8 <0.001 CON: group without supplemented diet; BC-2.5: group supplemented with 2.5% of bovine colostrum; BC-5: group supplemented with 5.0% of bovine colostrum; GAE: gallic acid equivalent; TE: Trolox equivalent; TPC: total phenol content; DPPH: 2,2-diphenyl-1-picrylhydrazyl; ABTS: 2,20-azinobis(3-ethylbenzthiazoline-6-sulfonic acid); ORAC: oxygen radical absorbance capacity; SD: standard deviation; ns: not significant (p > 0.05). animals-13-00850-t004_Table 4 Table 4 Effect of increasing levels of bovine colostrum supplementation on plasma CAT, GPx and SOD enzymes (values are given as means +- SD). CON BC-2.5 BC-5 p-Value Plasma CAT (U/mL) 11.68 +- 1.86 9.82 +- 1.75 7.66 +- 2.11 0.347 Plasma GPx (U/mL) 196.21 +- 16.67 253.46 +- 33.39 237.27 +- 21.85 0.265 Plasma SOD (U/L) 91.72 +- 8.69 87.38 +- 7.83 77.82 +- 6.85 0.449 CON: group without supplemented diet; BC-2.5: group supplemented with 2.5% of bovine colostrum; BC-5: group supplemented with 5.0% of bovine colostrum; CAT: catalase; GPx: glutathione peroxidase; SOD: superoxide dismutase; SD: standard deviation. animals-13-00850-t005_Table 5 Table 5 mRNA relative quantification of the antioxidant enzymes CAT, GPx and SOD in the liver and LD muscle of rabbits (values are given as means +- SD). Liver LD muscle p-Value CON BC-2.5 BC-5 CON BC-2.5 BC-5 Diet Tissue CAT 2.067 +- 0.79 1.995 +- 0.56 1.786 +- 0.35 1.568 +- 1.08 2.659 +- 1.28 2.459 +- 1.18 0.237 0.257 GPx 1.439 +- 0.72 1.244 +- 0.61 0.997 +- 0.24 0.739 +- 0.31 0.806 +- 0.35 0.886 +- 0.31 0.606 0.001 SOD 1.384 +- 1.25 0.722 +- 0.15 0.657 +- 0.15 1.086 +- 0.40 1.315 +- 0.44 1.449 +- 0.37 0.471 0.022 CON: group without supplement diet; BC-2.5: group supplemented with 2.5% of bovine colostrum; BC-5: group supplemented with 5.0% of bovine colostrum; CAT: catalase; GPx: glutathione peroxidase; SOD: superoxide dismutase; SD: standard deviation. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000082

Data on 4487 Turkish Saanen kids from 176 bucks and 1318 dam-goats, obtained from the Turkish Saanen goats in the Izmir region and collected between 2018 and 2019, were analyzed to examine the effect of genetic and non-genetic factors on growth traits. The average birth weight of the kids was determined as 3.33 +- 0.68 kg, the average W60 was 13.06 +- 2.94 kg, the average WW was 18.38 +- 4.14 kg, and the average PreWDG until weaning was 0.17 +- 0.04 g. Model 1, which does not account for the maternal effect, and Model 2, which includes the maternal effect, were used in the estimation of genetic parameters. The heritability estimates of BW, W60, WW, and PreWDG ranged from 0.05 to 0.59 in both models. It is recommended to consider the maternal effect as well as the environmental factors in the selection program for the best early breeder selection of kids growing alongside their mothers until the weaning period. Turkish Saanen growth traits maternal genetic effect heritability This research received no external funding. pmc1. Introduction The Turkish Saanen breed was developed by crossing the Saanen breed with native Turkish breeds (e.g., Saanen x Hair, Saanen x Kilis). Turkish Saanen goats are known for their high milk production and fertility efficiency and are commonly used in goat husbandry in Turkey, especially in Western Anatolia. The male kids are generally marketed for meat . As a result, growth characteristics of goats are important biological factors that affect the sustainability and profitability of businesses, and these are economically significant for early breeding of breeding goats. In addition, improving growth performance is an important way of increasing meat output in a kid production system . In particular, body weight and number of kids at birth are known to be associated with environmental factors as well as genetic traits . The potential for genetic improvement is dependent largely on the heritability of the trait measured and its relationship with other traits of economic significance . Birth weight is an important parameter of economic significance in animals due to its high and positive correlation with growth rate, weaning weight, and adult live weight, as well as its effect on the viability of newborn animals . For this reason, it is necessary to examine the growth and morphometric characteristics of goats in order to evaluate their growth potential more accurately. Thus, profitability can be increased in goat breeding by incorporating these characteristics into breeding programs. In other words, to provide the development of an optimal breeding program, knowledge of parameters for growth traits is vital. On the other hand, by revealing the effect of genetic and environmental factors on growth, a more accurate estimation of adult live weight is possible, and rapid genetic progress can be achieved in the herd with the application of selection in this direction . Accurate performance evaluation and estimation of genetic parameters across various traits are prerequisites for creating successful selection strategies and breeding programs . In addition, genetic and maternal effects on pre-weaning growth characteristics are important in defining variation among individuals . Although maternal effects reflecting maternity ability are more important in the early stages of life, they have effects that can be transferred to later periods of life . Especially in the period from birth until weaning, keeping the kids with their mothers makes the maternal effect even more impactful. Therefore, there are many studies that demonstrate that the maternal effect should be taken into account in order to reach more reliable genetic predictions in selection programs . Although there are studies on the genetic parameters and factors affecting the growth characteristics before and after weaning in goats, there are not enough studies on the genetic parameters of growth characteristics in Turkish Saanen kids. The goal of this study was to determine the most appropriate time, accounting for maternal effects, to determine early selection based on the growth characteristics of Turkish Saanen kids. Therefore, genetic parameters for pre-weaning growth traits in Turkish Saanen kids were estimated. Based on their effects on pre-weaning growth traits, herd, gender, dam age, type of birth and birth year, and maternal genetic effect were all considered. 2. Materials and Methods 2.1. Animals and Experimental Procedure The dataset collected from the Turkish Saanen Goat subproject in the Izmir region as part of the National Sheep and Goat Improvement Project was examined in this study. A total of 4487 Turkish Saanen kids, including 176 bucks and 1318 dams, were studied. Growth trait records of Turkish Saanen kids were collected from 15 herds between the years 2018 and 2019. Birth weight (BW), 60th day live weight (W60), weaning weight (WW), and pre-weaning daily live weight gain (PreWDG) were employed as the growth traits in kids. The kids were fed alongside their mothers until weaning and were weaned on the 90th day. 2.2. Statistical Analyses Herd, gender, type of birth, age of dam, and birth year effects on BW, W60, WW, and PreWDG of Turkish Saanen kids were analyzed. Analyses of variance components and heritability were carried out for BW, W60, WW, and PreWDG records. For this purpose, the model is structured with herd, gender, age of dam, type of birth, and birth year being the fixed effects; BW as a covariate effect for W60, WW, and PreWDG; and age of dam also as a covariate effect for BW. Animals' additive genetic, maternal genetic, and residual effects were considered to be random effects. Descriptive statistics of the growth traits of Saanen kids are given in Table 1. BW, W60, WW, and PreWDG were 3.33 +- 0.68 kg (CV = 20.51%), 13.06 +- 2.94 kg (CV = 22.51%), 18.38 +- 4.14 kg (CV = 22.51%), and 0.17 +- 0.04 g (CV = 26.89%), respectively (Table 1). The following single trait and single record animal models were used:Y = Xb + Za + e(1) Y = Xb + Za + Zm + e(2) where, Y: a vector of BW, W60, WW and PreWDG; b: a vector of herd, gender, type of birth, age of dam, and birth year as fixed effects; BW as a covariate for W60, WW, and PreWDG; and age of dam also as a covariate effect for BW; a: direct additive genetic effects; m: maternal genetic effects; e: residual effect, X, and Z are incidence matrices relating observations to b, a, and m, respectively. The estimates of variance components for each trait were estimated using the Average Information-Restricted Maximum Likelihood (AI-REML) method by Meyer . 3. Results For all growth traits, the effects of herd, gender, type of birth, and birth year were found to be significant (p < 0.01). On the other hand, the effects of birth type on PreWDG were not significant (p = 0.39). Therefore, all effects were included in the model to obtain estimates of variance components. When the flocks were examined, the average BW was 3.26 +- 0.68 kg, the highest BW was 3.63 kg in the 6th herd, and the lowest BW was in the 10th flock at 2.74 kg. It was observed that W60 averaged 13.06 +- 2.94 kg and ranged between 8.53-16.04 kg, and WW averaged 18.38 +- 4.14 kg and varied between 11.64-21.65 kg. The average daily live weight gain was 0.17 +- 0.05 kg. Male kids were found to be heavier than female kids in all growth stages (p < 0.01). It was observed that twin births were the most common in Turkish Saanen kids, followed by singleton and triplet births, and quadruplet births had the lowest rate of 3%. The weights of single-born kids were found to be higher than those of multiple-born kids in all growth periods. For BW, 5-year-old mothers were found to have heavier kids than mothers of other ages, and for W60 and WW, kids of 7-year-old mothers were found to be heavier. The effect of the year of kids on live weights was found to be higher and more significant in 2018 than in 2019 (Table 2). Variance components of BW, W60, WW, and PreWDG traits that were affected by herd, gender, birth type, the age of dam, and birth years were estimated. The additive, phenotypic, residual variances and heritability estimations are given in Table 3. In Model 1, the estimates of additive genetic variance were the lowest for BW (0.02). The additive genetic variance estimates increased rapidly by age--they reached 2.77 for WW. Phenotypic variance estimates have also shown the same tendency as the additive genetic variances; while the variance was 0.43 in the BW, it was estimated to be 4.70 in the WW. In this research, major differences have been identified between the heritability estimations obtained from BW to WW for different ages. Estimates of heritability were found to be 0.05 +- 0.02, 0.57 +- 0.002, 0.59 +- 0.04, and 0.33 +- 0.002 for BW, W60, WW, and PreWDG, respectively, in Model 1. In the present study, the maternal effect was not included in Model 1 of Turkish Saanen goats for all growth traits, and additive genetic variances ranged from 0.02 to 2.77. Additive genetic variances ranged from 0.02 to 0.43 in Model 2, in which the maternal genetic effect was also considered. Moreover, maternal genetic variances ranged from 0.001 to 0.22 in Model 2. Additive genetic variances and, therefore, direct heritability estimates caused overestimation--the heritability estimates obtained in the present study were 0.57 +- 0.002 and 0.59 +- 0.04 for W60 and WW in Model 1, respectively. The heritability estimate for PreWDG was lower with the maternal model than the direct animal model. PreWDG heritability estimates were lower in Model 2 (0.22 +- 0.001), where the maternal effect was better accounted for than in Model 1 (0.33 +- 0.002). 4. Discussion Many studies have been conducted to determine the factors that influence live weight in kids at various stages of development. Thiruvenkadan et al. stated that the effect of birth year, birth season, birth type, and gender are important. According to Mio et al. , mean daily body weight gain and WW weight are significantly higher (p < 0.01) in males than in females. Birth type was determined as effective on BW (p < 0.01); weaning age with WW as effective on daily body weight gain (p < 0.05), gender (excluding BW (p < 0.01)), WW (p < 0.05), and daily body weight gain (p < 0.01); and the season of birth as effective on BW, WA, and daily live weight gain (p < 0.01), excluding WW. Supakorn and Pralomkarn found that the effect of birth year, birth type, and gender on WW was significant (p < 0.05). Atoui et al. reported that birth year, birth type, gender, maternal age, and maternal weight had a significant (p < 0.05) effect on BW. Alade et al. discovered the effects of genotype, birth type, and gender to be significant in all growth periods (p < 0.01). Cappai et al. investigated the interpretation of the metabolic profile of the transition goat raised in an extensive farming system. They found that metabolic patterns related to pre-partum, post-partum, and single - twin gestation. In this study, singletons and males had significantly higher body weights compared to multiples and females at all growth stages (p < 0.01). These results were found to be similar to the study by Tozlu and Olfaz , which reported that the effects of genotype, birth type, and gender on live weights at 30, 75, and 180 days were significant. On the other hand, it was stated that only genotype had a significant effect (p < 0.05) on the daily live weight gains of kids between birth and after 30 days, the viability of kids at the age of 30 days was significantly affected by gender, and the viability of males was higher than that of females (p < 0.01). Ghimir et al. found the effect of genotype and gender on live weight to be significant (p < 0.05). They did not, however, find any significant effect of dam age or season on body weight or daily body weight gain at any period (p > 0.05). Similarly, they reported that WW and PreWDG were not affected by any factor. According to Alade et al. , Akdag et al. , and Tozlu and Olfaz , male kids were significantly heavier than females (p < 0.01) among singletons compared to those born with multiples. They found that kids born to mothers aged five years had the highest BW (p < 0.01). According to Atoui et al. , fixed effects, such as gender, species, and age of dam, which have significant effects on the body weight selection to be applied, should always be taken into account. The estimates of heritability for BW, W60, WW, and PreWDG in our current study ranged from 0.05 to 0.59. These results were higher than those reported by Rashidi et al. for Markhoz goats (0.02), Kasap et al. for Saanen goats (0.04), and Menezes et al. for Boer goats (0.01). Moreover, our estimate of direct heritability for WW was higher than that estimated by Rashidi et al. for Markhoz goats (0.03), Maghsoudi et al. for Iranian Cashmere goats (0.07), and Mokhtari et al. for Raeini Cashmere goats (0.15). On the other hand, Otuma and Osakwe found the estimated heritability values of BW and body weight at 90 and 360 days to be 0.41 +- 0.08, 0.45 +- 0.31, and 0.45 +- 0.28, respectively. They claimed that by selecting individuals with high body weights at a young age, they could achieve higher body weights at weaning age. According to Thiruvenkadan et al. , the heritability of live weight tends to increase from birth to age 160 days. Heritability estimates for pre- and post-weaning weight gain were found to be 0.29 +- 0.12 and 0.39 +- 0.17, respectively. Onder et al. estimated the genetic parameters of sex and birth type, which are thought to be effective on live weights from birth to 180 days of age, and found the genetic variance to be 0.14 and heritability to be 0.27 for BW. It was stated that the selection to be made for any of these traits will also result in significant improvement for other traits. Kuthu et al. found heritability estimates for BW, W60, WW, and PreWDG of 0.28 +- 0.23, 0.26 +- 0.44, 0.23 +- 0.32, and 0.21 +- 0.32, respectively. They reported that the growth traits considered in selection should have low to moderate heritability and the growth traits should be based on estimated breeding values in selecting the animals that are future parents of the herd. Jawasreh et al. determined their heritability estimates as 0.30 +- 0.04 for BW, 0.19 +- 0.04 for WW and PreWDG, and as 0.2 +- 0.04 for WW. Tesema et al. reported their h2 estimates for BW, WW, and PreWDG as 0.38, 0.12, and 0.09, respectively. Kosum et al. found heritability for BW and WW as 0.43 and 0.05, respectively. Gunia et al. estimated heritability for live weight of Capricorn goats at 70 days and 11 months as 0.20 and 0.32, respectively. In their study, Rashidi et al. estimated direct heritability to be at a moderate level of 0.21 for daily live weight gain, 0.22 for BW, and 0.16 for WW. Dashtizadeh et al. estimated heritability as 0.55, 0.18, 0.47, and 0.43; Oseni and Ajayi as 0.50-0.59, 0.14-0.4, 0.29, and 0.11, for BW, WW, and live weight at 90 and 180 days, respectively; and Mohammadi et al. found values of 0.22, 0.25, and 0.29 for BW, WW, and W180. Bhattarai et al. determined the heritability estimate for BW as 0.37 +- 0.12. In the present study, heritability estimates in growth traits were found to be higher in Model 1, in which maternal effect was not taken into consideration. On the other hand, in Model 2, where maternal effect was also included, estimates were observed to be lower. Therefore, model 2 resulted in lower, and possibly more realistic, variance components. Maternal effects play a significant role in the expression of pre-weaning traits. As a result of this, it is important to know the extent of both maternal genetic and environmental effects, in addition to the additive genetic effects. Hammoud and Salem reported that maternal effects should be taken into consideration when carrying out genetic evaluations of pre-weaning growth traits. Similarly, Magotra et al. highlighted the considerable role of maternal effects on early growth traits. Supakorn and Pralomkarn found direct heritability estimates to be 0.44 and 0.51, and maternal heritability estimates as 0.15 and 0.16 for BW and WW, respectively, in kids of four different goat species. Supakorn and Pralomkarn recorded direct heritability estimates as 0.26 and 0.38, and maternal heritability estimates as 0.09 and 0.12 for BW and WW, and highlighted that the most suitable model to achieve rapid progress in WW in the herd would be one that included maternal genetic effect and excluded the direct maternal genetic covariance. Bedhane et al. found direct additive heritability, additive maternal heritability, and maternal environmental effects values of 0.04-0.39 and 0.02-0.08, 0.09-0.20 and 0.03-0.07, and 0.12-0.21 and 0.05-0.09, respectively. They reported additive genetic variance, maternal permanent environmental variance, and phenotypic variances for BW and WW as 0.02-0.05 and 0.09-0.22, 0.03-0.04 and 0.15-0.26, and 0.21-0.26 and 2.49-2.70, respectively. They stated that maternal environmental effects and maternal permanent environmental effects were critical variation sources for body weights of young kids. In addition, they suggested considering the non-genetic effects on growth traits in any breeding program. In the study in which they estimated the variance components and genetic parameters for birth and weaning weights, Gholizadeh et al. found the maternal effect on the examined traits to be significant and suggested considering maternal effects in a future selection program. Barazandeh et al. estimated genetic parameters of effective factors on BW, WW, and PreWDG, and reported that the model including additive direct genetic and permanent maternal environmental effects was the most suitable. Bangar et al. and Bangar et al. obtained low levels of heritability estimates for BW and WW. When compared with our study, these findings are similar in terms of BW, but higher than our findings for WW. Tesema et al. recommended that both the direct additive genetic effect and maternal effects should be considered as environmental variation sources. Schoeman et al. examined heritability estimates and the variance components of the factors that are thought to be effective on BW and WW and found that lower but more accurate variance components were obtained when maternal genetic effects were included in the individual model. Roy et al. used models in which maternal genetic or maternal permanent environmental effects were excluded and included. According to the results of the study, heritability estimates were higher for all traits when maternal effects were ignored. They reported heritability estimates for BW and WW to be 0.12 and 0.18, respectively. Based on the heritability estimates obtained from the present study, it is possible to select kids prior to weaning for breeding. 5. Conclusions In conclusion, the effects of environmental factors such as herd, birth year, gender, birth type, and age of dam were significant for all growth traits in Turkish Saanen kids. At the same time, the maternal effect is of great importance due to its effect on the growth characteristics of Turkish Saanen kids. As a result of comprehensive knowledge of these traits, it is possible to design the successful inheritance of economically significant traits in goat breeding. This information is considered an important step in the planning and implementation of any successful selection or breeding program aimed at improving the genetic gain of animals. While Turkish Saanen goats, of which there is a substantial population, are used for milk production, some of the offspring born are used for breeding and a significant amount are used for butchery. Therefore, it is recommended that early selection that considers environmental factors as well as maternal effects at the pre-weaning age may be delivered for genetic progress in Turkish Saanen kids growing with their mother from birth to weaning. This effect should also be considered in fertility programs. In addition to these, further studies examining the primary effect at the F2 level are required. Acknowledgments We would like to thank the Republic of Turkey Ministry of Agriculture and Forestry General Directorate of Agricultural Research and Policies National Sheep and Goat Improvement Project (Project Code 35 TSK 2015-01) for supporting this research and providing this dataset. Author Contributions The study planning and methodology, F.E.A.; collection of data F.E.A.; data analysis, F.E.A., C.T. and Y.G.; preparation and writing--review and editing F.E.A., C.T., Y.G., S.O.A. and T.A. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement In this study, there was no need for ethical approval due to the lack of blood sampling from the animals and the absence of any surgical procedures. Data Availability Statement All datasets collected and analyzed during the current study are available from the corresponding author on fair request. Conflicts of Interest The authors declare no conflict of interest. animals-13-00940-t001_Table 1 Table 1 Descriptive statistics of the growth traits of Turkish Saanen kids. Traits Mean Std. Deviation Coefficient of Variation (%) BW 3.33 0.68 20.51 W60 13.06 2.94 22.51 WW 18.38 4.14 22.51 PreWDG 0.17 0.04 26.89 BW: birth weight; W60: weight at 60th day; WW: weaning weight; PreWDG: pre-weaning daily gain. animals-13-00940-t002_Table 2 Table 2 Analysis of variance of non-genetic affecting growth traits in Turkish Saanen kids. Factors n (BW) (kg) W60 (kg) WW (kg) PreWDG (g/day) 3.26 +- 0.68 13.06 +- 2.94 18.38 +- 4.14 0.17 +- 0.05 Herd 1 588 3.42 +- 0.03 ab 10.18 +- 0.10 f 14.72 +- 0.14 h 0.13 +- 0.002 g 2 430 3.62 +- 0.03 a 12.75 +- 0.11 cd 16.60 +- 0.17 g 0.14 +- 0.003 f 3 44 2.99 +- 0.10 def 8.53 +- 0.36 g 11.64 +- 0.52 i 0.10 +- 0.006 h 4 23 3.55 +- 0.13 a 11.21 +- 0.50 def 17.24 +- 0.72 fg 0.15 +- 0.008 cdefg 5 141 3.34 +- 0.05 abc 12.29 +- 0.20 de 18.80 +- 0.29 bcde 0.17 +- 0.003 bcde 6 65 3.63 +- 0.08 a 11.60 +- 0.30 e 16.03 +- 0.43 g 0.14 +- 0.005 fg 7 245 3.08 +- 0.04 cde 13.28 +- 0.15 bc 18.46 +- 0.22 def 0.17 +- 0.003 bcde 8 44 3.21 +- 0.10 bcd 12.96 +- 0.36 bcde 16.82 +- 0.52 g 0.15 +- 0.006 ef 9 115 3.43 +- 0.06 ab 9.79 +- 0.22 fg 14.74 +- 0.32 h 0.13 +- 0.004 g 10 134 2.74 +- 0.06 g 12.29 +- 0.21 de 19.02 +- 0.30 cd 0.18 +- 0.004 bc 11 225 3.40 +- 0.04 ab 13.29 +- 0.16 bc 21.65 +- 0.23 a 0.20 +- 0.003 a 12 56 2.89 +- 0.09 a 16.04 +- 0.32 a 19.87 +- 0.46 c 0.19 +- 0.002 ab 13 1051 3.53 +- 0.02 bcde 15.23 +- 0.07 a 21.23 +- 0.11 b 0.19 +- 0.002 a 14 654 3.16 +- 0.03 cde 13.81 +- 0.09 b 18.68 +- 0.14 d 0.17 +- 0.002 bcd 15 668 3.09 +- 0.02 12.61 +- 0.09 de 18.20 +- 0.13 ef 0.17 +- 0.02 de p-value 0.01 0.01 0.01 0.01 Gender Male 2046 3.39 +- 0.04 a 13.08 +- 0.14 a 18.71 +- 0.21 a 0.17 +- 0.002 a Female 2441 3.12 +- 0.04 b 12.47 +- 0.14 b 17.73 +- 0.21 b 0.16 +- 0.002 b p-value 0.01 0.01 0.01 0.01 Type of birth Single 1099 3.61 +- 0.04 a 13.32 +- 0.14 a 18.80 +- 0.21 a 0.17 +- 0.002 Twin 2482 3.28 +- 0.04 b 12.99 +- 0.13 ab 18.53 +- 0.20 ab 0.17 +- 0.002 Triplet 765 3.05 +- 0.04 c 12.49 +- 0.15 ab 17.99 +- 0.22 b 0.16 +- 0.002 Quadruplet 141 3.08 +- 0.06 c 12.36 +- 0.22 b 17.57 +- 0.34 b 0.16 +- 0.004 p-value 0.01 0.01 0.01 0.39 Age of dam 1 639 3.19 +- 0.04 a 12.81 +- 0.13 a 18.11 +- 0.19 ab 0.16 +- 0.002 cd 2 1315 3.22 +- 0.03 bc 12.55 +- 0.11 c 18.03 +- 0.17 c 0.16 +- 0.002 de 3 977 3.28 +- 0.03 b 12.31 +- 0.11 bc 17.56 +- 0.17 c 0.16 +- 0.002 e 4 835 3.24 +- 0.03 bc 12.46 +- 0.12 bc 17.59 +- 0.18 bc 0.15 +- 0.002 de 5 364 3.30 +- 0.04 bc 12.70 +- 0.15 bc 18.51 +- 0.22 a 0.17 +- 0.002 abc 6 173 3.19 +- 0.05 cd 12.49 +- 0.19 ab 17.95 +- 0.28 a 0.16 +- 0.003 ab 7 104 3.27 +- 0.07 bcd 13.14 +- 0.23 abc 19.20 +- 0.35 a 0.18 +- 0.004 a 8 80 3.25 +- 0.08 d 12.78 +- 0.28 abc 17.71 +- 0.42 abc 0.16 +- 0.005 bcde p-value 0.01 0.01 0.01 0.01 Birth year 2018 1987 3.37 +- 0.04 a 13.99 +- 0.15 a 19.62 +- 0.22 a 0.18 +- 0.002 a 2019 2500 3.14 +- 0.04 b 11.60 +- 0.14 b 16.83 +- 0.21 b 0.15 +- 0.002 b p-value 0.01 0.01 0.01 0.01 Values with different letters in the same row are different (p < 0.01). animals-13-00940-t003_Table 3 Table 3 Variance components and heritability values of growth traits. Model 1 Traits sa2 sp2 se2 ha2 +- S.E. BW 0.02 0.43 0.41 0.05 +- 0.02 W60 0.42 0.73 0.31 0.57 +- 0.002 WW 2.77 4.70 1.92 0.59 +- 0.04 PreWDG 0.31 0.94 0.62 0.33 +- 0.002 Model 2 Traits sa2 sp2 se2 sm2 ha2 +- S.E. hm2 +- S.E. BW 0.02 0.43 0.41 0.001 0.05 +- 0.02 0.002 +- 0.018 W60 0.43 0.86 0.32 0.13 0.50 +- 0.005 0.13 +- 0.002 WW 0.11 1.05 0.70 0.22 0.11 +- 0.003 0.22 +- 0.002 PreWDG 0.10 0.94 0.62 0.22 0.11 +- 0.001 0.22 +- 0.001 BW: birth weight; W60: weight at 60th day; WW: weaning weight; PreWDG: pre-weaning daily gain; sp2: phenotypic, sa2: additive genetic, sm2:maternal genetic, and se2: residual variances, ha2: heritability of direct additive; hm2: heritability of maternal genetic effects; and S.E.: standard errors. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000083

Large-scale pig farming is associated with the production of large amounts of animal excrement, which, after processing into the form of, e.g., slurry, are managed on agricultural land as natural fertilizers. The utilization of pig manure on agricultural land in an excessive and uncontrolled manner may pose a threat to zoonoses due to the significant amounts of potentially pathogenic microorganisms within its content. This study aims to determine the impact of the methane fermentation process carried out in two agricultural biogas plants on the efficiency of sanitization of pig slurry, input biomass, and digestate. The biogas plants differed in terms of the substrate used; one used pig slurry from a maternal (breeding) farm (BP-M), and the other utilized pig slurry from a fattening farm (BP-F). The physicochemical analyses showed that the slurry, input biomass, and digestate from the BP-F were characterized by a significantly higher contents of organic dry matter, ash, and ammonium nitrogen than the slurry, input biomass, and digestate from the BP-M. The parameters of the methane fermentation process, including temperature and pH, reached higher values in the BP-F compared to the BP-M. The microbiological analyses led to the conclusion that the efficiency of sanitization of input biomass, including pig slurry, was significantly higher in the BP-F compared to the BP-M. Due to the above findings, locating biogas plants near pig fattening farms should be recommended. biogas plants biomass sanitization pig slurry methane fermentation This research received no external funding. pmc1. Introduction Continuous, intensive pig production is associated with a significant concentration of a large number of animals in one place and, thus, with the production of large amounts of slurry, which harms the environment. In this context, Buelna et al. estimated that the average amount of slurry produced by one animal per day is about 7% of its body weight. This translates into 150 million tons of pig slurry produced annually in Europe . The management of slurry is mainly based on its use as fertilizer in large agricultural areas. It should be emphasized that such use of slurry can be microbiologically unsafe. Pig slurry contains a high load of microorganisms, especially of fecal origin (natural intestinal microbiota of animals), including saprophytic bacteria and pathogens that are dangerous to humans and animals. Due to the large amounts of urea, slurry also contains ammonifying bacteria-producing urease, e.g., bacteria of the genera Pseudomonas, Proteus, and Azotobacter . Although slurry from healthy animals does not contain significant amounts of absolute pathogens, opportunistic pathogens constitute a significant part of the natural intestinal microbiota . The most abundant bacteria in slurry are Enterobacteriaceae, in particular various strains of Escherichia coli, including the O157:H7 serotype . In addition, bacteria of the genera Klebsiella, Enterobacter, Citrobacter, and Salmonella, including the serotypes S. Enteritidis and S. Typhimurium, and occasionally Shigella spp., are also isolated from this material . Hafnia spp. and Proteus spp. , as well as Campylobacter spp. and Yersinia spp. , may also be present in relatively high amounts in slurry. Streptococci, staphylococci, and Enterococcus spp. are other large groups of relatively pathogenic bacteria inhabiting slurry . Moreover, the ratio of isolation of antibiotic-resistant strains, including dangerous vancomycin-resistant enterococci (VRE), is constantly growing . Other Gram-positive bacteria isolated from pig slurry include Bacillus spp., Corynebacterium spp., Clostridium spp., and Listeria spp. . Anaerobic digestion is increasingly used worldwide to generate energy from biogas and brings significant economic and environmental benefits . Biogas (also called landfill gas) is a combustible mixture of gases produced by fermentation and putrefaction of stored organic wastes in anaerobic conditions. Biogas is an environmentally friendly, renewable energy source . China is currently the largest producer of biogas in Asia (and globally), along with Germany being the largest producer in Europe, and the USA being the largest producer in North America . The list of raw materials that can be used for biogas production is extensive, ranging from sewage sludge and animal feces, through plant biomass, to wastes from the agricultural and food industry and slaughterhouse wastes. The direct by-product of methane fermentation is referred to as a fermentation residue or a digestate. It is a homogenized biomass with good fertilizing properties. Due to the high degree of mineralization caused by microorganisms, post-fermentation residues are a better fertilizer than, for example, raw slurry . Furthermore, the use of post-fermentation biomass as a fertilizer significantly reduces the emission of greenhouse gases and odors. It is also worth noticing that the methane fermentation process reduces the number of bacteria found in the substrates used for biogas production, including zoonotic pathogens, which are harmful to humans and animals . Currently, numerous factors are known that determine the rate and efficiency of bacterial elimination from biomass involved in methane fermentation, e.g., temperature, fermentation time, pH, concentration of volatile fatty acids, type of fermentation, and factors related to natural antagonism and competition between different species of bacteria . Previously published reports on the effectiveness of sanitization of substrates used in the biogas production process included the level of reduction of various groups of microorganisms . However, based on a thorough review of the literature, no reports on the reduction of microorganisms depending on the type of slurry from different pig farms were found. Nevertheless, the potential impact of different types of substrates, including different types of slurry (produced by sows with piglets and produced by fatteners), on the efficiency of biomass sanitization has not been clarified. Presumably, the different chemical compositions of slurry, which are the results of different feeding and the use of different prevention and treatments of both production groups of pigs, may affect the degree of biomass sanitization. Therefore, the current study aimed to determine the effect of different types of pig slurry on the efficiency of biomass sanitization in agricultural biogas plants. 2. Materials and Methods 2.1. Biological Samples The biological samples included pig slurry, input biomass, and post-fermentation biomass collected from two agricultural biogas plants located at two large-scale pig farms in Poland. The pig slurry produced by the farms was used as a substrate, while co-fermentation with the addition of plant biomass was used to stabilize it. The installations were built with the same technology and applied in correspondence to the technical solutions throughout the production process. One biogas plant was located at a fattening farm (this biogas plant is referred to as the "BP-F" in this paper), and the other was located at a maternal farm with sows and piglets (this biogas plant is referred to as the "BP-M"). Thus, the biogas plants constituted an experimental system that differed in the type of pig slurry used. 2.2. Biogas Plant Characteristics The BP-F included the following characteristics:A preliminary tank with a capacity of 962 m3, in which slurry from the farm was stored. A component tank with a capacity of 962 m3. A fermentation tank with a capacity of 3990 m3. A post-fermentation tank with a capacity of 2490 m3, which was also a biogas storage. The BP-F was located at a fattening farm where pigs weighing from 24 to 114 kg were kept. During the research period, the average annual production of the farm was 39,500 pigs. There were 16 livestock buildings (piggeries) on the fattening farm, with a total of 13,480 fattening places. The fattening pigs were kept in a bedding-free system (without straw), in group pens varying in size. Four piggeries had 1700 places for fatteners, another four had 750 places, and the remaining eight had 460 places. The fattening pigs were fed with granulated feed mixtures. In total, three different feed mixtures supplemented with exogenous amino acids were used (Table 1). The annual nitrogen load from the feed was close to 250,000 kg. The slurry was delivered from the fattening farm to the biogas plant in the amount of approx. 31,500 tons per year. In addition to the slurry, approximately 9000 tons of corn silage were used in the biogas production process every year. The biogas plant operated a two-stage, thermophilic methane fermentation. Its estimated capacity was approximately 2,500,000 m3 of biogas per year. The BP-M included the following characteristics:A preliminary tank with a capacity of 950 m3, in which slurry from the farm was stored. Two stirred fermentation tanks, both with a capacity of 3884 m3. A post-fermentation tank with a capacity of 2490 m3, which was also a biogas storage. The BP-M used slurry from a maternal farm where sows with piglets were kept. The foundation herd consisted of 1300 sows and 350 gilts. The annual production on this farm was over 47,500 piglets weighing 6.5 kg. The farm consisted of 6 piggeries with 1919 places for sows, including 396 farrowing pens for sows with piglets. In addition, one of the pig houses had 480 places for weaned piglets. Sows were housed in group pens (pregnant section) and individual pens (mating and farrowing section). Weaned piglets were kept in group pens. These animals were kept in a bedding-free system. Throughout the production cycle, six types of granulated feed mixtures supplemented with exogenous amino acids were used (Table 1). The annual nitrogen load from the feed was close to 57,000 kg. Approximately 13,000 tons of manure per year were used to produce biogas. The other substrates included maize and grass silage in the amount of 18,000 tons per year. The biogas plant operated a two-stage, thermophilic methane fermentation with an annual biogas production of approximately 4,015,000 m3. 2.3. Biological Sample Collection A total of three samples were taken from each biogas plant at intervals of one month, including one sample of raw slurry and two samples of biomass. The first biomass sample was taken before the fermentation tank (before participation in the methane fermentation process carried out by a symbiotic consortium of microorganisms), and the second one was taken after the post-fermentation tank (after the process was completed). The sample of biomass taken before the fermentation tank was a mixture of pig slurry, various types of silage (corn or maize and grass, depending on the biogas plant), and recirculation from the post-fermentation tank. The sample collected after the post-fermentation tank constituted fermented biomass. An indicative diagram of the sampling locations in the context of biomass circulation in both biogas plants is presented in Figure 1. The samples were taken from specially adapted discharge taps to sterile containers with a capacity of 500 mL. To ensure the representativeness of the samples, the cocks were rinsed with biomass in the amount of at least 5 L before collection. The collected samples were transported in containers with cooling inserts to the laboratory, where they were stored at 4 degC and analyzed within 24 h. In total, 15 collection cycles were performed, which gave a total of 90 biological samples. 2.4. Parameters of the Methane Fermentation Process The parameters of the methane fermentation process were constantly monitored and were made available by the biogas plants' laboratories. The parameters included the following:Daily average temperature; pH in fermentation chambers; Hydraulic retention time (HRT); Concentration of volatile fatty acids (VFA) in fermenting biomass. 2.5. Physicochemical Analyses Analyses of the basic chemical composition of the slurry and biomass samples were performed following the AOAC Official Analytical Methods (2017) . 2.5.1. Contents of Dry Matter, Dry Organic Matter, and Crude Ash The content of dry matter (DM, the mass remaining as a result of water evaporation from a fresh mass sample) of each sample was determined by drying a given biomass portion placed in a ceramic crucible at 105 degC until a constant weight was obtained at a laboratory dryer (Memmert, Buchenbach, Germany) . The DM for each sample, expressed as % of fresh weight, was calculated using Equation (1):(1) DM=(W2-W0)W1-W0x100 where W0-the weight of the empty, dry crucible [g]; W1-the weight of the crucible with the sample weight before drying [g]; W2-the weight of the crucible with the sample weight after drying [g]. The dry samples were then used for the determination of crude ash and organic dry matter. For this purpose, the dried samples were burned at 550 degC in a muffle furnace (Czylok, Jastrzebie-Zdroj, Poland) until a constant weight was obtained. Crude ash (CA) for each sample was calculated according to Equation (2):(2) CA=(W3-W0)W1-W0x100 [%] where W0-the weight of the empty, dry crucible [g]; W1-the weight of the crucible with the sample weight before drying [g]; W3-the weight of the crucible with the sample weight after combustion [g]. Dry organic matter (DOM) for each sample was calculated according to Equation (3):(3) DOM=(W2-W3)W2-W0x100 [% DM] where W0-the weight of the empty, dry crucible [g]; W2-the weight of the crucible with the sample weight after drying [g]; W3-the weight of the crucible with the sample weight after combustion [g]. The above analysis were performed in duplicate for each sample. 2.5.2. Ammonium Nitrogen Content The content of ammonium nitrogen in the samples was determined using the distillation method according to Waring and Bremner with modifications. A 100 g portion of the sample was diluted in a sealed glass jar by adding 900 mL of distilled water and was thoroughly mixed. The resulting suspension was then incubated for 24 h with stirring. In the next stage, the suspension was filtered using quality filters. Next, 40 mL of the filtrate was distilled in a distillation apparatus B-324 (BUCHI, Flawil, Switzerland). The device automatically dosed a 30% sodium hydroxide (NaOH) solution to strongly increase the pH of the samples, which changed ammonium ions (NH4+) to ammonia (NH3). The next step was a 5 min distillation of ammonia into a receiver containing 50 mL of a 2% solution of boric acid (H3BO3). The amount of ammonium nitrogen was then determined by titration of the distillate with a standard 0.05 mol solution of sulfuric acid (H2SO4) in the presence of a Tashiro's indicator (Avantor, Gliwice, Poland). Taking into account that 1 mL of a 0.05 mol H2SO4 solution binds 0.0014 g of NH4, the ammonium nitrogen content in the samples was calculated using Equation (4):(4) N-NH4=VH2SO4x0.0014 Vodsxk x1000 [g/kg] where VH2SO4-the volume of sulfuric acid used [mL]; Vods-the volume of filtrate used for distillation [mL]; k-the sample dilution factor; 0.0014-the weight of ammonia that binds 1 mL of sulfuric acid (0.05 mol). 2.6. Microbiological Analyses Before performing the microbiological analyses, the biological samples were mixed (initial suspension) and used to prepare successive decimal dilutions based on the PN-EN ISO 6887-1:2000 standard. From each sample, 10 mL of the suspension was transferred with a sterile pipette to a previously prepared conical flask with 90 mL of sterile saline solution (0.85% NaCl solution) and then shaken for 1 h on an orbital shaker (Biosan, Pila, Poland). Subsequently, successive decimal dilutions were prepared from the initial suspension. 2.6.1. Quantification of Selected Groups of Bacteria For the quantitative determination of bacteria, the method of surface inoculation of successive decimal dilutions of the samples on solid microbiological media was used. From each dilution of the sample, 100 mL of the suspension was spread on the surface of the enriched, selective, and differential media. The Brain Heart Infusion agar medium (BHI, BioMaxima, Lublin, Poland) was used to determine the total number of bacteria. The MacConkey agar medium was used to determine the number of bacteria from the Enterobacteriaceae and Hafniaceae families, while the medium containing esculin and sodium azide (BEA, BioMaxima, Lublin, Poland) was used to determine the number of Enterococcus spp. The cultures were incubated in a laboratory incubator for 24 h at 37degC. The selectively differentiating Chromagar ECC medium (BioMaxima, Lublin, Poland) and the RapIDTM ONE (Thermo Fisher Scientific, Santa Fe, NM, USA) biochemical tests were additionally used to confirm the correct identification of the bacteria grown on the MacConkey agar. Prior to biochemical testing, selected colonies were plated on the BHI and incubated 24 h at 37 degC. Furthermore, all characteristic colonies grown on the MacConkey and BEA agar media were subjected to basic microbiological analyses, including microscopy and detection of cytochrome oxidase and catalase. The results of the analyses were presented as the total number of microorganisms and the number of Enterobacteriaceae, E. coli, H. alvei, or Enterococcus spp., expressed in colony-forming units (CFU) per 1 mL of the test sample and calculated according to Equation (5):(5) Nx=aV(n1+0.1n2)d [CFU/mL], where Nx-the number of a given group of microorganisms; a-the sum of colonies on all plates from two consecutive dilutions; n1-the number of plates from the first counted dilution; n2-the number of plates from the second counted dilution; V-the volume of the sample applied to the plate; d-the dilution factor corresponding to the first dilution to be counted. 2.6.2. Effectiveness of Sanitization in a Biogas Plant The effectiveness of the biomass sanitization process in the biogas plants was determined based on the calculated reduction degrees for individual groups of microorganisms. The degree of bacterial reduction (RB) was expressed as the percentage of microorganisms removed by the methane fermentation process, calculated using Equation (6):(6) RB=100%-(YbXbx100%) where RB-the degree of bacterial reduction; Xb-the number of bacteria in the biomass before methane fermentation (input biomass); Yb-the number of bacteria in the biomass after methane fermentation (digestate). 2.7. Statistical Analyses Statistical analyses were performed using the STATISTICA 13.1 (StatSoft(r)) software. The data were checked for normality using the Shapiro-Wilk test. Because the data were not normally distributed, the non-parametric Mann-Whitney U-test or Kruskal-Wallis test was used. 3. Results and Discussion 3.1. Basic Chemical Composition of Slurry It was found that the composition of the slurry samples differed substantially with regard to their source. The slurry produced by fatteners was characterized by over two times higher in the contents of dry matter and crude ash, over 10% higher in the content of dry organic matter, and 40% higher in the content of ammonium nitrogen compared to the slurry from the maternal farm (Table 2). The higher values of the analyzed parameters in the slurry from the fattening farm most likely resulted from the differences in the way of feeding and in the technological procedures used for rearing the two groups of animals. The abovementioned differences were also reflected in the higher content of NH4-N in the slurry produced by fattening pigs. The results obtained in the current work differ from the values reported by other authors. Marszalek et al. reported that the average dry matter content of slurry from sows was more than two times higher than from fatteners, and the content of ash was more than 3 percentage points higher. Similarly, Kowalski et al. reported higher contents of dry organic matter and ash than the values found in our study. It can be explained that the basic method used in pig nutrition to lower nitrogen excretion is to reduce the level of total protein in feed rations , whereas an effective way to decrease the amount of nitrogen excreted is the use of phase feeding that takes into account the different nutritional requirements of individual pig production groups. According to Guillou et al. and Everts , this strategy can result in a 20-25% reduction in nitrogen excretion in pregnant and lactating sows. The amount of nitrogen excreted is also determined by retention, i.e., the amount of nitrogen retained in the pigs' bodies. Thus, it should be noted that the amount of nitrogen retention significantly differs between different production groups of pigs, ranging from 10 to 47% . As reported by Smith et al. , sows excrete more nitrogen than fattening pigs, but the composition of their slurry depends also on the degree of dilution with water. The frequent washing of pens in a maternal farm and the high excretion of urine by sows result in lower contents of ammonium nitrogen, dry matter, dry organic matter, and ash in the slurry they produce . 3.2. Input Biomass Composition The analysis of biomass composition used in both biogas plants showed that in the case of the BP-F, the biomass was characterized by statistically significantly higher parameters, i.e., higher content of slurry, dry matter, organic dry matter, crude ash and ammonium nitrogen than in the case of the BP-M (Table 3). The key differentiating factor, in this case, was the type of slurry applied to prepare the biomass used in the biogas production process. The slurry from the BP-M, which was produced in the maternal farm, contained significantly more water and was used as a substrate for biogas production in a lesser amount. The procedure was carried out so as not to underestimate the dry matter content of the input biomass. As reported by Igoni et al. , extremely low dry matter content in fermenting biomass decreases the efficiency of biogas production. Significantly lower water content in the slurry from fatteners allowed the BP-F to achieve a substrate mixture composition typical for wet fermentation . 3.3. Composition of Fermentation Residues Table 4 shows the results of the basic composition of fermentation residues from both biogas plants. The residues from the BP-M and the BP-F did not differ significantly (p <= 0.05) only in terms of dry matter content (DM). The lower content of dry organic matter and higher contents of ash and ammonium nitrogen indicated a more effective decomposition process of organic substances in the BP-F. The fermentation residues from both biogas plants were characterized by a lower dry matter content than reported by other authors. On the other hand, the contents of dry organic matter, ash, and ammonium nitrogen were similar to those presented by Holm-Nielsen et al. , Tambone et al. , and Qi et al. . 3.4. Parameters of Methane Fermentation in the Biogas Plants Table 5 presents the parameters of the biogas production process in the individual biogas plants . Both the temperature and pH were significantly higher in the BP-F. According to Gunaseelan , the highest efficiency of methane production occurs at pH values in the range of 7.5 to 8.5. This is later confirmed by the results of Kawai et. al , who indicated that to obtain a relatively high content of methane in biogas (50-70%), the biomass pH should not be lower than 7.5. It is also worth noting that during the biofermentation process, the pH of biomass increases from 6.33-6.73 to 7.52-7.67 . The low values of the standard deviation testify to the high stability of these parameters and the course of the process. The concentration of VFA achieved in the fermentation tanks of both installations did not show statistically significant differences. In turn, the difference of about 48 days concerning the hydraulic retention time of both biogas plants was confirmed statistically and could have resulted from the different working capacities of the biogas plants. 3.5. Efficiency of Sanitization of Slurry, Input Biomass, and Digestate Residues in the Biogas Plants The microbiological analyses of the biomass from the biogas plants showed significant differences in the number of microorganisms between the samples from different stages of the biogas production process. Table 6 presents the results of the analyses of the samples from the BP-M . The total number of microorganisms and the numbers of Enterobacteriaceae, E. coli, and Enterococcus spp. in the fermentation residues were significantly lower than in the input biomass. The digested biomass also contained a lower number of H. alvei, although the difference was not statistically significant. Table 7 shows the number of microorganisms in the biomass samples collected from various stages of the biogas production process in the BP-F, which uses slurry from a fattening farm . As in the case of the samples from the BP-M, the number of microorganisms (all groups) was significantly lower in the digested biomass than in the input biomass. In this case, significantly lower numbers of microorganisms were also found in the slurry samples used to prepare the input biomass. In both biogas plants, the process of preparing the input biomass had an impact on the number of microorganisms in it. The observed increase in the number of microorganisms in the input biomass when compared to the slurry was caused by the introduction of the microbial load found in the maize silage, as well as the addition of inoculum from the post-fermentation tank (the inoculum brought a load of methane-forming bacteria to start the process). A greater share of inoculum in the initial phase of fermentation significantly increases the efficiency of methane production . As reported by Barlaz et al. , the most intense proliferation of facultatively anaerobic bacteria, including those from the Enterobacteriaceae family or lactic acid bacteria, occurs in the initial hydrolytic stage of methane fermentation. Similar observations were also reported by Strauber et al. , who found an increase in the number of these microorganisms in the first, hydrolytic-acidic phase of methane fermentation and a decrease in acetogenesis/methanogenesis (the second phase). In the final stages of fermentation, homoacetate bacteria and methane-forming archaea begin to predominate, and as a result of their metabolism, the environmental conditions change, which in turn leads to a rapid reduction of Enterobacteriaceae and lactic acid bacteria. The current study also revealed that the total number of microorganisms in the slurry and input biomass samples differed significantly between the biogas plants (Table 6 and Table 7). Similar trends were observed for H. alvei and Enterococcus spp. A greater number of microorganisms was observed in the slurry from fatteners, which might have resulted from a lower dilution and a higher proportion of feces in its composition . In contrast, the slurries did not differ significantly in terms of the content of E. coli and Enterobacteriaceae. However, the numbers of these bacteria in the input biomass obtained from different biogas plants were significantly different. In turn, in the case of digestate obtained from different biogas plants, statistically significant differences in numberof microorganisms were not demonstrated. Therefore, it could be assumed that the methane fermentation process in both biogas plants reduced the number of microorganisms to equally low levels, regardless of the differences in the substrates used. In this respect, our observations are consistent with those of Cote et al. , who fermented 20 different pig manures and observed a significant reduction in the number of microorganisms in each of these samples. Similarly, Costa et al. reported that the fermentation of pig and bovine slurries, which initially differed in the content of microorganisms, ultimately resulted in a similar bacterial reduction. In summary, the current study showed that a greater degree of bacterial reduction occurred in the BP-F, where slurry from fatteners and maize silage were used as the substrates, compared to the BP-M, where slurry from sows and maize silage with grasses were utilized (Table 8). The greater degree of microorganism reduction in the BP-F compared to the BP-M probably resulted from more favorable conditions of the methane fermentation process in terms of temperature and pH. Kim et al. showed that the production of biogas and methane increased in the temperature range between 40 and 50 degC, whereas at 55 degC, it significantly decreased. A similar relationship was also observed for methane gas yield. To the largest extent, statistically significant differences were demonstrated for the reduction in the total number of microorganisms. In the BP-M, the average degree of the reduction in the total number of microorganisms was 13.65% lower when compared to the BP-F . It was most likely related to more frequent destabilizations of the biogas production process in the BP-M . In turn, the degree of reduction of bacteria from the family of Enterobacteriaceae was not statistically different between the biogas plants. However, a greater reduction of Enterobacteriaceae was observed in the BP-F. On the other hand, in the BP-M, these bacteria were less frequently isolated from the post-fermentation biomass . Similar results in psychrophilic conditions were obtained by Cote et al. , where the reduction of bacteria from the Enterobacteriaceae family was in the range of 97.94-100.00%, and in 8 out of 20 examined biogas plants, these bacteria were not detected. In the study by Termorshuizen et al. , who analyzed the process of biogas production in mesophilic conditions with the use of fruit and vegetable wastes as substrates, the obtained bacterial reduction was in the range of 99.99-100.00%. Thus, the reduction of Enterobacteriaceae in the BP-M was several percentage points lower than the results presented by the above-mentioned authors. In turn, the reduction of E. coli was at a high level and did not differ significantly between the biogas plants . However, it was noticed that these bacteria were not found in the post-fermentation residues only in the BP-F. The level of E. coli reduction during various stages of the methane fermentation process has been widely described in the literature, and the results obtained by different authors are similar to those obtained in the current study. As an example, Horan et al. reported that the reduction of E. coli during anaerobic fermentation reached 99.95%, while Cote et al. reported that methane fermentation under psychrophilic conditions reduced the amount of E. coli by 99.67-100.00%. Similar results were also obtained by Masse et al. , who reported a 99.87% reduction of E. coli. The average level of H. alvei reduction was significantly higher in the BP-F, which showed a greater stability of the anaerobic fermentation process. Therefore, it can be assumed that this species of bacteria can be a good indicator of the efficiency of the biomass sanitization process during the biogas production process. This assumption is further confirmed by the threefold presence of this bacterium in the post-fermentation biomass obtained from the BP-F, in the months when the stability of the process was found to be disturbed . In this context, it can also be assumed that H. alvei can be a good indicator of the efficiency of biomass sanitization, especially in thermophilic biogas production conditions. The BP-M had a lower process temperature, which could translate into greater instability of fermentation and more frequent presence of this bacterium in the fermentation residues. The degree of reduction of Enterococcus spp. in both biogas plants reached relatively high values . In the BP-M, the reduction of this bacterium exceeded 94%, and in the BP-F, it was equal to 97%. Comparable results of fecal streptococci reduction in fermented pig slurry were also obtained by McCarthy et al. . In contrast, Juris et al. reported a slightly higher level of reduction of these microorganisms (range 99.99-100.00%) during mesophilic methane fermentation. Watcharasukarn et al. and De Luca et al. indicated that bacteria of the Enterococcus genus can be an important indicator of sanitization for biogas plants operating a thermophilic fermentation process. 4. Conclusions In summary, the obtained results indicated that the efficiency of sanitization of input biomass, including pig slurry, was significantly higher in the biogas plant that used pig slurry from the fattening farm as a substrate (BP-F, with bacterial reduction of 94-99%), when compared to the biogas plant that utilized pig slurry from the maternal (breeding) farm as a substrate (BP-M, with bacterial reduction of 80-97%). Therefore, in order to improve the degree of reduction of microorganisms in the process of biogas production in agricultural biogas plants, it would be advisable to use biomass containing a higher proportion of pig slurry from fattening than from maternal farms. Acknowledgments We would like to thank Michal Grudzinski for his help with sample collection and analyses. Supplementary Materials The following supporting information can be downloaded at Figure S1: Selected parameters of the biogas production process in the BP-M; Figure S2: Selected parameters of the biogas production process in the BP-F; Figure S3: Total number of microorganisms in the samples from the BP-M in monthly periods; Figure S4: The number of Enterobacteriaceae in the samples from the BP-M in monthly periods; Figure S5: The number of E. coli in the samples from the BP-M in monthly periods; Figure S6: The number of H. alvei in the samples from the BP-M in monthly periods; Figure S7: The number of Enterococcus spp. in the samples from the BP-M in monthly periods; Figure S8: Total number of microorganisms in the samples from the BP-F in monthly periods; Figure S9: The number of Enterobacteriaceae in the samples from the BP-F in monthly periods; Figure S10: The number of E. coli in the samples from the BP-F in monthly periods; Figure S11: The number of H. alvei in the samples from the BP-F in monthly periods; Figure S12: The number of Enterococcus spp. in the samples from the BP-F in monthly periods. Click here for additional data file. Author Contributions Conceptualization, A.P.; methodology, A.P.; investigation, A.P.; data curation, D.C.-J.; writing-original draft preparation, A.P., M.M. and D.C.-J.; writing--review and editing, A.P., M.M. and D.C.-J.; visualization, M.M. and D.C.-J.; supervision, A.P. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available from the corresponding author upon request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Scheme of sampling in the biogas plants: I-pig slurry; II-biomass intake; and III-fermented biomass (digestate). The black arrows represent biomass circulation in biogas plants. animals-13-00855-t001_Table 1 Table 1 The content of amino acids in the feed mixtures from individual pig farms. Total Protein Content [%] Amino Acid Content [g/kg] Lysine Methionine with Cystine Threonine Tryptophan Fattening farm 15.5 8.31 4.83 5.46 1.61 17.6 9.50 5.33 6.12 1.93 18.2 12.5 6.51 7.47 2.57 Maternal farm 13.5 5.01 3.82 3.61 1.20 13.7 6.46 4.18 4.32 1.23 15.5 8.84 5.08 5.71 1.66 18.2 12.5 6.51 7.47 2.57 18.5 13.0 6.86 8.06 2.73 19.4 14.1 6.86 8.48 2.75 animals-13-00855-t002_Table 2 Table 2 The basic composition of pig slurry from the maternal and fattening farms. Parameter BP-M BP-F DM [%] 2.25 A +- 1.92 5.32 B +- 1.66 DOM [%] 66.0 A +- 11.24 74.4 B +- 3.51 CA [%] 0.600 A +- 0.371 1.31 B +- 0.290 NH4-N [g/kg] 1.91 A +- 0.290 3.44 B +- 0.390 BP-M-biogas plant located at the maternal farm; BP-F-biogas plant located at the fattening farm. Values in one row marked with different superscript letters show statistically significant differences (p <= 0.05). animals-13-00855-t003_Table 3 Table 3 The basic composition of the input biomass in the biogas plants. Parameter BP-M BP-F Slurry content [%] 44.4 A +- 6.57 71.2 B +- 6.60 DM [%] 5.39 A +- 1.33 9.44 B +- 1.46 DOM [%] 80.8 A +- 4.22 85.2 B +- 1.53 CA [%] 0.991 A +- 0.130 1.38 B +- 0.210 NH4-N [g/kg] 2.01 A +- 0.260 3.12 B +- 0.272 BP-M-biogas plant located at the maternal farm; BP-F-biogas plant located at the fattening farm. Values in one row marked with different superscript letters show statistically significant differences (p <= 0.05). animals-13-00855-t004_Table 4 Table 4 The basic composition of post-fermentation residues from the biogas plants. Parameter BP-M BP-F DM [%] 4.50 A +- 1.17 4.36 A +- 1.11 DOM [%] 75.6 A +- 4.06 71.7 B +- 2.32 CA [%] 1.09 A +- 0.351 1.22 B +- 0.253 NH4-N [g/kg] 2.17 A +- 0.212 3.53 B +- 0.271 BP-M-biogas plant located at the maternal farm; BP-F-biogas plant located at the fattening farm. Values in one row marked with different superscript letters show statistically significant differences (p <= 0.05). animals-13-00855-t005_Table 5 Table 5 Selected parameters of the methane fermentation process in the fermentation tanks of the biogas plants. Parameter BP-M BP-F Temperature [degC] 46.7 A +- 3.61 50.2 B +- 2.62 pH 7.51 A +- 0.390 7.96 B +- 0.0301 VFA [mg/L] 3716 A +- 1000 4542 A +- 1493 HRT [days] 121 A +- 15.5 73.6 B +- 15.4 BP-M-biogas plant located at the maternal farm; BP-F-biogas plant located at the fattening farm. Values in one row marked with different superscript letters show statistically significant differences (p <= 0.05). animals-13-00855-t006_Table 6 Table 6 The numbers of selected groups of microorganisms in the slurry, input biomass, and fermentation residues from the BP-M (log CFU/mL). Group of Microorganisms Slurry Input Biomass Fermentation Residues Total number of microorganisms 7.01 AB +- 0.924 7.72 A +- 0.424 6.63 B +- 0.840 Enterobacteriaceae 4.89 A +- 0.454 4.68 A +- 1.04 2.26 B +- 1.86 E. coli 4.20 A +- 0.635 3.67 A +- 0.802 0.410 B +- 1.06 H. alvei 3.46 A +- 0.612 3.98 A +- 0.953 1.54 A +- 1.82 Enterococcus spp. 5.61 A +- 0.901 5.16 A +- 1.51 2.93 B +- 2.18 Values in one row marked with different superscript letters show statistically significant differences (p <= 0.05). animals-13-00855-t007_Table 7 Table 7 The numbers of selected groups of microorganisms in the slurry, input biomass, and fermentation residues from the BP-F (log CFU/mL). Group of Microorganisms Slurry Input Biomass Fermentation Residues Total number of microorganisms 8.35 A +- 0.601 8.40 A +- 0.501 6.40 B +- 0.891 Enterobacteriaceae 4.93 A +- 0.492 5.44 B +- 0.512 2.45 C +- 1.26 E. coli 4.34 A +- 0.483 4.47 A +- 0.643 0.00 B +- 0.00 H. alvei 4.24 A +- 0.412 5.02 B +- 0.584 0.194 C +- 0.413 Enterococcus spp. 7.29 A +- 0.851 7.08 A +- 0.753 3.94 C +- 2.03 Values in one row marked with different superscript letters show statistically significant differences (p <= 0.05). animals-13-00855-t008_Table 8 Table 8 Comparison of the degrees of reduction in selected microorganisms in the individual biogas plants. Parameter BP-M BP-F Rtotal [%] 80.3 A +- 26.4 94.3 B +- 11.4 REnterobacteriaceae [%] 92.9 A +- 18.2 99.3 B +- 1.39 RE. coli [%] 97.3 A +- 7.70 >99.9 A RH. alvei [%] 90.1 A +- 21.7 99.9 B +- 0.041 REnterococcus [%] 94.8 A +- 6.44 97.0 A +- 7.02 BP-M-biogas plant located at the maternal farm; BP-F-biogas plant located at the fattening farm. Values in one row marked with different superscript letters show statistically significant differences (p <= 0.05). Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000084

Schisandra chinensis polysaccharide (SCP) is an experimental therapeutic for the treatment of intestinal injury. Selenium nanoparticle modification can improve the bioactivity of polysaccharides. In this study, SCP was firstly extracted and purified by a DEAE-52 column, then SCP-Selenium nanoparticles (SCP-Se NPs) were prepared, and the procedure was optimized. Thereafter, the obtained SCP-Se NPs were characterized by transmission electron microscope, X-ray diffraction, energy-dispersive X-ray spectroscopy, and Fourier transform infrared spectroscopy. The influence of different storage environments on the stability of colloidal SCP-Se NPs was also investigated. Finally, the therapeutic effects of SCP-Se NPs on LPS-induced intestinal inflammatory injuries in mice were evaluated. Results showed that the optimized SCP-Se NPs were amorphous, uniform, spherical particles with a diameter of 121 nm, and the colloidal solution was stable at 4 degC for at least 14 d. Moreover, SCP-Se NPs could more effectively alleviate LPS-induced diarrhea, intestinal tissue injury, and tight junction destruction and decrease the elevated expression levels of TNF-a, IL-1b, and IL-6 compared with SCP. These results demonstrate that SCP-Se NPs may alleviate LPS-induced enteritis through their anti-inflammatory effects, indicating that SCP-Se NPs can serve as a good candidate for preventing and treating enteritis in the livestock and poultry industry. Schisandra chinensis polysaccharide selenium nanoparticle intestinal inflammatory injury Natural Science Foundation of Chongqing, ChinaCSTB2022NSCQ-MSX0470 Open Project Program of Beijing Key Laboratory of Traditional Chinese Veterinary Medicine at Beijing University of AgricultureBUAPSP202205 Fundamental Research Funds for the Central UniversitiesSWU120002 SWU-KT22012 Natural Science Foundation of Chongqingcstc2021jcyj-msxmX0982 This work was supported by Natural Science Foundation of Chongqing, China (CSTB2022NSCQ-MSX0470), the Open Project Program of Beijing Key Laboratory of Traditional Chinese Veterinary Medicine at Beijing University of Agriculture (BUAPSP202205), Fundamental Research Funds for the Central Universities (SWU120002, SWU-KT22012), and the Natural Science Foundation of Chongqing (cstc2021jcyj-msxmX0982). pmc1. Introduction The intestinal tract is the main organ for the digestion and absorption of nutrients . Therefore, its health status directly determines the nutritional supply and overall growth of the animal. The intestinal tract is also a crucial barrier for defending against external pathogenic factors. Animal enteritis, caused by Gram-negative bacterial infection, is one of the most common and harmful diseases in the livestock and poultry industry . In production practice, antibiotic treatment is the current standard of care for enteritis. However, with the rise of drug residues, antibiotic resistance, and food safety concerns, the development of new safe antibacterial alternatives to alleviate animal enteritis has become an urgent matter for the development of the current livestock and poultry industry. Traditional Chinese veterinary medicine has a long history of safeguarding the health of livestock and poultry in China and other Asian countries . Schisandra chinensis polysaccharide (SCP) is one of the main active components of the traditional Chinese medicine Schisandra chinensis (Turcz.) Baill and has beneficial therapeutic potential for a variety of enteritis diseases . For example, SCP has been reported to alleviate antibiotic-associated enteritis caused by lincomycin hydrochloride by inhibiting pro-inflammatory factors and increasing the expression of anti-inflammatory factors . Additionally, SCP also has a therapeutic effect on mice with ulcerative colitis by restoring the intestinal structure, regulating cytokine levels, and improving the diversity of intestinal flora . However, due to the macromolecular structure of the polysaccharide, its bioavailability is extremely low, which limits its clinical application. Selenium is an essential trace element for animals, as shown with its anti-inflammatory, antioxidant, and antiviral properties . Selenium supplementation can alleviate intestinal inflammation, improve the structure of intestinal flora, and enrich the diversity of intestinal flora . Currently, inorganic and organic selenium are important sources of selenium supplements, but the narrow therapeutic window limits its clinical application . As a new form of selenium, nano-selenium has attracted much attention for its higher biological activity and safety compared with traditional inorganic and organic selenium . However, due to high surface energy, bare selenium nanoparticles (Se NPs) quickly aggregate into black or gray elemental selenium, reducing their bioactivity, biocompatibility, and bioavailability . It has been reported that polysaccharide molecules can react with the surface of Se NPs to form stable amorphous nanoparticles through intermolecular hydrogen bonds, seleno-oxygen bonds, and selenium-nitrogen bonds . The polysaccharide-Se NPs conjugate preparation using the polysaccharide as a soft template solves the drawback of easy agglomerative inactivation of Se NPs. It also improves the bioavailability and bioactivity of polysaccharides by nanosizing and selenization . Hence, the research on polysaccharide-based Se NPs derivatives has become a hot spot in the field of polysaccharide and nanomedicine in recent years. For example, it was found that the nano-selenium derivative of Ulva polysaccharide significantly enhanced its therapeutic effect on DSS-induced ulcerative colitis in mice by inhibiting the pro-inflammatory response mediated by the NF-kB pathway . However, whether SCP-Se NPs can be synthesized by using SCP as a template and whether the synthesized SCP-Se NPs have an effect on the intestinal injury induced by LPS in mice has not been reported. In this study, considering the bioactivities of SCP, we firstly extracted and purified SCP and then optimized the synthesis procedure of SCP-Se NPs using the particle diameter, polydispersity index (PDI), and Zeta potential examination analyzed by ultraviolet-visible (UV-Vis) spectroscopy and dynamic light scattering (DLS). Transmission electron microscopy (TEM), X-ray diffraction (XRD), energy-dispersive X-Ray spectroscopy (EDX), and Fourier transform infrared (FTIR) spectroscopy were used to further characterize the prepared SCP-Se NPs colloidal solution. Additionally, the stability of the colloidal solution in different storage conditions was studied. Finally, the intestinal protective effects of SCP-Se NPs were investigated based on the disease activity index (DAI) scoring analysis, hematoxylin-eosin (HE) staining, immunohistochemical (IHC) staining, and qRT-PCR examination of the pro-inflammation index in vivo. 2. Materials and Methods 2.1. Reagents and Materials Schisandra chinensis (Turcz.) Baill was obtained from Chongqing Zhong Miao medicine Co., Ltd. Lipopolysaccharide (LPS, Escherichia coli 055:B5), DAB substrate kit was purchased from Biosharp Beijing Labgic Technology Co., Ltd., Beijing, China. Hematoxylin-eosin staining solution was purchased from Changde BKMAM Biotechnology Co., Ltd., Changde, China. Bovine serum albumin (BSA) and DEAE-52 cellulose were purchased from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China. Monosaccharide references (mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, xylose, and arabinose) were purchased from Shanghai baomanbio Co., Ltd., Shanghai, China. Anti-ZO-1 rabbit polyclonal antibody and HRP-labeled goat anti-rabbit IgG antibody were purchased from Wuhan Servicebio Biotechnology Co., Ltd., Hubei, China. IL-1b, IL-6, and TNF-a Elisa kits were obtained from Multi Sciences Biotech, Co., Ltd., Hangzhou, China. RNAiso Plus, PrimerScriptTM RT reagent kit with gDNA Eraser, and SYBR(r) Premix Ex TaqTM II (Tli RNaseH Plus) kit were purchased from Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China. Sodium selenite, ascorbic acid, ethyl alcohol, xylene, paraformaldehyde, trifluoroacetic acid, 1-phenyl-3-methyl-5-pyrazolone, chloroform, and other reagents were purchased from Chengdu Cologne Chemical Co., Ltd., Chengdu, China. 2.2. Isolation and Purification of SCP SCP was extracted from Schisandra Chinensis (Turcz.) Baill according to methods described by previous studies . Briefly, dried Schisandra chinensis (Turcz.) Baill was defatted with 95% ethyl alcohol for 24 h, dehydrated with a drying oven, and boiled with distilled water (1:20, w/v) at 100 degC three times (2 h each time). After the supernatant was filtrated, collected, and concentrated with rotary evaporation at 60 degC under reduced pressure, three volumes of ethyl alcohol were slowly added and kept overnight at 4 degC. Subsequently, the precipitate was collected, dissolved with distilled water, and subjected to protein removal by the Sevage method. Then, the polysaccharide was further purified by using a DEAE-52 cellulose column (2.50 x 30.0 cm) and eluted with a 0.200 M distilled water and 0.400 M NaCl solution at a flow rate of 1.00 mL/min. Each 10.0 mL fraction was collected and monitored by the phenol-sulfuric acid method to plot the elution curve. The eluents in the same fraction were collected, combined, and dialyzed against distilled water within a molecular weight cutoff 3500 Da dialysis bag for 48 h. Finally, the purified SCP was concentrated and lyophilized with a lyophilizer (Scientz-10N, Ningbo Xinzhi Freeze-drying Equipment Co., Ltd., Ningbo, China). 2.3. Monosaccharide Composition Analysis of SCP The monosaccharide composition analysis of SCP was performed according to previous studies . Briefly, SCP was hydrolyzed with trifluoroacetic acid (TFA, 4 M), and the hydrolysate was added to 1-phenyl-3-methyl-5-pyrazolinone (PMP) solution (0.500 M, dissolved in methanol) for derivation and filtered by 0.450 mm filter for later use. Analysis was performed using a liquid chromatography pump (LC-20AD Shimadzu, Japan) under the following conditions: Shim-pack vp-ods C18 column (250 mm x 4.60 mm, 5.00 mm), column temperature 30 degC, mobile phase--acetonitrile: phosphate buffer = (17:83), flow rate of 1.00 mL/min, UV detector (spd-20a shimadzu, Kyoto, Japan), detection wavelength of 245 nm, injection volume of 10.0 mL, and acquisition time of 60 min. 2.4. Preparation of SCP-Se NPs SCP-Se NPs were prepared by the Na2SeO3/ascorbic acid chemical reduction approach. Based on the reaction principle and referring to previous relevant studies , the molar ratio of Na2SeO3 to ascorbic acid was fixed at 1:4. Firstly, a 3.00 mL SCP solution of different concentrations was mixed with 0.500 mL Na2SeO3 solution (50.0 mM) for 10 min, respectively. Subsequently, 1.00 mL of ascorbic acid solution (100 mM) was slowly added to the above mixture under magnetic stirring at a speed of 1200 rpm. After reacting for 3 h at room temperature (RT) in the dark, the final reaction solution was dialyzed with a dialysis bag against ultra-pure water until no Se was detected in the outer solution. Finally, the SCP-Se NPs colloidal solution was obtained and kept at 4 degC for the following experiments. The schematic diagram is shown in Figure 1. 2.5. Optimization of SCP-Se NPs Synthesis The obtained SCP-Se NPs were characterized by using multiple methods. Since SCP-Se NPs solution has the basic colloidal characteristics, dual-wavelength spectrophotometry was first carried out to monitor the particle size according to previous research . In brief, the absorbance of the SCP-Se NPs solution was measured at 410 nm and 490 nm by using a G-9 Series double beam UV-Vis spectrophotometer (G-9S, Nanjing FILA Instrumen, Nanjing, China), and the A410/A490 value was calculated. Meanwhile, the index of particle diameter, PDI, and Zeta potential were also determined by a DLS analyzer (Litesizer 500, Anton Paar, Graz, Austria). 2.6. Characterization of SCP-Se NPs To observe the morphology and distribution of SCP-Se NPs directly, TEM (JEM-2800, JEOL, Akishima, Japan) was applied. Briefly, 20.0 mL of the SCP-Se NPs colloidal solution was placed on the porous carbon film surface of the copper mesh and dried in an oven to avoid contamination by dust and other impurities. After drying, the sample area was observed by TEM. The crystal form of SCP-Se NPs was measured with an X-ray diffractometer from 5deg to 90deg at an angle of 2th and a speed of 10deg/min with the operating current and voltage set at 30 mA and 40 kV, respectively. The SCP-Se NPs sample was put into the test window, and the energy-dispersive X-ray spectroscopy was started to obtain the SCP-Se NP element analysis results. The infrared absorption spectra of SCP and SCP-Se NPs were determined by an infrared spectrometer (iS50 FTIR, Thermo Nicolet Corporation, Waltham, MA, USA). Briefly, dried samples were mixed with potassium bromide solid powder (spectrally pure grade) in a ratio of 1/100, thoroughly ground in a mortar, transferred to a mold, and made into sheets for analysis in the spectral range of 4000-400 cm-1. 2.7. Stability of SCP-Se NPs Colloidal Solution at Different Storage Conditions The stability of the SCP-Se NPs colloidal solution at different storage temperatures was determined by visual changes and the detection of particle diameter size, PDI, and Zeta potential. The SCP-Se NPs colloidal solution samples stored at 4 degC and RT were observed, and the particle size, PDI, and Zeta potentials of the SCP-Se NP samples were measured on days 0, 3, 7, and 14. 2.8. Anti-Intestinal Inflammatory Injury Activity of SCP-Se NPs In Vivo 2.8.1. Animal Feeding and Management Twenty-eight SPF male KM mice were obtained from Southwest Medical University Laboratory Animal Center. During the whole experiment, all the mice were housed in the Animal Experimental Center of Southwest University at a temperature of 25 degC, relative humidity of 50%, and a light and dark cycle of 12 h, and they had adaptive feeding for one week before the experiment. Meanwhile, the mental state, activity, diet, urine, and feces of the mice were observed every day. The animal study was conducted in accordance with the guidelines approved by the Southwest University Laboratory Animal Welfare Ethics Committee (Ethics NO.: IACUC-20210915-03). 2.8.2. Effects of SCP-Se NPs on LPS-Induced Enteritis After adaption, the mice were randomly divided into four groups: blank control group (BC group), LPS model group, SCP group, and SCP-Se NPs group. After 14 days of intragastric administration of the corresponding solutions (SCP group--10.0 mg/kg SCP solution; SCP-Se NPs group--10.0 mg/kg SCP-Se NPs solution ; and BC group and LPS group--equal volume of sterile water), mice in the LPS, SCP, and SCP-Se NPs groups were intraperitoneally injected with 20.0 mg/kg LPS solution , and the BC group was intraperitoneally injected with the equal volume of sterile saline. Thereafter, body weight, fecal occult blood, and diarrhea of mice within 6 h after intraperitoneal administration were observed and recorded for DAI score analysis . At the end of the experiment (6 h post-LPS injection), the mice in the different groups were sacrificed under anesthesia, serum was collected, part of the jejunum tissue was fixed in 4% paraformaldehyde solution, and the other part was rapidly collected and frozen in liquid nitrogen. 2.8.3. Histopathological Analysis of Jejunum After being fixed in 4% paraformaldehyde solution for 24 h, the jejunum tissue samples were dehydrated with gradient alcohol solutions, made transparent with xylene, embedded in paraffin, and sliced into sections of 4.00 mm thickness by a microtome (LEICA RM2016, Wetzlar, Germany). Subsequently, the slices were stained with HE-staining solution, and images were taken under an optical microscope (ZEISS Axiocam ERc 5S, Oberkochen, Germany). Meanwhile, the villi height (from the apex of the villi to the junction of the villi crypt) and crypt depth (the depth of the invagination between adjacent villi) were measured with the ZEISS image analysis system. Values are means from 20 complete villi, and only vertically oriented villi and crypts from each slide were measured. 2.8.4. IHC Staining Paraffin-embedded tissues were prepared as described in Section 2.8.3, cut into 4.00 mm thick sections, dewaxed with xylene, rehydrated with gradient alcohol solutions, and sealed with 5% BSA. Then, sections were incubated with the anti-ZO-1 antibody at 4 degC overnight and with HRP-labeled goat anti-rabbit IgG at RT for 30 min. Finally, the DAB working solution was used for color rendering, and the hematoxylin solution was incubated for 5 min. The images were analyzed after capture using ImageJ and the IHC Profiler plugin, and six different images were counted for each group. The average gray value (staining intensity) and percentage of the positive area (staining area) of positive cells were taken as IHC measurement indicators, and the final scores were given. 2.8.5. Inflammatory Cytokine Analysis To investigate the effect of different treatments to alleviate intestinal inflammatory injury, the serum cytokine contents and the relative mRNA expression levels of jejunum IL-1b, IL-6, and TNF-a were detected. The serum cytokine contents were tested by ELISA kits according to the manufacturer's instructions. The relative mRNA expression levels of the inflammatory cytokines were detected using qRT-PCR method. Briefly, total RNA was extracted from jejunum tissue homogenate with an RNAiso Plus kit. After the concentration was determined by BioDrop mLITE (BioDrop, UK), cDNA was synthesized using a PrimerScriptTM RT Reagent kit with gDNA Eraser. Hereafter, the mRNA expression level of each gene was determined by using a QuantStudio 3 real-time fluorescence quantitative PCR instrument (Thermo Fisher Scientific Co., Ltd. New York), and the relative gene expression level of each target gene was calculated by the 2-DDCt method. The primer sequences used in this experiment are shown in Table 1. The reaction procedures are as follows: 95 degC for 30 s, followed by 40 cycles at 95 degC for 5 s, and 60 degC for 30 s. All these above operations were strictly in accordance with the instructions. 2.9. Statistical Analysis All results were expressed as "Mean +- SD" from at least three independent experiments. The differences between multiple groups were analyzed by Duncan's multiple range test using SPSS 20.0 analysis software. Differences were considered significant at p < 0.05. 3. Results 3.1. Isolation and Purification of SCP In this study, SCP was successfully extracted by the water extraction and alcohol precipitation method and purified with DEAE-52 cellulose column chromatography. As shown in Figure 2, two main fractions were obtained, of which the elution curves were single peaks in distilled water and 0.200 M NaCl eluents. Meanwhile, there was no obvious elution peak in the 0.400 M NaCl eluent. Therefore, eluents of tubes 12-18 and 62-68 were collected and combined. After dialysis and lyophilization, the purity of SCP was determined using the carbohydrate content of 89.2%. 3.2. Monosaccharide Composition of SCP The monosaccharide composition results of SCP are shown in Figure 3. SCP was composed of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, xylose, and arabinose with the mole ratio of 6.93:1.00:14.7:1.71:29:13.0:21.3:23.1. 3.3. Preparation and Characterization of SCP-Se NPs To investigate the appropriate SCP concentration for SCP-Se NPs formation, 0 mg/mL, 0.0100 mg/mL, 0.0500 mg/mL, 0.100 mg/mL, 0.500 mg/mL, 1.00 mg/mL, and 2.00 mg/mL of SCP solution were added to the reaction system, respectively. As shown in Figure 4A, Se NPs quickly aggregated and precipitated when there was no SCP in the system and when the Se NPs solution with SCP modification was orange-red and evenly dispersed. Meanwhile, the absorbance values of the solution prepared with different SCP concentrations were also measured, and the values of A410/A490 were calculated . When SCP concentration reached 0.100 mg/mL, the value of A410/A490 was the highest and tended to be more stable at higher concentrations. Figure 4C,D shows the particle size distribution, PDI, and median particle size of the Se NPs prepared by different SCP concentrations. The particle size distribution of the bare Se NPs was polydisperse with a PDI of 0.430 and a median particle size of 1453 nm. However, the uniformity of the solution greatly changed when SCP was added to the reaction system. Especially for 0.100 mg/mL SCP, the nanoparticles showed the best uniformity with the PDI of 0.170 and the median particle size of 121 nm. Figure 4E,F illustrates the Zeta potential of SCP and SCP-Se NPs modified with different concentrations of SCP. As shown in Figure 4E, the Zeta potential of SCP was -28.9 mV. With the increase of the SCP concentration, the Zeta potential of the SCP-Se NPs first increased and then decreased . Moreover, at the SCP concentration of 0.1 mg/mL, SCP-Se NPs showed the highest potential of -28.9 mV. 3.4. Characterization of SCP-Se NPs Based on the above experiments, 0.100 mg/mL SCP was identified as the optimal concentration for SCP-Se NP preparation. TEM images showed that SCP-Se NPs presented a monodisperse uniform spherical structure with a particle diameter of almost 120 nm , similar to the above results shown in Figure 4D. The XRD result showed that there was only a broad diffraction peak at lower angles , indicating that SCP-Se NPs were amorphous according to the JCPDS database. Furthermore, the elemental composition of SCP-Se NPs was also analyzed by EDX, as shown in Figure 5C-F. It was found that five signals were obtained, in which the proportion of carbon was 65.5%, followed by selenium (18.5%), oxygen (15.3%), calcium (0.400%), and sodium (0.0200%). To clarify the possible banding mechanism between selenium and SCP, FTIR was employed to provide information on their interaction. As shown in Figure 6, SCP and SCP-Se NPs had similar spectra. The absorption peaks of SCP at 3393 cm-1, 2932 m-1, and 1623 cm-1 represent the stretching vibration of the O-H and C-H groups, which are the characteristic peaks of polysaccharides. It could be clearly seen that the characteristic absorption peaks of the O-H and C-H groups for SCP shifted to lower wavelengths of 3375 cm-1 and 2914 cm-1 for SCP-Se NPs. Meanwhile, the absorption peak at 1093 cm-1 of SCP belonging to the C-O group stretching vibration was slightly shifted to 1081 cm-1 in the SCP-Se NPs spectrum. These results may indicate that the interaction between O-H and C-O of SCP with the surface of Se NPs forms new O-H***Se bonds and C-O***Se bonds. 3.5. Stability of SCP-Se NPs at Different Storage Temperatures To investigate the influence of the temperature on the stability of the SCP-Se NPs colloidal solution, the well-prepared colloidal solution from the same reaction was divided into two parts. One was kept in a RT environment, and another was stored at 4 degC. During the monitoring period (lasting for 14 d), the visual changes, particle size, PDI, and Zeta potential of the SCP-Se NPs colloidal solution were recorded. As shown in Figure 7A, the solution remained uniformly dispersed for 14 d when it was kept at 4 degC, while visible precipitation occurred on the 14th day of storage at RT . Meanwhile, the particle size of SCP-Se NPs at 4 degC was relatively stable, with a particle size of 121 nm on day 0 and 112 nm on day 14, and the PDI remained at a rather low value . By contrast, the particle size and the PDI of SCP-Se NPs at RT gradually increased to 516 nm and 0.420 on the 14th day, respectively . Zeta potential results are shown in Figure 7D, and there was no obvious difference between these two different storage conditions. 3.6. Effects of SCP-Se NPs on LPS-Induced Enteritis in Mice The animal experiment flow chart is shown in Figure 8A. During the experiment, mice in the SCP and SCP-Se NPs groups were treated with the corresponding drug solutions for 14 d. As shown in Figure 8B, the body weights of SCP and SCP-Se NPs groups were slightly higher than that of the BC group, but each group was relatively the same amount. After LPS injection, mice in the BC group reacted flexibly and frequently moved with clean and smooth coats and formed stools . In contrast, mice in the LPS group were depressed, curled together, had bristled hair, diarrhea, perianal contamination, and were positive for fecal occult blood . Furthermore, the enteritis symptoms of mice treated with SCP and SCP-Se NPs were effectively alleviated . DAI scoring results are shown in Figure 8D. Among the four groups, the DAI score of the LPS group was the highest, significantly higher than that of the BC group (p < 0.05). Meanwhile, compared with the LPS group, the DAI scores of the SCP group and SCP-Se NPs group decreased significantly, and the difference between it and the SCP-Se NPs group was significant (p < 0.05). For the SCP-Se NPs group, the enteritis symptoms were reduced, and the DAI score was much lower than that of the SCP group, though there was no significant difference between them (p > 0.05). 3.7. Effects of SCP-Se NPs on LPS-Induced Intestinal Injuries in Mice In order to investigate the effects of SCP-Se NPs on LPS-induced intestinal injuries in mice, HE staining was performed on jejunum tissue sections. As shown in Figure 9A, the jejunum villus was complete in structure, orderly arranged, and had no obvious inflammatory cell infiltration in the BC group. Conversely, in the LPS group, a great amount of inflammatory cell infiltration appeared, the jejunum villus structure was destroyed with epithelial cell exfoliation, the villus was broken and atrophied, villus height significantly decreased (p < 0.05) , crypt depth significantly increased (p < 0.05) , and the ratio of villus height to crypt depth significantly decreased (p < 0.05) . Compared with the LPS group, in the SCP and SCP-Se NPs groups, the inflammatory cell infiltration was greatly alleviated, the villus structure was repaired with higher villus height and shallower crypt depth, and the ratio of villus height to crypt depth was significantly elevated (p < 0.05) . 3.8. Influence of SCP-Se NPs on Jejunum ZO-1 Expression The IHC results of jejunum ZO-1 expression in each group are illustrated in Figure 10A. The yellow-brown points indicate the position of the ZO-1 protein. Compared with the BC group, the jejunum ZO-1 expression area of the LPS group was much smaller, and the color of the yellow-brown points was much lighter. Meanwhile, the quantitative analysis results shown in Figure 10B also revealed the significantly decreased expression level of ZO-1 in the LPS group (p < 0.05). For the SCP and SCP-Se NPs groups, the ZO-1 expression area was obviously expanded, and the yellow-brown color got darker compared with the LPS group. Importantly, the expression level of ZO-1 in the SCP-Se NPs group was significantly increased (p < 0.05), which was close to that of the BC group . 3.9. Effects of SCP-Se NPs on the Expression of Inflammatory Cytokines To investigate the effects of SCP-Se NPs on alleviating LPS-induced intestinal inflammation, the levels of IL-1b, IL-6, and TNF-a were determined. As shown in Figure 11, the serum concentration and the relative mRNA expression levels of IL-1b, IL-6, and TNF-a of the LPS group were all significantly elevated compared with those of the BC group (p < 0.05). Administration of SCP and SCP-Se NPs significantly downregulated the expression of IL-1b, IL-6, and TNF-a (p < 0.05). For serum content, all these inflammatory cytokine contents of the SCP-Se NPs group were significantly lower than those of the LPS and SCP groups (p < 0.05). For mRNA expression, the IL-6 mRNA expression level of the SCP-Se NPs group was significantly lower than that of the SCP group (p < 0.05) and all three of these inflammatory cytokines' mRNA expression levels of the SCP-Se NPs group were back to normal, showing no significant difference with those of the BC group (p > 0.05). 4. Discussion Enteritis is a common disease in animal husbandry, which seriously affects the healthy development of the animal breeding industry. SCP has demonstrated good therapeutic potential on a variety of enteritis diseases . However, its macromolecular structural property causing low bioavailability limits its clinical application. Recent studies found that polysaccharides extracted from herbal medicine could be modified with Se NPs to improve their bioactivity and bioavailability . Therefore, SCP-Se NPs were synthesized for the first time by the sodium selenite reduction method, and their anti-enteritis effects were also investigated . In this experiment, purified SCP was firstly obtained by deproteinization and DEAE-52 cellulose column purification and then applied to SCP-Se NP synthesis. To investigate the optimal SCP concentration for SCP-Se NP formation, the particle diameter, PDI, and Zeta potential were examined by using dual-wavelength colorimetric and DLS methods. The results showed that bare Se NPs quickly aggregated into black precipitation in the reaction system, the particle diameter and PDI of SCP-Se NPs were the lowest, and the Zeta potential was the highest at the SCP concentration of 0.100 mg/mL . These results demonstrate that SCP acts as a stabilizer, and 0.100 mg/mL SCP is the optimal concentration for SCP-Se NP synthesis. Meanwhile, TEM, XRD, and EDX were applied to further characterize the SCP-Se NPs . We found that the SCP-Se NPs were amorphous, sphere-like particles with a particle diameter of almost 120 nm, consistent with the result detected by DLS , and the selenium content of SCP-Se was 18.5%, similar to the previous research . These results further confirm the successful synthesis of the SCP-Se NPs. FTIR spectroscopy analysis is an important means to identify the interaction between polysaccharide and Se NPs. Some studies have found that the O-H peak and C-O-H band of some polysaccharide-bound Se NPs did not change significantly, indicating that only a weak interaction between Se and hydroxyl groups occurred without breaking the chemical bond of the hydroxyl group on the Se surface . The red or blue shift could be observed in the O-H peak and C-O-H band of some polysaccharide-bound Se NPs, implying the formation of the new bonds . In this study, the hydroxyl peak at 3393 cm-1 in the SCP spectrum was red-shifted to 3375 cm-1 in the SCP-Se NP spectrum, indicating a reduction of free hydroxyl groups after Se linkage attributed to the formation of O-H***Se bonds. The peak at 1093 cm-1 shifted to 1081 cm-1, indicating the formation of C-O***Se bonds . This is similar to the combination mode of 1,6-a-D-glucan selenium nanoparticles (CPA-Se NPs) . As shown in Figure 4F, SCP-Se NPs were negatively charged and displayed stability in water. Furthermore, the stability analysis showed that SCP-Se NPs easily aggregated at RT , indicating that the proper storage environment of SCP-Se NPs should be at 4 degC. Gram-negative bacterial infection is one of the main pathogenic factors of enteritis in livestock and poultry production. LPS is the major cell wall component of the Gram-negative bacteria, commonly used to establish an animal enteritis model of Gram-negative bacterial infection . In this study, LPS induced typical symptoms of enteritis, such as diarrhea, weight loss, fecal occult blood, and intestinal tissue damage, indicating the animal intestinal injury model was successfully established . Interestingly, the symptoms of enteritis and the intestinal pathological injury were obviously attenuated when treated with SCP and SCP-Se NPs, and the effects of SCP-Se NPs were better than SCP . These results suggest that SCP-Se NPs have better anti-intestinal injury activity than SCP in vivo. The intestinal barrier plays a key role in preventing pathogen invasion and maintaining intestinal environmental homeostasis. Tight junction proteins (TJP) are the main proteins to maintain the function of the gastrointestinal barrier . Zonula occludens-1 (ZO-1) is an important member of the TJP family that acts as a scaffold and is the main component of TJPs . Loss of ZO-1 results in a significant increase in the permeability of the colonic mucosa, which allows endotoxin to enter the intestine and leads to marked intestinal inflammation in a mouse model of colitis . In this study, we found that both SCP-Se NPs and SCP treatment effectively alleviated LPS-induced impairment of ZO-1, and the effect of SCP-Se NPs was more pronounced . These results indicate that SCP-Se NPs could more effectively protect the intestinal barrier function than SCP. When pathogens invade the intestinal cavity of the body, they immediately active the innate immune response by regulating the corresponding signaling pathways, such as JAK-STAT, MAPKs, PI3K-AKT, and NF-kB, to produce a large number of cytokines, such as IL-1b, IL-6, and TNF-a . IL-1, mainly secreted by monocytes, macrophages, and epithelial cells, has strong pro-inflammatory activity and can induce a variety of pro-inflammatory mediators, such as cytokines and chemokines . IL-6 is mainly produced by macrophages and T helper cells and can exert pro-inflammatory functions by activating a variety of target cells, including antigen-presenting cells (APCs) and T cells . TNF-a is one of the earliest and most important mediators of the inflammatory response. During pathogen invasion, intestinal macrophages recruited from blood monocytes rapidly transform into a pro-inflammatory phenotype and produce large amounts of TNF-a once activated . Although the secretion of these cytokines is important for pathogen elimination, the excessive immune response is a key trigger for the impairment of intestinal barrier function. For example, TNF-a can significantly destroy the expression and distribution of TJPs, and IL-1b and IL-6 can not only regulate the reorganization of cytoskeleton proteins, but also can directly lead to the rearrangement of TJPs and weaken the barrier function . Furthermore, studies have also found that the reduction of IL-1b content in intestinal tissue can improve colitis . Anti-TNF-a targeting therapies can improve the clinical score and mucosal healing in IBD patients . The present study found that treatment of SCP-Se NPs more effectively reduced LPS-induced elevation of IL-1b, IL-6, and TNF-a relative to SCP , closely correlated with its anti-enteritis effects. These results may suggest that SCP-Se NPs can more effectively alleviate the excessive inflammatory response against LPS-induced intestinal injury than SCP, indicating that SCP-Se NPs may serve as good candidates in preventing and treating enteritis in the livestock and poultry industry. 5. Conclusions In conclusion, SCP-Se NPs were synthesized and characterized for the first time and showed a better effect against LPS-induced intestinal injury than SCP, suggesting that SCP-Se NPs may serve as a good candidate for preventing and treating enteritis in the livestock and poultry industry. Acknowledgments We are grateful to all other staff in the Department of Traditional Chinese Veterinary Medicine of Southwest University for their assistance in the experiments. Author Contributions Conceptualization, H.D. (Hongxu Du)., J.L. and L.C. methodology, H.D.(Hong Dong) and L.C.; software, X.T.; validation, X.T., Z.L. and H.D. (Hong Dong); formal analysis, X.T. and Z.L.; investigation, L.S. and Z.H.; resources, H.D. (Hong Dong); data curation, H.D. (Hong Dong); writing--original draft preparation, H.D. (Hongxu Du) and X.T.; writing--review and editing, H.D. (Hongxu Du), X.T., Z.L., H.D(Hong Dong)., L.S., Z.H.,Q.M., S.D., M.R. and J.L.; visualization, Q.M.; supervision, L.C.; project administration, H.D.(Hongxu Du); funding acquisition, H.D(Hongxu Du)., S.D. and Q.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study was conducted in accordance with the guidelines approved by the Southwest University Laboratory Animal Welfare Ethics Committee (Ethics NO.: IACUC-20210915-03). Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The schematic diagram of SCP-Se NPs preparation. Figure 2 The elution curve of SCP on DEAE-52 cellulose column. Figure 3 Monosaccharide composition analysis of SCP by HPLC. (A) HPLC chromatograms of mixed standards. (B) HPLC chromatograms of SCP. Monosaccharide corresponding to each peak: 1. mannose, 2. ribose, 3. rhamnose, 4. glucuronic acid, 5. galacturonic acid, 6. glucose, 7. xylose, 8. arabinose. Figure 4 Optimization of SCP-Se NPs preparation process. (A) The appearance of SCP-Se NPs modified with different concentrations of SCP. (B) The A410, A490, and A410/A490 values of SCP-Se NPs solution at different SCP concentrations. (C) The particle size distribution of SCP-Se NPs at different SCP concentrations. (D) The median particle size and PDI of SCP-Se NPs at different SCP concentrations. (E) Zeta potential of SCP solution. (F) Zeta potential of SCP-Se NPs at different SCP concentrations. Figure 5 SCP-Se NP characterization. (A) TEM image of SCP-Se NPs. (B) XRD spectrum of SCP-Se NPs. (C) The elemental composition of SCP-Se NPs. (D) Carbon. (E) Selenium. (F) Oxygen. Figure 6 FTIR spectra of SCP and SCP-Se NPs. Figure 7 Stability of SCP-Se NPs at different storage temperatures. (A) Appearance changes of SCP-Se NPs colloidal solution at 4 degC. (B) Appearance changes of SCP-Se NPs colloidal solution at room temperature (RT). (C) Changes in particle size and PDI of SCP-Se NPs at 4 degC and RT. (D) Changes of Zeta potential of SCP-Se NPs at 4 degC and RT. Figure 8 Effects of SCP-Se NPs on LPS-induced enteritis in mice. (A) Flow chart of animal experiment. (B) Body weight changes of each group during the experiment. "#" indicates the time point of 6 h post-LPS injection. (C) Hair and diarrhea in mice after different treatments. (D) DAI scores in each group. Different lowercase letters (a-c) indicate significant differences (p < 0.05). Figure 9 Effect of SCP-Se NPs on LPS-induced intestinal injuries in mice. (A) Effect of SCP-Se NPs on the histopathological changes in the jejunum (x100 magnification). (B) Jejunum villus height in each group. (C) Jejunum crypt depth in each group. (D) The ratio of jejunum villus height to crypt depth in each group. Different lowercase letters (a-c) indicate significant differences (p < 0.05). Figure 10 Influence of SCP-Se NPs on ZO-1 expression in LPS-induced intestinal injury. (A) IHC image results of jejunum ZO-1 expression in each group (x200 magnification). (B) Quantitative analysis of jejunum ZO-1 expression in each group. Different lowercase letters (a-c) indicate significant differences (p < 0.05). Figure 11 Effects of SCP-Se NPs on inflammatory cytokine (IL-1b, IL-6, and TNF-a) expression levels in LPS-treated mice. (A) Serum inflammatory cytokine (IL-1b, IL-6, and TNF-a) content. (B) Jejunum inflammation cytokine (IL-1b, IL-6, and TNF-a) relative expression level. Different lowercase letters (a-c) indicate significant differences (p < 0.05). animals-13-00930-t001_Table 1 Table 1 Primer sequence information. Gene Primer Sequence (5'-3') Product Size (bp) IL-1b Forward CACTACAGGCTCCGAGATGAACAAC Reverse TGTCGTTGCTTGGTTCTCCTTGTAC 145 145 IL-6 Forward CTTCTTGGGACTGATGCTGGTGAC Reverse TCTGTTGGGAGTGGTATCCTCTGTG 91 91 TNF-a Forward CGCTCTTCTGTCTACTGAACTTCGG Reverse GTGGTTTGTGAGTGTGAGGGTCTG 113 113 b-actin Forward TTCCTTCCTGGGTATGGAAT Reverse GAGGAGCAATGATCTTGATC 206 206 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000085

Milk is a complex liquid, and the concentrations of many of its components are under genetic control. Many genes and pathways are known to regulate milk composition, and the purpose of this review is to highlight how the discoveries of quantitative trait loci (QTL) for milk phenotypes can elucidate these pathways. The main body of this review focuses primarily on QTL discovered in cattle (Bos taurus) as a model species for the biology of lactation, and there are occasional references to sheep genetics. The following section describes a range of techniques that can be used to help identify the causative genes underlying QTL when the underlying mechanism involves the regulation of gene expression. As genotype and phenotype databases continue to grow and diversify, new QTL will continue to be discovered, and although proving the causality of underlying genes and variants remains difficult, these new data sets will further enhance our understanding of the biology of lactation. mammary biology mammogenesis lactation lactogenesis quantitative trait loci Ministry for Business Innovation and Employment Endeavour FundLICX1802 Funding was provided by the Ministry for Business Innovation and Employment Endeavour Fund (contract ID LICX1802). pmc1. Introduction The composition of milk is complex, featuring an emulsion of fat globules and a colloidal dispersion of casein micelles in an aqueous solution of lactose (and other carbohydrates), whey proteins, and minerals. Although milk from different species contains the same basic constituents, their proportions can vary greatly. In cattle, the typically average percentages (g/100 g) of fat, caseins, whey proteins, and lactose are 3.9%, 2.6%, 0.6%, and 4.6% respectively; in humans, the corresponding percentages are 4.5%, 0.4%, 0.5%, and 7.1% . Even more extreme differences can be seen in other species. Some seal species, for example, producing little to no lactose, resulting in highly concentrated milk with fat percentages of 50% . Less-extreme differences in milk composition are also visible within species. In many cases, the differences in composition among individuals are under partial genetic control. Regions of the genome where the genotypes of genetic variants are associated with phenotypes such as milk composition are known as quantitative trait loci (QTL). When they can be identified, the causative genes underlying these QTL can help elucidate the pathways involved in milk production. The aims of this review were to describe some of the major pathways required for milk production in terms of the QTL and genes that have helped to identify them and to note some of the methods that can be used to identify causative genes underlying QTL. We focus primarily on cattle (Bos taurus) as a model species, though there are some references to QTL identified in sheep; additionally, some comparisons with human milk composition are also presented where there is a major difference between the two species. 2. QTL for Major Pathways Involved in Milk Production Many QTL have been observed for both milk yield and milk composition traits in cattle (see Table 1). These QTL have been identified using a number of different techniques, including linkage-based approaches, such as the transmission disequilibrium test (TDT); and association-based approaches, such as genome-wide association studies (GWAS) and transcriptome-wide association scans (TWAS). The genes attributed to these QTL have a variety of functions. Some, such as the hormones prolactin and growth hormone, and their associated signalling pathways, are involved in mammogenesis (the development of the mammary gland during puberty and pregnancy), lactogenesis (the onset of milk secretion), and galactopoiesis (the continued production of milk). Other pathways, such as those for fat and protein synthesis, and ion channels, are involved directly in milk production. The following sections list some of the pathways involved in mammogenesis and lactation, as identified by QTL for milk production, where the candidate causal gene encodes a protein that sits within those pathways . Methods for identifying candidate causative genes underlying QTL are discussed in Section 3. 2.1. Milk Proteins QTL have been mapped to many milk proteins, i.e., those expressed directly in milk. In cattle, the largest proportion of milk protein content (80% ) consists of the four casein proteins, encoded by a cluster of genes mapping to BTA6: casein alpha-S1 (CSN1S1), encoding aS1-casein, casein alpha-S2 (CSN1S2) for aS2-casein, casein beta (CSN2) for b-casein, and casein kappa (CSN3) for k-casein. The casein proteins aggregate into micelles in the milk, sequestering calcium phosphate as a nutrient source for the neonate. K-casein seems to be particularly important for successful lactation, as knockout mice deficient in k-casein fail to lactate, due to destabilisation of the casein micelles . Like b-lactoglobulin, the various casein proteins all have a range of coding variants, although, with the exception of CSN1S1*G , these have not been intrinsically linked to lower rates of gene expression, such as the b-lactoglobulin B variants discussed below. However, there is evidence that the SNP responsible for the A2 variant of b-casein is associated with both milk yield and protein yield , and QTL for milk protein phenotypes have also been detected at this locus in other studies . These QTL can overlap with, but are not in linkage disequilibrium with, other nearby QTL assigned to the GC gene : this gene encodes the protein group-specific component (vitamin D binding) and maps around one megabase from the casein gene cluster. Outside the mammary gland, casein expression in human CD14+ monocytes has been observed to upregulate expression of the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1b (IL-1b), and IL-6 via the p38-MAPK pathway . This suggests a possible role for caseins in regulating the innate immune response in the neonate intestine following milk consumption. The major whey protein in cattle, accounting for around 50% of whey protein and 10% of total protein, is b-lactoglobulin, which is encoded by the progestagen-associated endometrial protein (PAEP) on BTA11. A QTL has been detected to map to this gene. It has pleiotropic effects on milk fat yield, protein yield, and volume . Other studies looking at individual milk proteins have also shown that genetic variants associated with low b-lactoglobulin concentrations yield high concentrations of a-, b-, and k-caseins . Variation in milk b-lactoglobulin concentrations are primarily driven by differences between the A and B protein variants . The B variant showed lower expression than the A variant. Other variants mapping to the B variant background, such as B* and B' , cause further reductions in b-lactoglobulin expression. Milk with low b-lactoglobulin concentrations has potential uses in infant formula (as human milk lacks this protein), and the associated higher concentrations of caseins would also be expected to provide better properties for cheese making. The dominant whey protein in humans, which is second in cattle (18.5% of total whey protein ), is a-lactalbumin. It is encoded by the gene lactalbumin alpha (LALBA) on BTA5. The QTL associated with milk protein concentration have been identified at this locus in several dairy populations and breeds . The a-lactalbumin protein is required for lactose synthesis, modifying the enzyme b-1,4-galactosyltransferase (encoded by B4GALT1 on BTA8): in most tissues, B4GALT1 adds galactose moieties to N-acetylglucosamine (GlcNAc) residues. However, the binding of a-lactalbumin to B4GALT1 changes the latter to instead add galactose to glucose, forming lactose: this binary enzyme is known as lactose synthase. Given the importance of these two genes for lactose synthesis, it is no surprise that a QTL for milk lactose concentration has also been identified at the LALBA locus , along with a milk yield at the B4GALT1 locus . The importance of lactose synthesis for milk production was shown in a-lactalbumin knockout mice, which produce highly viscous milk with otherwise normal fat and protein composition that cannot be extracted by the pups from the mammary gland . It has been shown in vitro that multimeric a-lactalbumin is cytotoxic to stem cells and transformed cell lines, though it does not harm healthy epithelial cells , suggesting that a-lactalbumin may also have a protective function in either the mammary gland or the neotate digestive system. Another whey protein is the iron-binding antimicrobial protein lactoferrin, encoded by the lactotransferrin (LTF) gene on BTA22. In cattle, this protein exists in bovine milk at low but variable concentrations (average 115.4 mg/mL in but ranging from 31.8 to 485.6), but reaches much higher concentrations in human milk--around 2 g/L in mature milk . Interestingly, another antimicrobial protein, lysozyme, which is present at lower levels in human milk , is also present at relatively low concentrations in cattle . Levels of lysozyme in Bovidae are reported to be as low as 1/1000th of those of other mammalian species . Although lactoferrin expression can be induced by mastitic infection , it is also under partial genetic control , and genetic variants affecting expression have been identified at the LTF locus . QTL at this locus have also been associated with casein number and lactose concentration . A third antibacterial protein present in milk is lactoperoxidase, encoded by the LPO gene on BTA19. In contrast to lysozyme and lactoferrin, lactoperoxidase activity was found to be around 20x higher in bovine milk compared to that of humans . A trans-eQTL for LPO, overlapping a QTL for milk protein concentration, has been identified on BTA20 at the locus of the C6 and C7 genes : these two genes encode proteins that comprise part of the complement pathway in the innate immune system, suggesting that lactoperoxidase may be co-regulated with this system. 2.2. Fat Synthesis Pathways Fat is one of the major components of milk, forming membrane-bound droplets known as milk fat globules (MFG), mostly in the form of triglycerides. MFG membranes also contain a range of proteins, such as butyrophilin, adipophilin, mucin, lactadherin, lactoferrin, and xanthine oxidase . Two of these, butyrophilin (encoded by butyrophilin subfamily 1 member A1; BTN1A1) and xanthine oxidase (XOR; encoded by xanthine dehydrogenase XDH), are required for enveloping the MFGs with the apical cell membrane, and therefore, for secretion of the MFG into milk . Heterozygous knockout mice for XOR are unable to maintain lactation . Many of the QTL for fat yield or milk concentration in cattle map to genes encoding fat synthesis or metabolic enzymes . One of the most prominent QTL detected in cattle for milk volume, fat, and protein phenotypes in cattle maps to the DGAT1 locus on BTA14 . This gene encodes the enzyme diacylglycerol O-acyltransferase 1, which is responsible for the final stage triglyceride fat synthesis . The causative variant for this QTL is a non-conservative lysine-to-alanine substitution at position 232 . More recently, this same variant has been shown to cause an expression QTL for the DGAT1 gene by disrupting an exon splice enhancer, which in turn alters the splicing efficiency of several introns in the transcript . Another enzyme sitting earlier in the same trigyceride synthesis pathway is glycerol-3-phosphate acyltransferase 4, encoded by the GPAT4 gene (formerly known as AGPAT6) on BTA27. Like DGAT1, a highly pleiotropic QTL has been detected at this locus for many milk phenotypes, including volume, fat, protein, and lactose traits . The enzyme lipin 1 sits in a related pathway, catalysing the conversion of phosphatidate into diacylglycerol and functioning as a transcriptional coactivator for genes involved in fatty acid oxidation . QTL for milk protein and casein concentrations and milk yield have been detected at the LPIN1 locus, which encodes this enzyme, in BTA11, in cattle. Before they can be assembled into triglycerides, fatty acids first need to be obtained either from the diet or from de novo synthesis. Several genes involved in this latter pathway have also shown QTL for milk-related phenotypes. The rate-limiting step in fatty acid synthesis is the carboxylation of acetyl-CoA to malonyl-CoA, catalysed by the enzyme acetyl-CoA carboxylase (ACC). The alpha form of this enzyme is encoded by the gene acetyl-CoA carboxylase alpha (ACACA) on BTA19, and QTL for fatty acid composition and somatic cell score (a proxy phenotype for mastitis) have been mapped to this locus. Another important enzyme in the fatty acid synthesis pathway is fatty acid synthase (FAS), encoded by the gene FASN on BTA19, dimers of which are responsible for synthesising the saturated C16 fatty acid palmitic acid from acetyl-CoA and malonyl-CoA . QTL attributed to this gene have been identified for milk fat yield , fat concentration , and fatty acid composition in cattle. The fatty acid synthase gene will produce only saturated fatty acids. To generate monounsaturated or polyunsaturated fatty acids, desaturase enzymes are required. One cluster of fatty acid desaturase genes on BTA29 includes the two genes fatty acid desaturases 1 and 2 (FADS1 and FADS2); these enzymes are responsible for the final, rate-limiting steps in omega-3 and -6 fatty acid syntheses . Variants mapping in cattle to these two genes have been associated with concentrations of a range of polyunsaturated fatty acids in milk , and similar associations are also observed in human milk . A third desaturase enzyme involved in fatty acid synthesis is stearoyl-CoA desaturase, which is encoded by the gene SCD on BTA26 and responsible for oxidising the C16 and C18 saturated fatty acid compounds palmitoyl- and stearoyl-CoA into the monounsaturated compounds palmitoleoyl- and oleoyl-CoA, respectively . This enzyme is also important in regulating metabolism: knockout mice exhibited lower levels of tissue triglycerides and low-density lipoproteins , and showed increased insulin signalling and glucose uptake in muscle tissue . In cattle, a QTL assigned to SCD has been identified for milk fat yield . 2.3. Hormones and Signalling Hormones, and the receptors and signalling pathways they activate, are important in most if not all biological functions, and milk production is no exception. For example, knocking out the SCD gene discussed in the previous section leads to an increase in tyrosine phosphorylation of the insulin receptor, which has downstream effects on the PI3K-Akt signalling pathway, leading to increased levels of the glucose transporter GLUT4 in the plasma membrane and increased glucose uptake in muscle . These knockout mice also showed lower levels of the hormone leptin, which is involved in regulating energy intake and partitioning. In cattle, leptin (encoded by the gene LEP on BTA4) has been associated with both milk yield and feed intake . No milk phenotype QTL have been reported for the leptin receptor (LEPR on BTA3), though an association has been reported with body size . In both humans and mice , leptin resistance is associated with obesity. Another gene associated with obesity is FTO, encoding the enzyme FTO alpha-ketoglutarate-dependent dioxygenase. This enzyme is involved in DNA repair; specifically, it demethylates 3-methylthymidine . It can also demethylate bases in RNA, including 3-methyluracil and 6-methyladenosine . Via this latter mechanism, FTO can inhibit adipogenesis by demethylating cyclin A2 and cyclin-dependent kinase 2 mRNA, reducing the expression of these genes and thereby prolonging the cell cycle . In cattle, variants at the FTO locus on BTA18 have been associated with milk fat yield . One hormone of particular importance for lactation is prolactin, a peptide hormone secreted by the anterior pituitary gland. In cattle, this peptide is encoded by the PRL gene on BTA23. The importance of this hormone in cattle was underlined by the discovery of a dominant missense mutation that caused, among other phenotypes, a failure to lactate . Prolactin promotes development of the mammary gland during pregnancy, in conjunction with progesterone--generating ductal branching and alveolar buds . Prolactin is detected by cells using prolactin receptor (PRLR; BTA20) and acts via the PRLR/JAK2/STAT5 signalling pathway to promote mammary gland development and milk protein expression , and it induces the expression of the enzyme UDP-glucose pyrophosphorylase 2 (UGP2) and the transporter UDP-galactose transporter 2 (SLC35A2), thereby promoting the synthesis of lactose . In parallel, prolactin receptor also acts via the PI3K/Akt pathway to downregulate repressors of PRLR/JAK2/STAT5 signalling . Akt1 also upregulates fat synthesis and glucose uptake into the cell for lactose production . More recently, it has been shown that Akt signalling induces developing mammary epithelial cells to express prolactin, which in turn acts in an autocrine manner via STAT5 to cause terminal differentiation of the mammary epithelium . Given the importance of these pathways, it is not surprising that QTL for milk yield have been widely identified at the PRLR locus and for somatic cell score . Likewise, many studies have reported milk-related QTL at the signal transducer and activator of transcription 5 (STAT5) locus on BTA19 (genes STAT5A and STAT5B) . Beyond the leptin and prolactin pathways, other hormones have also been linked to lactation. One example is growth hormone (GH), also known as somatotropin, which comprises part of the same family of protein hormones as prolactin . Growth hormone acts in the lactating animal to partition nutrients and energy towards the mammary gland , which is thought to be mediated by increased serum insulin-like growth factor (IGF-1) levels . In the cow, growth hormone is encoded by the gene growth hormone 1 (GH1) on BTA19, and its receptor, encoded by growth hormone receptor (GHR), maps to BTA20. Both the GH1 locus , and especially the GHR locus , have been associated with milk, fat, and protein yield phenotypes in a range of cattle populations. Another important family of signalling molecules is the interleukin family of cytokines, a group of primarily immunomodulatory proteins that operate in a paracrine or autocrine manner to affect cell growth and differentiation during immune responses . The gene colony stimulating factor 2 receptor subunit beta (CSF2RB) on BTA5 encodes the b-chain of GM-CSF (also known as CSF2) receptor, and also forms a common subunit with the receptors for interleukin-3 (IL-3) and IL-5. QTL for milk, fat, and protein volume; and fat and protein concentrations, have been identified at this locus . CSF2RB is suggested as the most likely candidate causative gene, although the neighbouring genes neutrophil cytosolic factor 4 (NCF4) and thiosulfate sulfurtransferase (TST) have also been put forward as candidates. The GM-CSF receptor operates via the JAK/STAT signalling pathway--specifically, JAK2 and STAT5 , the same pathway as used by prolactin signalling. In contrast to STAT5, which is linked to increased milk protein and lactose expression, STAT3 has been identified as a mediator of involution and apoptosis in the mammary gland , which is primarily activated by the cytokine leukaemia inhibitory factor (LIF) . STAT3 phosphorylation is upregulated within one hour of physical distension of the mammary gland in cattle, ultimately leading to the cessation of milk production when the gland is not emptied . Mice where either STAT3 or LIF is knocked out show delayed involution and reduced levels of apoptosis , raising the possibility of improving efficiency in dairy herds, especially those milking once a day, by breeding for animals lacking one or more of these genes. STAT3 is also involved in leptin signalling, interacting with the long form of the leptin receptor . It is difficult to assign QTL unambiguously to the STAT3 or STAT5 loci, as STAT3 maps on BTA19 directly between the two STAT5 genes STAT5A and STAT5B. Yet another STAT protein, STAT1, is believed to be involved in the development of the mammary gland . A QTL mapping to the STAT1 locus on BTA2 has been associated with milk, fat, and protein yield . One group of proteins that is upregulated by the JAK/STAT pathway is the suppressors of the cytokine signalling (SOCS) gene family . Proteins in this family downregulate JAK/STAT-mediated signalling. For example, SOCS1 (SOCS1 on BTA25) expression is induced by prolactin signalling, and in turn binds to JAK2, inhibiting its association with STAT5 and dampening signal transmission . SOCS2 (SOCS2 on BTA5) negatively regulates GH signalling. SOCS2 knockout mice showed higher body weights and skeletal dimensions than control mice . As SOCS proteins ultimately downregulate milk protein expression via prolactin and growth hormone signalling pathways, there is the potential that knocking out the genes encoding them could improve lactation phenotypes in dairy animals. For example, mice with homozygous Socs1 knockout genotypes showed enhanced alveolar development , and a point mutation in the ovine Socs2 gene has been associated with higher milk production in dairy sheep, albeit with increased susceptibility to mastitis . In cattle, several different SOCS genes have been associated with milk volume, fat, and protein phenotypes . 2.4. Transporters and Ion Channels Another important mechanism affecting milk production involves trans-membrane transport and ion channels. All major milk components need to either be produced within the mammary epithelial cells or transported across them from the blood, and in both cases need to cross the apical membrane into the lumen. While some small molecules, such as urea, can simply diffuse across the membrane, in most cases either passive or active transport channels are required. The volume of water excreted into the milk is driven by osmotic pressure, which is in turn created by exporting lactose and ions across the cell membrane against their concentration gradients. This means that QTL for milk phenotypes frequently map to genes encoding transporters. One important group is the sugar transporters. The gene solute carrier family 37 member 1 (SLC37A1) (on BTA1) encodes a phosphate-linked, glucose-6-phosphate antiporter , which is responsible for importing glucose into the cell, and QTL for milk volume have been detected at this locus in several populations . Another glucose transporter, SWEET1, is encoded by the gene SLC50A1 on BTA3. SWEET1 has been observed in the Golgi in mammary cells and is possibly responsible for importing glucose into the Golgi for lactose synthesis . QTL for lactose concentration and protein concentration have been identified near this gene, though the latter QTL has been assigned to GBA1, which encodes the lysosomal protein glucosylceramidase beta 1. Other glucose transporters have also been implicated in milk production, such as GLUT1 (SLC2A1 on BTA3) , the Na+/glucose co-transporter SGLT1 (SLC5A1 on BTA17) , and GLUT12 (SLC2A12 on BTA9) . While the mammary epithelial cells use osmotic pressure to secrete milk, it is important that they maintain their own cell volumes correctly. One mechanism by which they can do this is using voltage-regulated anion channels (VRACs), which help regulate cell volume by exporting Cl- ions, and small organic anions such as taurine . VRACs are comprised of heteromers of leucine-rich repeat containing 8 (LRRC8) proteins A to E, encoded by the genes LRRC8A-LRRC8E. In cattle, the locus on BTA3 containing LRRC8B-LRRC8D has been associated with milk lactose concentration . LRRC8C is suggested as the likely causative gene on the basis of gene-expression data. Interestingly, LRRC8C has been associated with adipocyte differentiation (under the name fad158) and is also present in the membranes of MFGs , suggesting it may also have a role in the storage or export of fat. As stated above, it is important that water be able to move across the cell membrane to balance osmotic pressure as milk is synthesised. However, the lipid bilayer of the cell membrane is impermeable to water, requiring channels to facilitate the crossing. These channels are provided by aquaporin proteins (AQPs). At least seven aquaporins are expressed in the mammary gland , where they are believed to play a role in gland development and in transporting water to the lactating gland for milk synthesis and secretion. For example, AQP1 is expressed in the capillary endothelial cells, and may be involved in oestrogen-mediated angiogenesis in the developing mammary gland , and AQP5 is expressed in mammary epithelial cells, and the protein pores are moved from the cytoplasm to the apical cell membrane under the regulation of prolactin , suggesting a role in milk production. Genetic effects mapping to aquaporin genes have been reported, such as a study in sheep that mapped QTL for milk fat and protein concentrations to a window on OAR3 that contains the genes AQP2, AQP5, and AQP6, albeit alongside LALBA, which is also a strong candidate causal gene for milk-related traits, as described in the milk protein section above. Another important class of transporter is the potassium channel, a type of widely expressed ion channel found in the majority of cell types. The majority of these transporters are encoded by genes named KCN for the potassium channel, followed by a letter representing the subfamily. A number of different families of potassium channel have been identified. The largest family, the voltage-gated potassium channels (Kv, reviewed in ), responds to voltage changes in the cell's membrane potential. This family includes the subfamilies KCNA, KCNB, KCNC, KCND, KCNF, KCNG, KCNH, KCNQ, KCNS, and KCNV . One channel, Kv3.3, which is encoded by the gene KCNC3 on BTA18, has been associated with milk yield in cattle . Another QTL for both milk volume and fat yield maps to the gene KCNS2, encoding Kv9.2, on BTA14 . A third gene in the same family, KCNH4, encodes the channel Kv12.3, and is a potential candidate for a lactose concentration QTL on BTA19 , although the QTL also encompass the genes STAT3, STAT5A, and STAT5B, which are also candidates. A second large family is the potassium inwardly rectifying channel family (Kir, reviewed in ), comprising lipid-gated channels that are activated by phosphatidylinositol 4,5-bisphosphate (PIP2). These transporters correspond to the gene family KCNJ . In cattle, a locus on BTA19 has been associated with QTL for fat and protein concentrations , lactose concentration , and milk yield . This locus contains two genes encoding Kir channels: KCNJ2 (Kir2.1) and KCNJ16 (Kir5.1), and both genes have been proposed as candidate causatives underlying the QTL. A third, smaller family of potassium channels is the two-pore-domain potassium channels (K2P), known as leak channels , corresponding to the KCNK family. A QTL for milk fat, protein, and volume has been identified on BTA26 at the locus of the KCNK18 gene, encoding the potassium channel K2P18.1 . The fourth family is the calcium- and sodium-activated potassium channels , of which the most well-known member is KCa1.1, also known as BK. The activity of KCa1.1 is modulated by auxiliary b and g subunits , including g1, encoded by the gene LRRC26 on BTA11, where a QTL for fat concentration has been detected . The channels discussed so far are limited to moving solutes along an existing concentration gradient. Another class of transporter requires energy in the form of ATP to concentrate solutes to establish a gradient or membrane potential. Many of these belong to the ATP-binding cassette family (ABC). One important example from this family is ATP binding cassette subfamily G member 2 (ABCG2). Initially identified as a xenobiotic drug transporter , ABCG2 also functions as a transporter of riboflavin into the milk and a urate transporter in the kidneys . QTL for several milk phenotypes have been mapped to the ABCG2 locus on BTA6, including milk yield , fat and protein concentration , lactose concentration , and aS1-CN concentration . The causative variant at this QTL is generally believed to be a tyrosine-to-serine substitution (Y581S), identified by Cohen-Zinder et al. ; however, the adjacent gene SPP1, encoding the protein osteopontin (involved in bone remodelling), has also been proposed as a candidate . A second member of the ABC family is SUR2 (encoded by ABCC9 on BTA5), which forms a component of the ATP-sensitive potassium channel KATP, alongside SUR1 (ABCC8) and the inward rectifying channels Kir6.1 (KCNJ8) and Kir6.2 (KCNJ11) . The exact composition varies by tissue . The channel is inhibited by ATP and activated by MgADP. The KATP transporter is important for glucose-level sensing to control insulin release in pancreatic beta cells : at low glucose levels, ATP levels are low and ADP levels are elevated, so the channel is open; and at high glucose levels, ATP closes the channel. This polarises the plasma membrane, a change that is detected by voltage-gated calcium channels, which then open, causing calcium to enter the cell and trigger the release of insulin. In cattle, a QTL for milk fat yield has been identified at the ABCC9 locus . A third ATP-binding transporter is the calcium transporter SERCA2, encoded by the gene ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (ATP2A2) on BTA17. This transporter pumps Ca2+ from the cytosol into the endoplasmic reticulum, whence it can be exported into milk . A QTL at this locus has been detected for milk and protein yield, and for milk calcium . Another ion transporter, showing widely reported associations with milk phenotypes, is inorganic pyrophosphate transport regulator, encoded by the gene ANKH on BTA20. The ANKH transporter controls extracellular mineralisation by regulating the levels of inorganic pyrophosphate in the extracellular matrix. In humans and mice, mutations in this transporter have been associated with arthritis and bone growth disorders linked to tissue calcification . This transporter has previously been assumed to be a pyrophosphate transporter; however, recent work has shown that the channel in fact transports ATP, and that the production of extracellular pyrophosphate from ATP requires the enzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) . In cattle, QTL mapping to the ANKH locus have been identified for milk yield , milk lactose concentration , and a-lactalbumin concentration . Another recent study discovered that the ANKH protein cycles between the plasma membrane and trans-Golgi network using clathrin-coated vesicles mediated by clathrin adaptors AP1 and AP2. The phosphatidylinositol binding clathrin assembly protein also interacts with AP2 , binding to the signalling molecule phosphatidylinositol and recruiting the AP2 complex to form clathrin-coated pits. This protein is encoded by the gene PICALM on BTA29 and is associated with QTL for aS1-CN and lactose concentration . 3. Identifying Candidate Causative Genes The previous section may have given the impression that identifying the causative genes, or even variants underlying QTL, is easy. However, in many cases, there will be no obvious candidate genes in the QTL, possibly because the causative variant sits in a long-range regulatory element for a distant gene. In other cases, there may be many potential candidate genes, and different selection methods may highlight different candidates. For example, the window from 42.2 to 42.5 Mbp on BTA19 envelopes several candidate genes for milk phenotypes. A 2015 study by Raven et al. highlighted the genes GH3-domain containing (GHDC), STAT5A, and STAT5B on the basis of differential expression and enrichment of significant variants, and proposed STAT5A as causative on the basis of knockout studies in mice. In contrast, later work by our group used eQTL data and missense variants in strong LD with the top QTL variant to highlight the genes DExH-box helicase 58 (DHX58), GHDC, lysine acetyltransferase 2A (KAT2A), KCNH4, STAT5A, and STAT5B. The two STAT5 genes were again proposed as the most likely candidates. As discussed above, potassium channels, such as that encoded by KCNH4, have been associated with milk at a number of loci, and the histone deacetylase and transcription activator KAT2A2 is also a possible candidate, based on the high levels of gene expression required in the lactating mammary gland. It is possible that multiple QTL are segregated at this locus, and therefore, that more than one gene is causative. Another region with two strong candidate causal genes maps to between 15.3 and 15.6 Mbp on BTA3. At this locus, Raven et al. identified a QTL for milk protein concentration and proposed the epithelial mucin gene MUC1 as the best candidate. The mucin 1 protein coded by this gene is considered a "metabolic master regulator" , regulating tyrosine-kinase signalling and the expression of metabolic genes. Work by our group has identified a QTL for milk lactose concentration at the same locus, and proposed the gene SLC50A1, encoding a sugar transporter. Again, it is possible that these are two separate QTL, and that both genes are causative for the corresponding phenotypes; however, another possibility is that the QTL is pleiotropic, and only one QTL is present controlling both phenotypes. Distinguishing between these two possibilities is difficult or impossible using purely statistical or bioinformatic means, and additional experiments are likely to be required. 3.1. Molecular Phenotypes Widely used phenotypes, such as fat and protein, effectively aggregate signals from a large number of milk components: individual fatty acids and other lipids for fat, and a number of casein and whey proteins for protein, for example. It is likely that these different components are not all under the same genetic regulation, and some correlations may even be negative. For example, Stoop et al. observed genetic correlations of as low as -0.84 between milk fatty acid measures (C14:0 and C16:0), and correlations over 0.9. This suggests that using finer composition measures, such as individual fatty acids or proteins, should give cleaner, and possibly a larger number of genetic signals compared to complex phenotypes such as fat or protein. This approach has been successfully applied to use milk minerals and individual protein measurements to identify novel QTL and to highlight SLC37A1 as involved in lactation . These phenotypes can be considered "molecular", as measurements for a single molecule, for example, using gas or liquid chromatography with mass spectroscopy, provide the phenotype. Molecular phenotypes in milk are often measured using Fourier-transform mid-infrared spectroscopy, as this method is commonly used for commercial herd testing and is cheaper to perform on a large scale than spectroscopy. This method uses the absorbance of around 900 different frequencies of infrared light (known as wavenumbers), then uses these values in a model to predict the phenotypes of interest. In commercial herd testing, the predicted phenotypes are typically fat, protein, and lactose concentrations; however, models have been developed for a range of other phenotypes, such as individual fatty acids and proteins , and phenotypes more remote from milk, such as methane production and fertility . However, because FT-MIR phenotypes are predicted rather than measured, their utility for QTL discovery can be variable. For example, our recent work showed that both HPLC measured and FT-MIR predicted phenotypes could detect significant cis-QTL for a-casein, k-casein, and b-lactoglobulin. However, FT-MIR was unable to identify the highly significant cis-QTL for lactoferrin, which was identified using HPLC, and identified a QTL at the locus of the fat synthesis gene DGAT1 for a-casein. The latter may be due to signal crossover from fat, which is highly correlated with protein in milk. This form of crossover, if present, would complicate the task of identifying pleiotropy. In addition to predicting concentrations of milk components, it is also possible to use the individual wavenumber data themselves as phenotypes : different wavenumbers represent different chemical bonds that absorb MIR light at the corresponding frequencies, so wavenumber phenotypes effectively measure the concentrations in milk of compounds that contain specific chemical bonds. Wavenumber phenotypes have been shown to detect stronger and more numerous genetic signals compared to the predicted phenotypes such as fat and protein . Examining the expression level of each gene in a relevant tissue sample provides an alternative to measuring or estimating protein concentrations. In milk, for example, expression levels of the genes encoding milk proteins in the lactating mammary epithelial tissue can give proxy measurements for milk proteins. Traditionally, this could be done using techniques such as qPCR or expression microarrays. More recently, RNA sequencing (RNA-seq) being used to sequence and count (relatively) mRNA molecules has become common. As RNA-seq captures data for every expressed gene, it is possible to search for expression QTL (eQTL) and allele-specific expression (ASE) for every gene, including those not expressed in the milk, such as fat synthesis enzymes and hormone receptors. The presence of a QTL for a given gene can provide evidence on the causality (or otherwise) of the gene at an overlapping QTL: when the QTL is caused by an underlying eQTL, we expect that the causal variant or variants will be strongly associated with both the QTL and eQTL, and that associations for other variants will decay away in proportion to linkage disequilibrium with the causal variants. This pattern can be identified by examining the correlations between variants regarding the QTL and eQTL variants' effects , or p values (on a logarithmic scale) , or correlations between local genomic estimated breeding values (GEBVs) and gene expression . Other methods used to associate eQTL with GWAS results include transcriptome-wide association scans (TWAS) , Mendelian randomisation , and Bayesian colocalisation methods . Molecular phenotypes and their QTL have assisted in highlighting candidate causative genes in cases where no obvious candidates existed. For example, the gene MGST1 on BTA5 encodes microsomal glutathione S-transferase 1, which belongs to a family of detoxification enzymes . It has no obvious role in milk production. Nevertheless, QTL for milk traits mapping to this locus have been reported in many different studies . The neighbouring gene EPS8 (epidermal growth factor receptor kinase substrate 8) is sometimes proposed as a candidate. However, gene-expression data from lactating bovine mammary tissue have shown that the milk QTL at this locus co-segregate with an eQTL for MGST1, whereas EPS8 is barely expressed in this tissue. Similarly, a QTL for milk-fat percentage on the distal end of BTA11, has been linked to the gene encoding the ABO blood group, which is also named ABO (alpha 1-3-N-acetylgalactosaminyltransferase and alpha 1-3-galactosyltransferase), using RNA-seq data to show that a splice donor site mutation causes aberrant splicing of the ABO transcript, in turn causing an eQTL that co-segregates with the fat percentage QTL . 3.2. Chromatin Structure Phenotypes Chromatin is the name given to the compound comprising DNA wound around nucleosomes, which are themselves composed of histone proteins. The three-dimensional folded structure of chromatin can have an important impact on gene expression. On a proximal scale, the structure of the chromatin region surrounding a gene can be in open or closed configurations. The former provides access for transcription factors and RNA polymerase components to reach the promoter region and trigger gene expression, and the latter, closed, configuration blocks expression. Variants sitting within an open chromatin region in the appropriate cell type are more likely to have a regulatory impact on gene expression , and therefore, are more likely to present good candidate causal variants for a given trait, compared to other non-coding variants. One method commonly used to study chromatin state is chromatin immunoprecipitation (ChIP) , which can be used to identify transcription-factor binding sites or histone modifications. Histone proteins feature long tails, and a wide variety of post-translational modifications can be made to these tails to alter the chromatin state. For example, the modifications H3K4me2 (histone 3, lysine 4 dimethylation), H3K4Me3, and K3K27ac (lysine 27 acetylation) are associated with open chromatin in active promoters and enhancers; and closed, repressed regions can be marked by H3K9me2 or H3K27me3 . More recent versions of ChIP, using DNA sequencing techniques to target the whole genome in a single experiment, include ChIP-seq and CUT&RUN-seq . Recent work has identified cis-QTL for ChIP-seq phenotypes; and demonstrated that these QTL frequently exhibit strong correlation with nearby eQTL, and that eQTL tag variants are significantly enriched within ChIP-seq-identified open chromatin windows; this illustrates the utility of this type of data for understanding regulatory variation in the genome. Open chromatin is accessible to transcription factors and other proteins required for gene expression. However, it is also more accessible than other regions to nuclease enzymes, and this fact is used in another approach to identifying open chromatin regions. One commonly used nuclease is DNase 1, which can be used in conjunction with high-throughput sequencing via DNase-seq . The hypersensitivity sites identified using this technique highlight the positions of regulatory elements such as promoters, silencers, and enhancers. A similar technique, using the enzyme micrococcal nuclease, is called MNase-seq , and identifies the positions of nucleosomes. As nucleosomes are scarcer in open chromatin, these data give an inverse signal to hypersensitivity sites identified by DNase-seq. A more recent technique called the assay for transposase accessible chromatin (ATAC-seq) uses a modified hyperactive transposase enzyme Tn5 to fragment and load sequencing adaptors to open chromatin regions. ATAC-seq typically requires fewer cells and less time to perform compared to DNase-seq, although single-cell protocols have now been developed for both methods . On a larger scale, the 3D folded structure allows distal enhancer and silencer elements to enter close contact with the gene to perform their respective functions on gene expression . Identifying this structure allows linkages between genes and distal regulatory elements to be identified. These sorts of long-range interactions can be studied using chromosome conformation capture (3C) , and related methods, such as 3C-on-chip (4C) and 3C carbon copy (5C) . These older techniques can study only small parts of the genome. A more recent technique, Hi-C , could originally study the structure of the whole genome only at low resolution (1Mbp), but later refinements of the method have allowed for improved resolution and signal-to-noise ratios. 3.3. From Candidate Genes to Causative Variants The results of GWAS and similar experiments are typically genomic intervals containing several associated variants that are in strong LD with one another. Although candidate causal genes may be selected using known pathways, gene-expression data, or chromatin structure, as discussed above, for some applications it is useful or necessary to know the causal variant underlying the QTL. These applications include improving the accuracy of genomic selection, performing genetic testing, or creating gene-edited animals . However, identifying the causal variant using purely statistical or bioinformatics approaches is typically not possible, as variants in strong LD cannot be distinguished from one other statistically. In some cases, variant annotation (using tools such as SnpEff or Ensembl's Variant Effect Predictor ) can highlight missense or nonsense mutations that will make stronger candidates , but many QTL are driven by regulatory effects rather than coding ones. This means that other tools will be needed to resolve QTL with underlying regulatory effects. One commonly used technique for discovering cis-regulatory elements (CREs) is transcription factor binding site (TFBS) prediction. Several tools have been developed for this, generally using information from databases containing binding site motifs for large numbers of transcription factors, such as TRANSFAC and JASPAR . A second approach to investigating regulatory effects is to use a reporter assay, where the effects of putative promoter variants can be tested against the expression of a reporter gene such as GFP. This method has recently been scaled up to test thousands of variants simultaneously using a massively parallel reporter assay (MPRA) . A third method is to use CRISPR-Cas9 or other gene-editing technologies to test putative regulatory sequences, either by deleting them using NHEJ, or by editing in alleles of interest using HDR and then observing the resulting effect on gene expression, often using single-cell RNA-seq. Methods incorporating CRISPR include Perturb-seq , CROP-seq , and HCR-Flowfish . 4. Conclusions Over the last twenty years since the seminal work of Grisart et al. showed that the DGAT1 gene underlies the large milk QTL on BTA14, a large number of milk-related QTL have been identified in cattle, and candidate causative genes have been proposed for many of them. In the coming years, we can expect that sequence-resolution data sets will continue both to grow and diversify to include additional breeds from around the world. These larger, more diverse data sets will likely empower the discovery of many novel QTL. Although proving the causality of genes and variants underlying these QTL will likely remain difficult, the use of molecular phenotypes, massively parallel reporter assays, and CRISPR will extend the list of proven causatives. This new information should provide greater insights into the genes and pathways underlying the initiation and maintenance of lactation in mammals. Knowledge of causative genes and variants may also improve the accuracy of genomic selection in animal breeding, which would accelerate genetic gain and improve on-farm productivity. Causal variants will also provide for gene editing to rapidly spread beneficial variants through the population, should future regulatory environments allow it. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement No new data were created or analysed in this study. Data sharing is not applicable to this article. Conflicts of Interest T.J.L. is an employee of the Livestock Improvement Corporation, a commercial provider of bovine germplasm. The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Abbreviations The following abbreviations are used in this manuscript: BTA Bos taurus (cow) chromosome CRISPR Clustered regularly interspaced short palindromic repeats eQTL Expression quantitative trait locus FT-MIR Fourier-transform mid-infrared GM-CSF Granulocyte-macrophage colony-stimulating factor GWAS Genome-wide association study HDR Homology-directed repair HPLC High-performance liquid chromatography IL Interleukin LD Linkage disequilibrium Mbp Million base pairs MFG Milk fat globules MPRA Massively parallel reporter assay NHEJ Non-homologous end joining OAR Ovis aries (sheep) chromosome qPCR Quantitative polymerase chain reaction QTL Quantitative trait locus TDT Transmission disequilibrium test TFBS Transcription factor binding site TWAS Transcriptome-wide association scan Figure 1 Locations on the 29 bovine autosomes of the QTL discussed in this paper. Colors indicate which of the sections the QTL is discussed under. The "other" category represents the two genes, MGST1 and ABO. Positions are based on the ARS-UCD1.2 reference genome. Figure 2 An overview of fatty acid and triglyceride synthesis, highlighting the enzymes discussed in Section 2.2. Enzymes are shown in blue, and other products in yellow. The AGPAT enzyme group is shown in grey, as the only AGPAT gene exhibiting a significant QTL (AGPAT6) has been renamed to GPAT4. Figure 3 JAK/STAT signalling pathways with effects on mammary gland growth or involution. Signalling proteins, receptors, and transducers with known milk QTL are shown in blue, and selected other components are shown in grey. Blue arrows represent activation, and orange arrows indicate suppression. The green band represents the plasma membrane. animals-13-00911-t001_Table 1 Table 1 Top milk yield and composition QTL counts for cattle by trait. Data from CattleQTLdb release 49 (28 December 2022) . Trait Number of QTL Variants Milk fat percentage 11,911 Milk protein percentage 9958 Milk fat yield 9255 Milk yield 7383 Milk C14 index 4847 Milk kappa-casein percentage 4275 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000086

Bovine parainfluenza virus type 3 (BPIV3) is a common respiratory pathogen that causes respiratory illness in cattle and makes a major contribution to the bovine respiratory disease complex (BRDC); however, data on the prevalence and molecular features of BPIV3 are still scarce in China. To investigate the epidemiological characteristics of BPIV3 in China, between September 2020 and June 2022, 776 respiratory samples were received from 58 BRDC-affected farms located in 16 provinces and one municipality. Those were screened for BPIV3 using a reverse transcription insulated isothermal PCR (RT-iiPCR) assay. Meanwhile, the HN gene and complete genome sequence of strains from different provinces were amplified, sequenced, and analyzed. The tests showed that 18.17% (141/776) of samples tested were positive for BPIV3, which originated from 21 farms in 6 provinces. Moreover, 22 complete HN gene sequences and 9 nearly complete genome sequences were obtained from the positive samples. Phylogenetic analysis based on the HN gene and complete genome sequences revealed that the sequences were clustered in one large clade for all Chinese BPIV3 genotype C strains, while overseas strain sequences of BPIV3 genotype C clustered into other clades. Moving beyond the known complete genome sequences of BPIV3 in GenBank, a total of five unique amino acid mutations were found in N protein, F protein, and HN protein in Chinese BPIV3 genotype C strains. Taken together, this study reveals that BPIV3 genotype C strains, the dominant strains in China, have a broad geographical distribution and some unique genetic characteristics. These findings contribute to our understanding of the epidemiological characteristics and genetic evolution of BPIV3 in China. bovine parainfluenza virus type 3 China prevalence molecular characterization phylogenetic analysis evolution National Key Research and Development Program of the 14th Five-Year Plan, China2021YFD1600203 Key Laboratory of Colleges and Universities of Sichuan Province--Laboratory of Animal Medicine China2021PTJS34 Innovation team for emerging animal diseases, Southwest Minzu University2020NTD02 This work was funded by the National Key Research and Development Program of the 14th Five-Year Plan, China (2021YFD1600203), and the Key Laboratory of Colleges and Universities of Sichuan Province--Laboratory of Animal Medicine China (2021PTJS34); Innovation team for emerging animal diseases, Southwest Minzu University (grant number 2020NTD02). pmc1. Introduction Bovine parainfluenza virus type 3 (BPIV3) is a respiratory tract pathogen of the genus Respirovirus belonging to the family Paramyxoviridae . Infection of cattle with BPIV3 causes tissue damage, immunosuppression, and heightened susceptibility to secondary bacterial infections, leading to more severe clinical signs. The resulting disease was described as bovine respiratory disease complex (BRDC), which causes significant economic losses to producers each year . The BPIV3 genome is a negative-strand RNA of 15434-15504 bases in length, encoding six structural proteins (N, P, L, M, F, and HN) and three non-structural proteins (C, D, and V) . According to HN gene sequence or complete genome sequence phylogenetic analysis, BPIV3 can be divided into three genotypes, A, B, and C, and genotype C was first identified in China . Genotype C has since been found in Japan, the US, Korea, and Turkey . So far, three BPIV3 genotypes have been found in China . A seroepidemiological study of BPIV3 showed that the antibody positive rates of BPIV3 in the Jilin, Xinjiang, Inner Mongolia, and Shanxi regions were more than 80%, indicating that BPIV3 was widespread in northern China. A recent study showed that genotype C was the dominant strain in Inner Mongolia ; however, the prevalence and molecular characteristics of BPIV3 in China remain largely unknown. To better understand the epidemiology and genomic characteristics of BPIV3, we have systematically surveyed BPIV3 prevalence in China and analyzed its molecular features. In this study, a total of 776 respiratory samples of beef cattle and yak were collected from 16 provinces and one municipality in China between September 2020 and June 2022 to detect the prevalence of BPIV3. Moreover, 22 HN gene and 9 near-complete genome sequences were obtained from BPIV3-positive samples and analyzed, laying the foundation for further studies on genetic variation of BPIV3 genotype C strains in China. The findings help improve our understanding of the evolution and molecular characteristics of BPIV3. 2. Materials and Methods 2.1. Sample Collection From September 2020 to June 2022, a total of 776 respiratory samples were collected from beef cattle and yak at 2-6 months of age and with BRDC from 16 provinces and one municipality in China (Table 1). The typical clinical characteristics of beef cattle and yak with BRDC are a runny nose, cough, and dyspnea. All samples were transported on ice to the laboratory and stored at -80 degC. 2.2. RNA Extraction and cDNA Synthesis The nasal swabs were placed in 1 mL phosphate buffer saline (PBS), vigorously vortexed, and centrifuged at 10,000x g for 10 min. The lung tissues were fully ground, and the homogenates were diluted 1:3 (w/v) with PBS. Then, the samples were prepared by freeze-thawing three times, and the lysed samples were centrifuged (10,000x g, 10 min) and the supernatants collected. According to the kit instructions, viral RNA was extracted from 300 mL of the sample suspension using a Nucleic Acid Co-Prep Kit (GeneRadar, Xiamen, China), and a PrimeScript RT Reagent Kit (TaKaRa, Kyoto, Japan) was used to synthesize cDNA, which was then stored at -20 degC. 2.3. Detection of BPIV3 A total of 776 respiratory samples were screened for BPIV3 detection by a specific reverse transcription insulated isothermal PCR (RT-iiPCR) assay established at our laboratory. A POCKITTM device (GeneRadar, Xiamen, China) was used for BPIV3 detection, where the program was run with the default parameters, and the tubes were incubated at 95 degC for 58 min to complete the reaction. The sequences of primers used were the following: F: 5'-ARAGGACACAGAAGAGAGCACT-3'; R: 5'-TRGCCACACATACAACTCTCT-3', and the sequence of the probe was FAM-TTACAGAAAGGGCGATTACATTATTACAGA-BHQ1 (targeting the P gene; amplification length 125 bp). The reaction system was as follows: 3 mL forward primer (10 mM), 3 mL reverse primer (10 mM), 0.25 mL probe (10 mM), 2 mL cDNA, 16.75 mL double distilled water, and 25 mL Premix Ex TaqTM (Probe qPCR) Kit (5 U/mL) (TaKaRa, Dalian, China). 2.4. Amplification of HN and Genome Sequences Based on known BPIV3 genomes, a pair of primers (HN-F: 5'-CGACAATAGCAATGAACCC-3'; HN-R:5'-TGTTGATGCCTGTCTTCTGT-3') was designed to amplify the complete HN gene sequences. Subsequently, a panel of primers was designed for BPIV3 genome amplification from BPIV3-positive samples (Table 2). PCR products were purified and cloned into the pMDTM 19T-vector (TaKaRa, Dalian, China) for sequencing. 2.5. Sequence, Phylogenetic, and Recombination Analysis Using the MegAlign program of DNASTAR 7.0 software (DNASTAR Inc., Madison, WI, USA), the homologies of the nucleotide and amino acid sequences were determined. Utilizing SeqMan software (version 7.0; DNA Star), the gene sequences were assembled. A phylogenetic tree was constructed with MEGA version 7.0 using the maximum likelihood (ML) method. The Bayesian phylogenetic tree was constructed using MrBayes software. Recombination analyses were conducted using the RDP4 program . The AlphaFold v2.0 was used to predict the protein structure , and the protein domains were predicted by SMART ), accessed on 7 February 2023. 3. Results 3.1. Detection of BPIV3 For the total sample, the detection rate of BPIV3 was 18.17% (141/776). Of the beef cattle nasal swab samples, 107 of the 645 (16.59%) samples were detected as BPIV3-positive by RT-iiPCR and distributed in six provinces : Sichuan (60/173, 34.68%), Ningxia (4/30, 13.33%), Inner Mongolia (16/58, 27.59%), Hebei (12/61, 19.67%), Shanxi (12/52, 23.08%), and Jilin (3/10, 30.00%). The overall positive rate of BPIV3 in the yak samples was 25.59% (34/131). The detection rates of BPIV3 in nasal swab samples and lung tissue samples from yaks in Sichuan were 16.90% (12/71) and 44.00% (22/50), respectively (Table 1). 3.2. Bioinformatics Analysis of HN Gene Sequences A total of 22 HN gene sequences (GenBank numbers: OP908132-OP908153) were amplified from BPIV3-positive samples from 13 farms in 6 provinces. Among them, 19 HN genes and the 3 HN genes were amplified from beef cattle and yak, respectively. The 22 HN gene sequences had a length of 1719 bp and encoded 573 amino acids (aa). The ML phylogenetic tree, which was constructed using all available complete HN gene sequences of BPIV3 in GenBank, revealed that the 22 strains belonged to genotype C and clustered with sequences from other Chinese genotype C strains. Furthermore, all overseas BPIV3 genotype C strains clustered into distinct clusters . Multiple sequence alignments indicated that the HN gene sequences identified in this study displayed an 81.10-99.80% nucleotide (nt) identity and 97.40-99.80% aa sequence similarity with all 61 BPIV3 genotype C complete HN gene sequences in GenBank. Further sequence analysis revealed that, compared to the HN gene sequences of the 61 available BPIV3 genotype C strains from GenBank, the Chinese strain HN sequences had a unique aa mutation (T84A). Structural modeling of the HN protein showed that the mutation was located in the a-helix and the predicted structural domain of the HN protein ranged from 34-571 aa and had only one transmembrane region, which was located at 34-56 aa . 3.3. Bioinformatics Analysis of the Complete Genomes Nine near-complete genomes were obtained from the clinical samples (GenBank numbers: OP718792-OP718797, OM621819, OM782290, and OM782291), which were all 15,474 bp in length. Among them, six genomes and three genomes were amplified from beef cattle and yak, respectively. All the complete BPIV3 sequences were used to create a Bayesian phylogenetic tree that showed the nine strains belonged to genotype C and clustered with all the Chinese strains in one large clade, while the seven overseas strains of BPIV3 genotype C clustered into another clade . Multiple sequence alignments indicated that the nine near-complete genomes shared 99.2%-99.8% nt identity (99.3-99.8% aa identity) with one another and shared 97.4-99.8% nt identity (97.5-99.8% aa identity) with all the complete genomes of overseas BPIV3 genotype C strains in GenBank. Meanwhile, no recombination events were found in any of them. Fascinatingly, all the Chinese strains (including the nine strains in this study and others from the uploaded three strains) shared 45 nt mutations (Table 3), which were not demonstrated by the seven known genomes of overseas BPIV3 genotype C strains. Five mutations occurred in non-coding regions, accompanied by thirty-five nonsense mutations and five sense mutations, which resulted in aa mutations in the N (P439L, T494A), F (N452D, M497I), and HN (T84A) proteins. 4. Discussion BRDC is a multifactorial disease complex of cattle that restricts the healthy development of cattle breeding worldwide ; however, the prevalence and molecular features of BPIV3, one of the major causative agents of BRDC, are still mostly unknown in most Chinese provinces. The findings of this study showed that 16.59% (107/645) of the beef cattle nasal swab samples were positive for BPIV3, and the positive samples were scattered over 14 farms located in 6 provinces. The reason for this may be the rapid development of the cattle industry in these provinces in recent years and the frequent transportation of live cattle, thus leading to the spread of the virus. In addition, the geographic distances between sampling localities were large (in some cases over 2500 km), which indicates that BPIV3 has a broad geographical distribution in China. We previously demonstrated the presence of BPIV3 in yak, but the prevalence remains unknown . In this study, 25.95% (34/131) of yak respiratory samples were detected as BPIV3-positive, which suggests that BPIV3 is widely present in yak. Yak (Bos grunniens) are members of the family Bovidae and genus Bos ,. There are more than 14 million yak worldwide, and they are found at high altitudes (above 2500-6000 m) in China, India, Nepal, Pakistan, Kyrgyzstan, Mongolia, and Russia , representing the main income source for herdsmen in plateau areas . The finding will aid in yak respiratory disease diagnosis and prevention. In addition, BPIV3 genotype C was found to be the dominant BPIV3 strain in cattle herds in China in the present study, with some unique genetic characteristics. These findings help improve our understanding of the evolution and molecular characteristics of BPIV3. Based on the genome sequence analysis, the Chinese BPIV3 genotype C strain shares five unique aa mutations in the N (P439L, T494A), F (N452D, M497I), and HN (T84A) proteins. The N protein in Paramyxovirinae is highly conserved and immunogenic. Both ends of the BPIV3 N protein (aa 9-157, N-terminal; aa 391-500, C-terminal) were identified to have good immunogenicity, and the C-terminal domain epitopes can be used to develop a diagnostic analysis that can discriminate between different genotypes . Fascinatingly, comparing to the N gene sequences of BPIV3, the aa mutations (P439L, T494A) were found in all Chinese BPIV3 genotype C strains, and these mutations were also found in genotype A strains. This phenomenon is significant as it may add to the difficulties in developing diagnostic methods that differentiate between genotypes of BPIV3. In addition, sequence comparison of the viral genomes with known paramyxoviruses showed that BPIV3 and human parainfluenza type 3 virus (HPIV3) were the most similar . In the study of HPIV3, an immunodominant region was also found in the C-terminal 397-486 region of N protein, and the above antigenic sites on N protein were found to be important in triggering the humoral immune response against HPIV3 . This indicates that further study of the biological significance of the N protein mutation sites (P439L, T494A) of BPIV3 genotype C strains in China should help better diagnose, prevent, and control BRDC. HN and F are two glycoproteins on the viral envelope, which play a crucial role in virus entry into host cells . The paramyxovirus F protein belongs to the class I viral fusion protein type and is produced as inactive precursors (F0), which are then split into two disulfide-linked domains during cellular processing (F1 and F2) . Currently, there is a paucity of research on the BPIV3 F0 protein, but there are many studies on a very similar HPIV3, and this information can provide a reference for future studies on the BPIV3 F0 protein. For instance, in the secondary structure of HPIV3, F1 is mainly composed of HRA (129-192aa) and HRB (447-484aa), of domains DIII (193-284aa), DI (285-368aa), and DII (375-428aa) . In this study, it was found that one of the two mutation sites of the F protein of BPIV3 (N452D) may be located in the HRB junction region of F1. Related research has shown that this HRB structure has a significant impact on virus and host cell fusion performance . For example, in the NDV study, four amino acids (L436, E439, I450, and S453) in the HRB region were identified, which can regulate the fusion ability or the expression of the active form to a certain extent . In HPIV3, T429 and N446 were identified as the key amino acids of the HRB region affecting the fusion process . Additionally, several studies indicated that point mutations in the F protein might impact the virus virulence . Therefore, the effects of F protein mutations in BPIV3 genotype C of the Chinese strain on the function of the HRB region and viral virulence are worthy of further study. HN glycoprotein is a multifunctional protein that induces efficient membrane fusion of the virus with the host cell and mediates virus entry into the host cell through a complex mechanism . The predicted structure of the HN protein showed that the mutation at 84 sites changed from alanine to threonine. Alanine was a hydrophobic aa and threonine was a hydrophilic aa. A hydrophobic aa is generally located within proteins and plays an important role in maintaining the protein structure and antibody-antigen interactions; further studies are needed to determine the effect of mutations on the viral protein structure and function. In addition, previous studies showed that aa mutations in HN protein affect the formation of binding sites to sialic-acid-containing receptors, for instance, the loss of the binding site caused by the Arg516 mutation in NDV HN significantly reduced the fusion properties and growth rate of the virus, demonstrating that the binding site on the NDV HN protein is closely related to virus infection and replication . Moreover, previous findings indicated antigenicity differences between BPIV3 isolates, mainly in the epitope envelope glycoprotein HN, differences that are related to their pathogenicity and virulence in animals . Meanwhile, in a study of the HN protein of NDV in Paramyxoviridae, it was found that a single nucleotide mutation in the HN protein either made the virus a highly pathogenic strain or reduced the virulence of the virus . Therefore, the effects of the unique mutation of the HN protein in BPIV3 genotype C of the Chinese strain on the fusion performance, pathogenicity, and virulence of the virus must be further studied. 5. Conclusions In this study, we detected the prevalence of BPIV3 on beef cattle and yak farms in different regions of China and further analyzed the genetic evolution of the HN gene and genome of BPIV3 strains. We confirmed that BPIV3 genotype C strains, the dominant strains in China, have a broad geographical distribution, and the Chinese BPIV3 genotype C strains considered showed some unique genetic characteristics, which improve our knowledge of the prevalence and genetic variation of BPIV3. Author Contributions Conceptualization, C.T. and H.Y.; methodology, Y.R.; software, Y.R.; validation, C.T., H.Y. and Y.R.; formal analysis, C.T. and H.Y.; investigation, Y.R.; resources, H.Y.; data curation, Y.R.; writing--original draft preparation, Y.R.; writing--review and editing, H.Y.; visualization, C.T.; supervision, C.T.; project administration, H.Y.; funding acquisition, H.Y. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The experimental protocol was approved by the Animal Management and Use Committee of Southwest Minzu University (SYXK2011-043). Local farmers sent samples to our laboratory for pathogen detection, and no animal experiments were conducted in this study. Informed Consent Statement Not applicable. Data Availability Statement The HN gene sequences and near-complete genomes were uploaded to GenBank (GenBank numbers: OP908132-OP908153, OP718792-OP718797, OM621819, OM782290, and OM782291). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Map of China displaying the areas of sample collection. The 17 sampled regions are indicated in gray. The red triangles () represent the sampling areas that tested positive for BPIV3. Figure 2 Phylogenetic tree based on HN gene sequences. MEGA 7.0 software was used to conduct multiple sequence alignments, and a phylogenetic tree was created using the ML method. The black circular marker sequences were obtained by amplification in this study; the black box markers are the sequences of other genotype C strains in China; the black triangle represents the set of HN sequences of BPIV3 genotype A sequences that are not shown in the phylogenetic tree. Figure 3 Structural model of HN protein. (A) The sequences from this study (GenBank number: OP908143). (B) Sequence as a reference (GenBank number: MH357343). Red bars represent mutation sites and orange bars represent transmembrane domains. Figure 4 Phylogenetic tree based on complete genomes. A phylogenetic tree was created using the MrBayes software. The orange font represents human parainfluenza virus type 3 sequences; the green font represents overseas BPIV3 genotype C strains; the blue font represents BPIV3 genotype C strains uploaded by others in China; and the red font represents the Chinese strain sequences in this study. animals-13-00793-t001_Table 1 Table 1 Prevalence of BPIV3 in respiratory samples from different regions of China. Region Sample Source Sample Type Number of Farms Number of Samples Positive Rate (%) Sichuan Beef cattle Nasal swab 10 173 34.68% (60/173) Fujian Beef cattle Nasal swab 1 10 0.00% (0/10) Ningxia Beef cattle Nasal swab 2 30 13.33% (4/30) Inner Mongolia Beef cattle Nasal swab 5 58 27.59% (16/58) Jiangsu Beef cattle Nasal swab 1 15 0.00% (0/15) Henan Beef cattle Nasal swab 4 40 0.00% (0/40) Hebei Beef cattle Nasal swab 4 61 19.67% (12/61) Shanxi Beef cattle Nasal swab 4 52 23.08% (12/52) Chongqing Beef cattle Nasal swab 4 83 0.00% (0/83) Shandong Beef cattle Nasal swab 1 30 0.00% (0/30) Jilin Beef cattle Nasal swab 1 10 30.00% (3/10) Qinghai Beef cattle Nasal swab 2 16 0.00% (0/16) Anhui Beef cattle Nasal swab 1 15 0.00% (0/15) Yunnan Beef cattle Nasal swab 1 10 0.00% (0/10) Heilongjiang Beef cattle Nasal swab 1 12 0.00% (0/12) Shaanxi Beef cattle Nasal swab 1 10 0.00% (0/10) Gansu Beef cattle Nasal swab 2 20 0.00% (0/20) Sichuan Yak Nasal swab 7 71 16.9% (12/71) Qinghai Yak Nasal swab 1 10 0.00% (0/10) Sichuan Yak Lung tissue 5 50 44.00% (22/50) animals-13-00793-t002_Table 2 Table 2 PCR primers for complete genome amplification of BPIV3. Name Primer Sequence (5'-3') Position BPIV3-1F ACCAAACAAGAGGAGAGACTTG 1-1963 BPIV3-1R ATTGTTGTGCTGAGCCTTGT BPIV3-2F AGACTCCATCCACAACCCA 1750-3697 BPIV3-2R CTTGTGTCTGGGAACTACTGTG BPIV3-3F TCAAAGGCAAAACAGTCATACAT 3482-5728 BPIV3-3R TCCTACTGAGCTTTGAATTGACTGT BPIV3-4F AACAGTACTAGTTCCAGGAAGAAGC 5396-7884 BPIV3-4R GGACAGCCAGTTAAATTGCATATTAC BPIV3-5F CAGTAGGACCGGGGATTTA 7880-9902 BPIV3-5R TGTCCTCCGTGTCTTTCTCTA BPIV3-6F CCTTTTTCCGAACTTTTGG 9734-12,509 BPIV3-6R GTAGTCACTGGTGTCAGAATCTTTA BPIV3-7F AGGAGGAAGAATGATAAATGG 12,105-13,822 BPIV3-7R TTCTTGGATTATCGTCACAGTTA BPIV3-8F GTGTGTTGTTTAGCAGAAATAGC 13,501-15,474 BPIV3-8R ACCAAACAAGAGAAAAACTCTGT animals-13-00793-t003_Table 3 Table 3 Nucleotide and amino acid differences between the complete genomes of the Chinese strains in this study and all BPIV3 genotype C overseas strains. Nucleotide Position Nucleotide Mutations Amino Acid Mutations Chinese Strains Overseas Strains Chinese Strains Overseas Strains 1088 T C - - 1169 A G - - 1355 T C - - 1426 C T P L 1436 C T - - 1590 A G T A 1666 A G / / 3659 A C / / 4096 T C - - 4234 C T - - 4357 C T - - 4603 T C - - 4795 A G - - 4938 A G / / 4968 A C / / 5654 T C - - 6474 A G N D 6611 G A M I 6767 A G / / 7082 C T - - 7104 A G T A 7310 T C - - 7847 A G - - 7916 C T - - 7943 C T - - 7946 C T - - 7952 C T - - 7991 T C - - 8180 T C - - 8282 A G - - 8453 G A - - 8701 A C / / 9424 C T - - 9784 C T - - 10,342 A G - - 11,815 T C - - 12,082 A G - - 12,853 T C - - 13,853 C T - - 14,261 C T - - 14,367 T C - - 14,607 C A - - 14,637 A G - - 14,904 G A - - 14,961 A C - - Note: - nonsense mutation; / noncoding region. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000087

This study aimed to investigate the potential of Bacillus coagulans (BC) as an inoculant in alfalfa silage fermentation. Fresh alfalfa was harvested at a dry matter (DM) content of 329.60 g/kg fresh weight (FW), and inoculated without (CON) or with BC (1 x 106 CFU/g FW), Lactobacillus plantarum (LP, 1 x 106 CFU/g FW), and their combinations (LP+BC, 1 x 106 CFU/g FW, respectively). Samples were taken at 3, 7, 14, 30, and 60 d, with three replicates for each. The prolonged ensiling period resulted in a decrease in pH values and an increase in lactic acid (LA) concentrations in alfalfa silages. After 60 d of fermentation, the application of BC and LP decreased the pH values and increased LA concentrations in treated silages, especially when their combination was applied. Application of BC preserved more water-soluble carbohydrates (WSC), and further application of BC increased WSC in LP+BC-treated silage compared to LP-treated silage. There was no significant difference in the crude protein (CP) content between the CON and treated silages, however, the BC and LP treatments reduced the ammonia nitrogen (NH3-N) concentration, especially when their combination was applied. Additionally, the BC and LP-treated silages had lower neutral detergent fiber (NDF) and acid detergent fiber (ADF) when compared to the CON silage (p < 0.001). Inoculants also increased Lactobacillus abundance and decreased Enterococcus abundance after 60 d of fermentation. Spearman's rank correlation analysis revealed a positive correlation between LA concentration and Lactobacillus abundance. It was noteworthy that LP, BC, and their combination increased the relative abundances of carbohydrate metabolism, energy metabolism, cofactors, and vitamin metabolism, decreasing the relative abundances of amino acid metabolism and drug resistance: antimicrobial. Therefore, the inclusion of BC increased the fermentation quality of alfalfa silage, with the optimal combination being LP+BC. According to the findings, BC could be considered a viable bioresource for improving fermentation quality. alfalfa Bacillus coagulans fermentation composition microbial diversity Natural Science Foundation of Ningxia Hui Autonomous Region2020AAC03079 National Natural Science Foundation of China31660694 the top Discipline Construction Project of Pratacultural ScienceNXYLXK2017A01 the Key Research and Development Program of Ningxia Hui Autonomous Region2021BBF02034 2021BEG02042 This study was supported by the Natural Science Foundation of Ningxia Hui Autonomous Region (2020AAC03079), the National Natural Science Foundation of China (31660694), the top Discipline Construction Project of Pratacultural Science (NXYLXK2017A01), and the Key Research and Development Program of Ningxia Hui Autonomous Region (2021BBF02034 and 2021BEG02042). pmc1. Introduction During each stage of silage production, there is a certain amount of dry matter loss (DM loss), which reduces the silage quality. Notably, the DM loss of aerobic respiration could be up to 4-20% , and even 2% higher in alfalfa silage than in grass silage due to the lower water-soluble carbohydrates (WSC) and higher buffering capacity . Although a number of lactic acid bacteria (LAB) have been developed to reduce DM loss in silage fermentation, traditional LAB inoculants primarily target the anaerobic fermentation phase and have little effect on DM loss in the aerobic stage . The development of silage inoculants is still ongoing, shifting from homofermentative LABs such as Lactobacillus plantarum and heterofermentative Lactobacillus buchneri to LAB strains with prebiotic functions . Therefore, the strains that could consume oxygen, shorten the aerobic stage, and reduce DM loss also have the potential to be silage inoculants. Previous studies have demonstrated that Bacillus could consume free oxygen and generate an anaerobic environment, which favors the growth of anaerobic beneficial intestinal bacteria, such as Bifidobacteria and Lactobacilli, and improves animal performance when administered orally . Currently, Bacillus have been used as a silage additive to enhance the fermentation quality and aerobic stability of alfalfa and corn silage . Of the Bacillus species, Bacillus coagulans (BC) has become a focus of research due to its lactic acid-producing capability, degradation of lignin model compounds , and strong resistance to acid, heat, and pressure . Additionally, BC has also been used as a feed supplement for monogastric animals, with satisfactory growth performances observed in pigs, fishes, and chickens . However, few studies have assessed its effects on silage fermentation characteristics. We hypothesized that BC could shorten the aerobic phase of silage by consuming free oxygen during the initial stage, finally improving silage quality. Silage fermentation is a process involving the interactions of microbes, and a better understanding could help to regulate silage fermentation. With the progress of science and technology, microbiomics technology has been used to provide specific microbial information in the silage ecosystem . It has only been in recent years that this new method has been widely applied to the study of silage microbial communities . To the best of our knowledge, few studies have confirmed the potential of microbes to be inoculants by using microbiomics technology. Therefore, this study investigated the effects of BC on silage fermentation characteristics and microbial community, confirming the potential of BC as an inoculant in alfalfa silage fermentation. 2. Materials and Methods 2.1. Materials and Silage Preparation Alfalfa (cultivar "WL 440") was harvested at the early bloom stage from three randomly selected plots of Ningxia University located in Yinchuan city, Ningxia province, China. Fresh alfalfa was wilted naturally to the dry matter (DM) content of 329.60 g/kg (within 8 h) and chopped into 1-2 cm pieces immediately using a hay cutter. The chopped forages from each plot were divided into 21 equal piles (each weighing approximately 500 g). One pile was immediately frozen at -20 degC for further analysis. The remaining 60 piles (5 ensiling durations x 4 treatments x 3 replications) were randomly assigned to one of the following treatment groups: (1) distilled water control (CON), (2) Lactobacillus Plantarum (LP, provided by Ningxia University, Yinchuan, China), (3) Bacillus coagulans (BC, provided by China center of industrial culture collection, identification number: CICC21735, Beijing, China), and (4) L. Plantarum plus B. Coagulans (LP+BC). The inoculants were applied at a rate of 1 x 106 CFU/g FW, and an equal volume of distilled water was used for the CON. All silage were vacuum packaged in polyethylene plastic bags using a vacuum packaging machine (Zhucheng Yizhong machinery Ltd., Shandong, China). The alfalfa silage bags were stored at room temperature (25 +- 2 degC) and sampled at 3, 7, 14, 30, and 60 d. 2.2. Analysis of Chemical Composition, Fermentation Characteristics and Microbial Composition The DM content was determined by drying the fresh and ensiled forages at 65 degC in an oven for 72 h and then ground with a mill (1 mm screen). Crude protein (CP), ether extract (EE), neutral detergent fiber (NDF), and acid detergent fiber (ADF) were analyzed according to the methods of the Association of Official Analytical Chemists (AOAC) , while ammonia nitrogen (NH3-N) and water-soluble carbohydrate (WSC) were measured according to Cai . A 20 g of sample was placed in a juice extractor (BA-828, Mannengda Plasthetics Co., Ltd., Guangdong, China) and squeezed for 30 s with 180 mL of distilled water. The filtrate was then filtered through four layers of cheesecloth, and the pH was measured using a glass electrochemical pH meter. Lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA) were measured by high-performance liquid chromatography (KC-811, column, Shodex, Shimadzu, Japan; oven temperature, 50 degC; flow rate, 1 mL/min; SPD, 210 nm) . Samples (5 g) of fresh forage and silage were blended with 45 mL of sterile water and diluted (10-1 to 10-5) after being shaken for 0.5 h at room temperature. To analyze the microbial composition, LAB was cultured on MRS medium in an anaerobic box (ANX-1; Hirosawa Ltd., Tokyo, Japan) at 37 degC for 48 h, whereas yeasts and molds were cultured on potato Dsandy AGAR at 28 degC for 72 h . The number of viable microorganisms per gram of fresh matter (FM) was calculated in log10 CFU. 2.3. Microbial Diversity Analysis For molecular analysis of microbial communities, genomic DNA was extracted using a FastDNA(r) SPIN Kit (Tiangen, DP302-02, Tiangen, China). Th e DNA quality was verified by an ultramicro spectrophotometer (Thermo, NanoDrop 2000, Waltham, MA, USA). The DNA used for high-throughput sequencing was amplified using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'), targeting the V3-V4 regions of 16S rRNA. The PCR was conducted in a 20 mL mixture containing 1.6 mL primer mix (5 mM), 10 ng template DNA, 4 mL 5 x FastPfu Buffer, 2 mL dNTPs (2.5 mM each), 0.4 mL FastPfu Polymerase and 0.2 mL BSA, with dd H2O added to reach 20 mL. The PCR products were evaluated with 1% agarose gel and purified with AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). Amplified fragments were sequenced on an Illumina MiSeq PE300 platform (Majorbio Bio-pharm Technology Co., Ltd., Shanghai, China). All raw reads were cleaned by discarding those <50 bp and those not matching standard barcodes. The OTUs were clustered, and chimeras were removed based on 97% similarity. Each sequence was annotated for species classification using the RDP classifier, and compared to the Silva database, setting a comparison threshold of 70%, and counting each sample at the taxonomic level of community species composition. Principal coordinates analysis (PCoA) was drawn using the Vegan and Python packages. The linear discriminant analysis effect size (LEfSe) was used to calculate the communities or species with significant differences among the four groups. To investigate the relationship between silage quality and the bacterial community, Spearman's rank correlation coefficients were generated using R language (version 3.0.2). Using the relative abundances of microbes at the species level, the top 50 independent variables were calculated, and the dependent variables were calculated based on pH, NH3-N, LA, and AA. The bacterial function was predicted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database Phylogenetic Survey of Communities in Unobserved States Reconstruction (PICRUSt2, v2.3.0_b, accessed on 21 August 2022), which predicts the functional abundance of a sample based on the abundance of tagged gene sequences in the samples . 2.4. Statistical Analysis Data was analyzed using One-Way ANOVA of SPSS 21.0 (SPSS Inc., Chicago, IL, USA) for chemical composition and fermentation quality. Tukey's test was used for multiple comparisons and p < 0.05 was considered significant. Comparisons of CON vs. LP, CON vs. BC, and LP vs. LP+BC were conducted to evaluate the effects of LP, BC, and their combination on silage fermentation quality and chemical composition using the Kruskal-Wallis test. Comparisons of inoculant treatments were analyzed using t-tests. Differences were considered significant when p < 0.05. 3. Results 3.1. Chemical Composition and Microbial Population of Raw Materials The chemical composition and microbial populations of fresh materials are presented in Table 1. The pH, WSC, CP, NDF, and ADF contents in wilted alfalfa were 6.49, 19.61 g/kg DM, 201.29 g/kg DM, 476.73 g/kg DM, 311.48 g/kg DM, respectively. 3.2. Fermentation Characteristics of Alfalfa Silage Table 2 shows the fermentation properties of silages. The pH values decreased with prolonged ensiling time. After 3 d of ensiling, the application of LP and BC alone resulted in lower pH values in treated silages compared with the CON silage. However, further application of BC to LP treatment did not lead to any improvement in pH value, with comparable pH values observed in LP and LP+BC-treated silages. On the contrary, LA concentrations increased with prolonged ensiling time, and greater LA concentrations were observed in LP and LP+BC-treated silages. Application of BC did not show significant difference on LA concentration from the CON group regards 3 to 30 days, but increased it after 60 d of fermentation. Compared with LP-treated silages, LP+BC-treated silage had moderately greater LA concentrations at the same ensiling time, significant after 60 d of fermentation. Additionally, the application of LP and BC resulted in decreased AA in treated silages, particularly in LP+BC-treated silages, which being significant lower AA than the CON silage after 14 d of fermentation. 3.3. Chemical Composition and Microbial Population of Alfalfa The chemical composition and microbial population of silages are shown in Table 3. Application of LPor BC had no effects on the contents of DM and DM loss. The treated silages had lower WSC concentration when only LP was applied, while greater WSC concentration was observed when BC was added alone. Compared with LP-treated silage, further application of BC increased WSC concentration in LP+BC-treated silage. Neither LP nor BC affected CP contents but limited proteolysis in treated silages, with lower NH3-N concentrations found. In comparison to LP-treated silage, BC-treated silages had comparable NH3-N concentrations, while lower NH3-N concentration was observed in LP+BC-treated silage when further BC was applied. Both LP and BC decreased NDF and ADF concentrations in the treated silage compared to the CON silage, especially when LP was applied. Moreover, further application resulted in lower ADF in LP+BC-treated silage relative to LP-treated. The application of LP and BC promoted the growth of LAB in treated silages, especially when their combination was applied. Applying BC had no effects on the population of the yeasts, whereas decreased yeasts were observed in LP-treated silage in comparison to the CON silage. Additionally, molds were not detected in this study. 3.4. Microbial Community Composition and Diversity in Alfalfa Silage The principal coordinates analysis (PCoA) of the beta diversity revealed that the bacterial communities of fresh alfalfa and each treatment group were distinct after fermentation . The first two principal coordinates (PC1 and PC2) accounted for 42.05% and 25.89% of the total variance, respectively. The FM was grouped into a single category, whereas the CON- and BC-treated silages were clustered together and separated from the LP- and LP+BC-treated silages. The bacterial communities of fresh and silage samples were mainly composed of four phyla . Before ensiling, Proteobacteria was the most abundant phylum (75.97%), followed by Actinobacteriota (15.53%), Firmicutes (4.98%), and Bacteroidota (2.44%). After fermentation, Firmicutes became the dominant phylum, reaching up to 80.34%, 87.41%, 92.87%, and 88.69% in CON silage, LP-, BC-, and LP+BC-treated silages, respectively. Proteobacteria were more abundant in CON- and LP-treated silages than in BC- and LP+BC-treated silages. At genus level, the predominant genera in FM were Pseudomonas (30.50%), Methylobacterium (11.48%), Sphingomonas (7.91%), Enterobacter (7.00%) . The relative abundance of Lactobacillus increased in the treatment group, whereas Pseudomonas, Sphingomonas, and Enterobacter decreased. Weissella (41.59%) and Enterococcus (27.99%) were the leading genera in CON silage, whereas Lactobacillus dominated in LP- and LP+BC-treated silages, reaching 80.73% and 74.98%, respectively. Conversely, BC-treated silages had lower concentrations of Lactobacillus (15.28%), but greater concentrations of Weissella (42.88%). Linear discriminant analysis (LDA) effect size (LEfSe) was used to compare different treatments (LDA > 2.0) . There were 13 microbes that differed among the treatments. Thermoleophilia, Stappiaceae and Solirubrobacterales were more abundant in CON silages (LDA > 4.5). The LP-treated silage had higher relative abundances of Lactobacillus and Lactobacillaceae (LDA > 5.5), whereas the BC-treated silage had greater concentrations of Enterococcaceae and Enterococcus (LDA > 5.0). 3.5. Fermentation Characteristics and Microbial Community Correlation Analysis The Spearman's rank correlation between fermentation parameters and microbial strains at genus level is visualized in the form of a heatmap in Figure 4. There was a positive correlation between pH and Lactococcus, Enterococcus, and Aerococcus, but a negative correlation with Lactobacillus. The NH3-N concentration was positively correlated with the abundance of norank-f-Solirubrobacteraceae. Additionally, the LA concentration was positively correlated with Lactobacillus, but negatively correlated with Lactococcus, Enterococcus, Aerococcus, Weissella, and Methylobacterium-Methylorubrum. Conversely, Lactococcus, Brachybacterium, Rubellimicrobium, Brevundimonas, unclassified-f-Rhizobiaceae, Leuvobacter, and unclassified-f-Microbacteriaceae were negatively correlated with AA concentrations. 3.6. Bacterial Metabolic Functions and Enzyme Shifts during Ensiling in Alfalfa Silage The PICRUSt2 was used to predict the potential functions and enzymes of bacterial communities of the four groups . The predominant metabolism was the carbohydrate metabolism in silages, followed by amino acid metabolism. Compared to the CON silage, LP and LP+BC- treated silages showed a greater abundance of carbohydrate metabolism and amino acid metabolism, respectively . The majority of predicted functions explained by KEGG pathways in FM and silages were classified into organismal systems (8 pathways), human diseases (12 pathways), environmental information processing (2 pathways), genetic information processing (4 pathways), and metabolism (11 pathways). Compared with FM, alfalfa silages had a higher proportion of amino acid metabolism and lower proportions of carbohydrate metabolism. The carbohydrate metabolism, energy metabolism, cofactors, and vitamin metabolism abundances were lower in treated silages when compared with the CON silage . The LP+BC-treated silage had the lowest abundances of amino acid metabolism and drug resistance: antimicrobial, the LP-treated and BC-treated silages had comparable abundances. Compared with CON-treated silages, LP-and LP+BC-treated silages had a higher abundance of peptidase, whereas no significant difference was observed between CON- and BC-treated silages . The LP- and LP+BC-treated silages had a lower abundance of cellulose than the control group, and the LP+BC-treated silages had the lowest abundance. 4. Discussion 4.1. Fermentation Characteristics and Chemical Composition of ALFALFA Silage It is usually difficult to ensile alfalfa due to its high buffering capacity and low WSC concentration , resulting in more significant DM loss in the aerobic phase compared to corn and grass silages . Therefore, reducing the aerobic phase is essential for preserving more nutrients. This study found applying BC resulted in a reduced pH value in treated silage at each sampling time, particularly at 3 d, which is likely due to the acceleration of LA fermentation caused by oxygen consumption. Bai et al. also observed similar results when alfalfa silage was treated with Bacillus and Lactobacillus buchneri. At 60 d of silage, compared to CON silage, further application of BC resulted in lower pH levels and greater concentrations of LA in treated silage. This result confirms the hypothesis and suggests that BC could be an excellent supplement to the current inoculants. After 14 d of ensiling, when LP and BC were used in combination decreased AA concentrations in treated silages. After 60 d of ensiling, a greater LAB population was observed in treated silages than that in the CON silages, which was correlated with the results of lower pH and greater LA concentration. Moreover, lower levels of yeast were found in silages when BC and LP were applied, particularly in LP+BC silage. It appears that both BC and LP used in the study had advantages in nutrition competition over undesirable microbes. To produce high-quality silage, a WSC is an essential component . The study showed that silages inoculated with LP and its combination with BC had lower WSC concentrations after 60 d of storage; however, greater amounts of WSC were observed in BC-treated silage. It seems that the application of BC could shorten the aerobic phase and preserve more fermentation substrates. For legumes, proteolysis has been identified as a crucial problem in silage fermentation with a greater proportion of non-protein nitrogen, ranging from 440-880 g/kg total nitrogen . The proteolysis could result in lower protein utilization efficiency. Therefore, there have been numerous studies evaluating the effects of inoculants on proteolysis, with positive results in legume silage fermentation . The concentration of NH3-N, which is an indicator of proteolysis , was found to be lower in LP and BC-treated silages when compared to the CON silage, particularly when both BC and LP were applied. This could be attributed to the lower pH levels in treated silages. Additionally, lower NDF and ADF concentrations were found in treated silages when LP and BC were applied. These results could be explained by the acid degradation of hemicellulose. Furthermore, Bacillus could produce enzymes such as cellulase and feruloyl esterase, and it has been demonstrated that BC could degrade the lignin-related compounds ferulic acid and vanillin to vanillic acid . This could be the reason for lower ADF concentrations in LP+BC-treated silage when compared with silage inoculated with LP alone. 4.2. Diversity and Composition of Microbial Community Silage fermentation involves the interactions of microbes, and a better understanding of the dynamic of microbial communities could be beneficial for regulating the fermentation process . In this study, we investigated the effects of LP and BC on the bacterial communities of ensiled alfalfa. The results showed that fresh alfalfa was dominated by Proteobacteria and Firmicutes, which is in agreement with previous findings . Li et al. reported similar results for Cassava foliage after 60 days of ensiling with an increase in the relative abundance of Firmicutes to 88.69%. To further evaluate the effects of BC on the bacterial community during fermentation, we also analyzed the bacterial structures of alfalfa at the genus level. The dominant bacteria in the fresh alfalfa were Gram-negative bacteria, including Pseudomonas, Methylobacterium-Methylorubrum, and Sphingomonas. According to a previous report, the microbial flora of the alfalfa phyllosphere was predominately composed of Erwinia, Escherichia, Pseudomonas, and Pantoea . This might be because the settling of bacteria on the plant surface is affected by plant species, climate, geographical location, and fertilizer type . Lactobacillus and Weissella are the most common bacteria involved in the LA fermentation of silage . The researchers discovered that, as compared to the CON silage, treated silages showed greater relative abundances of Lactobacillus after 60 d, especially in LP and LP+BC-treated silages. This could be attributed to the lower pH and greater lactic acid production. Similar results were reported by Bai et al. , who found that the abundance of Lactobacillus was higher in Bacillus subtilis and Bacillus amyloliquefaciens-treated silages than in the CON silage. Weissella, which was considered an early colonizer, had a lower acid tolerance, and was unable to survive when the pH was below 4.5, yet the relative abundance of Weissella in BC-treated silage increased from 41.59% to 42.88% after 60 d of ensiling . This suggested that BC was not as effective in competing for nutrients with Weissella. 4.3. Relationships between Fermentation Parameters A correlation analysis was conducted between the composition of microorganisms and end-products after ensiling. It was observed that Methylobacterium-Methylorubrum, an aerobic bacterium utilizing the serine pathway to consume methanol and other reduced one-carbon compounds , was gradually replaced by Lactobacillus in this study as the pH of the silage decreased, resulting in a positive correlation between pH and Methylobacterium-Methylorubrum and a negative correlation between pH and Lactobacillus. Lactobacillus is essential in inhibiting harmful microorganisms by rapidly acidifying silage in the late stages of ensiling . In this study, a positive correlation was observed between NH3-N concentration and norank-f-Solirubrobacteraceae. This is likely due to the ability of certain species of Solirubrobacteraceae to thrive in anaerobic conditions and competes with Lactobacillus for nutrients . These results also agreed with the greater NH3-N concentration and relative abundance of norank-f-Solirubrobacteraceae in CON silage. Additionally, LA and AA concentrations were shown to be positively connected with Lactobacillus and negatively correlated with Enterobacter, which is likely a result of the production of LA and AA by Lactobacillus. These findings were further supported by the higher NH3-N concentration and relative abundance of norank-f-Solirubrobacteraceae in CON silage. 4.4. Bacterial Metabolic Functions and Enzyme Shifts in the Silage Alfalfa silage bacterial community functions were predicted using the KEGG pathways database and PICRUSt2. All samples contained a high proportion of the KEGG metabolism pathways, including the carbohydrate metabolism and amino acid metabolism. It was observed that four metabolic categories had a significant impact on microbial metabolisms. These categories were secondary metabolite biosynthesis, microbial metabolism in various environments, carbon metabolism, 2-oxocarboxylic acid metabolism, fatty acid metabolism, amino acid biosynthesis, and aromatic compound degradation . After ensiling, microbial metabolism was inhibited in the anaerobic environment, resulting in weaker amino acid metabolism. However, LP-treated silage had a greater abundance of Lactobacillus than LP+BC-treated silages, which contributed to a higher microbial metabolism capacity. We concentrated our efforts on the metabolic pathways of carbohydrates, amino acids, energy, cofactors, vitamins, and drug resistance, as these are all associated with changes in fermentation, chemical composition, and human health . Du et al. determined that the relative abundance of total LAB in the microbial community affected the abundance of carbohydrates metabolism pathways. However, this study found that entire LAB was lower in silage with more carbohydrate metabolic pathways. Because carbohydrate metabolism is primarily glycolysis and gluconeogenesis, Enterococcus are more capable of metabolizing WSC in CON silage and LP+BC-treated silages than beneficial species of LAB . Amino acids are integral to proteins and peptides, and play a substantial role in the energy metabolism and environmental tolerance of lactic acid bacteria . The application of LP, BC, and their combination resulted in lower amino acid metabolism abundances, which correlates with the reduced NH3-N concentrations observed in the treated silages compared to the CON silage. The relative abundance of cofactor and vitamin metabolism was higher in treated silages, suggesting that BC could accelerate vitamin production or produce vitamins directly during ensiling. Similar results were reported by Bai et al. who found that inoculating alfalfa silage with Enterococcus faecalis enhanced the relative abundance of cofactor and vitamin metabolism. Abuse of antibiotics leads to the development of multidrug-resistant bacteria, one of the greatest health threats . The antimicrobial drug resistance relative abundance was found to be the greatest in the CON silage, whereas the LP+BC-treated silage had the lowest relative abundance, followed by LP-treated silages. Antimicrobial drug resistance is primarily found in undesirable bacteria, such as Salmonella and Escherichia coli . The inhibition of undesirable and hazardous bacteria may be related to the lower pH in LP+BC-treated silages, which reduces resistance genes. 5. Conclusions The addition of BC increased the fermentation quality of alfalfa silages, indicated by lower pH values and greater LA concentrations, especially when BC was applied together with LP. Furthermore, the application of BC decreased DM loss, NDF, ADF and NH3-N contents, while increasing WSC. The microbial analysis demonstrated that the application of BC increasing Lactobacillus abundance and decreasing Enterococcus abundance in treated silages after 60 d of fermentation. Additionally, the application of LP, BC, and their combinations increased cofactor and vitamin metabolism abundance while decreasing drug resistance: antimicrobial pathway abundance. Thus, BC could be considered a viable bioresource for improving fermentation quality. Acknowledgments We sincerely thank the Key Laboratory of High-efficiency Production Model Innovation of Forage of the Ministry of Agriculture for their technical support for this research. Author Contributions Conceptualization, G.Z.; methodology, Y.W.; software, Y.W.; validation, Y.W. and W.K.; formal analysis, Q.L.; investigation, Y.W. and W.K.; resources, G.Z.; data curation, Y.W. and W.K.; writing--original draft preparation, Y.W., W.K. and Q.L.; writing--review and editing, G.Z., Y.W., W.K. and Q.L. visualization, Y.W.; supervision, G.Z.; project administration, G.Z.; funding acquisition, G.Z. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement All the sequence data of this study were deposited in the NCBI Sequence Read Archive (SRA) database under the accession number PRJNA935541. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Bacteria community principal coordinates analysis (PCoA) on genus level for alfalfa silage. FM, fresh shrub; CON, control silage; LP, silages inoculated with Lactobacillus plantarum; BC, silages inoculated with Bacillus coagulans; and LP+BC, silages combined with Lactobacillus plantarum and Bacillus coagulans. Figure 2 Bacterial communities and relative abundance by phylum level (A) and genus level (B) for alfalfa silage after 60 d ensiling. FM, Fresh material; CON, control silage; LP, silages inoculated with Lactobacillus plantarum; BC, silages inoculated with Bacillus coagulans; and LP+BC, silages combined with Lactobacillus plantarum and Bacillus coagulans. Figure 3 Comparison of microbial variations after 60 d of ensiling using the LEfSe analysis. CON, control silage; LP, silages inoculated with Lactobacillus plantarum; BC, silages inoculated with Bacillus coagulans; and LP+BC, silages combined with Lactobacillus plantarum and Bacillus coagulans. Figure 4 Correlation analysis between bacterial community and fermentation characteristics of alfalfa. LA, lactic acid; AA, acetic acid; NH3-N, ammonia nitrogen. * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001. Different color ranges represent different correlation coefficients in the right legend. Figure 5 Bacterial alterations that contribute to functional shifts after fermentation in different groups. CON, control silage; LP, silages inoculated with Lactobacillus plantarum; BC, silages inoculated with Bacillus coagulans; and LP+BC, silages combined with Lactobacillus plantarum and Bacillus coagulans. The Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) predicts functional shifts. The second level of the predicted functional shift for each Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway is shown for each group. (A). Alfalfa functional abundance (top 50 abundant functions) before and after ensiling. In the middle heat map, the color corresponds to the Z value calculated after normalizing the relative abundance of the function. The closer the color is to red, the greater the abundance. (B). Level 2 KEGG orthologue functional predictions explained by PICRUSt2. (C). Significant differences in the 5 functional pathways among the four groups at p < 0.05. Different lowercase letters shows difference among treatments (p < 0.05). Figure 6 Bacterial alterations that contribute to enzyme shifts after fermentation in different groups. CON, control silage; LP, silages inoculated with Lactobacillus plantarum; BC, silages inoculated with Bacillus coagulans; and LP+BC, silages combined with Lactobacillus plantarum and Bacillus coagulans. Different lowercase letters indicate differences between treatments in cellulase and protease (p < 0.05). animals-13-00932-t001_Table 1 Table 1 Chemical composition and microbial population of alfalfa before ensiling. Item 1 Alfalfa DM, g/kg FW 329.60 pH 6.49 WSC, g/kg DM 19.61 CP, g/kg DM 201.29 EE, g/kg DM 14.75 NDF, g/kg DM 476.73 ADF, g/kg DM 311.48 LAB, log10 CFU/g FW 6.41 Yeasts, log10 CFU/g FW 3.28 Molds, log10 CFU/g FW 2.26 1 DM, dry matter; WSC, water-soluble carbohydrate; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; EE, ether extract; CFU, colony-forming unit; LAB, lactic acid bacteria; FM, fresh matter. animals-13-00932-t002_Table 2 Table 2 Fermentation characteristics of the alfalfa silage during ensiling. Items 1 Ensilage Time, d Treatments 2 SEM 3 p-Value CON LP BC LP+BC CON vs. LP CON vs. BC LP vs. LP+BC I 4 pH 3 6.13 Aa 5.42 Ac 5.79 Aa 5.36 Ac 0.04 <0.001 0.034 0.84 <0.001 7 6.04 Aa 5.40 Ac 5.80 Ab 5.33 Ac 0.04 <0.001 <0.001 0.31 <0.001 14 5.83 Ba 5.18 Bc 5.68 Ab 5.17 Bc 0.03 <0.001 0.05 0.90 <0.001 30 5.69 Ba 4.96 Cc 5.27 Bb 4.94 Cc 0.03 <0.001 <0.001 0.54 <0.001 60 5.38 Ca 4.89 Cc 5.14 Cb 4.85 Cc 0.04 <0.001 <0.001 0.57 <0.001 LA, g/kg DM 3 15.41 Db 25.45 Ca 18.51 Cb 26.48 Ca 0.12 0.001 0.24 0.64 <0.001 7 31.92 Cc 59.01 Bb 58.23 Bb 63.84 Ba 0.54 <0.001 <0.001 0.69 0.004 14 52.87 Bb 79.48 Aa 57.03 Bb 85.06 ABa 0.46 0.01 0.30 0.54 0.001 30 67.06 ABc 87.25 Aab 72.64 Abc 90.66 Aa 0.21 0.02 0.46 0.57 0.003 60 74.88 Ac 91.55 Aab 82.12 Ab 93.83 Aa 0.19 0.02 0.05 0.04 <0.001 AA, g/kg DM 3 20.28 C 9.80 C 10.78 C 8.05 B 0.30 0.02 0.09 0.67 0.06 7 24.88 BC 17.96 BC 23.23 BC 14.60 AB 0.29 0.21 0.74 0.31 0.09 14 35.50 ABa 27.09 Bab 32.26 ABa 16.82 ABb 0.29 0.03 0.56 0.01 0.004 30 47.32 Aa 38.89 Aa 45.03 Aa 19.10 ABb 0.32 0.09 0.53 0.013 <0.001 60 47.19 Aa 42.19 Aa 43.59 Aa 23.87 Ab 0.29 0.34 0.49 <0.001 <0.001 Means with different uppercase superscripts in the same column differ significantly (p < 0.05); Means with different lowercase superscripts in the same row differ significantly (p < 0.05). 1 LA, lactic acid; AA, acetic acid; DM, dry matter; 2 CON, control silage; LP, silage inoculated with Lactobacillus plantarum, BC, silage inoculated with Bacillus coagulans; LP+BC, silage inoculated with the conbination of Lactobacillus plantarum and Bacillus coagulans; 3 SEM, error of the means; 4 I, inoculants treatment. animals-13-00932-t003_Table 3 Table 3 Chemical compositions and microbial population of the alfalfa after 60 d of ensiling. Item 1 Treatments 2 SEM 3 p-Value CON LP BC LP+BC CON vs. LP CON vs. BC LP vs. LP+BC I 4 DM, g/kg 432.47 388.31 367.11 394.80 0.18 0.26 0.13 0.51 0.17 DM loss, % 10.61 8.25 9.65 7.98 1.45 0.52 0.84 0.95 0.93 WSC, g/kg 12.88 7.93 14.15 12.10 0.06 0.01 0.33 0.01 0.001 CP, g/kg DM 208.34 211.09 210.45 216.65 3.54 0.18 0.69 0.31 0.42 NDF, g/kg DM 391.29 363.00 374.00 361.78 0.18 0.01 0.10 0.11 <0.001 ADF, g/kg DM 269.53 247.00 254.00 214.83 0.14 0.004 0.02 <0.001 <0.001 NH3-N, g/kg total N 142.00 126.51 130.91 110.90 256 0.003 0.03 0.004 <0.001 LAB, log10 CFU/g 7.41 7.71 7.61 7.91 0.02 <0.001 <0.001 <0.001 <0.001 Yeasts, log10 CFU/g 2.79 2.62 2.71 2.53 0.02 0.002 0.06 0.01 <0.001 Molds, log10 CFU/g <2.0 <2.0 <2.0 <2.0 - 1 DM, dry matter; DM loss, dry matter loss; WSC, water-soluble carbohydrate; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; NH3-N, ammonia nitrogen; CFU, colony-forming unit; LAB, lactic acid bacteria; 2 CON, control silage; LP, silage inoculated with Lactobacillus plantarum, BC, silage inoculated with Bacillus coagulans; LP+BC, silage inoculated with the conbination of Lactobacillus plantarum and Bacillus coagulans; 3 SEM, error of the means; 4 I, inoculants treatment. 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PMC10000088

Sound communication is important for underwater species. The wild population of the Chinese soft-shelled turtle (Pelodiscus sinensis) is listed as vulnerable. However, its vocalization, which can serve as the basis for ecological and evolutionary research, has not been studied. Here, we performed underwater recordings of 23 Chinese soft-shelled turtles of different ages and sexes and identified 720 underwater calls. The turtle calls were manually divided into 10 call types according to visual and aural inspection properties. The similarity test indicated that the manual division was reliable. We described the acoustic properties of the calls and the statistical analysis showed that the peak frequency of calls was significantly different between adult females and males, and also between subadults and adults. Similar to other aquatic turtles that prefer to live in deep water, Chinese soft-shelled turtles have a high vocal diversity and many harmonic calls, indicating that this highly aquatic species developed a variety of vocalizations to enhance their underwater communication, which helped them adapt to the complex and dim underwater environment. Furthermore, the turtles showed a tendency for vocalization to become more diverse with age. Pelodiscus sinensis vocalization diversity underwater recordings call types age-sex difference Postdoctoral Research Project of Hainan ProvinceRC2100007855 Natural Science Foundation of Hainan Province2019RC175 Academician Working Platform Project in Hainan ProvinceThis research was funded by the Natural Science Foundation of Hainan Province under Grant No. 2019RC175, Postdoctoral Research Project of Hainan Province under Grant No. RC2100007855 and the Academician Working Platform Project in Hainan Province. pmc1. Introduction As an indispensable source of information, sound is important for underwater species . Vocalization plays an important role in a turtle's life, conveying a large amount of individual information , and is associated with mating and incubation . Female Testudo marginata prefer fast-rate and high-pitched calls in males . Vocalization of T. hermanni can transmit information about individuals' size and health status and is related to their mating success . Moreover, the vocalizations of Chelonia mydas and Eretmochelys imbricata hatchlings in a nest are related to the synchronization of emergence . According to previous studies, turtle vocalizations are relatively simple compared to those of other groups of species, mostly coming in the form of pulses and harmonics . Early studies on the vocalization of turtles were mainly in the air, such as Dermochelys coriacea and Testudinidae . However, in recent years, an increasing number of studies have focused on the underwater vocalizations of turtles. Chelonida oblonga produces 17 call types in semi-natural pools . Three call types of Carettochelys insculpta , 11 call types of Podocnemis expansa , and six call types of Lepidochelys kempii were recorded in the field. Cultured Mauremys sinensis and Trachemys scripta elegans can produce 10 and 12 underwater sounds, respectively , and even juvenile green turtles (C. mydas) in field can produce 11 types of underwater sounds . Researchers believe that reptiles intermittently regulate the passage of air by expulsing large amounts of air and changing the size of the glottis . According to the anatomy of the turtle's throat, the structure and position of the hyoid cartilage and two bands of elastic fibers in the tortoise's larynx suggest that these bands are capable of vibrating, and two diverticula supported by the cricoid form a resonating chamber, all of which may function as vocal cords . This may explain why turtles can make underwater sounds. The most common method for turtle call classification is to record vocalizations and manually classify them according to visual and auditory differences . Turtles make sounds when they move underwater in tanks, such as crawling, stroking water, releasing bubbles, sucking water, scratching claws against the tank bottom, and scratching the tank wall with claws, and spectrograms of the sounds produced by these behaviors have been reported . These sounds can be easily distinguished from the vocalizations of turtles . The correlation coefficient test of signals is a traditional method for checking the degree of similarity between two signals . A method that combines manual classification with a signal similarity test can decrease the subjectivity of the manual division of calls, and it performed well in a previous study for turtle call classification . Chinese soft-shelled turtles (Pelodiscus sinensis) live in many countries in East and Southeast Asia . In China, they are bred in large numbers on farms, but their wild population continues to decline, and it is listed as a "Vulnerable species" by the International Union for Conservation of Nature Red List of Threatened Species . The physiology, ecology, and molecular biology of this species have been extensively studied , but its vocalizations have not been studied. The Chinese soft-shelled turtle inhabits inland fresh water and is a highly aquatic turtle (spends most of its life in water). These turtles prefer to live in deep water, normally 1-2 m and they dive into the mud at the bottom of the water most of the time . Thus, underwater vocalizations may be crucial for communication between individuals of the Chinese soft-shelled turtle species. Therefore, vocalization research can provide the basis for the study of the behaviors and ecology of Chinese soft-shelled turtles. In this study, passive acoustic monitoring was used to investigate the diversity of underwater vocalizations of Chinese soft-shelled turtles in a laboratory. We aimed to classify calls and describe their acoustic characteristics. Moreover, we used a similarity test to verify the objectivity of classification. We also analyzed differences in vocalizations across sex and age groups. 2. Materials & Methods 2.1. Data Collection Twenty-three healthy, captive Chinese soft-shelled turtles were used for the recordings: four adult females, four adult males, four subadult females, four subadult males, and seven hatchlings. These turtles live in semi-natural pools in a feedlot. We recorded the five groups of turtles. In addition, eight adult male and female turtles were mixed and recorded to determine whether there were any vocalizations associated with mating. The recordings were performed at Hainan Normal University in July 2021, in a professional soundproof room, free from environmental noise. Turtles were recorded in an inflatable plastic circular tank, which effectively reduced the sound reflection. The diameter of the tank was 1.2 m, and the water depth was 30 cm. The turtles could move freely through the water, and the hydrophone was placed in the center of the tank, 15 cm below the surface. Vocalizations of turtles were recorded using a Song Meter SM4 underwater sound recorder (Wildlife Acoustics, Inc., Maynard, MA, USA) (omnidirectional hydrophone bandwidth: 2-30,000 Hz +- 3 dB; sensitivity: -165 dB Re: 1 V/mPa; sampling rate: 44,100 Hz; gain: 16 dB). The hydrophone was calibrated by the company (Wildlife Acoustics, Inc.) before purchase. Each group was continuously recorded for 24 h, and 144 h of data were obtained. 2.2. Data Analysis Raven Pro 1.6 software (The Cornell Lab of Ornithology, USA) was used to visualize the waveforms and spectra of the recorded data. Sounds with spectral shapes similar to published turtle sounds were selected by researchers with experience in turtle vocalizations. Only calls with a high signal-to-noise ratio were selected. Before the selection of turtle calls, we identified the sounds produced by the common behaviors of turtles in water based on studies of other freshwater turtles . Turtle calls were classified based on differences in auditory sensing and spectrograms . Two calls that showed extremely similar spectral properties and aural characteristics were classified as the same type. Sennheiser IE300 earphones (Sennheiser, Hanover, Germany) were used to analyze the data. The low frequency (lower limit of a call, Hz), high frequency (upper limit of a call, Hz), peak frequency (frequency of a call at its highest energy point, Hz), signal duration (the time difference between end time and start time of a call, ms), and number of harmonics of calls were extracted using the Raven Pro 1.6 software. We chose the Pearson correlation coefficient to calculate the similarity between calls to judge the accuracy of the manual division . This included the similarity between calls of different call types and similarity between calls of the same call type. Pearson's linear correlation coefficient was calculated using MATLAB R2021a (MathWorks Inc., Natick, MA, USA). 2.3. Statistical Analyses The median and interquartile range (IQR) for all call types were calculated. We evaluated the differences in two acoustic parameters between age and sex groups: peak frequency and duration. First, the parameters of the sounds for each group were estimated using the Kolmogorov-Smirnov test to determine if they followed a normal distribution. If the distribution was normal (p > 0.05), the differences in the parameters of the calls among groups were tested using the least significant difference test (post hoc test algorithms); if not (p < 0.05), the differences were tested using Kruskal-Wallis analysis (non-parametric tests algorithms) . Second, if the differences in the parameters of the calls among the groups were significant (p < 0.05), paired comparisons were conducted between every two groups to estimate the differences. Differences were considered statistically significant at p < 0.05. These analyses were completed using IBM SPSS Statistics 26.0 (IBM, Armonk, NY, USA). 3. Results We found a total of 720 calls, of which 143 were produced by subadult males, 117 by subadult females, 192 by adult males, 133 by adult females, 135 by mixed-sex adults, and none by juveniles (Table 1). No courtship behavior was detected during the recording of the mixed-sex adult group. Turtle calls were classified into 10 types . Audio samples of each type are available in Supplementary Audio A-J. Acoustical parameters for each call are shown in Supplementary Table S1. 3.1. Description of Call Types Ten call types were identified, of which five were high-frequency calls and five were low-frequency calls. The statistics of their acoustic parameters are listed in Table 2 and their spectra are shown in Figure 1. Type A was a high-frequency call. It is a short vertical line on the spectrum, looking like a pulse with a very short duration. It was the most common call type of the Chinese soft-shelled turtle; more than half of them were produced by subadults. Type B was also a high-frequency call. The spectrum is composed of two parts: the front is a vertical pulse signal, followed by a point signal . This call type can be found in all age and sex groups, and more than half were produced by subadult males. Type C is a high-frequency call that has a fan-shaped spectrum , and its peak frequency is stable at approximately 14 kHz; Approximately half of the calls were emitted by adult females, but very few by adult males. Type D is a high-frequency call with an oblique vertical line on the spectrum, the top half of which is vertical and the bottom half rightward . It has a wide bandwidth. More than half of the calls were emitted by subadult females but none by subadult males. Type E is a medium-high frequency call that appears M-shaped or inverted V-shaped on the spectrogram, with one or three inflection points . This call has a long duration. Half of the calls were produced by subadult males. Type F is a low-frequency call with a long duration. It appeared as a straight line in the spectrum . This type occurred in all age and sex groups, with adult females accounting for more than 30 % of the calls. Type G is a low-frequency call with 1-11 down-sweep harmonics on the spectrogram for a long duration. The peak frequency is stable at approximately 100 Hz. More than two-thirds of calls were produced by adult males, but none by adult females. Type H is a low-frequency call, and the spectrum diagram shows 1-7 up-sweep harmonics with a turning point . More than half of these calls were produced by adult males. Type I is a low-frequency multi-harmonic call, whose spectrum diagram is a parallel horizontal line with 1-7 harmonics, most are 2-3 harmonics . Most calls of this type were produced by adults, with very few produced by subadults. This call type is the most common type of harmonic call in Chinese soft-shell turtles. Type J is a low-frequency multi-harmonic call, and the spectrum diagram shows a curved shape with 2-12 harmonics and multiple inflection points . Its peak frequency is 100 Hz. Only 10 calls of this type were found, all emitted by adults. 3.2. Similarity Calculation All the correlation coefficients between the calls within each call type were larger than 0.6, averaging 0.73, which means that the similarity between calls in each call type was high, and the difference was small. All the correlation coefficients between the 10 call types were lower than 0.22, which means that the call similarity between each type was low, and the difference was large. Thus, the manual classification of calls by Chinese soft-shell turtles had high reliability. The specific correlation coefficients are presented in Table 3. 3.3. Differences in Vocalizations between Sexes and Age Groups 3.3.1. Peak Frequency Three of the call types (Types A, C, and I) showed significant differences in the peak frequencies among the five groups. There is no significant differences in their peak frequencies among groups in other call types . In Type A, the peak frequency of subadult males showed significantly higher than that in the other groups (p < 0.05). No significant differences in peak frequency were detected between subadult females, adult males, and mixed-sex adults (p > 0.05). In Type C, subadult females showed a significantly higher peak frequency than mixed-sex adults (p < 0.05). There were no significant differences among the subadult males, adult males, and adult females (p > 0.05). In Type I, adult females showed significantly higher peak frequencies than adult males and mixed-sex adults (p < 0.05). There were no significant differences among subadult males, subadult females, adult males, and mixed-sex adults. (p > 0.05). In addition, there were no significant differences between the adult females and subadult groups (p > 0.05). 3.3.2. Duration Seven call types (Types A, B, C, F, G, H, and I) showed significant differences in call duration among the five groups. . In type A, adult females and males showed significantly longer call duration than subadult males and mixed-sex adults (p < 0.05). There were no significant differences between adult females and males, between subadult males, subadult females and mixed-sex adults (p > 0.05). In Type B, the adult female group had a significantly longer call duration than the subadult male group (p < 0.05). No significant differences were detected between adult females and adult males or between subadult females and subadult males (p > 0.05). In Type C, adult females showed significantly longer call durations than subadult males and subadult females (p < 0.05). There were no significant differences between adult females and adult males or between subadult females and subadult males (p > 0.05). Mixed-sex adults showed no significant differences from other groups (p > 0.05). In Type F, adult females showed a significantly longer call duration than mixed-sex adults (p < 0.05). No significant differences were detected among the other groups (p > 0.05). In Type G, adult males had a significantly longer call duration than mixed-sex adults (p < 0.05). No significant differences were detected among the other groups (p > 0.05). In Types H and I, adult males presented significantly longer call durations than mixed-sex adults (p < 0.05). No significant differences were detected among the other groups (p > 0.05). 4. Discussion 4.1. Vocalization Diversity The Chinese striped-neck turtle (M. sinensis), a sympatric species of the Chinese soft-shelled turtle, can produce 10 call types (three types are harmonics) . Of these call types, only one is a high-frequency pulse call type. In contrast, Chinese soft-shelled turtle can produce four high-frequency pulse call types, with much higher peak frequencies than those of Chinese striped-neck turtles. Both species vocalize down-sweep harmonics, but the peak frequency and high frequency of the Chinese striped-neck turtle's harmonics are higher than those of the Chinese soft-shelled turtle (the harmonics of the Chinese striped-neck turtle can reach 7 kHz, while the harmonics of the Chinese soft-shelled turtle are below 2 kHz). In addition, the Chinese striped-neck turtle has a low-frequency call type that is very similar to call type F of the Chinese soft-shelled turtle (spectrogram and auditory), but the peak frequency of Chinese soft-shelled turtles is approximately twice as high as that of Chinese striped-neck turtles. Whether the very similar vocalizations between the two species are due to their similar living environments and whether they have the same communication function requires further study. Of the 12 call types produced by the red-eared turtle (T. scripta elegans), another sympatric species of Chinese soft-shelled turtle, none resemble those produced by the Chinese soft-shelled turtle . Among the harmonic calls of long-necked turtles (Chelodina oblonga), three call types are very similar to call types G, I, and J of Chinese soft-shelled turtles . Although the habitats of the two species do not overlap, they are both carnivorous and often dive into the sediments of water . This finding may be explained by their similar lifestyles. Many tortoises, freshwater turtles, and sea turtles can vocalize, and most have been shown to produce multi-harmonic calls . Chinese soft-shelled turtles can produce 10 call types underwater, four of which are multi-harmonic calls. Many turtle species can produce multi-harmonic calls, which may be determined by the similar larynx structure of turtles . The structure and position of the larynx of the turtle are similar to those of the mammalian vocal cords, and the two diverticula held by the cricoid might function as low-frequency resonating chambers, improving the harmonic structure of tortoise calls . Similar to the Chinese soft-shelled turtle, other identified turtle species with more call types and more harmonic calls are highly aquatic species that prefer to live in deep water, such as P. expansa (11 call types, six of which are harmonics) and C. oblonga (17 call types, nine of which are harmonics) . These species might have developed a greater variety to adapt to dim underwater environments. The matched filter hypothesis proposes that tuning of the receiver's auditory sensitivity will evolve to closely match the dominant frequency of species-specific calls . Several studies on insects, fish, birds, and anurans have verified this hypothesis . However, because of the influences of natural and sexual selection, a mismatched relationship might arise between the sender's signals and the receiver's auditory sensitivity . Existing research has revealed that the hearing range of turtles is concentrated at low-frequencies ; however, aquatic turtle species have been found to produce high-frequency calls that can reach 6000-15,000 Hz . The Chinese soft-shelled turtle can produce high-frequency calls underwater, similar to its sympatric red-eared turtle (T. scripta elegans) in China . The advantage of high-frequency calls is that they help resist interference from low-frequency noise in communication, since natural waters are full of low-frequency noise . For instance, frogs (Odorrana tormota and O. graminea) and birds (Abroscopus albogularis) inhabiting aquatic environments produce calls containing ultrasonic components to avoid masking by wideband noise . As there have been no studies on the hearing threshold of Chinese soft-shelled turtles, we cannot be sure whether they can hear such high calls or whether these high-frequency calls have communication functions. In some reptiles, high-frequency calls are often used to intimidate heterospecific intruders but are not heard by conspecifics . Vocalization has been found to change with age in bats (Myotis nattereri bombinus and Rhinolophus ferrumequinum) , and this kind of change for Lonchura striata swinhoei was shown to be associated with the development of neural modulation mechanisms . The differences in vocalization and auditory sensitivity between Lusitanian toadfish (Halobatrachus didactylus) juveniles and adults demonstrates that acoustic communication may be absent in their early developmental stages . As Melopsitiacus undelafusp grows, its calls become more complex and higher in amplitude; as they grow, male birds have more complex calls than female birds . American Coots (Fulica americana) change their call types as they grow . The harmonic number of the song of the lesser horseshoe bat (R. cornutus) increases with age after birth . In rhesus monkeys (Macaca mulatta), the pitch of the "cooing" drops and the amplitude becomes more constant as they age . Green sea turtle (C. mydas) hatchlings produce only four call types, whereas subadults can emit 11 call types . In this study, juvenile Chinese soft-shelled turtles were not found to produce vocalizations; the subadults produced a large number of high-frequency calls, but only a very small number of low-frequency harmonic call types; and adults produce a more balanced number of different types of calls. This shows a tendency for the vocalization of Chinese soft-shelled turtles to become more diverse with age. Whether this phenomenon is related to the degree of development of the physiological function of the vocal organs requires further study. 4.2. Sex and Age Differences in Vocalization Diverse vocalizations among individuals in an animal population are often caused by differences in the structure of the vocal organs . A larger body size usually indicates a larger vocal organ that produces lower frequencies . Many birds and frogs have verified that their dominant frequencies of vocalization are inversely related to body size . The vocal frequency of tortoises is found inversely proportional to their size . In our recordings, the Chinese soft-shelled subadults had higher peak frequencies and shorter durations in two high-frequency call types. This may be because the size of subadults is smaller than that of adults, or it may be due to age differences in the neural modulation of vocalizations . However, the frequency of animal calls is not always inversely related to their body size. In several owl species, females produce higher calls than males, but females are larger than males . Male baboons (Papio cynocephalus), gibbons (Hylobates lar), and chimpanzees (Pan troglodytes) are larger than their respective females but have higher peak frequencies than their females in some call types . Jacana spinosa is larger than J. jacana but emits higher frequency calls than J. jacana . Adult females of red-eared turtles (T. scripta elegans) are much larger than adult males of the same age but produce more high-frequency calls than adult males . For call type I, which was the most frequent low-frequency call, the peak frequencies of adult females were significantly higher than that of adult males; however, the body sizes of the two groups were similar. For type A, there was also a difference in the peak frequency between male and female subadults of similar body size. Recent research has revealed that freshwater turtles show differences in sexually dimorphic hearing sensitivity , which is associated with the amount of gene expression in neural inhibitory pathways for hub genes related to the inner ear and tympanic membrane . Hence, sex differences in hearing sensitivity and auditory genes may be one reason for sex differences in vocalization. The potential function of turtle calls remains unclear, but they may be important for inter-specific communication . Research has shown that individual-specific vocalizations are used for the individual recognition of sea turtles . It must be noted that because these calls were recorded in an artificial environment, the sound descriptions might not completely reflect free-field recordings . 5. Conclusions We identified 720 underwater calls of Chinese soft-shelled turtles in recordings and divided them into 10 call types. These results provide basic data for other bioacoustics and evolutionary studies of Chinese soft-shelled turtles. Chinese soft-shelled turtles have a high vocal diversity and many harmonic calls, indicating that they may have developed a greater variety to adapt to dim underwater environments. In addition, Chinese soft-shelled turtles show a tendency for vocalization to become more diverse with age. The peak frequency of calls in Chinese soft-shelled turtles was significantly different between adult females and males and between subadults and adults. These findings suggest that further studies on the ecology and anatomy of Chinese soft-shelled turtle should be conducted to explain the mechanisms behind these phenomena. Further behavioral and auditory studies are needed to determine the intra-specific communication function and ecological implications of Chinese soft-shelled turtle vocalizations. Acknowledgments We thank the graduate students Yuanyu Qing and Shuo Cui at Hainan Normal University for their substantial assistance during data recording. We thank Liang Tao from Nanjing Forestry University and Haoran Ji from Hunan University for their help with the data analysis. Supplementary Materials The following supporting information can be downloaded at: Table S1: Acoustic parameters of each call produced by Chinese soft-shelled turtles; Table S2: Difference analysis of peak frequencies for 10 call types between five groups (Kruskal-Wallis tests); Table S3: Difference analysis of call durations for 10 call types between five groups (Kruskal-Wallis tests); Audio A: Sample of call type A; Audio B: Sample of call type B; Audio C: Sample of call type C; Audio D: Sample of call type D; Audio E: Sample of call type E; Audio F: Sample of call type F; Audio G: Sample of call type G; Audio H: Sample of call type H; Audio I: Sample of call type I; Audio J: Sample of call type J. Click here for additional data file. Author Contributions Conceptualization, L.Z., H.S. and J.W.; methodology, L.Z. and J.W.; software, L.Z.; formal analysis, L.Z.; investigation, L.Z., J.L., X.Z. and N.L.; resources, L.Z., H.S. and J.W.; data curation, L.Z. and J.W.; writing--original draft preparation, L.Z.; writing--review and editing, L.Z. and J.W.; visualization, L.Z.; supervision, J.W.; project administration, L.Z. and J.W.; funding acquisition, L.Z. and J.W. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The research was reviewed and approved by the Animal Research Ethics Committee of the Hainan Provincial Education Centre for Ecology and Environment, Hainan Normal University (HNECEE-2021-002). Informed Consent Statement Not applicable. Data Availability Statement The data set of vocal parameters of the turtles used in this study can be found in Supplementary Table S1. Conflicts of Interest All authors declare no conflicts of interest. Figure 1 Spectrogram and waveform views of underwater sounds produced by Pelodiscus sinensis. All spectrograms were obtained with Raven Pro 1.5 using Hamming windows with 1024 pt fast Fourier transform. The waveforms were drawn in MATLAB R2021a. Capital letters A-J represent call types A-J. Figure 2 Differences analysis in peak frequencies between five groups. Capital letters A-J represent call types A-J. Boxplots with different lowercase letters indicate significant results (p < 0.05). Boxplots with the same lowercase letters indicate no significant difference (p > 0.05). When boxplots are marked with only one letter, the boxplot marked "a" is significantly higher than the boxplot marked "b" in a call type (p < 0.05). Call types without letters on the boxplots means no significant differences between groups. Groups: 1 represents subadult males, 2 represents subadult females, 3 represents adult males, 4 represents adult females and 5 represents mixed-sex adults. Figure 3 Differences analysis of call durations between five groups. Capital letters A-J represent call types A-J. Boxplots with different lowercase letters indicate significant results (p < 0.05). Boxplots with the same lowercase letters indicate no significant difference (p > 0.05). When boxplots are marked with only one letter, the boxplot marked "a" is significantly higher than the boxplot marked "b" in a call type (p < 0.05). Call types without letters on the boxplots means no significant differences between groups. Groups: 1 represents subadult males, 2 represents subadult females, 3 represents adult males, 4 represents adult females and 5 represents mixed-sex adults. animals-13-00812-t001_Table 1 Table 1 Number of call types produced by Pelodiscus sinensis in each group. Type Adult Females Ratio (%) Adult Males Ratio (%) Subadult Females Ratio (%) Subadult Males Ratio (%) Mixed-Sex Adults Ratio (%) Sum A 27 11.49 21 8.94 65 27.66 69 29.36 53 22.55 235 B 8 22.22 2 5.56 5 13.89 19 52.78 2 5.56 36 C 26 49.06 1 1.89 6 11.32 15 28.30 5 9.43 53 D 3 10.00 6 20.00 16 53.33 5 16.67 30 E 3 9.38 4 12.50 5 15.63 16 50.00 4 12.50 32 F 27 30.68 14 15.91 16 18.18 19 21.59 12 13.64 88 G 0.00 69 77.53 2 2.25 3 3.37 15 16.85 89 H 2 6.25 19 59.38 1 3.13 1 3.13 9 28.13 32 I 34 29.57 52 45.22 1 0.87 1 0.87 27 23.48 115 J 3 30.00 4 40.00 3 30.00 10 Total 133 192 117 143 135 720 Note: This ratio is equal to the number of calls of a call type made by a group divided by the total number of calls of that call type. animals-13-00812-t002_Table 2 Table 2 Descriptive statistics of the acoustic parameters of all call types. Type Low Frequency Median +- IQR (Hz) High Frequency Median +- IQR (Hz) Peak Frequency Median +- IQR (Hz) Duration Median +- IQR (ms) Bandwidth (Hz) No. of Harmonics A 10,656 +- 6218 11,567 +- 6324 10,745 +- 6180 31.57 +- 30.45 689 +- 947 B 13,230 +- 5811 15,749 +- 1929 14,729 +- 3553 95.58 +- 92.17 1497 +- 1975 C 13,714 +- 2248 14,539 +- 1811 14,320 +- 2068 122.86 +- 108.89 538 +- 527 D 8572 +- 6323 10,262 +- 5093 9055 +- 6013 49.15 +- 32.49 722 +- 1098 E 6506 +- 5939 7086 +- 5836 6406 +- 3726 137.92 +- 102.55 367 +- 333 F 331 +- 303.25 426 +- 334 366 +- 367 244.76 +- 186.78 86 +- 43 G 86 +- 56 385 +- 228 129 +- 108 284.08 +- 240.57 129 +- 86 1~11 H 275 +- 162 721 +- 503 345 +- 210 182.43 +- 264.17 129 +- 183 1~7 I 151 +- 158 424 +- 405 215 +- 172 327.62 +- 275.51 86 +- 21 1~7 J 84 +- 45 1058 +- 1039 140 +- 54 231.95 +- 232.79 216 +- 215 2~12 Note: "High frequency" indicates the upper limit of the frequency for a call type; "low frequency" indicates the lower limit of the frequency for a call type; "IQR" is the interquartile range of the call type. animals-13-00812-t003_Table 3 Table 3 Average value of correlation coefficients for calls within each call type and between call types. Call Types A B C D E F G H I J Average value of correlation coefficients between call types A 0.71 B 0.16 0.77 C 0.15 0.21 0.78 D 0.15 0.22 0.21 0.71 E 0.12 0.15 0.17 0.17 0.80 F 0.14 0.18 0.20 0.20 0.16 0.77 G 0.11 0.15 0.15 0.16 0.12 0.14 0.77 H 0.10 0.14 0.15 0.14 0.11 0.13 0.11 0.60 I 0.11 0.15 0.16 0.15 0.13 0.15 0.11 0.10 0.71 J 0.14 0.18 0.19 0.19 0.15 0.17 0.14 0.12 0.14 0.63 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000089

Animal welfare is a complex subject; as such, it requires a multidimensional approach with the main aim of providing the animals with the "five freedoms". The violations of any one of these freedoms could have an influence on animal wellbeing on different levels. Over the years, many welfare quality protocols were developed in the EU thanks to the Welfare Quality(r) project. Unfortunately, there is a lack of such summarized information about bull welfare assessment in artificial insemination stations or about how disturbed welfare can be reflected in their productivity. Animal reproduction is the basis for the production of meat and milk; therefore, factors contributing to reduced fertility in bulls are not only indicators of animal welfare but also have implications for human health and the environment. Optimizing the reproductive efficiency of bulls at an early age can help to reduce greenhouse gas emissions. In this review, welfare quality assessment will be evaluated for these production animals using reproduction efficiency as a key area, focusing on stress as a main effect of poor animal welfare and, thereby, reduced fertility. We will address various welfare aspects and possible changes in resources or management to improve outcomes. animal welfare bulls welfare assessment reproduction sperm quality This research received no external funding. pmc1. Introduction Modern animal husbandry relies on artificial insemination (AI) as a substitute for natural mating in some species, such as dairy cattle. One of the reasons for the introduction of AI was to control the transmission of diseases between animals. Historically, AI has offered several advantages over natural mating. The most recently advocated one is an increased rate of genetic improvement in intensive animal husbandry . Other historical advantages of AI include decreasing the bull-cow ratio and decreasing the risk of bull injury compared with extensive herd breeding . Since the aim of animal production is to produce meat and milk for human consumption, which is globally considered to be a One Health benefit, improving bull fertility could help to alleviate malnutrition due to insufficient proteins of animal origin . However, ruminants can contribute to greenhouse gas (GHG) emissions, especially methane. Genetic improvement can help select animals with less environmental impact, improving the reproductive efficiency of these animals and thereby reducing GHG output per kg of meat or milk produced . Keeping breeding bulls in Artificial Insemination Centers (AICs) decreases the number of sires that are needed to cover all the females. One aim of holding bulls in AICs (AI bulls) is sperm production. Housing bulls in AICs varies between and within countries, but their objectives are to minimize the spreading of sexually transmitted diseases and to maximize the reproductive efficiency of the animals. Thus, a large number of males of different ages are kept in relatively static conditions at an AIC, without access to females. Compromised welfare can manifest in several ways, depending on the severity of the stressor and its duration of action. For example, long-term impairment of animal welfare can have profound effects on the animal, such as sub-fertility, reduced life expectancy, impaired growth, body damage, disease, immunosuppression, adrenal activity, behavior anomalies, and self-narcotization . One of the key components affecting reproduction is animal welfare, which can be affected by animal housing and management, including semen collection. Therefore, it would be useful to have an early indicator of an incipient welfare problem so that countermeasures could be taken in time to prevent such long-term effects on the animals. For example, penned bulls cannot exhibit avoidance behavior by removing themselves from the vicinity of a dominant male. Changes in public attitudes towards conditions under which domestic animals are kept have led to questions being raised about animal husbandry and its impact on animal welfare. Therefore, various protocols aimed at raising welfare standards have been introduced during the past few decades. However, there is a lack of published information about factors affecting the welfare of bulls in AICs. The concept of AICs is based on keeping male animals isolated from animals of the opposite sex. Due to the fact that breeding bulls in AICs is different than in natural conditions and that the process of semen collection is not normal reproductive behavior, requiring close human-animal interaction, there are many critical points in the semen production process that can cause animals to express inappropriate behavior. On the other hand, inadequate space and insufficient nutrition can cause health problems that further decrease animal quality of life. Therefore, the structure of this review paper is based on the main principles and requirements for welfare standards. The welfare assessment recommendations are based on indicators in the Welfare Quality Assessment Protocol for Cattle , which is grouped into 12 criteria based on the principles of good feeding, good housing, good health, and appropriate behavior (Supplementary Table S1). Therefore, in this review, we will discuss how disturbance of these criteria can lead to increased stress in AI bulls as a main cause of poor animal welfare, and thus lead to decreased bull semen quality. We will give a short account of stress factors and stress responses and basic bull behavior, as well as how husbandry can inhibit the expression of bull behavior and therefore act as a stressor. Then, we will summarize requirements for bulls kept as production animals (in this case, semen production) and determine objectives that are important to fulfill in order to improve welfare. The articles considered in this review were identified using the main search engines. The abstracts of potentially relevant articles were evaluated, and, finally, 80 articles were included and reviewed. The relevant keywords, such as animal welfare, bull welfare, poor welfare, bull reproduction, etc., were first used in the search. In the case of a lack of such literature for this specific production group, we linked it with other production groups, with animals of the opposite sex from the same species, and, eventually, with other species in a similar production group. 2. Main Principles, Application, and Animal Welfare Assessment The application of animal welfare science occurs at three levels of research: (i) fundamental research that provides a basic understanding of animal welfare, (ii) research that develops ways of assessing animal welfare, and (iii) practice-oriented research, the actions that are operationalized in welfare standards or criteria . Application of these levels of research can have a practical influence on animal welfare standards through requirements that contain key elements for good animal welfare. Blokhius et al. classified requirements for welfare standards into three main types. First are requirements that are resource-based, which usually set out minimum standards for the animal's environment and other resources, such as bedding, space, air quality, temperature, and access to food and water. Second are management-based requirements, which describe managers' activities concerning animal health. They include requirements for pain management, inspecting animals and feed at a certain frequency, and having established protocols for health care and euthanasia. The final requirements are animal-based that specify the outcomes that should be achieved. These requirements include health-related markers such as maximum prevalence of lameness and injuries, allowable rates of mortality, and minimum body condition scores. In animal-based requirements, animal behavioral outcomes, focusing on low levels of aggression and stereotyped behavior, and the ability to move freely and lie down comfortably are also included . These requirements were guidelines for developing different welfare assessment protocols . The requirements in animal welfare standards can be viewed as serving four broad objectives , reflecting beliefs about the important factors for animals to have satisfactory welfare. The first objective is to maintain the basic health and bodily functioning of animals, reflected by a low incidence of disease and high rates of survival, reproduction, and growth. The second objective is focused on the "affective states" of animals, especially to prevent or minimize unpleasant states such as pain, distress, and hunger, and to allow animals to experience positive states such as comfort and contentment. The third objective is to provide animals with the opportunity to carry out elements of their natural behavior, especially types of behavior that animals are highly motivated to perform, such as the drive to reproduce. The final objective is to provide animals with access to natural elements in their environment such as natural light, fresh air, and the outdoors. This direct transfer of science into practice occurs especially in cases where an innovation simplifies animal management, improves productivity, or reduces production costs. If we think of animal welfare as a complex outcome that depends on a match between the genetic make-up of the animals, the production system in which they are kept, and the ability of the people to manage these animals in that system, then improving animal welfare needs to involve coordinated action in all three domains. These objectives were the basis for the welfare principles of the Welfare Quality(r) (WQ) assessment protocols (WAPs), created and recommended to be used by EU countries, and which we chose to follow in this review. Different WQ protocols have been developed for cattle welfare measurement on farms and in slaughterhouses, but those do not include AI bulls. The main focus for these objectives and principles evaluated in the WQ protocols was to satisfy the five freedoms point of view, which is the guiding principle that advises the World Organization for Animal Health's work on the welfare of terrestrial animals. Since the beginning, the Five Domains Model was developed alongside the Five Freedoms Concept . The latter concept was that animals should be free from "negative" experiences (free from hunger, disease, etc.), while the Five Domains Model not only focuses on these "negative" animal welfare perspectives but also on "positive" ones. The five freedoms and five domains contain essentially the same five elements (see Supplementary Table S2). However, the five domains focus more on how an animal feels, i.e. its mental state, and distinguish between the physical and functional factors that affect its mental state. The latest updated version of the Five Domains Model highlights the evaluation of negative and/or positive impacts of human behavior on animal welfare. Since there is little research conducted on bull welfare, specifically on AI bulls, our work in this review will be mostly based on the Five Freedoms Concept through the WQ principles and criteria. We indicate husbandry conditions for AI bulls and summarize basic knowledge of potential negative effects, mostly on animal-based requirements. However, we found the human-animal interaction in the Five Domains Model to be an important assessment criterion for future research because AI bulls are animals that require close interaction with humans, due to the semen collection procedure to which the animals are regularly exposed. Therefore, we will incorporate some actions in such interactions that can promote positive welfare states. 3. Animal Physiological Stress Response Due to Poor Animal Welfare Stress appears when environmental demands exceed the regulatory capacity of an organism, particularly when an animal perceives a given situation as unpredictable and uncontrollable . The two main components of the stress response are the hypothalamic-pituitary-adrenal (HPA) axis and the sympatho-adrenomedullary (SAM) system. Both plasma levels of glucocorticoids and behavior changes have been used as markers of stress. The physiological response to stress can be described by measures of HPA axis activity. Earlier in stress research, it was assumed that increased HPA activity was a common response to all stressors, both internal and external . However, Pacak and Palkovits stated that there are other peripheral physiological responses to stress besides HPA axis activity. They introduced stressor-specific pathways and showed that each stressor has a neurochemical signature. This is important for further understanding of the pathogenesis and introduction of proper treatment for stress-related disorders. In the review article of Ralph et al., , the effects of various impacts of psychosocial stress on gonadotrophins and sexual behavior in female ruminants were discussed. Besides HPA activation, a group of neurons in the hypothalamus (arcuate nucleus) co-synthesize mediators such as kissproteins, neurokinin B, and dynorphin (KNDy cells), which interfere in the negative effect of cortisol on reproduction and are also involved in the metabolic control of puberty . These neuropeptides have stimulatory (kissprotein and neurokinin B) and inhibitory (dynorphin) effects on GnRH secretion regulation . The neuroendocrine regulation of female reproduction starts with the synthesizing of gonadotropin-releasing hormone (GnRH) in the hypothalamus (GnRH neurons), which further stimulates gonadotrophins (luteinizing hormone (LH) and follicle-stimulating hormone (FSH)) synthesis and secretion. However, there have been claims that KNDy neurons have control over the reproductive system by integrating information about the animals' external and internal environments and influencing the release of GnRH (acting on GnRH neurons). During the stress reaction, high levels of cortisone act on KNDy cells to increase dynorphin or/and decrease kissproteins and neurokinin B. Furthermore, these changes increase the inhibition of GnRH neurons and their pulsatile secretion and eventually result in the inhibition of sexual behavior and ovulation in females . The effect of this neuroendocrine regulation of reproduction has not yet been evaluated in bulls. The interaction between the HPA and the hypothalamic-pituitary-gonadal (HPG) axis is presented in Figure 1. The hypothalamus secretes corticotropin-releasing hormone (CRH), which stimulates the secretion of adrenocorticotropic hormone (ACTH) via the pituitary gland and, consequently, the synthesis and secretion of cortisol from the adrenal cortex. In cattle, as in humans, the main glucocorticoid (GC) released in stress is cortisol; it regulates the basal activity of the HPA axis and the response to stress via negative feedback at the hypothalamus in reducing CRH release. Both CRH and cortisol inhibit GnRH secretion from the hypothalamus (cited by ). In addition, cortisol has a direct inhibitory effect on the secretion of LH from the pituitary gland and testosterone from the testis . Based on the duration, stress can be acute, which lasts for minutes or days, or chronic, which lasts for weeks or months (and even years) . Blood sampling for cortisol or catecholamine assays may be an appropriate means of detecting HPA or SAM responses to acute stress that have a well-defined beginning and end . However, this method is not reliable when dealing with prolonged stress (such as that which occurs with the effects of housing) or when the end of the stress is not well defined (e.g., where there may be prolonged effects, such as chronic pain following nose-ringing, dehorning, or tail docking) . Animals subject to chronic stress generally suffer from metabolic disturbances associated with reduced feed intake, a negative energy balance, an increased metabolic rate, and, subsequently, loss of body weight or reduced growth . On the other hand, obesity in humans leads to dysregulation of the HPG axis and can result in hypogonadotropic hyperestrogenic hypoandrogenemia, a condition that affects fertility by modifying spermatogenesis, reducing testicular function, or reducing libido . Although activation of the HPA axis has a wide range of effects on animals, including various pathological histological changes in a number of organs, research on cattle has concentrated mainly on documenting metabolic effects and effects on the immune system. Liestner and Menke reviewed the literature on sex differences in the function of the HPA in humans and rodents. Preclinical models in numerous studies have observed sex differences in the stress response; thus, the HPA axis in females was activated more rapidly and produced a larger output of stress hormones than in males. However, studies with humans often produced inconsistent findings because of some associated factors, such as menstrual cycles. We will now consider the three main types of requirements for animal welfare standards, according to the classification by Blokhius et al. , in the context of AI bulls. 4. Resource-Based Requirements For resource-based requirements, research often tries to identify critical levels or thresholds beyond which animal welfare indicators are affected. Such work has typically found that each production system has its specific welfare challenges and that outcomes differ considerably within the same type of system . 4.1. Space and Bedding (Good Housing--Comfort around Resting and Ease of Movement) There are studies showing that space allowance and type of bedding have an influence on animal production . To our knowledge, no studies have been conducted to explore the effects of housing conditions on the welfare and productivity specifically of breeding bulls. Mossberg reviewed the welfare of growing bulls in different housing conditions. She concluded that buildings with slatted floors, and, likewise, small space allowances, or hard and slippery floor surfaces, can have a negative effect on young bulls health and behavior, and thus on their welfare. It was suggested that concrete and slatted floor surfaces should be softened by rubbing a coating on the slats. Animal health, especially lameness, depends on the type of floor. Haufe et al. evaluated the influence of four different types of floor surface, as well as access to pasture, on claw health and concluded that the effect of floor type was slight. Likewise, access to pasture did not have an effect on floor-associated claw lesions. However, it is likely that bulls kept in AICs could reach an older age and heavier weight than the animals used in these studies, emphasizing the need for a study on AI bulls. Some AICs use sand in the semen collection area to reduce slipping. However, this can lead to accidental insertion of sand into the preputium, which later on can cause pain associated with erection and rapid loss of the mounting impulse. There are different housing systems for bulls in AICs, but currently, bulls are frequently kept in individual pens with access to outdoor space. The UK codes of recommendations for the welfare of cattle livestock , give specific recommendations for the pen size for bulls. The accommodation for a single adult bull of average size should include a sleeping area of at least 16 m2. For bulls weighing over one ton, the sleeping area should be at least 1 m2 for every 60 kg of live weight. If a bull is not regularly and routinely exercised outside the bull pen, the pen should include an exercise area at least twice as large as the sleeping area. In some countries, tethered housing was previously widely used. However, this system was shown to induce abnormal behavior in young bulls, such as abnormal licking of equipment or leaning against equipment, compared with loose-housed bulls. Sufficient bedding and/or rubber matting for stall flooring helps to prevent and/or minimize foot, leg, back, and spinal column discomfort . 4.2. Temperature and Air Quality (Good Housing--Thermal Comfort) Since the WQ protocol for dairy cattle has no developed measure of thermal comfort, we will introduce sperm quality evaluation as a potential method based on extensive research conducted on thermal discomfort and sperm quality results. A high testicular temperature has an effect on sperm quality; an increased testicular temperature leads to increased production of defective spermatozoa. Physiological mechanisms to avoid an increase in testis temperature include heat loss from the scrotum via scrotal sweating, heat exchange in the testicular vascular cone, relaxation of scrotal muscles, and whole-body responses. For the production of fertile spermatozoa, testicular temperatures should be between two and six degrees Celsius lower than the bull body temperature . When the testicular temperature increases, testes metabolism increases, leading to testicular hypoxia . The optimal environmental temperature for sperm production varies between 15 and 18 degC during the whole period of spermatogenesis of 65 to 70 days . Since spermatogenesis takes approximately 60 days, environmentally induced alterations in sperm quality might not become apparent for several weeks . There are many studies reviewing the effect of increased ambient temperature on semen quality. Most of those studies include humidity as well, in the temperature humidity index (THI), and heat as prolonged heat stress, forcing animals to adapt. Berian et al. evaluated the effect of heat stress (based on the THI) on the physiological and hematobiochemical profile of cattle. Increased levels of some blood parameters, such as red blood cell count, white blood cell count, packed cell volume, and hemoglobin, were followed via an increase in levels of cholesterol, creatinine, alanine transaminase (ALT), aspartate aminotransferase (AST), cortisol, and blood urea nitrogen (BUN). Physiological parameters such as respiratory and heart rate were also observed as significantly raised with an increased environmental temperature. This indicates that the THI is a sensitive indicator of heat stress. Furthermore, differences between bull species (Bos taurus and Bos indicus) were noted. Bos taurus bulls are more sensitive to high ambient temperatures than Bos indicus bulls. Even the semen quality parameters of crossbred bulls (Bos taurus x Bos indicus) recover more rapidly after heat stress than for Bos taurus bulls. Morrell indicated that differences in temperature between seasons might be equally important, in the short term, inducing heat stress in animals with consequences on sperm quality. Therefore, providing bulls with comfortable stalls that are heated or cooled to maintain a temperate environment will positively affect sperm output, sperm quality, and bull well-being . On the other hand, few studies have been conducted to explore the influence of air pollution on fertility in farm animals. There are systematic reviews that link the impact of air pollution with reproduction in women and laboratory animals , as well as fertility and spermatogenesis in humans . In the majority of the studies, reproductive and fertility parameters were negatively affected by air pollutants. Good air quality is affected by numerous factors, such as gases, dust, and microorganisms, and the management of an intensive animal breeding system that includes stocking density, the size of the cattle, flooring, bedding, waste management, building design, and ventilation system . Similar studies on the effects of these pollutants on bull fertility are needed. 4.3. Access to Food and Water (Good Feeding) Access to clean drinking water and a diet that is appropriate to a species and can keep them healthy is one of the five freedoms and therefore needs to be assessed to meet animal welfare demands. It is known that stress influences feed intake in animals, but there are few reports on how feeding time and frequency influence animal welfare by causing a stress response. Calamari et al. conducted a study on the influence of frequency and feeding time on metabolic condition and milk production in heat-stressed dairy cows. The animals were divided into three groups, and a total mixed food ration was delivered either once daily in the morning or evening, or twice daily in the morning and evening. Different plasma metabolites were analyzed, and rectal temperature and respiratory rate were noted. The results showed that feeding once daily, especially in the morning, was less suitable to provide adequate cow welfare during the hot season than the other feeding regimens. In detail, plasma glucose levels were lower in the morning feeding routine, while plasma urea was lower in the evening feeding routine. Rectal temperature and respiratory rate values were higher in the group of cows receiving food just in the morning. Milk production was not affected by feeding management. The bull husbandry requirements for optimum productivity may be different from those of dairy cows; therefore, the feeding management and production performance of AI bulls needs to be evaluated. Recent studies on the nutritional control of puberty in female cattle demonstrate that maternal nutrition during gestation can induce morphological and functional changes in the hypothalamus system, which can persist long after the birth of female offspring, influencing reproductive performance even in adulthood . Furthermore, the hormone leptin was shown to be linked to metabolic status and puberty, acting through arcuate nucleus neurons, transducing the nutritional signal into input (influencing GnRH neurons) in sheep and other species . The fertility of breeding bulls is also a key factor in sustainable cattle production; proper management and nutrition of the bulls are essential to maximize reproduction efficiency . Feed restriction in young bulls altered the onset of puberty in relation to plasma insulin-like growth factor-I (IGF-I) and IGF-binding proteins in . The study showed a progressive increase in IGF-I in a group of bulls fed a balanced diet compared with two groups with restricted feeding, which did not show changes in IGF-I levels. The levels of IGF-binding proteins varied between groups depending on the laboratory methods that were used: radioimmunoassay (RIA) or Western ligand blotting (WLB). Previous studies by the same group of authors showed an association between puberty and plasma testosterone, insulin-like growth factor-I (IGF-I), and IGF-binding proteins . Insulin-like factor 3 (ILF-3) is one of the peptide hormones (a member of the IGF family) that is produced mostly by Leydig cells in mammals , together with testosterone, and is another important reproductive hormone. This hormone was found to be crucial for testicular descent but also the survival of rete germ cells , and recently, low levels of ILF-3 were associated with malnutrition in children . The serum concentration of ILF-3 increases from birth to puberty when it reaches a plateau . In adulthood, ILF-3 secretion depends on the differentiation stage of Leydig cells, which are dependent on LH . However, the regulation of ILF-3 secretion during puberty differs from that of testosterone, which is regulated by the HPG axis as well as LH . Therefore, serum concentrations of ILF-3 can be used as a biomarker of testis status, especially the status and number of Leydig cells. Similar findings were reported in a study on bulls . 5. Animal-Based Requirements For many animal-based measures, such as the proportion of lame or injured animals, there are no non-zero values where welfare is not affected . Assessing behavioral and health indicators as a main animal-based requirement of animal welfare can be used indirectly for resource-based and management requirements . 5.1. Health-Related Measures (Good Health) Absence of pain is one of the main principles of good animal welfare, as well as absence of illness. There are two groups of markers of farm animal welfare that can be measured and that are health-related. The first group comprises physical indicators including cut injury, body damage, abscess formation, swelling of the joints, and loss of hair or wool. The other group consists of physiological indicators, which include cortisol level, reduced feed intake, immunosuppression, adrenal activity, and altered physiological responses, e.g. depressed reproductive parameters. Health indicators included in the welfare assessment of bulls in AICs have not been described. Physical indicators modified from the protocol for dairy cows are presented in Table 1; these indicators might be used for bulls as well. The Office International des Epizooties (OIE) Terrestrial Animal Health Code, which implements improvement standards for animal and public health and animal welfare from a veterinary viewpoint, summarized general considerations and conditions applicable to AICs and to breeding bulls. Instructions for general hygiene in AICs are provided in Chapter 4.5 of the Health Code, although the requirements that bulls should fulfill before entering AICs are based only on potentially pathogenic infectious agents. Furthermore, recommendations are given that would reduce semen contamination with common microorganisms. However, additional details are needed, which could include an assessment of welfare biomarkers, if sufficiently sensitive indicators could be identified. The study by Cojkic et al. indicates that differences in the seminal microbiota of healthy bulls occur and might be correlated with fertility. The influence of bacteria on fertility may depend on their effect on sperm quality parameters during storage, and also on their interaction with other bacteria. Some bacteria decrease sperm quality parameters directly or indirectly, negatively influencing fertility . Therefore, sperm quality evaluation could be a good indirect parameter of bull welfare. Identification of health problems, even at the individual level, can be a useful indicator of a problem at the herd level as well. Early diagnosis of diseases is important for welfare based on their association with negative experiences such as pain, distress, and discomfort . Excellent hoof condition is one of the most important physical aspects of pain-free semen collection. Lameness, whether from sole abscesses, foot rot, interdigital dermatitis, hoof cracks, laminitis, improper hoof trimming, etc., will often result in bulls being reluctant to mount. Furthermore, lame bulls often overcompensate for the foot pain and exert abnormal pressure on other bone and muscle groups, placing the bull at risk for greater injury . 5.2. Bull Behavior and Libido Measurements (Appropriate Behavior) Puberty and social and sexual development in bulls are under strong genetic regulation. There are breed differences, as well as age differences, in the beginning of the manifestation of, and changes in, sexual behavior . Young calves already display aspects of sexual behavior by mounting during play. Sexual behavior in bulls is differentiated into two components: libido (sexual drive) and mating ability . Both can be negatively influenced by external factors, particularly stress. Rearing calves to become bulls in Artificial Insemination Centers (AICs) is quite unlike a situation in the wild or under extensive farm husbandry conditions. In the wild, or under extensive farm husbandry conditions, the social ranking of bulls is based on size and age within the group . In a herd with multiple bulls, all tend to mate with the same sexually active cows. It is recommended to group the bulls together before the breeding season to allow them to determine their social ranking before intensive mating occurs, thus avoiding stress and possible injuries due to competition between bulls. There are certain behaviors that indicate a problem with animal welfare. Some of these include an increased respiratory rate or panting, decreased feed intake, and demonstration of aggressive, depressive, stereotypic, and/or other abdominal behaviors . Physiological stress leads to hormonal imbalance and contributes to infertility by suppressing the hypothalamic-pituitary-ovarian axis (HPO) in rats. The mechanism of inhibition is through the inhibition of GnRH secretion, thereby suppressing LH release from the pituitary . In the same study on acute stress, significant elevations in corticosterone, plasma adrenocorticotropic hormone, prolactin, and progesterone were observed, as well as alterations in sexual behavior, decreased FSH, and immunoreactive inhibin. In a similar study in men, psychological stress decreased serum testosterone levels, leading to a secondary rise in LH and FSH. Serum LH and FSH levels were negatively correlated with sperm count and positively correlated with abnormal sperm motility and morphology . On the other hand, a positive correlation between serum testosterone concentration and expression of sexual behavior was noted for young and adult bulls, although the relationship was not significant . However, expression of sexual behavior and serum testosterone levels were significantly higher in adult bulls compared with the younger group. In the study by Lange et al. , a good human-animal relationship had a positive influence on heifers even during restraint. The results of gentle interaction were reflected in physiological parameters by decreasing heart rate and via behaviors such as longer durations of neck stretching. However, similar studies have not been conducted for bulls. During bull semen collection, personnel actively lead the animals. Therefore, previous positive experiences and a lack of fear of humans might improve libido during sexual stimulation prior to collection. Change of personnel can have a negative effect on semen quality or, on the other hand, may help to resolve a problem, if there are no other underlying causes. Apparently, similar environments may be managed differently by a stock person, further affecting animals' experience of a particular situation . Mellor et al. provided some examples that can generate positive effects in animals. The companionable presence of persons who provide feelings of safety and preferred foods, with tactical contacts and/or reinforcements, results in an increased score during welfare assessment. Furthermore, personnel who participate in enjoyable routines and engaging activities initiate a positive reaction in bulls; the calming presence of familiar persons in threatening circumstances, and taking action to end periods of deprivation, inhibition, and harm, all help to improve animal welfare. These positive impacts of interaction can be graded separately according to the frequency, variety, duration, and form of congenial contact. The frequency of semen collection is positively correlated with bull reaction time and novelty . Management-based semen collection procedures should be reviewed for an individual bull if production goals are not met. If the bull does not show a response to stimulation in 5 min, a change in stimulus should be made . One of the most effective approaches for sexual preparation is false mounting. Allowing three false mounts, followed by a final fourth mount for semen collection, was found to maximize sperm harvest. Furthermore, false mounting in combination with active restraining increased sperm concentration per ejaculate. However, these mounting opportunities need to be reduced for bulls with low libido and physical limitations. 6. Management-Based Requirements For management-based requirements, science has been used in two main ways. One is simply to assess how animal welfare is affected by management practices, and the second application is to develop and test practices that improve animal welfare. For example, in the "Welfare Quality" project, a large cooperative research program that developed standards for cattle, pigs, and chickens, the scoring systems rely heavily on animal-based measures such as body condition, lameness, lesions, and agonistic behavior (e.g., Welfare Quality, 2009). Such results contribute to a growing recognition that good animal welfare is a complex outcome of different factors including animal genetics, management, and environment. During the last 70 years, efforts in the bull semen industry have been made to maximize reproduction efficiencies to meet demands. For this to be achieved, there is a need for management schemes to be efficient to exploit bulls' reproductive potential and, at the same time, to minimize the risk of injury associated with it. At present, bulls younger than 15 months old are commonly included in intensive semen collection . However, a comprehensive study of lifetime productivity has not been carried out for these young bulls. Management requirements should be evaluated based on welfare principles and criteria. The welfare quality assessment protocol for cattle summarized four main welfare principles and provided criteria that can be used even for the welfare assessment of bulls in insemination centers. Antimicrobial use is one of the management requirements in the process of semen preservation since the antibiotics are used without previous bacterial identification and testing of antimicrobial susceptibility. The sources of bacterial contamination in bull semen are the animals themselves, their environment, and the process of semen collection and preservation. The European Union provides government directives on the use of antibiotics in semen for international trade , aiming to avoid the transmission of potential pathogens and disease outbreaks. This non-therapeutic use of antibiotics may increase the risk for antimicrobial resistance (AMR). Currently, the Food and Agriculture Organization of the United Nations (FAO), the World Health Organization (WHO), and the Office International des Epizooties (OIE) have created the "One Health" concept with transdisciplinary public policies to minimize AMR and promote responsible antibiotic use through the aspects of human, animal, and environmental health. Albernaz-Goncalves et al. linked antibiotic use in pig farming with animal welfare. They summarized the major disadvantages of husbandry procedures used in intensive pig farming that lead to stress and that are consequential to diseases and increased use of antibiotics. They emphasize that the adoption of good management practices based on animals' holistic needs is essential for the rational use of antibiotics. A similar approach is now needed in the bull breeding industry. 7. Discussion The main aim of this review was to summarize the critical points in bull semen production where animal welfare could be compromised. Furthermore, we explained how insufficiently welfare-oriented production criteria lead to stress and, subsequently, decreased reproduction efficiency. Assessment of animal welfare can be challenging due to its complexity and multifactorial nature. Poor animal welfare can be easily missed due to a lack of knowledge of the species and insufficient evaluation. This can be avoided by using appropriate methods for animal welfare assessment. Two primary methods which can be used are (1) objective evaluation by scoring specific criteria and (2) use of subjective judgments and evaluation. There are differences in the scoring systems between different countries , which should be normalized to enable effective comparisons to be made. There are also differences in welfare assessment based on the animals used in different programs, for example, animals used in research programs versus those in agricultural production programs. For animal agriculture programs, six basic assessment categories were given: performance standards (animal-based measurements or outcome criteria), prohibited practices, input-based standards (engineering or design standards), five freedoms, record-based standards, and subjective evaluation. These standards can be used as a template for other animal programs and for different animal groups within the same program. The development of systems to evaluate animal welfare was based on scientific interest as well as public concern about the raising conditions and treatment of animals. The first versions were developed for animals used in research, teaching, and testing (RTT). In subsequent years, questions were raised about other animals and their keeping conditions. Thereafter, hundreds of welfare assessment protocols were developed around the world; all differ from one another based not only on the animal species and production group but also on the place where the animals are kept at the time of evaluation. Since AI technology is mainly used in the dairy industry, dairy bulls are usually the cattle kept in AICs. Krueger A et al. summarized the similarities and differences between different welfare evaluation programs for dairy cattle on farm levels used in three continents: the European Welfare Quality(r) Assessment Protocol dairy cattle (WQ), the U.S. National Dairy Farmers Assuring Responsible Management Program (FARM), and the New Zealand Code of Welfare: Dairy Cattle (The Code). The fourth, upcoming program, the Integrated Diagnostic System Welfare (IDSW), was also discussed. Some of them are voluntary (FARM, WQ, and IDSW), and some of them are required to be followed by law (The Code). Some are more animal-oriented (WQ), and others include environmental and management-based requirements (FARM, The Code, and IDSW). Looking at all of them from the bull perspective, just two of them include bull breeding (FARM and The Code), and only one protocol includes reproduction data (IDSW) for the welfare evaluation but is not complete. The FARM program includes bull breeding, based on the fact that all bulls being raised as dairy steers should be castrated, and how the procedure should be performed, with the remainder being on pain management. On the other hand, The Code gives more recommendations about natural service bulls, regarding the number of cows that needs to be served, nutrition, and environmental requirements. Some of them can be implemented for AI bulls, such as the size of the sleeping and exercise area requirement, but the rest cannot, based on the environmental conditions that animals are kept in and the level of social interaction between humans and other animals. Animals experience the same environment in different ways, based on their genetics, early experiences, and temperament . Bulls kept in AICs are unique in many ways; they are mostly dairy breeds with specific metabolic requirements due to the intensive selection for milk production. Furthermore, husbandry conditions for AI bulls differ slightly between countries at present but have huge differences compared with other animals from the same species and between production groups (beef and dairy). In addition, semen collection is not a natural way of expressing reproduction behavior. All of these differences and specifics require detailed knowledge of AI bull physiology, behavior, and husbandry before defining criteria for welfare assessment. It might be possible to create a unique WAP for the specific conditions of AI bulls by analyzing different existing welfare assessment protocols and approaches to welfare assessment. Subjective evaluation is difficult to eliminate in total because of the observers projection of their own impression. This can be minimized by the personnel responsible for these assessments attending training programs and by establishing criteria that define good welfare customized to the species, sex, age of animals, and production purpose. The whole goal of assessment programs should focus on minimizing the subjective component. One objective assessment system, which has been developed during the past few decades, was Welfare Quality(r) designed to determine the welfare status of cattle, pigs, and poultry in animal units. However, creating such an assessment system for other animal types, in our case for bulls in AICs, has its difficulties due to the multidimensional and multidisciplinary approach of animal welfare . The focus of all further assessment protocols should be the same, i.e., reducing stress in animals from the beginning but also promoting positive stimulants. The reason why this demand is important for the bull industry is that, apart from the known consequences of stress on reproductive parameters and performance, stress can also influence the onset of puberty . Delayed puberty increases the production time for highly genetically valuable bulls and indirectly contributes to increased greenhouse gas emission in terms of methane and nitrous oxide and, as such, is implicated in the One Health concept. Currently, welfare regulations stipulating the conditions that apply to bulls kept in AICs are lacking. The current regulation of conditions that apply to the keeping of calves confined for rearing and fattening can be applied to a certain point to male calves that are going to be used for semen collection. With the onset of puberty, the rearing conditions for young bulls require more consideration due to the beginning of social ranking within the group at the same time. The major factor influencing social ranking in the wild or under extensive farming conditions is bull seniority , and then dominance is controlled by the size of the bull. However, there is little scientific evidence on how rearing groups of bulls, of the same age and similar in size, in the same pen influences animal welfare and, consequentially, fertility results. In multiple-sire breeding groups, all bulls tend to breed with the same sexually active cows, which increases the risk of rivalry. This can be avoided by introducing young bulls into a group of mature bulls to establish the social ranking prior to turning them out into the herd containing sexually active females . 8. Conclusions There is a lack of information on bull welfare assessment in AICs or on how disturbed welfare can be reflected in bull productivity. Production systems for breeding bulls should be described and evaluated to meet the standards of good animal welfare. More research is needed on welfare throughout an animal s life, i.e., from when calves are selected to become breeding bulls to the end of the bulls' productive lives. Furthermore, specific protocols for welfare quality evaluation for this specific production group of animals should be created, focusing on their physiological and behavioral needs, with the aim of achieving optimal productivity. Supplementary Materials The following supporting information can be downloaded at: Table S1: Principles and criteria that are the basis for the Welfare Quality(r) assessment protocols; Table S2: Five Freedoms and Five-Domain Model. Click here for additional data file. Author Contributions Conceptualization, literature review, and writing of the first draft, review and editing A.C.; supervision, review and editing, funding acquisition, J.M.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Schematic representation of hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axis. Black arrows represent inhibitory effect of each hormone. CRH: corticotropin-releasing hormone, GnRH: gonadotropin-releasing hormone, ACTH: adrenocorticotropic hormone, LH: luteinizing hormone, FSH: follicle-stimulating hormone, and GCs: glucocorticoids. animals-13-00942-t001_Table 1 Table 1 Health indicators, which might be included in bull welfare assessment (modified from Rousing et al. ). Body Part Clinical Parameter Welfare Relevance Bull Welfare Principle to Apply General appearance Body condition score Poor body condition may cause long-term discomfort and an increase in disease susceptibility because of impaired immune competence; it indicates metabolic disorders, sub-optimal management, or chronic coping difficulties. Good feeding Good health Skin Skin parasites Skin infection pressure sores Pruritic skin disorders result in long-term discomfort and increase the risk of secondary self-inflicted lesions. Skin injury and infection cause acute and chronic pain. Provides information about problems regarding housing system, management, or underlying diseases. Good housing Good health Legs Lameness Hoof care Lameness indicates a painful leg condition and affects the freedom of movement and the performance of behavior. Overgrown or deformed hooves might indicate foot disorders causing pain and discomfort. The resulting changes in leg conformation might evolve into chronic articular damage. Good housing Good health Appropriate behavior Systemic diseases General condition Clinical diseases Clinical diseases typically involve pain and discomfort. The welfare implications vary according to the intensity and duration of the disease condition and welfare. General condition is affected. Good feeding Good housing Good health Appropriate behavior Mortality Case history of culled animals This information points out specific problem areas in the herd and provides details for tackling serious health problems. Good feeding Good health Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000090

Turfgrass in equine sports has clear advantages over other types of reinforcement but adds complexity to the management. This study investigates factors that influence the turfgrass' surface performance and how the use of a drainage package and a geotextile reinforcement affect quantitative measurements of turfgrass. The measurements are made using affordable, lightweight testing tools that are readily available or easily constructed. Eight boxes with turfgrass over a mix of the arena with peat at a consistent depth were tested for volumetric moisture content (VMC %) with time-domain reflectometry (TDR), the rotational peak shear device (RPS), the impact test device (ITD), soil cone penetrometer (SCP), and the Going Stick (GS). Results obtained using TDR, RPS, ITD, SCP, and GS indicate that the presence of the geotextile and drainage package was mainly detected by VMC (%), SCP detected geotextile addition, and GS detected the interaction of geotextile x drainage package. Linear regression showed SCP and GS are related to geotextile and was positively correlated between them and negatively with VMC (%). The testing showed some limitations of these devices, mainly related to moisture content and sod composition, but the potential exists to utilize these devices for quality control as well as for the monitoring of maintenance of the surfaces when controlling the range of both VMC (%) and sod constitution. equine arenas turfgrass base layers portable tools safety equine welfare Secretary of Science and Technology from the University of Moron, and the Racing Surface Testing LaboratoryPI3-18-06-MB-005 This research was funded by a biennial research project PI3-18-06-MB-005 of the Secretary of Science and Technology from the University of Moron, and the Racing Surface Testing Laboratory. pmc1. Introduction For most equine sports surfaces, the competition surfaces are composed of sand, sand reinforced with fiber, or turfgrass . The use of turfgrass as an equine sports surface adds complexity to the interaction of horseshoe and foot with the surface. Turf allows the hoof and shoe to penetrate into the surface while the root system provides additional reinforcement to support the hoof to avoid a shear failure, or divot, in the surface Turf also has clear advantages over other types of surfaces like sand stabilized with fibers, since, unlike added fibers, the damaged roots will regrow. Furthermore, the roots tend to be preferentially oriented in a vertical direction which increases shear strength but does not inhibit vertical penetration. The vertical root orientation allows the toe to penetrate the surface between the roots while, at the same time, the roots reinforce the surface during propulsion when large horizontal forces are applied. As in other athletic sports fields root- zones are constructed using a high proportion of sand to improve growth and performance . However, this can cause a lack of cohesion and a poor support to the horseshoe, so stabilization of the root zone is needed. The stabilization of the surface can be achieved by reinforcement of the root zone with synthetic materials . Recent research has considered the effect of the type of surface and condition on the incidence of injuries and performance . Horse racing has a primary focus on safety. The availability of injury data and the sensitivity of the public to rider and horse safety are an opportunity to keep the social license on horseracing . The epidemiological research associated with risk due to surface conditions is generally based on ratings on the turf. In general, the ratings of surface conditions are qualitative judgments from racing officials . Quantitative measurements may be obtained from the track, but the publicly available ratings are adjusted according to the experience of racing official and may not match the quantitative measurements. A notable exception is the data from New Zealand which is strictly quantitative . New Zealand uses the Longchamp Penetrometer to quantify the surface effect on race times, but the same data has also been recently shown to be useful in quantifying risk associated with the surface condition . Ratings in Australia, Japan, and the United Kingdom are a combination of qualitative and quantitative data which are interpreted by racing officials that have been shown to predict risk in some cases, although results are mixed . Existing tools used for handicapping and measurement of the performance of the turf surface may provide a promising basis for research. Currently, available track ratings may not be comparable between racing jurisdictions or even within a single racing jurisdiction because of qualitative adjustment of the quantitative measurements. In other equine sports like showjumping or dressage, the effect of condition or rating of the surface on injuries is less readily available since both surface ratings and injury data are less consistently available. However, it is generally accepted that understanding the risk to the horse (and rider) may require an improved understanding of the condition of equestrian turf surfaces . The focus of this paper was on use of several commercially available or easily constructed portable instruments which are used in equine sports to test surface conditions. Since surface moisture content is closely related to surface quality a moisture probe based on an older ASTM standard D6565 was included in the work to test volumetric moisture content (VMC %). The rotational peak shear (RPS), based on ASTM F2333, was used in a previous study to test sand surfaces with variable results. In turfgrasses a similar device described by Canaway and Bell was used successfully to differentiate species and cultivars , reinforcement materials in the root zone , and biomass , but it was less sensitive to soil condition resulting of traffic and compaction . The impact test device (ITD) based on ASTM D5874-16 is similar to the Clegg hammer, the Impact Test Index (ITI) express the deformation of the surface after dropping. Guertal and Han found that Clegg hammer could differentiate between traffic and non-traffic areas in turf tracks but could not distinguish different levels of traffic. Two commonly used measurement devices, the Longchamp penetrometer and the Going Stick, were also considered. While no testing standard exists for the Longchamp penetrometer, it is the one device in current use which is supported by data that correlates the measurements to the performance of the horse through a general descriptive model as well as being correlated with injury . A similar tool, the dynamic cone penetrometer, is used instead of the Longchamp penetrometer since it is already included as a standard with only slight modifications from an international standard . The Going Stick is commonly used in some countries, and while it is currently not a standard test and is only provided through a commercial entity, it was included in the current form. The objective of this investigation is to identify if these tools provide independent measures of the turfgrass surface condition. Based on the outcome, the tools required to characterize the surface would be able for use in future large scale epidemiological research. 2. Materials and Methods 2.1. General Overview The effects of two turfgrass building systems, which consisted of a drainage package (Dr) above the limestone base and the addition of geotextile chips (G) as reinforcement of the root zone, were tested with five portable devices. The surface was prepared with and without a drainage package over the limestone to establish if there was an effect that would modify the mechanical properties of the surface. The drainage package was a three-layer system consisting of two layers of geotextile with a geomesh layer between the layers of geotextile. Since the profile was built with sand substrate geotextile chips were added to stabilize or reinforce the profile. 2.2. Study Design The design was a randomized block with two factors (23) with two-level with two repetitions (eight boxes). The factors were the addition of geotextile to the sand, which constitutes a depth cushion and the inclusion of a drainage layer over the limestone base. The absence of geotextile was designated as G1, with the inclusion of 2 kgm2 of geotextile chips mixed through the profile, designated as G2. In both cases, the depth cushion is 10 cm deep over the base. The base was a compacted limestone with 0.7% of cross-slope either directly under the depth cushion, designated as Dr1, or a drainage package was placed between the depth cushion and the limestone base, designated as Dr2 . The drainage package consisted of two layers of geotextile fabric with a geomesh layer placed between them. Testing was performed on two dates: 3 March 2018 and 30 March 2018. The two dates were considered as blocks in the analysis. Gravimetric moisture content was determined in the laboratory for both dates treatments on 3 March 2018; it was 44.6% +- 9.40, and on 30 March 2018 was 47.6% +- 8.94 . The testing box dimensions were 1 m x 1 m with an overall prepared depth of 0.20 m. Boxes were constructed as described in a previously published study with the dimensions established from prior testing . The 10 cm of sand over the 30 cm base material was applied in two layers. Each 5 cm layer was compacted separately using a 4 kg mass dropped three times from a height of 0.30 m onto an area of 0.20 m by 0.17 m for all treatments. Above the sand, sphagnum moss peat and Bermudagrass Tifway 419 sods were placed. All boxes were fertilized with controlled-release complex fertilizers, which were fully coated and with slow solubility. The fertilizer applied was a mixture of nitrogen, phosphorous, and potassium (NPK) with 30 g/m2 of Basacote (Compo Expert(r) GmbH, Munster, Germany) and 50 g/m2 of Floranid permanent (Compo Expert(r) GmbH, Munster, Germany) . Irrigation was supplied during the whole experiment. The mowing height was kept to 30-50 mm in every box and across the experiment. The growing medium was 92.3% sand, 2.6% silt, and 5.1% clay (Testing by Racing Surfaces Testing Laboratory, Lexington, Kentucky) . The 2 cm sod was not of the same composition as the profile and was acquired from a turfgrass producer. The geotextile was 100% polyester (Lab Cor Materials, LLC, Seattle, WA, USA) . The sand was 90.6% quartz, 2.7% potassium feldspar, 2.5% plagioclase, 0.2% calcite, and 4% phyllosilicates (KT Geoservices, Gunnison, CO, USA). The Geomesh was high-density polyethylene. The materials characterization, sand, and geotextile used are the same as a prior study on arena surfaces without turfgrass . Because of the living nature of turfgrass and also as a soil pore system after the experiment on 1 April 2018, samples of depth cushion from both geotextile and drainage package treatments were taken for water-air availability testing (Laboratory of Substrates of the Faculty of Agronomy of University of Buenos Aires) in accordance with European standards CEN EN-3041 , results are in Appendix A. 2.3. In Situ Measurement of Test Boxes All testing was performed on both testing dates using five in situ measurement tools. The five measurement tools were: a volumetric moisture content tester (VMC) consistent with ASTM D6780 , a rotational traction tester, which was used to measure the rotational peak shear (RPS) based on ASTM F2333 , an impact test device which measured the impact test index (ITI) based on ASTM D5874, , a dynamic penetrometer also called the soil cone penetrometer (SCP) consistent with ASTM D 6951-03 and the Going Stick, a commercial instrument which measures the Going Stick index (GSI). To the extent possible, these devices were used in accordance with all applicable standards. While no current standard exists for the Going Stick, the use and design was consistent with other published literature . Three repetitions of all measurements were achieved during the first date, and five on the second date (Table 1). The field moisture probe (Spectrum Field Scout TDR-100, Aurora, IL, USA) using time-domain reflectometry was used to measure the volumetric moisture content (VMC %). The probe was equipped with two measuring rods of 8 cm in length. The rotational peak shear (RPS) was recorded using a torsional shear tester based on a modification of ASTM F2333-04 . The standard device was modified by eliminating the cleats on the disk and replacing them with a horseshoe. The horseshoe included two 2.5 cm long cleats. The rotational peak shear (RPS) load was measured with a digital torque wrench with a range of 4-200 NM and precision of 0.08 Nm (Model ARM602-4, ACDelco, Detroit, MI, USA). In order to minimize operator variability, the same person, shown in Figure 2, performed all of the testing, which was consistent with best practice . The surface hardness and resistance to compaction was measured using an impact test device (ITD) based on ASTM D5874-16. The deformation of the surface in cm is used to describe the surface and will be referred to as the Impact Test Index (ITI). The soil cone penetrometer (SCP) testing was based on ASTM D6951-03 . The SCP test uses repeated drops of a mass onto a rod to drive a cone-shaped penetrator into the soil a fixed distance . The test method is used with a number of different configurations, which depend both on the application and on the soil type. While the current testing was a typical agricultural application, a smaller mass dropped was dropped at a shorter distance to account for the finite depth of the test box and the sandy soil . A hammer with a mass of 2 kg was dropped from a height of 50 cm to the impact surface on the anvil. The number of drops required to press the end of the rod to a depth of 100 mm is measured. The end of the rod was a right circular cone with a 20 mm diameter base and 15 mm height, and the shaft mass was 3.5 kg. While previous research had correlated this apparatus to both the shear strength of the soil and the California Bearing Ratio , these relationships would depend on the exact configuration of the instrument. The Going Stick measures the penetration resistance and the resistance to rotation of the blade in the turf . The GSI is an integrated proxy of the two measured values. The GSI is commonly used in Thoroughbred racing to describe the surface conditions in a number of jurisdictions . 2.4. Statistical Analysis For all the data obtained, the analysis of variance was performed using commercial statistical analysis software (Infostat version 2, Buenos Aires, Argentina). For comparison of marginal means the t-test was performed. Values of p < 0.05 were considered statistically significant. Linear regression analysis between dependent variables and independent variables and Pearson's coefficients of correlation were calculated to identify the degree of association between dependent variables. The proposed model for the testing of the boxes is:Yijk=m+Dri+Gj+Bk+DrGij+DrBik+(GBjk)+DrGBiJ k+eijk Dr: Drainage (1,2)G: Geotextile (1,2)B (1,2): Blocks. 3. Results 3.1. In Situ Measurement of Test Boxes All measurements obtained from the five devices showed to be significant for some of the treatments, drainage (Dr), and the addition of geotextile (G) and Blocks (Dates). Results of variables tested for each treatment (Table 2 and Table 3). Significant effects are shown through f and p values (Table 3). Linear regression analysis (Table 4) demonstrated that VMC (%) was positively associated with Dr and G (R2 = 0.54), SCP was associated with G and GSI was associated with G. Correlations between devices (Table 5) showed that VMC (%) was negative correlated with penetrometer (p < 0.0216; p = -0.29) and GSI was positive correlated with SCP (p < 0.0025; p = 0.37). 3.2. Moisture Probe The volumetric moisture content (VMC %) was significant for triple interaction block x drainage x geotextile . The block effect showed higher values of VMC on date 2. When geotextile was present, VMC % was the lowest on both dates. Drainage packages preserved higher VMC % when geotextile was not present on both dates. Regression analysis identified VMC was significantly associated (R2 = 0.54) with Dr (p < 0.001) and with G (p < 0.001) (Table 4). Correlation between devices showed that VMC (%) was negative correlated with SCP (r = -0.29, p < 0.0216). 3.3. Rotational Peak Shear The rotational peak shear (RPS) was not significant for both treatments, but it was significant to block effect, which means that the moment of testing showed a different result (B1 (3 March 2018): 44.41 Nm +- 5.56 and B2 (30 March 2018): 47.64 +- 5.49; respectively p < 0.0274) (Table 2). 3.4. Impact Test Device The measurements made with the impact test device (ITD) were not statistically significant for all two factors and blocks (Table 2). 3.5. Cone Penetration The soil cone penetrometer (SCP) was significant for geotextile (p < 0.0118) (Table 2). Geotextile addition had the highest mean values of SCP (G1 = 2.78 +- 0.75 and G2 = 3.38 +- 0.94). When regression was run SCP, R2 was 0.17 and was significantly associated with G (p < 0.0061) (Table 4). 3.6. Going Stick GSI was statistically significant for drainage package (Dr) (Table 2 and Table 3). GSI showed higher values during the second date (B2) and with the drainage package as well as being significant for the interaction between the drainage package and geotextile addition (Dr x G) . The treatments B2Dr2G2 (6.57 +- 2.65) and B2Dr2G1 (5.53 +- 0.88) showed higher GS index values. When linear regression was run, GSI was associated with geotextile (Table 4). Correlation between devices showed that GSI was correlated with SCP (p < 0.0025 r = 0.37) (Table 5). 4. Discussion To be useful, the basic tools being used in this study should be sensitive to the treatments of the surfaces in the boxes that are known to have an impact on the performance of an equestrian turf surface, such as drainage and geotextile . The insensitivity of some of the measurements to the treatments is at least as notable as the sensitivity of other measurements. Moisture content is generally understood as one of the most important factors in the dynamic properties of equestrian surfaces . What is most notable about the results from the VMC (%) measure is that the treatments of geotextile and the drainage package are most clearly identified simply by monitoring the moisture content (Table 2). The dynamic of this effect seems to be influenced by pluviometry, soil temperature (B1 = 23 mm/month and average temperature 26 degC; B2 = 35 mm/month and average temperature 22.1 degC), and evapotranspiration occurred during both periods (end of summer). Drainage and geotextile combined seem to reduce VMC (%), while drainage seems to reduce the flow of water . Looking at the interactions, the most notable conclusion is that VMC (%) is sensitive to nearly all the effects that are known to influence performance. The measurement of RPS was notable for the insensitivity to the treatments and lacking repeatability between test days. The RPS was previously showed significant differences in a sand-geotextile mix . However, in this study, the profile includes a warm-season turfgrass, Bermuda Hybrid "Tifway 419", which has a high shoot density and horizontal stems. In sand surfaces, the effect of geotextile added on the response of the surface is related to the relative motion between the horseshoe and the surface. This resistance results in higher forces during a pivoting movement closely related to the RPS measurement . The turfgrass might modify the relative motion between the horseshoe and the surface, such that the turf, and not the presence of geotextile, dominates the response. The magnitude of the shear readings in the current experiment were 20 points above the previous testing in sand. The differences between dates (B1 = 44.41 Nm +- 5.56; B2 = 47.64 Nm +- 5.49) were detected, but if the turf itself and not the profile dominates the response, then the additional growth of the horizontal stems could account for the differences between days. This should be a useful measurement and would represent a characteristic of turfgrass arenas of warm-grasses which may be important for performance. Bermuda Hybrid "Tifway 419" has been reported as an intermediate traction species (42.9 Nm) in comparison with the other two warm grasses. Both the ITI and SCP do not appear to provide any additional information from the test boxes. Not only was the ITI insensitive to the two treatments it also did not correlate to any of the other measurement tools (Table 5). In contrast to our previous work, ITD was not sensitive to any factor in this experiment. Like with the RPS, the additional components of turfgrass profile, such as horizontal stem dispositions, shoot density, leaves thickness, and roots, may interfere with the displacement of a low weight of the drop; thus, mowing height may be obscuring drainage and geotextile effects. Other authors have found vertical displacement with a Clegg Hammer of 5.8 mm for Bermuda "Tifway 419" in field capacity at a mowing height of 15 mm; in this experiment, the range of vertical displacement get from ITD was from 8 to 13 mm at a mowing height of 30-50 mm and a higher VMC (%). The mowing height chosen here is more related to the racetrack than other sports like polo playgrounds. The same authors also found a negative correlation between dry matter and vertical deformation in football pitches. Our preliminary data on the partition of the dry matter (shoot/root) showed that turfgrass growing with drainage package or geotextile improves partitioning to shoot, these conditions and the addition of high moisture content achieved in the study may have operated on the lack of sensitivity shown by ITD. Some authors found a well-defined exponential trend of decreasing compressive strength with increasing soil saturation regardless of the soil type. The lack of sensitivity of ITD may be a combination of decreased compressive strength with increased volumetric moisture and turfgrass biomass that change the configuration. SCP showed higher values when geotextile is present (Table 2). SCP, as well as GSI, have also shown sensitivity to date of testing but detecting the effect of Dr and G. Bermuda Hybrid "Tifway 419" has also been reported as a high penetration resistance in soil playground surfaces , although in this experiment resistance to penetration recorded by SCP was an average of 3.08 drops (min = 1 and max:6), which means a lower value in comparison with other studies, and may be related to the high VMC (%), regarding present correlations as variance between VMC (%) and SCP explained (p < 0.0216, r = -0.29). This result is in accordance with the decreasing of the values of the dynamic cone penetrometer as a CBR% as saturation increased . The GSI was sensitive to different aspects of surface design. GSI was shown to consistently detect the presence of the geotextile when the drainage package was also present. The surface with geotextile had higher GSI, and alternative when drainage package is present. In both moments of testing, the GSI value was in a range equivalent as a good to soft surface rating, with lower values in the first testing (B1) and higher values in the second testing moment (B2). Although a correlation exists between the GSI and SCP results, the GSI includes a combination of longitudinal shear and penetration resistance. Because of the effect of turf, both properties were captured by the GSI under the conditions included in this study. The correlation shown between devices suggests that geotextile is modifying the penetration resistance force. Both results would be compatible with firmness and cushioning surface, although they don't measure peak load. The drainage package may be acting as a subsurface water storage, as shown by the water availability test . This is consistent with the results of McInnes and Thomas, where the drainage package on top of limestone reduced the water flux rate and may also provide a more consistent interface . This means that the reduction in water flux promotes a special arrangement of particle distribution, even though all of the treatments had the same size initial particle distribution. This finding is relevant to turfgrass health, although, for the experimental conditions considered, VMC's (%) was high. 5. Conclusions Many turfgrasses equine sports surfaces have different components than sand surfaces. However, in this experiment, the composition of the topsoil is closely related to a sand surface. The turfgrasses canopy adds another dimension to the interaction of the hoof-surface. The effect and the interaction of drainage and geotextile were tested with five devices. Previous studies on sand boxes were confirmed with the effect of drainage package and geotextile addition able to be detected with some of the simple instruments used in this experiment. In this study, TDR proved to be the most reliable tool, able to detect differences promoted by geotextile or drainage package despite the high moisture content. The effect of drainage on surface properties is likely to be related to the presence of both a vertical and horizontal water movement and the subsequent effect on packing particles that lead to higher VMC at higher water tension values and, in this sense, adding consistency. As in our previous study, the ability to test functional properties of the surface may be limited, however, due to the lower loads and lower load rates from these instruments. In addition, the turfgrasses canopy adds complexity through sward structure and variations in the height of mowing. These turfgrass properties contribute to the complex surface-hoof features and limit the ability to detect differences with simpler devices such as RPS, ITD, SCP, or GS. The different sensitivity of some tools like RPS, SPC, and GSI between dates highlights the interest in changes in the growth rate of shoots and roots and thickness of leaves regarding the season and affecting hoof-surface interaction. The lack of sensitivity of some devices may also be influenced by the method of turfgrass establishment. The turf was applied as sod, which included a 2 cm thick layer of silty clay soil which may be a barrier to the detection of drainage and geotextile treatments by the RPS and ITD. While future research may include washing the sod, this is not typically done in most installations. Moisture also plays a role in the lack of sensitivity of some devices. However, since the arenas or racetracks are outdoors, some combination of factors like drainage package and high volumetric moisture content may lead to turf with an off condition. Understanding the consequences of injury risk is the primary objective of the use of these tools. Future work should consider alternative approaches that are also suited for monitoring the proper maintenance of the turfgrass surfaces. Methods such as vertical cutting and aeration are critical to ensuring that a high-quality surface can be provided that results in a consistent performance over time. Acknowledgments The authors would like to acknowledge support from the Racing Surfaces Testing Laboratory (RSTL), the equestrian training center Solaguayre for allowing the study to be undertaken on the center, Marcelo Soria, from the Graduate School of Faculty of Agronomy of the University of Buenos Aires, for his statistician support, Juan Jose Crespi for supplying and providing technical support for the ITD and RPS and to TurfTrax Ltd. for providing Going Stick(c). Author Contributions M.A.B. conceived and supervised the study and had substantial inputs into the analysis and all drafts and created the original manuscript preparation. F.N.D.R. conducted the field camp data acquisition under supervision, M.P. had substantial inputs into conceptualization, data curation, and reviewing of the original manuscript of the paper. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Abbreviations B1 and B2 indication of the two blocks (dates of testing) Dr1 and Dr2 indication of the absence, 1, or existence, 2, of a drainage system G1 and G2 indication of the absence, 1, or addition, 2 of a geotextile GMC gravimetric moisture content GSI Going Stick Index ITI Impact Test Index RPS rotational peak shear SCP soil cone penetrometer TDR time domain reflectometry VMC volumetric moisture content WRC water retention capacity (10 hPa) AC aeration capacity EAW easily available water AWS available water storage RAW readily available water (100 hPa) Dd dry density (kg/m3) TP total porosity Appendix A Test of water-air availability CEN EN-13041 . animals-13-00811-t0A1_Table A1 Table A1 Table of Mean Test Comparison. Group 1 Group 2 Media 1 Media 2 T p-Value Dds (Kg/m3) Dr1:G1 Dr1:G2 1197.47 1083.88 2.75 0.1105 Dds (Kg/m3) Dr1:G1 Dr2:G1 1197.47 973.81 4.14 0.0536 Dds (Kg/m3) Dr1:G1 Dr2:G2 1197.47 982.27 4.00 0.0572 Dds (Kg/m3) Dr1:G2 Dr2:G1 1083.88 973.81 3.00 0.0956 Dds (Kg/m3) Dr1:G2 Dr2:G2 1083.88 982.27 2.79 0.1080 Dds (Kg/m3) Dr2:G1 Dr2:G2 973.81 982.27 -0.17 0.8821 TP Dr1:G1 Dr1:G2 0.47 0.52 -2.75 0.1105 TP Dr1:G1 Dr2:G1 0.47 0.57 -4.14 0.0536 TP Dr1:G1 Dr2:G2 0.47 0.56 -4.00 0.0572 TP Dr1:G2 Dr2:G1 0.52 0.57 -3.00 0.0956 TP Dr1:G2 Dr2:G2 0.52 0.56 -2.79 0.1080 TP Dr2:G1 Dr2:G2 0.57 0.56 0.17 0.8821 WRC Dr1:G1 Dr1:G2 0.43 0.43 0.29 0.7998 WRC Dr1:G1 Dr2:G1 0.43 0.49 -1.86 0.2036 WRC Dr1:G1 Dr2:G2 0.43 0.51 -1.90 0.1983 WRC Dr1:G2 Dr2:G1 0.43 0.49 -2.23 0.1558 WRC Dr1:G2 Dr2:G2 0.43 0.51 -2.16 0.1628 WRC Dr2:G1 Dr2:G2 0.49 0.51 -0.49 0.6712 20 hPa Dr1:G1 Dr1:G2 0.40 0.39 0.45 0.6988 20 hPa Dr1:G1 Dr2:G1 0.40 0.45 -2.03 0.1798 20 hPa Dr1:G1 Dr2:G2 0.40 0.48 -2.10 0.1706 20 hPa Dr1:G2 Dr2:G1 0.39 0.45 -2.49 0.1304 20 hPa Dr1:G2 Dr2:G2 0.39 0.48 -2.42 0.1367 20 hPa Dr2:G1 Dr2:G2 0.45 0.48 -0.78 0.5194 30 hPa Dr1:G1 Dr1:G2 0.39 0.37 1.18 0.3583 30 hPa Dr1:G1 Dr2:G1 0.39 0.43 -2.31 0.1475 30 hPa Dr1:G1 Dr2:G2 0.39 0.46 -1.99 0.1847 30 hPa Dr1:G2 Dr2:G1 0.37 0.43 -4.10 0.0547 30 hPa Dr1:G2 Dr2:G2 0.37 0.46 -2.67 0.1164 30 hPa Dr2:G1 Dr2:G2 0.43 0.46 -0.89 0.4678 40 hPa Dr1:G1 Dr1:G2 0.37 0.34 1.64 0.2435 40 hPa Dr1:G1 Dr2:G1 0.37 0.40 -1.67 0.2372 40 hPa Dr1:G1 Dr2:G2 0.37 0.43 -1.99 0.1854 40 hPa Dr1:G2 Dr2:G1 0.34 0.40 -4.68 0.0427 * 40 hPa Dr1:G2 Dr2:G2 0.34 0.43 -3.31 0.0803 40 hPa Dr2:G1 Dr2:G2 0.40 0.43 -1.02 0.4142 50 hPa Dr1:G1 Dr1:G2 0.36 0.33 2.15 0.1641 50 hPa Dr1:G1 Dr2:G1 0.36 0.39 -2.12 0.1677 50 hPa Dr1:G1 Dr2:G2 0.36 0.43 -2.16 0.1637 50 hPa Dr1:G2 Dr2:G1 0.33 0.39 -6.82 0.0208 * 50 hPa Dr1:G2 Dr2:G2 0.33 0.43 -3.41 0.0762 50 hPa Dr2:G1 Dr2:G2 0.39 0.43 -1.18 0.3597 60 hPa Dr1:G1 Dr1:G2 0.35 0.32 3.08 0.0912 60 hPa Dr1:G1 Dr2:G1 0.35 0.38 -3.73 0.0651 60 hPa Dr1:G1 Dr2:G2 0.35 0.42 -2.61 0.1209 60 hPa Dr1:G2 Dr2:G1 0.32 0.38 -11.60 0.0073 * 60 hPa Dr1:G2 Dr2:G2 0.32 0.42 -3.76 0.0641 60 hPa Dr2:G1 Dr2:G2 0.38 0.42 -1.30 0.3228 80 hPa Dr1:G1 Dr1:G2 0.34 0.31 4.09 0.0548 80 hPa Dr1:G1 Dr2:G1 0.34 0.38 -4.50 0.0459 80 hPa Dr1:G1 Dr2:G2 0.34 0.42 -2.75 0.1106 80 hPa Dr1:G2 Dr2:G1 0.31 0.38 -10.50 0.0089 * 80 hPa Dr1:G2 Dr2:G2 0.31 0.42 -3.78 0.0635 80 hPa Dr2:G1 Dr2:G2 0.38 0.42 -1.34 0.3117 RAW Dr1:G1 Dr1:G2 0.33 0.30 3.90 0.0599 RAW Dr1:G1 Dr2:G1 0.33 0.37 -4.88 0.0395 * RAW Dr1:G1 Dr2:G2 0.33 0.41 -2.83 0.1052 RAW Dr1:G2 Dr2:G1 0.30 0.37 -10.49 0.0090 * RAW Dr1:G2 Dr2:G2 0.30 0.41 -3.81 0.0625 RAW Dr2:G1 Dr2:G2 0.37 0.41 -1.38 0.3007 EAW Dr1:G1 Dr1:G2 0.07 0.10 -1.19 0.3564 EAW Dr1:G1 Dr2:G1 0.07 0.09 -1.51 0.2703 EAW Dr1:G1 Dr2:G2 0.07 0.08 -0.43 0.7120 EAW Dr1:G2 Dr2:G1 0.10 0.09 0.04 0.9682 EAW Dr1:G2 Dr2:G2 0.10 0.08 1.11 0.3840 EAW Dr2:G1 Dr2:G2 0.09 0.08 1.56 0.2593 AWS Dr1:G1 Dr1:G2 0.03 0.03 0.30 0.7904 AWS Dr1:G1 Dr2:G1 0.03 0.02 1.63 0.2452 AWS Dr1:G1 Dr2:G2 0.03 0.01 2.70 0.1141 AWS Dr1:G2 Dr2:G1 0.03 0.02 1.06 0.4008 AWS Dr1:G2 Dr2:G2 0.03 0.01 2.01 0.1816 AWS Dr2:G1 Dr2:G2 0.02 0.01 3.49 0.0732 AC Dr1:G1 Dr1:G2 0.03 0.09 -2.38 0.1407 AC Dr1:G1 Dr2:G1 0.03 0.08 -9.61 0.0107 * AC Dr1:G1 Dr2:G2 0.03 0.06 -0.53 0.6474 AC Dr1:G2 Dr2:G1 0.09 0.08 0.54 0.6438 AC Dr1:G2 Dr2:G2 0.09 0.06 0.66 0.5758 AC Dr2:G1 Dr2:G2 0.08 0.06 0.46 0.6904 (*) t-test significant difference p < 0.05. Figure A1 Whisker plot of water availability test of the samples of depth cushioning after the treatments of drainage package (D1: without drainage package and D2: with drainage package) and geotextile addition (G1: 0 addition and G2: 2 kg/m2). t-test significant difference p < 0.05. Figure 1 Scheme of boxes design, including the depth of the top cushion material consisting of sand (G1), or sand with geotextile (G2 = 2 kgm2) over limestone (Dr1), or over drainage package (Dr2). Figure 2 One of the members of the team stood over the boxes of turfgrasses at the moment of the RPS testing (3 March 2018), (c)2018 Maria Alejandra Blanco. Figure 3 The mean of triple interaction blocks x drainage x geotextile for volumetric moisture content (VMC %) B1: 3 March; B2: 30 March; Dr1: without drainage package, Dr2: with drainage package; G1: without geotextile; G2: with 2 kg/m2 of geotextile. The stars indicate significant statistical differences (*). (Numbers of asteriks *, **, ***, **** indicates p-value). Figure 4 The mean of triple interaction block x drainage x geotextile for Going Stick Index (GSI) B1: 3 March; B2: 30 March; Dr1: without drainage package, Dr2: with drainage package; G1: without geotextile; G2: with 2 kg/m2 of geotextile. The stars indicate significant statistical differences (*). (Numbers of asteriks *, **, *** indicates p-value). animals-13-00811-t001_Table 1 Table 1 Replications of treatments and variables from tools used in the experiment on both two dates: 3 March and 30 March 2018. Replications Drainage Package (Dr = 2 Repetitions) Geotextile (G = 2 Repetitions) Drainage Package x Geotextile Variables Dates 3 March 2018 30 March 2018 3 March 2018 30 March 2018 3 March 2018 30 March 2018 VMC 2 3 5 3 5 3 5 RPS 2 3 5 3 5 3 5 ITI 2 3 5 3 5 3 5 SCP 2 3 5 3 5 3 5 GSI 2 3 5 3 5 3 5 animals-13-00811-t002_Table 2 Table 2 Statistics for the VMC (%), RPS, ITI, SCP, and GSI and GSS (f and p-value), to three factors drainage, geotextile addition, and blocks. ANOVA parametric variables p < 0.05 statistic test f; 1 ANOVA for non-parametric variables: Kruskal-Wallis Test p < 0.05 statistic test H. Variable Drainage (Dr) Geotextile (G) Blocks (B) f (H) p f (H) p f (H) p VMC (%) 1 4.94 0.0262 * 27.92 0.0001 * 1.46 0.2275 RPS 2.46 0.1221 1.38 0.2445 5.13 0.0275 * ITI 1.01 0.3193 0.07 0.7975 0.22 0.6377 SCP 1 1.01 0.287 5.52 0.0118 * 2.50 0.094 GSI 1 4.79 0.0286 * 2.16 0.1413 10.04 0.0015 * Tukey test Alpha = 0.05 p < 0.05. (*) significant difference. animals-13-00811-t003_Table 3 Table 3 f (H) and p-values of interactions between factors: drainage x geotextile, drainage x blocks, geotextile x blocks, and drainage x geotextile x blocks over the five variables (VMC, RPS, ITI, SCP, and GSI) ANOVA p < 0.05 statistic test f; 1 ANOVA for non-parametric variables: Kruskal-Wallis Test p < 0.05 statistic test H. Variable Dr x G Dr x B G x B Dr x G x B f (H) p f (H) p f (H) p f (H) p VMC 1 (%) 33.75 0.0001 * 6.75 0.082 30.32 0.0001 * 37.87 0.0001 * RPS 0.04 0.8500 0.13 0.7161 0.99 0.3240 0.72 0.4001 ITI 0.57 0.4539 1.03 0.3152 0.16 0.6825 0.13 0.7237 SCP 1 6.76 0.055 3.73 0.2325 8.35 0.0225 * 9.72 0.132 GSI 1 10.46 0.015 * 14.84 0.0020 * 12.78 0.0054 * 21.19 0.0035 * Tukey test Alpha = 0.05 p < 0.05. (*) significant difference. animals-13-00811-t004_Table 4 Table 4 Regression coefficients (R2), and linear model coefficients for every variable VMC (%), RPS, ITI, SCP, and GSI. Significant values at p < 0.05 (*). Variables R2 Linear Model Coefficients Constant Dr G B VMC (%) 0.54 45.55 * 5.46 * -11.87 * 2.52 RPS 0.13 35.49 * 2.16 1.63 3.23 * ITI 0.02 0.02 * -0.0013 0.0003 -0.00006 SCP 0.17 1.67 * 0.30 0.82 * 0.54 GSI 0.22 -1.12 0.97 1.43 * 1.54 * animals-13-00811-t005_Table 5 Table 5 Pearson coefficients correlations r and p for every variable VMC (%), RPS, ITI, SCP, and GSI on treatments. (*) significant difference. Variables VMC (%) RPS ITI SCP GSI r p r p r p r p r p VMC (%) 1 0.001 -0.07 0.6083 0.07 0.5654 -0.29 0.0216 * -0.15 0.2439 RPS -0.07 0.6083 1 0.001 -0.01 0.9451 0.05 0.6807 0.17 0.1746 ITI 0.07 0.5654 -0.01 0.9451 1 0.001 -0.19 0.1418 -0.09 0.4655 SCP -0.29 0.0216 * 0.05 0.6807 -0.19 0.1418 1 0.001 0.37 0.0025 * GSI -0.15 0.2439 0.17 0.1746 -0.09 0.4655 0.37 0.0025 * 1 0.001 * Significant values at p < 0.05. 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PMC10000091

The study's main goal was to compare several data mining and machine learning algorithms to estimate body weight based on body measurements at a different share of Polish Merino in the genotype of crossbreds (share of Suffolk and Polish Merino genotypes). The study estimated the capabilities of CART, support vector regression and random forest regression algorithms. To compare the estimation performances of the evaluated algorithms and determine the best model for estimating body weight, various body measurements and sex and birth type characteristics were assessed. Data from 344 sheep were used to estimate the body weights. The root means square error, standard deviation ratio, Pearson's correlation coefficient, mean absolute percentage error, coefficient of determination and Akaike's information criterion were used to assess the algorithms. A random forest regression algorithm may help breeders obtain a unique Polish Merino Suffolk cross population that would increase meat production. random forest support vector machine CART biometric measurements Ministry of Education and Science of the Republic of Poland(funds for research activity BN-WHiBZ-4/2022) This work supported by the Ministry of Education and Science of the Republic of Poland (funds for research activity BN-WHiBZ-4/2022). pmc1. Introduction Sheep play an important role in both obtaining animal products and developing the rural economy among civilizations . In addition, sheep need a shorter time between generations than cattle. As in all farm animals, environmental factors play an essential role in the interaction and genetic potential of the sheep. Genotype, the environment and their interaction are the factors that must be considered to make an economical profit. To achieve a high-level yield, it may be necessary to use different genotypes or crossbreeds, taking into account environmental factors. In 2020, 31 breeds and lines of sheep were used in Poland, and the largest share in the breed structure was Polish Merino sheep . The origin of merino sheep breeding in Poland dates back to the 19th century. Merino sheep gradually evolved in terms of thicker wool and improved meat characteristics. Many years of breeding have resulted in breeds that have been genetically combined and used for meat and milk production. Polish Merino has a close and dense fleece. Additionally, Polish Merino sheep mature early and show an aseasonality in reproduction. Because of these characteristics, the Polish Merino is Poland's most common commercial breed. High-quality meat characteristics describe the Polish Merino sheep, and lambs can be used for dairy and medium-intensity and intensive fattening processes. The body weight of Polish Merino ewes and rams is 55-75 kg and 90-120 kg, respectively . The average fertility of ewes was 94.04% according to Piwczynski , and fertility was 152% according to Piwczynski and Mroczkowski . In the 1990s, the Polish Merino sheep breed was improved by crossbreeding with other breeds to enhance some characteristics; therefore, the number of native purebred sheep decreased considerably. Consequently, in 2008 the pure Polish Merino sheep breed was characterized, and the original breed pattern (maintains the breed purity) was described. From then, the breed was called Polish Merino sheep . The crossbreeds of Polish Merino ewes that appeared in the breeding stock books in 2015-2020 decreased from 3.93 to 1.71%, while the pure Polish Merino ewes were relatively stable (2015: 10.65%; and 2020: 10.69%) . The only condition for profitability in sheep farms in Poland is producing young lamb for slaughter. Unfortunately, Poland's sheep population structure does not meet the requirements for producing meat lambs. Sheep used for their wool are the most numerous, while the stock of meat breeds is scarce: in 2020, the number of Suffolk ewes under the utility assessment was only 151 . The use of displacement crossing is a breeding method that can change the breed structure in favor of meat breeds . The backcrossing of dams of Polish Merino sheep with meat breeds rams, among others Suffolk, might be an efficient way to increase the meat sheep population in Poland . The Suffolk breed originated in England and was created by crossing the Southdown rams with the Norfolk Horn ewes. The breed was recognized in 1810. In 1886, the Suffolk Breeders' Association started to keep a register of animals of this breed. Thanks to the intensive selection and proper selection of breeding pairs, animals with outstanding meat characteristics were produced. They were suited for crossing with other breeds to improve fattening and slaughter characteristics. The literature shows many examples of crossbreeding Polish Merino and Suffolk sheep . Growth and development are economically important features. Growth is determined by measurement and weighting, and its calculation is based on live weight. Furthermore, growth and weight gain can be calculated using various correlations between live weights and body measurements . Body weight is the most important feature for all animal species with an economic income, as it directly affects breeding income and meat production. Scientifically, more interest has been paid to defining the association between body measurements and body weight in improving meat production. Biometric measurements of all animals may indicate phenotypic and genotypic characteristics as well as growth characteristics . It has been reported that various biometric features taken throughout the early growth periods may be used as an early selection criterion to obtain offspring with superior body weight . In addition, body measurements are helpful in herd management in estimating body weight. Furthermore, being aware of body weight is essential in herd management practices such as calculating the amount of feed per animal, managing the medicinal drug doses and determining the optimum slaughter weight . For this reason, it may be used as an indirect selection criterion in making predictions based on body measurements . In this framework, the finest way to determine biometric measurements that directly affect body weight and define breed phenotypically is the application of trustworthy statistical procedures, such as multivariate statistical procedures for sheep . There may be differences in estimating live weight from body size from species to species and from breed to breed. Many studies evaluate body weight using measurements in several animal species, such as buffalo, sheep, dogs, cattle, goats, rabbits and camels . The three methods selected are the subject of multivariate statistics. Regression analysis is one of the multivariate statistical methods used to reveal the relationship between biometric features and animal weight. In multivariate statistical modelling, regression analysis is a process to estimate the relationship between explanatory and response variables. Many methods are used to estimate the response variable, with the most common being the Least Squares (LS) method. The LS method requires some assumptions to make an effective model estimation. Alternative methods are proposed when multicollinearity between explanatory variables is provided from these assumptions . Many studies in different species and breeds are based on estimating body weight using biometric features. They use multiple regression, Classification and Regression Tree (CART), Chi-square Automatic Interaction Detector (CHAID), Multivariate Adaptive Regression Splines (MARS) algorithms and artificial neural networks. Although there are different studies for other breeds and species, there is no such study for different shares of Polish Merino and Suffolk genotypes in crossbreeds, which is the subject of our study . In addition, there is no study for CART, SVR and RFR, apart from the scope of the aforementioned algorithms. In this framework, various statistical procedures can help produce more reliable estimates covered by indirect selection criteria in different animal species and expose the biometric features that influence body weight. The use of many methods such as CHAID, MARS, CART, Artificial Neural Networks (ANNs) and Exhaustive Chi-square Automatic Interaction Detector (Exhaustive CHAID) has gained importance in estimating body weight from biometric features in various sheep breeds . The use of these estimation methods, however, differs from breed to breed. To our knowledge, there is no research on using CART, Support Vector Regression and Random Forest Regression algorithms for body weight estimation of the different shares of Polish Merino in the genotype of crossbreeds. These three methods were selected to show a clear presentation of the results. The present study aims to fill this gap in the literature and evaluate the goodness of fit of these procedures. 2. Materials and Methods The numerical material used in the research came from the research carried out in 1990-1995 by Piwczynski as part of the topic of his master's and doctoral dissertation. The research material consisted of 344 animals, including 114 crossbreds R2 (75.0% Suffolk, 25.0% Polish Merino), 97 crossbreds R3 (87.5% Suffolk, 12.5% Polish Merino) and 133 animals of Suffolk breed. A total of 88 rams and 256 sheep were used in the study. The evaluated groups of crossbreds originated from the two subsequent stages of backcrossing of Polish Merino ewes with Suffolk rams. Implementation of crossbreeding started in 1986 in one flock in Bydgoszcz voivodship. The purpose of this crossbreeding was to obtain a meat-type sheep line. Suffolk sheep used for crossbreeding with Polish Merino were imported to the farm from Great Britain in 1985. The group of animals consisted of 40 ewes and five rams. All test animals were housed in litter-box buildings with running water and artificial lighting. Mothers and lambs were fed in accordance with the applicable nutrition standards declared by the National Research Institute of Animal Production, 1985. During the summer feeding period, the animals used a pasture. While on-site, they were fed a CJ mixture (for calves and lambs), dried corn, hay and green alfalfa, and during the winter feeding they were given a CJ mixture, beets, oats, dry pulp, briquette haylage and hay. To compose the data set, the genotype (share of Suffolk and Polish Merino genotypes), birth weight (BiW), sex, birth type and body weight at 12 months of age (LBW) and some body measurements such as withers height (WH), sacrum height (SH), chest depth (CD), chest width (CW), chest circumference (CC), shoulder width (SW) and rump width (RW) were used . Algorithms such as CART, CHAID and Exhaustive CHAID are tree-based algorithms used to evaluate a quantitative feature . For this purpose, Breiman et al. developed the first method called CART procedure. The CART algorithm is a binary decision tree structure created by recursively dividing a node into two child nodes. The algorithm covers the evolving process until many homogeneous nodes are obtained from a learning sample dataset, providing minimal error variance covered by training and test sets. The main purpose of a tree is to select new and homogeneous binary splits to obtain the purest child nodes. In the algorithm, each split is made for one estimator only. The variance-based method was used as the pruning rule in the tree construction, and the minimum size of a tree node was set to five and accepted as the stopping criterion. In addition, 10-fold cross-validation with a single standard error rule was applied to find the regression tree that fit the training data. In this way, it was warranted that there was no overfitting for the CART algorithm. A valuable part of the support vector machine (SVM), one of the most commonly used procedures among machine learning procedures, is the support vector regression (SVR) procedure . In the SVM algorithm, the part that deals with classification is known as support vector classification (SVC), and the part that deals with prediction is known as SVR . The SVR is one of the machine learning procedures that creates a linear model prediction to simultaneously minimize experimental risk and model complexity . While SVR is a regulated learning procedure, the presentation of SVR varies according to the training and test sets . The primary theory of SVR is to describe a function f(x) with the upper limit deviation (e) from the train set. The training set points are arranged inside the cutoff point between -e to +e . However, most studies cannot be modeled in a linear sense. Therefore, for conditions for which the solution is nonlinear, the input data of the SVR algorithm is mapped to a better dimensional Hilbert space (H), so the edge of the regression model can be linear . The regression hyperplane to be achieved under nonlinear conditions is presented below. y^=<w,ph(x)> +b where, w is the weights of the vector, ph(x) is the functions of the kernel, <.,.> implies an innermost vector result and b is a biased term. In addition, many kernel functions can be used to apply to nonlinear conditions. Although there are many kernel functions, the Gaussian radial basis function is used in this study. Random Forest is a standard procedure used between several multivariate statistical procedures in terms of its practicality for regression and classification form of the problems. The RFR algorithm consists of a process that includes a layer of casualness to the bagging algorithm. This procedure was presented by Breiman . The RFR algorithm is shown as a set of limitations utilized hierarchically from the tree's root to the leaf by merging clusters of the regression trees . The most significant benefit of this procedure is that it can be clearly utilized in nonlinearity. The procedure requires a method that consists of three stages . The first stage is constructing a number of trees (ntree) from the initial data. The second stage is to build an untrimmed regression or classification tree for every sample. The final step is predicting the recent data of the tree. In this aspect, for the Polish Merino sheep and Suffolk crossbreed sheep data set, the model parameters such as ntree and number of variables checked out for all splits are chosen (mtry) as 500 and 5, respectively. To compare the model performances, the goodness of fit of criteria such as the Pearson correlation coefficient (r), root mean square error (RMSE), determination of coefficient (R2), Akaike's information criterion (AIC), mean absolute percentage error (MAPE) and standard deviation ratio (SDratio) were used as shown below :Root-mean-square error (RMSE): RMSE=1ni=1n(yi-yip)2 Akaike information criterion (AIC): {AIC=n.ln[1ni=1n(yi-yip)2]+2k, if n/k>40 AICc=AIC+2k(k+1)n-k-1 otherwise Standard deviation ratio (SDR): SDratio=SmSd Global relative approximation error (RAE): RAE=i=1n(yi-yip)2i=1nyi2 Mean absolute percentage error (MAPE): MAPE=1n i=1n|yi-yipyi|*100 where, n is the number of the training data, yi is the actual amount of the response variable (BW), yip is the estimated amount for the response variable (BW), Sd is the standard deviation for the response variable (BW) and Sm is the standard deviation of the best model's errors. The aforementioned goodness of fit was used to compare the model performances, which were made along with the lowest RMSE, SDratio and MAPE. In addition, the model performances evaluated the highest r and R2 values . 3. Results In this study, the mean and standard deviation for each trait for Polish Merino sheep and Suffolk crossbreed sheep were calculated, and the descriptive statistics are presented in Table 1. The correlation coefficient was used to define the association between body measurements (birth weight, withers height, chest depth, sacrum height, chest width, shoulder width, rump width and chest circumference) and sex, birth type characteristics and LBW. Figure 1 shows the most significant correlation coefficient between CC and LBW (coefficient of 0.72). The other traits, except for the SH, show a similar correlation coefficient with LBW. Moreover, all coefficients were determined to be significant (p < 0.05). The tree diagram constructed using the CART algorithm is presented in Figure 2. The root node of the tree was recorded as 56 kg (LBW). In the case of CC, the initial depth was lower than 94 cm, and the average LBW of the crossbreed sheep was determined to be 49 kg. At the right side of the tree, the initial depth, in the case of CC, was greater than 94 cm, and the average LBW was determined as 63 kg. If the CC was less than 94 cm in the first split, the tree was divided into 2 parts. The first part was for the sex. If the sex was female, the tree was divided for CC < 89 and CC >= 89. In these cases, LBW was determined as 46 and 51 kg, respectively. If CC was >= 94 cm in the initial split, the tree was divided into 2 parts: CD < 32 cm and CD >= 32 cm. If CD was <32 cm, the tree was divided for Genotype = R2, WH < 63 cm, SW < 29 cm and RW < 26 cm. The average LBW was determined as 66 kg for the cases in which the Genotype = R2, WH < 63 cm, SW < 29 cm and RW < 26. In the case of the CD >= 32 cm, the average LBW was determined as 73 kg. This node was divided into two parts for Genotype = R2. If the genotype was R2, the average LBW was defined as 62 kg. However, when the genotype was not R2 (i.e., was R3 or Suffolk) and CD >= 34 cm, the LBW was determined as 88 kg (the node with the highest values of LBW). To contribute to the rural economy by increasing meat productivity, it has been determined that more profitable livestock can be made in cases when CC >= 94 cm, CD >= 32 cm, the genotype is not R2 and CD >= 34 cm. First, the SVR procedure was performed for the training set. After the training procedure, the SVR predicted the body weight of Suffolk sheep. The kernel function was estimated for the final body weight. The accessibility for the model is based on the selected factors such as epsilon and cost (C). The aforementioned factors were examined for several values, and the procedure was utilized for the epsilon and C values, which would provide a highly trustworthy model. Sensitivity analysis was used to estimate the model's virtual significance for explanatory variables for BW . CC had the most significant relative importance value obtained in the scope of the sensitivity analysis. The explanatory variable that produced the smallest relative importance value was genotype and birth type (twin). The RFR algorithm performance is presented in Table 2. Moreover, the sensitivity analysis was performed to predict virtual significance amounts of the explanatory variables LBW in RFR . For sensitivity analysis, CC, CD and SW had virtual significance higher than 10%. However, unlike the SVR algorithm, the explanatory variables that produced low virtual significance were determined to be CW and SH. The lowest virtual significance had BiW. The comparison of all algorithms and goodness of fit criteria are presented in Table 2. For all algorithms, the performances of the training and the test sets were evaluated. The performance values obtained from the test set for each model were weaker than those from the training data set. The best procedure for the test set was the RFR algorithm, although the most appropriate algorithm for the training set was SVR. RFR was determined as the most appropriate algorithm as it gave closer results for the training and test sets and gave the highest R2 and r values and the lowest RMSE, SDratio, CV, MAPE and AIC values in the test set. Because the training set memorized the SVR algorithm, the SVR algorithm gave unreliable test results. 4. Discussion Different characterization methods are used in the literature to investigate the relationship between biometric features and body weight in various animal species . The accuracy of statistical methods applied to predict BiW from biometric features that differ even between breeds is also very important. Many studies have been conducted on this subject in different animal species and breeds. This subject is very important, especially in rural areas and conditions where no weighing device is available . However, there is no study on this aim for the Suffolk sheep breed. In multivariate statistics, artificial neural networks, data mining, machine learning algorithms and the usage of the goodness of fit criteria have been suggested in selecting the finest model . Within this scope, the model performances are compared according to the goodness of fit criteria . CART, SVR and RFR algorithms were used to help determine the selection scheme for Polish Merino sheep and Suffolk crossbreed sheep. Various statistical methods can define effective variables for LBW estimation, which may be helpful for selecting farm animals; therefore, the basis for the sustainable animal breeding may be laid. Though the literature lacks studies on these algorithms, it has been established that similar to our study, only the RFR and SVR algorithms were used for the Thalli sheep breed . In that study, Tirink indicated that the MARS algorithm was superior to Bayesian Regularized Neural Network (BRNN), SVR and RFR. The results of this study showed that SVR was better than RFR. That study had the opposite results to ours. It means that the model selection depends on the genotype. Alonso et al. used Support Vector Machine Regression to estimate the carcass weight in Asturiana de los Valles beef. For this aim, 390 measurements for 144 animals were made. According to these results, the optimal carcass weight prediction was obtained 150 days before the slaughter time. They presented the use of SVR algorithm detailed. Ali et al. compared the CART, CHAID, ANNs and Exhaustive CHAID algorithms in this study for the Harnai sheep breed. The results were estimated as follows: Exhaustive CHAID 0.8421, CHAID 0.8377, ANNs 0.81999 and CART 0.82644. When the performance of the CART algorithm was evaluated against other algorithms within the scope of R2, the diversity of the algorithms used and the breed differences, the CART algorithm was the third-best algorithm. However, it gave results close to other algorithms in terms of performance. Our result showed that both SVR and RFR were better than CART algorithm. According to this, SVR and RFR algorithms had better fitting properties than CHAID and ANNs. Celik et al. aimed to compare CART, MLP, CHAID, MARS, Exhaustive CHAID, and RBF for Mengali rams. In the scope of the goodness of fit criteria such as R2, RMSE and SDratio, the finest estimation model was defined as the CART algorithm. The only comparable algorithm was CART, which we also used in our study. According to this, the SVR and RFR algorithms may be more appropriate, but it should be considered that the fitting performance may depend on the data. Hussain et al. compared the hybrid machine learning algorithms such as SVR and emotional ANNs for estimating the body fat percentage. They used anthropometric characteristics (BFP, sex, age, weight, height, WHR, abdominal C and BMI). RMSE, R2, and rRMSE were used for the model comparison criteria. According to the BFP results, SVR was 0.9682 for R2, 0.0245 for RMSE, and 7.6956 for rRMSE. However, the hybrid (SVR-EANN) method was the best algorithm for estimating the BFP. In our work, SVR was not the best one. The superiority of the RFR algorithm versus the SVR algorithm should be taken into account because the SVR algorithm gave unreliable test results due to it being memorized in the training set. Iqbal et al. aimed to compare the model performances for gradient boosting machine, regression tree, random forests and SVM algorithms. Beetal goats were used, and the explanatory variables such as sex, body length, shank circumference, neck length, head girth, rump height and belly sprung were evaluated. In this study, the model comparison criteria such as Pearson's correlation, R2, MAE, MAPE and RMSE were chosen. According to the results, the gradient boosting machine (GBM) was determined to be the best model for predicting the body weight of Beetal goats. However, the random forest regression algorithm was the second-best algorithm. RFR was one of the best algorithms, as indicated in this study's results. In our stuy, there is no evidence to compare GBM, which was the best for the Iqbal et al. study, but it is clear that RFR can be used reliably for model fitting. Marco et al. aimed to examine the AdaBoost ensemble learning method and RFR for different data sets. Several machine learning techniques, such as CART, kNN, MLP, SVR and RFR were used for different data sets. According to the results of most of the datasets applied in the study, they stated that RFR was the most reliable and successful algorithm for their study. The results matched to results of our study. Ahmad et al. aimed to compare various algorithms such as RFR, decision trees, extra trees and SVR. They wanted to predict solar thermal energy systems and revealed that the RFR and extra trees models gave more reliable results than variable selection tools. The matching Ahmad et al. and Marco et al. results with our study showed that RFR can be used instead of other algorithms. Coskun et al. aimed to compare the eXtreme Gradient Boosting (XGBoost), Random Forest (RFR) and Bayesian Regularization Neural Network (BRNN) data mining algorithms to predict the live weight at the end of fattening by using some of the body characteristics at the initiation of fattening in Anatolian Merino lambs. They indicated that the XGBoost algorithm gave a better fitting performance than BRNN and RFR according to the root mean square error (RMSE), standard deviation ratio (SDR), mean absolute percentage error (MAPE) and adjusted coefficient of determination (R2Adj). For the interpretation Coskun et al. focused on short-term fattening performance results. Even though not the best algorithm, the CART algorithm showed that the crossbreeding level should be at least R3 to reach the highest live body weight in Polish conditions. Compared to previous research results, several species and breeds were used for data mining and machine learning algorithms. The animal age, differences in flock management systems and differences in statistical methods applied can be attributed to the extensive variation in previous studies. When comparing our study with other results, the models we used according to the selected goodness-of-fit criteria give similar results as other studies. However, recommending several statistical procedures for BW estimation using biometric features is important in terms of both species and breed characterization for meat production industries. It reveals that more studies are needed on this subject. 5. Conclusions To conclude, the RFR procedure may help breeders improve the characteristics of great importance. Moreover, it shows BW as a criterion for establishing proper biometric measurements and flock organization principles. The study's outcomes showed that based on the goodness of fit criteria for choosing the most appropriate model, machine learning and data mining algorithms can be profitably utilized for body weight prediction based on measured body measurements. Author Contributions Conceptualization, C.T. and H.O.; methodology, C.T., D.P., M.K. and H.O.; validation, C.T., D.P. and H.O.; formal analysis, C.T., D.P. and H.O.; investigation, C.T. and D.P.; resources, C.T. and D.P.; data collection, D.P.; data analysis, H.O., C.T. and D.P.; writing--original draft preparation, C.T., D.P., M.K. and H.O.; writing--review and editing, C.T., D.P., M.K. and H.O.; visualization, C.T. and H.O.; supervision, C.T., D.P., M.K. and H.O.; project administration, C.T., D.P., M.K. and H.O. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. There is no need to take ethical approval because there is no clinical application on the animals. Informed Consent Statement Not applicable, as this research did not involve any humans. Data Availability Statement To reach the data please contact with the authors C.T. Conflicts of Interest The authors declare no conflict of interest and none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Figure 1 Correlation matrix. Figure 2 The constructed CART diagram. Figure 3 Relative importance for the SVR algorithm. Figure 4 Sensitivity analysis for RFR algorithm. animals-13-00798-t001_Table 1 Table 1 Descriptive statistics. Genotype Variables Mean +- Standard Deviation Suffolk N = 133 BiW 3.79 +- 0.92 LBW 58.32 +- 10.32 WH 62.07 +- 3.30 SH 64.49 +- 4.08 CD 28.86 +- 2.47 CW 22.95 +- 2.65 SW 24.65 +- 2.68 RR 26.54 +- 2.96 CC 94.16 +- 8.29 R2 N = 114 BiW 4.22 +- 0.82 LBW 52.70 +- 6.74 WH 63.18 +- 2.58 SH 63.61 +- 2.45 CD 28.50 +- 4.39 CW 23.12 +- 2.60 SW 22.71 +- 4.39 RR 25.20 +- 2.77 CC 92.64 +- 6.90 R3 N = 97 BiW 4.13 +- 0.96 LBW 58.77 +- 11.56 WH 62.66 +- 3.34 SH 63.35 +- 3.39 CD 29.52 +- 2.26 CW 23.20 +- 2.50 SW 24.90 +- 2.59 RR 26.35 +- 2.93 CC 95.93 +- 9.30 Birth weight (BiW), sex, birth type and 12th month of body weight (LBW) and some body measurements (cm) such as withers height (WH), sacrum height (SH), chest depth (CD), chest width (CW), chest circumference (CC), shoulder width (SW) and rump width (RW). animals-13-00798-t002_Table 2 Table 2 The results of the CART, SVR and RFR algorithms in the scope of the goodness of fit criteria. Criterion CART SVR RFR Training Set Test Set Training Set Test Set Training Set Test Set RMSE 20.304 49.750 16.100 33.745 24.271 31.279 SDratio 0.454 0.643 0.404 0.526 0.497 0.511 CV 8.000 12.320 7.110 10.080 8.740 9.780 r 0.891 0.774 0.918 0.852 0.869 0.860 MAPE 6.643 9.801 4.984 6.822 6.848 7.046 R2 0.793 0.578 0.836 0.714 0.753 0.735 AIC 830.985 265.677 766.951 239.280 880.245 234.121 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000092

Laminaria spp. and their extracts have preventative potential as dietary supplements during weaning in pigs. The first objective of this study was to evaluate increasing concentrations of four whole seaweed biomass samples from two different Laminaria species harvested in two different months in a weaned pig faecal batch fermentation assay. Particularly, February and November whole seaweed biomass samples of L. hyperborea (LHWB-F and LHWB-N) and L. digitata (LDWB-F and LDWB-N) were used. In the next part of the study, the increasing concentrations of four extracts produced from L. hyperborea (LHE1-4) and L. digitata (LDE1-4) were evaluated in individual pure-culture growth assays using a panel of beneficial and pathogenic bacterial strains (second objective). The LHE1-4 and LDE1-4 were obtained using different combinations of temperature, incubation time and volume of solvent within a hydrothermal-assisted extraction methodology (E1-4). In the batch fermentation assay, the L. hyperborea biomass samples, LHWB-F and LHWB-N, lowered Bifidobacterium spp. counts compared to the L. digitata biomass samples, LDWB-F and LDWB-N (p < 0.05). LHWB-F and LDWB-N reduced Enterobacteriaceae counts (p < 0.05). LHWB-F and LDWB-F were selected as the most and least promising sources of antibacterial extracts from which to produce LHE1-4 and LDE1-4. In the pure-culture growth assays, E1- and E4-produced extracts were predominantly associated with antibacterial and bifidogenic activities, respectively. LHE1 reduced both Salmonella Typhimurium and Enterotoxigenic Escherichia coli with LDE1 having a similar effect on both of these pathogenic strains, albeit to a lesser extent (p < 0.05). Both LHE1 and LDE1 reduced B. thermophilum counts (p < 0.05). LDE4 exhibited strong bifidogenic activity (p < 0.05), whereas LHE4 increased Bifidobacterium thermophilum and Lactiplantibacillus plantarum counts (p < 0.05). In conclusion, antibacterial and bifidogenic extracts of Laminaria spp. were identified in vitro with the potential to alleviate gastrointestinal dysbiosis in newly weaned pigs. brown macroalgae bifidogenic antibacterial seaweed polysaccharides weaned pig gastrointestinal microbiota batch fermentation Science Foundation Ireland (SFI)14/IA/2548 This research was funded by Science Foundation Ireland (SFI), grant number 14/IA/2548. pmc1. Introduction A healthy gut microbiota which is compositionally and functionally diverse and stable is essential to support host health and growth . Contrarily, dysbiosis represents a state of imbalance in the composition and function of this microbial community, characterised by decreases in beneficial microorganisms and/or overgrowth of pathogens and/or a loss of overall diversity, with a subsequent negative impact on gastrointestinal health . Commercial weaning in pigs is a typical example of dysbiosis, whereby the transition from milk to solid feed, coupled with emotional, social and environmental stressors leads to gastrointestinal dysfunction characterised by dysbiosis that predisposes them to intestinal infection and disease . In a recent review, the potential of marine macroalgae or seaweeds were considered as natural dietary supplements with which to promote gastrointestinal health and subsequently growth in weaned pigs . Brown seaweeds are rich in nondigestible polysaccharides, minerals, polyphenols and vitamins . Wide-ranging biological activities have been attributed to seaweed components, particularly fucoidan and laminarin, including prebiotic and antibacterial potential. However, various factors influence the concentration, structure and biological activity of seaweed-derived polysaccharides, such as seaweed species, harvest season, environmental conditions, and extraction methodologies . Recently, a multivariate statistic technique, response surface methodology, has been utilised to improve the extraction efficiency by optimising the extraction conditions for a selected seaweed polysaccharide and/or bioactivity . In that study, a novel hydrothermal-assisted extraction (HAE) methodology with combinations of temperature, time and solvent to seaweed ratio optimised for the best concentration of laminarin and/or fucoidan and/or antioxidant activity was developed using the response surface methodology. Seaweed extracts of Ascophyllum nodosum produced using this HAE methodology exhibited enhanced antibacterial and prebiotic activity compared to the conventional extraction methods . The brown seaweed Laminaria spp. is a rich source of biologically active nondigestible polysaccharides. Previous in vitro investigation has associated this seaweed species with various biological activities including anti-inflammatory, immunomodulatory, antioxidant, antitumor and antihypertensive effects, several of which have also been observed in in vivo studies with pigs . Concerning its effect on the gastrointestinal microbiota, dietary supplementation of pigs with crude Laminaria spp. extracts consistently led to a reduction in the numbers of the Enterobacteriaceae family which include several animal and human pathogens such as Salmonella enterica subsp. enterica serotype Typhimurium and pathogenic Escherichia coli . Furthermore, an increase in Enterobacteriaceae family is considered to be an indication of dysbiosis and a risk factor for post-weaning diarrhoea in pigs . Dietary supplementation of pigs with crude Laminaria spp. extracts led to a more variable response in the intestinal lactobacilli and Bifidobacterium spp. populations, as both increases and decreases in their counts have been reported. Lactobacilli are dominant members of the gastrointestinal microbiota in pigs and have an important role in growth and health due to their contributions to nutrient bioavailability, inhibition of pathogen colonisation and immunomodulation . Bifidobacterium spp. are considered a beneficial bacterial population due to their probiotic status , but are present in low abundance in the gastrointestinal tract of pigs . The current study focused on the antibacterial and prebiotic potential of two polysaccharide-rich members of the Laminaria spp., L. digitata and L. hyperborea, with an average total carbohydrate content of 70.7% and 65.5% of dry weight, respectively, as described in previous reports . Batch fermentation and pure-culture growth assays were useful screening tools when assessing the direct effects of whole biomass seaweed samples and their extracts on key bacterial populations and species in the porcine gastrointestinal tract . Thus, the first objective of this study was to assess the influence of seaweed species and harvest season on the effect of whole biomass samples of L. digitata and L. hyperborea with respect to selected faecal bacterial populations in a batch fermentation assay inoculated with pig faeces. The second objective was to investigate whether the different extraction conditions of the HAE methodology led to L. digitata and L. hyperborea extracts with improved antibacterial and prebiotic activities using a panel of pure-culture growth assays. 2. Materials and Methods 2.1. Laminaria spp.: Whole Biomass Samples and Extracts The whole biomass samples (WB) of L. digitata (LD) and L. hyperborea (LH) were harvested in February (LDWB-F and LHWB-F) and November (LDWB-N and LHWB-N) by Quality Sea Veg Ltd., Co. (Donegal, Ireland). For each seaweed species, whole biomass samples were collected at a single time point and from the same collection site. The preparation (oven-dying, milling) and compositional analysis (dry matter, ash, protein, crude lipids, polysaccharide content, total phenols) of the dried whole seaweed biomass samples was performed as previously described . LDWB-F, LDWB-N, LHWB-F and LHWB-N were stored at room temperature until their evaluation in the batch fermentation assay. A HAE methodology with optimised extraction conditions (temperature, incubation time and volume of solvent) was used to produce the extracts of LDWB-F and LHWB-F, as described previously by Garcia-Vaquero et al. and presented in Table 1. The parameters for each extraction condition were optimised towards the concentration of fucoidan for E1, laminarin for E2, antioxidant activity for E3 and all the above for E4. The produced extracts of L. digitata (LDE1-4) and L. hyperborea (LHE1-4) were freeze-dried and their laminarin and fucoidan content was determined as previously described . All extracts were analysed on two independent occasions (two biological replicates) with three readings each time. LDE1-4 and LHE1-4 were stored at -20 degC until their evaluation in the pure-culture growth assays. 2.2. Batch Fermentation Assay The preparation of the faecal inoculum and the batch fermentation assay were carried out as described previously . Briefly, faeces from 29 healthy newly weaned crossbred pigs (Large White x Landrace) fed a cereal- and milk-based diet were pooled, aliquoted and stored at -20 degC. One day prior to the batch fermentation assay, the pooled faeces were diluted (1:5 w/v) in phosphate-buffered saline (Sigma-Aldrich, St. Louis, MO, USA) after oxygen removal using oxyrase (Sigma-Aldrich, St. Louis, MO, USA) to prepare the faecal inoculum (FI) that was stored at 4 degC anaerobically. The FI was added to the fermentation medium at a 1:10 v/v ratio (21 mL final volume). The inclusion levels of LDWB-F, LHWB-F, LDWB-N and LHWB-N in the FI/fermentation medium were 0 (control tubes), 1, 2.5 and 5 mg/mL. The batch fermentation was carried out under anaerobic conditions using oxyrase and CO2 flushing at 39 degC for 24 h with gentle stirring (100 rpm). Sampling (5 mL fermentation broth) was performed at 0 and 24 h in duplicate. After centrifuging at 12,000x g for 5 min, the resultant pellets were stored in -20 degC until further analysis. All experiments were repeated on three independent occasions (biological replicates n = 3). 2.3. Quantification of Bacterial Groups Using Quantitative Real Time Polymerase Chain Reaction (QPCR) DNA extraction: Bacterial DNA was extracted using QIAamp Fast DNA stool mini kit (Qiagen, West Sussex, UK) according to the manufacturer's instructions, and its quantity and quality was evaluated spectrophotometrically (Nanodrop, Thermo Fisher Scientific, Waltham, MA, USA). Bacterial primers: The primers targeting the 16S rRNA gene of selected bacterial groups (total bacteria, lactobacilli and Bifidobacterium spp.) or the rplP gene (Enterobacteriaceae) are provided in Table 2. Primer design software, Primer3 (accessed on 26 June 2018)) and Primer ExpressTM (Applied Biosystems, Foster City, CA, USA) were used for larger amplicons (>150 bp) and smaller amplicons (<125 bp), respectively. Primer specificity was verified using Primer Basic Local Alignment Search Tool (Primer-BLAST), accessed on 26 June 2018. Bacterial enumeration by QPCR: The quantification of the above-mentioned bacterial groups was carried out using QPCR with plasmid-based standard curves as described previously . Briefly, Competent E. coli was transformed with a pCR4-TOPOTM TA vector containing each fragment of the targeted 16S rRNA genes for total bacteria, lactobacilli and Bifidobacterium spp. or the rplP gene for Enterobacteriaceae and the resistance to ampicillin gene using a TOPOTM TA CloningTM Kit for Sequencing (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) and stored in cryoprotective beads (TS/71-MX, Protect Multi-purpose, Technical Service Consultants Ltd., Lancashire, UK). Transformed E. coli was recultured in 200 mL LB Broth Base (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) containing ampicillin (100 mg/mL) at 37 degC for 18 h at 150 rpm. Plasmids were extracted on a large scale using the GenEluteTM HP Plasmid Maxiprep kit, (Sigma-Aldrich, St. Louis, MO, USA), linearised using APA1 restriction enzyme (Promega, Madison, WI, USA) and purified using GenEluteTM PCR Clean-Up kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturers' instructions. Following quantification, the plasmid copy number/mL was determined using the URI Genomics & Sequencing Centre online tool accessed on 14 May 2019). For the QPCR, the final reaction volume (20 mL) included 3 mL template DNA, 1 mL of forward primer (10 mM), 1 mL of reverse primer (10 mM), 5 mL nuclease-free water and 10 mL of Fast SYBR(r) Green Master Mix (Applied Biosystems, Foster City, CA, USA) for the lactobacilli or GoTaq(r) qPCR Master Mix (Promega, Madison, WI, USA) for the remaining bacterial groups. All QPCR reactions were performed in duplicate on the ABI 7500 Fast PCR System (Applied Biosystems, Foster City, CA, USA) using the following cycling conditions: a denaturation step (95 degC/10 min), 40 cycles of 95 degC for 15 s and 60 degC for 1 min. Dissociation curve analysis and visualisation on a 2% agarose gel stained with ethidium bromide were used to confirm the production of single and specific PCR products. All PCR reactions used in this study exhibited 90-110% efficiency established by plotting the threshold cycles (Ct) derived from the 5-fold serial dilutions of each plasmid against their arbitrary quantities. Bacterial counts were determined using the standard curve derived from the mean Ct value and the log-transformed gene copy number of the respective plasmid and expressed as log-transformed gene copy number per gram of digesta (logGCN/g digesta). animals-13-00823-t002_Table 2 Table 2 List of forward and reverse primers used for the bacterial quantification by QPCR. Target Bacterial Group Forward Primer (5'-3') Reverse Primer (5'-3') Amplicon Length (bp) Tm (degC) References Total bacteria F: GTGCCAGCMGCCGCGGTAA R: GACTACCAGGGTATCTAAT 291 64.2 52.4 Lactobacilli F: AGCAGTAGGGAATCTTCCA R: CACCGCTACACATGGAG 341 54.5 55.2 Bifidobacterium spp. F: GCGTGCTTAACACATGCAAGTC R: CACCCGTTTCCAGGAGCTATT 125 60.3 59.8 Enterobacteriaceae F: ATGTTACAACCAAAGCGTACA R: TTACCYTGACGCTTAACTGC 185 54.0 56.3 bp, base pairs; Tm, melting temperature. 2.4. Bacterial Strains and Pure-Culture Growth Assays Pure-culture growth assays using a panel of commensal strains Lactiplantibacillus plantarum subsp. plantarum (formerly Lactobacillus plantarum, DSMZ 20174), Limosilactobacillus reuteri (formerly Lactobacillus reuteri, DSMZ 20016) and Bifidobacterium thermophilum (DSMZ 20210) and pathogens S. typhimurium PT12 and enterotoxigenic E. coli (ETEC) O149A+ selected for their beneficial roles and negative impacts on pig and human health, respectively, were carried out as described in our previous work . Briefly, 24 h cultures of all bacterial strains were prepared using standard procedures and diluted in 10% medium: 10% de Man, Rogosa and Sharpe broth (MRS, Oxoid Ltd., Hampshire, UK) for L. plantarum, L. reuteri and B. thermophilum cultures; or 10% Tryptone Soya Broth (TSB, Oxoid Ltd., Hampshire, UK) for S. typhimurium and ETEC cultures, to obtain an inoculum of 106-107 CFU (colony-forming unit)/mL (verified for each assay). Two-fold dilutions (2-0.125 mg/mL) of LDE1-4 and LHE1-4 were performed in 10% MRS and 10% TSB prior to each assay from a working concentration of 4 mg/mL. 100 mL of each extract and each dilution and 100 mL of inoculum were added to duplicate wells of 96-well microtiter plates (CELLSTAR, Greiner Bio-One, Kremsmunster, Austria). Control wells were also included (bacterial inoculum only). Assay sterility was assessed using blank wells (no bacterial inoculum) for each dilution of each extract. After gentle agitation to ensure thorough mixing, plates were incubated aerobically at 37 degC for 18 h, apart from B. thermophilum, which was incubated anaerobically. Afterwards, bacterial enumeration was carried out by 10-fold serial dilution (10-1-10-8), spread plating onto MRS agar (Oxoid Ltd., Hampshire, UK) for L. plantarum, L. reuteri and B. thermophilum, and Tryptone Soya Agar (Oxoid Ltd., Hampshire, UK) for ETEC and S. typhimurium, and incubation aerobically at 37 degC for 24 h or anaerobically at 37 degC for 48 h for B. thermophilum. The dilution resulting in 5-50 colonies was selected for the calculation of CFU/mL using the formula, CFU/mL = average colony number x 50 x dilution factor. The bacterial counts were logarithmically transformed (logCFU/mL) for the subsequent statistical analysis. All experiments were carried out with technical replicates on three independent occasions (3 biological replicates n). 2.5. Statistical Analysis All data were statistically analysed using Statistical Analysis Software (SAS) 9.4 (SAS Institute, Cary, NC, USA). Normality tests were initially carried out using PROC UNIVARIATE procedure for each data set. Batch fermentation assay: For each bacterial group and tested compound, the bacterial counts (n = 12, 3 flasks/each compound concentration) were analysed using PROC GLM procedure (Tukey's test). The statistical model included the fixed effects of seaweed species (L. digitata, L. hyperborea), the season of collection (February, November), the concentration of whole biomass (0, 1, 2.5 and 5 mg/mL) and assay replicates (3 biological replicates) and their associated two- and three-way interactions with the bacterial counts at 0 h as a covariate. Pure-culture growth assay: To control for the natural variability in bacterial growth in the pure-culture assays, bacterial counts were expressed as the difference between the counts of each bacterial strain for each extract concentration and their respective control (0 mg/mL). The resulting positive or negative values representing the difference in bacterial counts were analysed using PROC GLM procedure (Tukey's test). The statistical model assessed the effects of seaweed species (L. digitata, L. hyperborea), extraction conditions (E1-4) and concentration of extracts (0.125, 0.25, 0.5, 1 and 2 mg/mL) and their associated two- and three-way interactions. The biological replicate was the experimental unit. Probability values of < 0.05 denote statistical significance. Results are presented as least-square mean values +- standard error of the means (SEM). 3. Results 3.1. Proximate Composition of L. digitata and L. hyperborea, and Laminarin and Fucoidan Content of Their Extracts The proximate composition of the whole seaweed biomass samples LDWB-F, LHWB-F, LDWB-N and LHWB-N is presented in Table 3, as reported previously . The laminarin and fucoidan contents in the L. digitata (LDE1-4) and L. hyperborea (LHE1-4) extracts are presented in Table 4. 3.2. Effects of the Whole Biomass Samples of L. digitata and L. hyperborea on Selected Bacterial Populations The effects of the whole biomass samples of L. digitata and L. hyperborea collected in February (LDWB-F and LHWB-F) and November (LDWB-N and LHWB-N) were evaluated on selected faecal bacterial populations in a batch fermentation assay. The effects of species, season and concentration and their interactions are presented in Table 5 and Table 6 and are described below. The species x concentration interaction and the season x concentration interaction were only significant for Bifidobacterium spp. (p < 0.05) and, as a result, were excluded from the statistical analysis of the other bacterial groups. Enterobacteriaceae: There was a species x season interaction whereby LHWB-F led to lower Enterobacteriaceae counts compared to LHWB-N, while the opposite was true for L. digitata (p < 0.05, Table 5). There was also a concentration effect on Enterobacteriaceae counts, whereby the 5 mg/mL reduced Enterobacteriaceae counts compared to the control (7.74 logGCN/g digesta (5 mg/mL) vs. 7.99 logGCN/g digesta (0 mg/mL) +- 0.056, p < 0.05). Bifidobacterium spp.: There was a species x season interaction, whereby LHWB-N led to lower Bifidobacterium spp. counts compared to LHWB-F, while harvest season had no effect on Bifidobacterium spp. counts with regard to L. digitata (p > 0.05, Table 5). There was a species x concentration interaction, whereby the concentrations of 2.5 and 5 mg/mL of L. hyperborea led to lower Bifidobacterium spp. counts compared to the control and 1 mg/mL (2.73 logGCN/g digesta (2.5 mg/mL) and below the limits of detection (5 mg/mL) vs. 6.72 (0 mg/mL) and 6.60 (1 mg/mL) logGCN/g digesta +- 0.056, p < 0.05)), while this effect was not as potent with the corresponding L. digitata concentrations (5.95 logGCN/g digesta (5 mg/mL) vs. 6.46 (0 mg/mL), 6.55 (1 mg/mL) and 6.37 (2.5 mg/mL) logGCN/g digesta +- 0.056, p < 0.05)). There was a season x concentration interaction, whereby the 5 mg/mL of both February and November suppressed Bifidobacterium spp. counts, while at 2.5 mg/mL, there was a greater reduction in the counts in November relative to February (p < 0.05, Table 6). Total bacteria: There was a species effect and a concentration effect on total bacterial counts. L. hyperborea increased total bacteria compared to L. digitata (9.83 logGCN/g digesta (L. hyperborea) vs. 9.72 logGCN/g digesta (L. digitata) +- 0.034, p < 0.05). The 1 and 2.5 mg/mL gave higher counts compared to the control (9.87 (1 mg/mL) and 9.87 (2.5 mg/mL) logGCN/g digesta vs. 9.64 logGCN/g digesta (0 mg/ mL) +- 0.048, p < 0.05). Lactobacilli: There was a species effect and a concentration effect on lactobacilli counts. L. hyperborea increased lactobacilli counts compared to L. digitata (8.79 logGCN/g digesta (L. hyperborea) vs. 8.45 logGCN/g digesta (L. digitata) +- 0.030, p < 0.05). The concentrations of 1 and 2.5 mg/mL were associated with higher counts compared to the control (8.66 logGCN/g digesta (1 mg/mL) and 8.69 logGCN/g digesta (2.5 mg/mL) vs. 8.56 logGCN/g digesta (0 mg/mL) +- 0.035, p < 0.05). In summary, whole seaweed biomass samples from L. hyperborea and L. digitata collected in February had the least negative impact on Bifidobacterium spp. counts. Furthermore, LHWB-F reduced Enterobacteriaceae counts to the greatest degree, while LDWB-F had no effect. Both LHWB-F and LDWB-F were selected to generate the extracts evaluated in the next part of the screening process to determine whether the extraction methodology could improve the bioactivity of two whole seaweed biomass samples with varying effects. 3.3. Identifying L. digitata and L. hyperborea Extracts with the Highest Antibacterial and Prebiotic Potential in Pure Bacterial Cultures The HAE methodology with four different extraction conditions (E1-4) was employed for producing the extracts from the L. digitata (LD) and L. hyperborea (LH) samples, collected in February, to investigate whether the extraction method could improve their biological properties. LDE1-4 and LHE1-4 were evaluated for their antibacterial and prebiotic activities in pure-culture growth assays with selected beneficial (L. plantarum, L. reuteri, B. thermophilum) and pathogenic (ETEC, S. typhimurium) bacterial strains. Bacterial counts were expressed as the difference between the counts of each bacterial strain for each extract concentration and their respective control (0 mg/mL). The effects of species, extraction condition and concentration and their interactions are presented in Table 7 and Table 8 and are described below. The species x concentration interaction was significant only for S. typhimurium (p < 0.05) and was excluded from the statistical analysis of the other bacterial species. 3.3.1. The Effect of the Different Extraction Conditions on the Antibacterial and Prebiotic Effects of L. hyperborea and L. digitata Extracts ETEC and S. typhimurium: There was a species x extraction condition interaction, whereby LHE1 had more potent antibacterial activity than LHE2, LHE3 and LHE4, whereas the effect of the E1 extraction condition was not as potent with L. digitata, despite being significant (p < 0.05, Table 7). B. thermophilum: There was a species x extraction condition interaction, whereby LDE4 was more bifidogenic than LDE1, LDE2 and LDE3, whereas the effect of E4 extraction condition was not as evident with L. hyperborea, despite being significant compared with E1 and E2 (p < 0.05, Table 7). L. plantarum: There was a species x extraction condition interaction, whereby LHE4 was more stimulating on L. plantarum growth than LHE1, LHE2 and LHE3 (p < 0.05) and there was no effect of the extraction condition on L. digitata (p > 0.05, Table 7). L. reuteri: There was a species effect and an extraction condition effect on L. reuteri counts. LH extracts led to higher L. reuteri counts compared to LD extracts (0.24 logCFU/mL (LH extracts) vs. 0.16 logCFU/mL (LD extracts) +- 0.029, p < 0.05). The extraction conditions E1 and E2 increased L. reuteri counts compared to E3 (0.30 logCFU/mL (E1) and 0.27 logCFU/mL (E2) vs. 0.07 logCFU/mL (E3) +- 0.041, p < 0.05). 3.3.2. The Effect of Concentration on the Antibacterial and Prebiotic Activity of the Different Extraction Conditions ETEC and S. typhimurium: There was a concentration x extraction condition interaction, whereby 2 mg/mL was more potent than 1, 0.5, 0.25 and 0.125 mg/mL for the E1 extraction condition (p < 0.05), whereas the effect of concentration was not as evident with E2, E3 and E4 extraction conditions (p > 0.05, Table 8). There was also a species x concentration interaction for S. typhimurium (p < 0.05), whereby the 2 mg/mL LH extracts led to lower counts compared to the 2 mg/mL LD extracts (-1.56 logCFU/mL (LH extracts) vs. -0.66 logCFU/mL (LD extracts) +- 0.058, p < 0.05), however, species had no effect at any of the other concentrations. B. thermophilum: There was a concentration x extraction condition interaction, whereby all concentrations of E4 were more bifidogenic than the equivalent concentrations in E1, E2 and E3, where some of the concentrations had no effect, while some were antibacterial (p < 0.05, Table 8). L. plantarum: There was a concentration effect on L. plantarum counts. The concentration of 1 and 2 mg/mL of all extracts increased L. plantarum counts compared to the 0.125 mg/mL (0.15 (1 mg/mL) and 0.15 (2 mg/mL) logCFU/mL vs. 0.02 logCFU/mL (0.125 mg/mL) +- 0.032, p < 0.05). L. reuteri: There was a concentration effect on L. reuteri counts. The concentration of 2 mg/mL of all extracts increased L. reuteri counts compared to 0.125 mg/mL (0.34 logCFU/mL (2 mg/mL) vs. 0.10 logCFU/mL (0.125 mg/mL) +- 0.045, p < 0.05). 4. Discussion The influence of seaweed species and harvest season on the effects of the whole biomass samples of L. hyperborea and L. digitata on selected bacterial markers of the porcine faecal microbiota were evaluated in a batch fermentation assay. In this study, seaweed species was the predominant factor affecting the growth of Bifidobacterium spp., Enterobacteriaceae, lactobacilli and total bacteria. Bifidobacterium spp. counts were also influenced by the harvest season. The February-harvested L. hyperborea biomass sample, LHWB-F, led to the lowest Enterobacteriaceae counts among all tested samples whilst also having a reduced negative impact on Bifidobacterium spp. compared to the November-harvested counterpart, LHWB-N. Contrarily, the February-harvested L. digitata biomass sample, LDWB-F, was the least promising in terms of its antibacterial properties, having no major effects on the tested bacterial groups. These two whole biomass seaweed samples were used to produce LHE1-4 and LDE1-4 using four extraction conditions (E1-4) of the HAE methodology. The extracts were assessed in a panel of pure-culture growth assays with selected beneficial and pathogenic bacterial strains, to evaluate whether the optimised extraction conditions could enhance their antibacterial and prebiotic activities. Regardless of the seaweed species, the extraction condition E1 was predominantly associated with improved antibacterial activity against S. typhimurium, ETEC and to a lesser extent B. thermophilum, while the E4 extraction condition was predominantly associated with bifidogenic activity. Total bacteria, lactobacilli, Bifidobacterium spp. and Enterobacteriaceae were monitored in the batch fermentation assay as part of the evaluation of the whole biomass of L. digitata and L. hyperborea collected in February (LDWB-F and LHWB-F) and November (LDWB-N and LHWB-N). The whole biomass of L. hyperborea, LHWB-N and LHWB-F, reduced the Bifidobacterium spp. counts in a concentration-dependent manner with LHWB-F having a lesser impact. The whole biomass of L. digitata, LDWB-F and LDWB-N, also showed evidence of minor reductions in this bacterial population. In addition, LHWB-F and LDWB-N were associated with reduced Enterobacteriaceae counts. Reductions in Bifidobacterium spp. and Enterobacteriaceae counts have been previously observed in the faeces and colonic and caecal digesta in pigs supplemented with crude extracts of L. hyperborea, L. digitata or Laminaria spp. . In this study, whole biomass samples of L. hyperborea were associated with minor increases in lactobacilli and total bacterial counts compared to whole biomass samples of L. digitata. Thus, bacterial growth was predominantly influenced by the seaweed species rather than harvest season, which only had a significant effect on the Bifidobacterium spp. population. It is interesting to hypothesise what the bioactive components within the whole seaweed biomass samples could be based on their proximate composition analysis and the results of the batch fermentation assay. The whole biomass samples of L. hyperborea had higher total polysaccharide content compared to L. digitata for both months. The main polysaccharides present in L. digitata and L. hyperborea are laminarin, mannitol, alginate and cellulose, of which laminarin and mannitol have been reported to exhibit significant seasonal variation in their concentration . This, along with the increase in total glucans (laminarin and cellulose combined) observed in November for both seaweed species in the current study suggests that the variation in the total carbohydrate content was due to laminarin. Fucoidan was confirmed to be a relatively minor polysaccharide in the whole biomass samples of L. digitata and L. hyperborea as expected for the Laminaria spp. and increased in November in both seaweed species. Previous research has reported that laminarin reduced the Enterobacteriaceae counts in the caecum and increased lactobacilli counts in the faeces and colon of weaned pigs , while fucoidan from whole A. nodosum biomass samples was considered to be the bioactive reducing Bifidobacterium spp. and Enterobacteriaceae counts in a batch fermentation assay inoculated with faeces from weaned pigs . The reduction in Bifidobacterium spp. counts could additionally be attributed to inhibitory effects due to the wide-ranging components within the extracts, including phenols, alginate, cellulose and fucoidan, on the activity of bacterial carbohydrate-degrading enzymes . As whole seaweed biomass samples are inherently complex, it is not possible to attribute the observed effects on the faecal microbiota to specific bioactive components within the whole biomass samples of L. hyperborea and L. digitata with certainty. For the second part of the study, LHE1-4 and LDE1-4 were produced from LHWB-F and LDWB-F, respectively, using the HAE methodology with four extraction conditions (E1-4). Of these, LHWB-F was identified as the most promising antibacterial sample in the batch fermentation assay and was selected for further analysis. In parallel, LDWB-F was included to investigate whether the extraction protocol could improve its limited bioactivity, an effect that was demonstrated in a previous study . LHE1-4 and LDE1-4 were evaluated for their antibacterial and prebiotic potential in a panel of pure-culture growth assays. The pathogens S. typhimurium and ETEC were selected as representatives of the Enterobacteriaceae family. While S. typhimurium infection in pigs is mostly asymptomatic, it is associated with intestinal inflammation and compositional changes in the gastrointestinal microbiota that can have a negative impact on animal health and performance . Furthermore, pigs and their meat products can become a reservoir for S. typhimurium, which can impact on human health . ETEC infection in newly weaned pigs contributes to the development of post-weaning diarrhoea, an economically significant disease characterised by diarrhoea, dehydration, stunted growth and significant mortality . The effects of the L. hyperborea and L. digitata extracts on representative beneficial bacterial strains, B. thermophilum, L. plantarum and L. reuteri, were also evaluated. These bacterial species commonly colonise the porcine gastrointestinal tract and exert a range of beneficial roles such as inhibition of intestinal pathogens, immunomodulation, improved composition in the gastrointestinal microbiota and enhanced health and growth . In the pure-culture growth assays, the E1 and E4 extraction conditions were predominantly associated with antibacterial and bifidogenic activities, respectively. LHE1 was the most potent extract in reducing S. typhimurium and ETEC counts. LDE1 also inhibited the growth of both pathogenic strains to a lesser extent. Additionally, LHE1 and LDE1 reduced B. thermophilum counts, whereas both extracts were also associated with a slight increase in L. reuteri counts. Interestingly, LDE4 followed by LHE4 increased B. thermophilum counts in a concentration-dependent manner, with LHE4 additionally stimulating the growth of L. plantarum. Based on the above, the use of the E1 and E4 extraction conditions of the HAE methodology produced antibacterial and bifidogenic extracts with the potential to promote a healthy composition in the gastrointestinal microbiota of pigs. The laminarin and fucoidan contents of LHE1-4 and LDE1-4 were determined to establish the concentrations of these polysaccharides achieved by each combination of extraction conditions of the HAE methodology . LHE1-4 extracts had higher concentrations of laminarin and fucoidan compared to LDE1-4, an expected outcome based on the proximate composition of the respective whole seaweed biomass. Interestingly, both sets of extracts had higher fucoidan content (12.76-14.68% for LHE1-4 and 3.84-5.80% for LDE1-4) than laminarin content (4.94-7.59% for LHE1-4 and <=0.70% for LDE1-4). The presence of laminarin is reported to be at lower concentrations during the winter months in these seaweed species, in agreement with our observation . Apart from laminarin and fucoidan, alginate is a polysaccharide which is present in high and relative stable concentrations throughout the year in both L. hyperborea and L. digitata , and could also be a significant component of the LHE1-4 and LDE1-4. While the alginate content of the tested extracts was not determined in the current study, this assumption is supported by the findings of a recent study evaluating an L. hyperborea extract produced using the E2 extraction conditions of HAE methodology . Furthermore, the different extraction conditions (Table 1) could affect not only the content but also the structure of these seaweed polysaccharides in the produced extracts, and hence, their bioactivity. For instance, the use of HCl and increasing temperatures in the extraction protocol was previously associated with changes in the chemical composition (monosaccharide content, sulphation level) and lower molecular weight due to partial hydrolysis of fucoidan and partial depolymerisation of alginate . Although we did not determine the antibacterial and bifidogenic components of the L. hyperborea and L. digitata E1 and E4 extracts, we hypothesise that fucoidan was likely the main bioactive, with the variation in bioactivities attributed to structural alterations due to the different extraction conditions (Table 1). Regarding the antibacterial activity, this assumption is supported by the following three facts: (1) LHE1 had both higher fucoidan content and stronger antibacterial activity against S. typhimurium and ETEC compared to LDE1, suggesting a connection between this bioactivity and fucoidan; (2) The fucoidan-rich A. nodosum extracts produced using the same E1 extraction protocol also led to significant reductions in S. typhimurium and ETEC counts in our previous studies ; (3) Depolymerised fucoidans from Laminaria spp., Sargassum spp. and Undaria spp. were reported to have improved antibacterial activity against various pathogenic strains including E. coli and S. typhimurium compared to the parent polysaccharide . The antibacterial activity of LHE1 and LDE1 against B. thermophilum indicate that bioactives other than fucoidan are involved. The bifidogenic effect of LHE4 and LDE4 may also be attributed to the depolymerised fucoidan fraction due to the similar effects on Bifidobacterium spp. growth of the fucoidan-rich A. nodosum extract produced using the same E4 extraction protocol and depolymerised fucoidans of Laminaria spp. and Sargassum spp. in previous in vitro studies . Alginate oligosaccharides have also exhibited a bifidogenic effect in pure-culture growth assays . Therefore, depolymerised alginate may have contributed to the increases in B. thermophilum, particularly in the case of LDE4. The slight increases in L. plantarum and L. reuteri counts with LHE4 counts and E1-produced extracts, respectively, indicate limited ability of these bacterial strains to utilise seaweed polysaccharides, most likely laminarin and alginate oligosaccharides . Taken together, all of the above results suggest a strong indication that fucoidan is the candidate bioactive responsible for the antibacterial and bifidogenic activities, although other seaweed constituents such as alginate may also contribute to the latter in the E4-produced extracts, particularly LDE4. In future studies, investigation into the chemical composition of LHE1, LDE1, LHE4 and LDE4 would provide better insight into the prebiotic and antibacterial bioactive components of these extracts, which was not possible at the laboratory-scale production of the extracts during the development of the HAE methodology. 5. Conclusions The species of seaweed was the main determinant of the growth of Bifidobacterium spp., Enterobacteriaceae and lactobacilli when whole seaweed biomass samples were tested in a porcine batch fermentation assay. Whole biomass samples of L. hyperborea (LHWB-F) and L. digitata (LDWB-F) harvested in February were then selected as the most and least promising sources, respectively, for the generation of antibacterial extracts, based on their effects on the Enterobacteriaceae counts in the batch fermentation assay. E1- and E4-produced extracts from both seaweed species were associated with antibacterial and bifidogenic activities, respectively, indicating that the extraction conditions were a more important determinant of bioactivity than seaweed species. Of these extracts, LHE1 was the most potent extract against S. typhimurium and ETEC, whereas LDE4 stimulated the growth of B. thermophilum to the greatest extent. Further compositional characterisation of these extracts is required to facilitate the identification and purification of the bioactive components involved in the observed bioactivities. Nevertheless. these crude extracts, particularly LHE1, merit further exploration in terms of their ability to promote a more beneficial microbiota and, thus, overall health and growth in weaned pigs, as a means of minimising the costs associated with the purification of the responsible bioactives from these extracts. Acknowledgments The authors acknowledge the contribution of the laboratory staff in Lyons Research Farm and the School of Veterinary Medicine at University College Dublin and thank Aine O'Doherty from the Pathology Division, Central Veterinary Research laboratory, DAFM-Laboratories Backweston, Co., Kildare, Ireland for kindly providing us with the ETEC O149 bacterial strain. Author Contributions Conceptualization, T.S. and J.V.O.; methodology, B.V., M.J.M., M.G.-V., G.R., M.T.R. and T.S.; formal analysis, B.V.; investigation, B.V., C.K. and M.G.-V.; data curation, B.V.; writing--original draft preparation, B.V.; writing--review and editing, B.V., T.S., J.V.O., M.G.-V., G.R., M.T.R., M.J.M. and C.K.; supervision, T.S. and J.V.O.; funding acquisition, T.S. and J.V.O. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement All data supporting the reported results are available in this article. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. animals-13-00823-t001_Table 1 Table 1 Extraction conditions employed to obtain the different L. digitata and L. hyperborea extracts. Laminaria spp. Extract Extraction Method * Solvent * Extraction Conditions * Optimised for Targeted Bioactives LDE1 LHE1 HAE 0.1 M HCl 120 degC 62.1 min 30 mL solvent/g seaweed Fucoidan LDE2 LHE2 HAE 0.1 M HCl 99.3 degC 30 min 21.3 mL solvent/g seaweed Laminarin LDE3 LHE3 HAE 0.1 M HCl 120 degC 76.06 min 10 mL solvent/g seaweed Antioxidant activity LDE4 LHE4 HAE 0.1 M HCl 120 degC 80.9 min 12.02 mL solvent/g seaweed For laminarin, fucoidan and antioxidant activity * Extraction methods and conditions used to produce L. digitata and L. hyperborea extracts as described by Garcia-Vaquero et al. . LDE1-4, L. digitata extract 1-4; LHE1-4, L. hyperborea extract 1-4; HAE, hydrothermal-assisted extraction. animals-13-00823-t003_Table 3 Table 3 Proximate composition (dry matter, ash, protein, crude lipids, total glucans, fucose and phenols) of whole L. digitata and L. hyperborea biomass (data published in ). Proximate Composition * Whole L. digitata Biomass Whole L. hyperborea Biomass LDWB-F LDWB-N LHWB-F LHWB-N Dry matter (%) 91.39 +- 0.01 95.93 +- 0.01 90.83 +- 0.00 95.75 +- 0.02 Ash (% DW basis) 34.84 +- 0.08 21.82 +- 0.00 30.01 +- 0.03 18.91 +- 0.16 Protein (% DW basis) 11.12 +- 0.76 4.01 +- 0.04 9.98 +- 0.01 3.57 +- 0.00 Ether extract (% DW basis) 0.26 +- 0.05 1.12 +- 0.05 0.76 +- 0.07 0.69 +- 0.06 Total soluble sugars (% DW basis) 11.88 +- 0.13 20.39 +- 0.56 14.49 +- 0.11 26.69 +- 0.05 Total glucans (% DW basis) 1.51 +- 0.02 17.68 +- 0.09 6.40 +- 0.09 25.70 +- 0.10 Fucose (% DW basis) 0.77 +- 0.09 4.83 +- 0.15 2.66 +- 0.03 4.86 +- 0.05 Total phenolic content (% DW basis) 0.06 +- 0.00 0.05 +- 0.00 0.06 +- 0.00 0.10 +-0.00 * Results are expressed as mean values +- standard deviation of the mean. DW, Dry weight. animals-13-00823-t004_Table 4 Table 4 Laminarin and fucoidan content of L. digitata and L. hyperborea extracts. Laminaria spp. Extract Laminarin (mg Laminarin/100 mg Freeze-Dried Extract) * Fucoidan (mg Fucoidan/100 mg Freeze-Dried Extract) * LDE1 0.52 +- 0.06 4.46 +- 0.03 LDE2 0.70 +- 0.07 3.84 +- 0.06 LDE3 0.44 +- 0.03 5.80 +- 0.05 LDE4 0.67 +- 0.08 5.74 +- 0.05 LHE1 4.94 +- 0.20 14.41 +- 0.46 LHE2 7.59 +- 0.02 12.76 +- 0.34 LHE3 6.17 +- 0.03 14.53 +- 0.12 LHE4 6.19 +- 0.03 14.68 +- 0.37 * Results are expressed as mean values +- standard deviation of the mean. LD1-4, L. digitata extract 1-4; LH1-4, L. hyperborea extract 1-4. animals-13-00823-t005_Table 5 Table 5 Effects of seaweed species and harvest season on the selected bacterial populations of the faecal microbiota in the batch fermentation assay (least-square means and standard error of the means). Bacterial Group (logGCN/g Faeces) Whole Seaweed Biomass samples SEM p-Value LDWB-F * LDWB-N * LHWB-F * LHWB-N * Species Season Species x Season Total bacteria 9.75 9.69 9.76 9.89 0.057 0.032 NS 0.058 Lactobacilli 8.40 8.50 8.78 8.81 0.041 <0.001 NS NS Bifidobacterium spp. 6.31 c 6.35 c 4.69 b 3.31 a 0.044 <0.001 <0.001 <0.001 Enterobacteriaceae 8.13 b 7.79 a 7.68 a 7.98 b 0.068 0.034 NS <0.001 * Average of least-square mean values at 0, 1, 2.5 and 5 mg/mL. a,b,c Mean values within a row with different superscript letter were significantly different (p < 0.05). NS, not significant (p > 0.10); logGCN/g faeces, log-transformed gene copy number per gram of faeces; SEM, standard error of the means. animals-13-00823-t006_Table 6 Table 6 Effect of harvest season and seaweed concentration on the selected bacterial populations of the faecal microbiota in the batch fermentation assay (least-square means and standard error of the means). Bacterial Group (logGCN/g Faeces) Season SEM p-Value February November Concentration (mg/mL) Season Concentration Season x Concentration 0 * 1 * 2.5 * 5 * 0 ++ 1 ++ 2.5 ++ 5 ++ Total bacteria 9.55 9.86 9.90 9.72 9.73 9.87 9.84 9.71 0.073 NS 0.003 NS Lactobacilli 8.50 8.67 8.68 8.53 8.62 8.66 8.69 8.64 0.052 NS 0.041 NS Bifidobacterium spp. 6.53 d 6.59 d 5.95 c 2.93 a 6.64 d 6.55 d 3.15 b 2.98 a 0.056 <0.001 <0.001 <0.001 Enterobacteriaceae 7.97 8.03 7.90 7.72 8.01 7.90 7.89 7.75 0.085 NS 0.012 NS * Average of least-square mean values of LDWB-F and LHWB-F. ++ Average of least-square mean values of LDWB-N and LHWB-N. a,b,c,d Mean values within a row with different superscript letter were significantly different (p < 0.05). NS, not significant (p > 0.10); logGCN/g faeces, log-transformed gene copy number per gram of faeces; SEM, standard error of the means. animals-13-00823-t007_Table 7 Table 7 Effects of seaweed species and extraction conditions on the antibacterial and prebiotic potential of the L. digitata and L. hyperborea extracts in the pure-culture growth assays (least-square means and standard error of the means). Laminaria spp. Extract Bacterial Strain (logCFU Difference/mL) * L. plantarum ++ L. reuteri ++ B. thermophilum ++ ETEC ++ S. typhimurium ++ LDE1 0.16 c 0.28 -0.21 a -0.45 b -0.18 b LDE2 0.15 c 0.25 0.09 b -0.08 c -0.01 c LDE3 0.02 ab 0.03 0.14 bc 0.02 cd -0.07 bc LDE4 0.05 bc 0.08 0.89 e 0.04 cd -0.03 c LHE1 -0.07 a 0.32 -0.28 a -1.02 a -1.14 a LHE2 0.06 bc 0.28 -0.24 a 0.01 cd -0.17 b LHE3 0.07 bc 0.10 0.30 cd 0.26 e 0.07 c LHE4 0.28 d 0.26 0.43 d 0.09 d 0.00 c SEM 0.040 0.058 0.073 0.055 0.052 p-value Species NS 0.048 0.001 NS <0.001 Extraction condition 0.013 <0.001 <0.001 <0.001 <0.001 Species x Extraction condition <0.001 NS <0.001 <0.001 <0.001 * Bacterial counts are expressed as the difference between the counts of each bacterial strain for each LD/LH extract concentration and their respective control (0 mg/mL). Bacterial counts at 0 mg/mL were as follows: 7.92 +- 0.030 logCFU/mL for L. plantarum, 7.40 +- 0.040 logCFU/mL for L. reuteri, 6.49 +- 0.055 logCFU/mL for B. thermophilum, 8.46 +- 0.046 logCFU/mL for ETEC and 8.93 +- 0.086 logCFU/mL for S. typhimurium. ++ Average of least-square mean values at 0.125, 0.25, 0.5, 1 and 2 mg/mL for each extract. a,b,c,d,e Mean values within a column with different superscript letter were significantly different (p < 0.05). LD1-4, L. digitata extract 1-4; LH1-4, L. hyperborea extract 1-4.; NS, not significant (p > 0.10); CFU, colony-forming unit; SEM, standard error of the means. animals-13-00823-t008_Table 8 Table 8 Effects of extraction conditions and increasing concentrations of L. digitata and L. hyperborea extracts on the counts of selected beneficial and pathogenic bacterial strains in the pure-culture growth assays (least-square means and standard error of the means). Extraction Condition LD/LH Extract Concentration (mg/mL) Bacterial Strains (logCFU Difference/mL) * L. plantarum ++ L. reuteri ++ B. thermophilum ++ ETEC ++ S. typhimurium ++ E1 2 0.09 0.35 -0.96 a -4.63 a -3.12 a 1 0.16 0.27 -0.44 b 0.17 cdef -0.14 d 0.5 -0.03 0.34 -0.04 cde 0.27 def -0.07 de 0.25 0.04 0.36 0.15 efg 0.36 f 0.00 def 0.125 -0.02 0.18 0.05 defg 0.17 cdef 0.03 def E2 2 0.19 0.47 -0.33 bc -0.25 b -0.78 b 1 0.12 0.34 0.12 defg -0.04 bc 0.05 def 0.5 0.07 0.34 0.01 def 0.05 cd 0.09 ef 0.25 0.08 0.06 -0.19 bcd 0.00 c 0.07 def 0.125 0.05 0.14 0.03 defg 0.06 cde 0.11 ef E3 2 0.19 0.24 0.33 fgh 0.07 cde -0.48c 1 0.10 0.04 0.28 efgh 0.29 ef 0.10 ef 0.5 0.04 0.03 0.25 efgh 0.19 cdef 0.07 def 0.25 -0.05 -0.03 0.15 efg 0.12 cdef 0.17 f 0.125 -0.05 0.05 0.08 defg 0.03 cd 0.14 ef E4 2 0.15 0.32 0.87 j 0.13 cdef -0.05 def 1 0.20 0.24 0.93 j 0.10 cde 0.00 def 0.5 0.20 0.18 0.67 ij 0.04 cd -0.01 def 0.25 0.17 0.07 0.47 hi 0.03 c 0.03 def 0.125 0.09 0.04 0.35 gh 0.04 cd -0.04 def SEM 0.064 0.091 0.115 0.087 0.082 p-value Concentration 0.012 0.002 0.022 <0.001 <0.001 Extraction condition 0.013 <0.001 <0.001 <0.001 <0.001 Concentration x Extraction condition NS NS <0.001 <0.001 <0.001 * Bacterial counts are expressed as the difference between the counts of each bacterial strain for each LD/LH extract concentration and their respective control (0 mg/mL). Bacterial counts at 0 mg/mL were as follows: 7.92 +- 0.030 logCFU/mL for L. plantarum, 7.40 +- 0.040 logCFU/mL for L. reuteri, 6.49 +- 0.055 logCFU/mL for B. thermophilum, 8.46 +- 0.046 logCFU/mL for ETEC and 8.93 +- 0.086 logCFU/mL for S. typhimurium. ++ Average of least-square mean values of LD and LH extracts for each extraction condition. a,b,c,d,e,f,g,h,i,j Mean values within a column with different superscript letter were significantly different (p < 0.05). LD, L. digitata; LH, L. hyperborea; NS, not significant (p > 0.10); CFU, colony-forming unit; SEM, standard error of the means. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000093

This study assessed the effects of the kernel extracts of apricot (AKE; Prunus armeniaca) and peach (PKE; Prunus persica), and their mixture (Mix) on growth efficiency, feed utilization, cecum activity, and health status, of growing rabbits. Weaned male New Zealand White rabbits at six weeks old [n = 84, 736 +- 24 SE g body weight (BW)] were randomly allotted to four dietary groups. The first group received no feed additives (control), the second and third groups received 0.3 mL/kg BW of AKE and PKE, respectively, and the fourth group received a mixture of AKE and PKE (1:1) at 0.3 mL/kg BW (Mix). Results indicated that 2(3h)-Furanone, 5-Heptyldihydro was found in abundance in both extracts, while 1,1-Dimethyl-2 Phenylethy L Butyrate and 1,3-Dioxolane, and 4-Methyl-2-Phenyl- were the most components detected in AKE and Cyclohexanol and 10-Methylundecan-4-olide were found in abundance in PKE. All the experimental extracts enhanced (p < 0.05) the growth performance, cecal fermentation parameters, and cecal L. acidiophilus and L. cellobiosus count, while PKE and the mixture treatments presented the highest (p = 0.001) total weight gain and average weight gain without affecting the feed intake. Rabbits that received the mix treatment had the highest (p < 0.05) nutrient digestibility and nitrogen retained, and the lowest (p = 0.001) cecal ammonia concentration. All the experimental extracts enhanced (p < 0.05) the blood antioxidant indicators (including total antioxidant capacity, catalase, and superoxide dismutase concentrations), and immune response of growing rabbits. In general, fruit kernel extracts are rich sources of bioactive substances that can be used as promising feed additives to promote the growth and health status of weaned rabbits. antioxidant status cecal fermentation cecal microbial counts fruit kernels natural feed additives phytochemicals weaned rabbits This research received no external funding. pmc1. Introduction Apricot (Prunus armeniaca) and Peach (Prunus persica) are the most important fruits grown and processed in Egypt. Apricot is primarily grown in Mediterranean nations, while it is also grown in Russia and the United States . In addition, peaches are the primary species for a variety of cultivars that are widely grown around the world . Thus, there are sustainable abundance amounts of both apricot and peach kernels worldwide, which are mostly neglected; however, these kernels are known to contain several biologically active chemicals (e.g., tocopherols, phenolic compounds, -carotene, vitamin E, oleic acid, linoleic acid, and different phytosterols) that possess antioxidant activities and antimicrobial against specific species, while enhancing the growth of fiber-degrading microbes . The natural feed additives derived from fruit kernels or their extracts are being included in animal nutrition on a larger scale due to their outstanding spectrum of these phytochemicals. Natic et al. recorded that kernel fruit extracts, such as apricot, cherry, nectarine, peach, and plum, are frequently utilized as supplements in the diets of both animals and humans. It has been established that most phytochemicals that possess antibacterial and antioxidant properties can promote the growth performance of growing rabbits . The cecal microbial ecosystem and digestive processes of growing rabbits are also altered by plant phytochemicals, which help in improving nutrient absorption while enhancing dietary nutrient utilization . Additionally, plant phytochemicals support the animal's immune system, and antioxidant state while protecting the feed lipids from oxidative damage . These findings provided a hypothesis that kernel extracts of apricot, peach, or their mixture can be used as efficient dietary feed additives for growing rabbits, in the context of high possible incidences of health problems and growth depression which can occur during the post-weaning period and limit the overall animal profitability . The objective of the current study was to investigate the effects of AKE, PKE, and their combination on growth performance, nutritional digestibility, cecal microbiota, antioxidant activity, and immunological status of growing rabbits. 2. Materials and Methods 2.1. Fruit Kernel Extracts, and Characterization The fruit kernel extracts of apricot (AKE) and peach (PKE) were provided commercially from the El Marwa Factory for natural seed extract, El-Nobaria, Behera, Egypt. The phytochemicals components of each extract were identified as described by Soltan et al. using a Thermo Scientific TRACE-1300 series gas chromatography/ mass spectrometry (GC/Mass; Thermo Fisher Scientific Inc., Waltham, MA, USA). The GC/Mass is equipped with a fused silica DB-5 capillary column (30 m x 0.32 mm, 0.25 mm film thickness) and coupled to a Triple Quadrupole Mass (TSQ 8000 Evo; Thermo Sci.). The Mass spectra were scanned at the range of 40-700 amu, and the scan time was 5 scans/s. The chemical components of the experimental AKE and PKE were detected by the combination of retention index data with mass spectra data using the Mainlib library. 2.2. Animal Management and Dietary Treatments Eighty-four weaned New Zealand White male rabbits (Oryctolagus cuniculus) at six weeks of age with an initial BW of 736 +- 23.9 SE g were used in this study. All animals were housed in galvanized wire battery cages with standard dimensions (50 x 45 x 40 cm3) located in a well-ventilated building. All cages were fitted with manual feeders and automatic systems of nipple drinkers to provide clean normal fresh water continuously. The animals were kept under similar hygienic, and environmental conditions. The relative humidity and ambient temperature during the experimental period were 60 to 65%, and 22 to 25 degC, respectively. The first group assisted as the control and received 0.3 mL/kg BW of distilled water without any feed additives, this was to control the rabbits' stress and fear when handled for the treatments . The second and third groups received 0.3 mL/kg BW of AKE and PKE, respectively, and the fourth group received 0.3 mL/kg BW of a mixture of AKE and PKE at a 1:1 ratio (Mix). The experimental extracts were orally administered daily for 56 days (8 weeks). The basal diet was formulated and pelleted to meet the nutrient requirements of growing rabbits . The rations were offered to rabbits ad libitum and free access to clean water was provided to the rabbits through stainless steel nipples that were fixed in each cage. The ingredients and chemical composition of the pelleted rations are presented in Table 1. The total protein (CP), crude fiber (CF), and ether extract (EE) recorded values of 17.2, 13.1, and 3.45%, respectively. 2.3. Growth Performance and Dry Matter Intake During the growth trial, all rabbits were weighed individually once a week before the morning feeding to determine the average daily gain (ADG). Feed refusals were collected and weighed daily to determine the dry matter intake (DMI). The feed conversion ratio (FCR) was calculated by dividing the DMI by the ADG. 2.4. Nutrient Digestibility and Nitrogen Balance At 87 days of animal age, we started the complete daily collection of feed refusals, urine, and feces for 5 consecutive days as a collection period. The excreted feces and urine of each cage were collected daily in bags before offering the morning meal. Representative samples of the daily amount of feces were stored at -20 degC and later pooled for each cage. At the end of the digestibility trial, the fecal samples were dried at 70 degC for 48 h in a forced air oven, ground to pass through a 1 mm screen on a Wiley mill grinder, and stored at -20 degC for further chemical analyses. Samples of the offered and residual diets and feces were analyzed chemically for dry matter (DM), organic matter (OM), CP = 6.25 x N, EE, and CF according to AOAC . For the nitrogen balance determination, urine samples were acidified with 10 mL sulfuric acid (H2SO4; 1M), frozen at -20 degC, and later pooled for each cage. By the end of the collection period, urine samples were thawed, centrifuged (15,000x g for 20 min) at room temperature, and analyzed for total nitrogen using the micro-Kjeldahl method . The contents of the non-fiber carbohydrate (NFC) were calculated as NFC (%) = 100 - (CP + Ash + EE + CF). 2.5. Blood Plasma Antioxidant and Immunological Indicators Ten animals were selected randomly from each treatment at age of 10 weeks and injected intramuscularly with 0.5 mL of a suspension of the sheep red blood cell counts (SRBCs; 10% concentration), as a T-dependent antigen . Plasma levels of hemagglutination antibodies against SRBCs were measured at 11, 12, and 13 weeks of animal age using a hemagglutination test, and the plasma antibody titers were calculated as log2 values . The same ten animals were subjected to blood plasma collections (after 12 h of fasting) for measuring the antioxidant biomarkers. At day 92 of the animal age (the last experimental day), the blood samples were harvested from the rabbit marginal ear vein using heparinized tubes (BD Vacutainer(r) Tubes, Jersey, NJ, USA). The blood samples were centrifuged at 2000x g for 20 min at 5 degC to obtain plasma. The collected plasma was stored at -20 degC for further antioxidant analyses. Malondialdehyde (MDA), catalase activity, superoxide dismutase activity, and total antioxidant capacity (TAC) were measured colorimetrically using commercial kits produced by Bio Diagnostic Inc., Dokki- Giza, Egypt. 2.6. Cecal Measurements, Fermentation Characteristics, and Microbial Count On the last day of the experiment (day 92 of age), the ten animals (that were subjected to blood sampling) were slaughtered as described by Lopez et al. . The cecum of each rabbit was removed and weighted (fully and empty). The cecal length was measured with a ruler. The pH of the cecal contents was measured immediately using a digital pH meter (Model 20, Digital pH meter for Orion Research, Alaska, Hawaii, Canada). The cecal contents were strained through three layers of cheesecloth and prepared for subsequent ammonia and short-chain fatty acids (SCFAs) determinations. Ammonia concentrations were measured by steam distillation (UDK 139- Semi-Automatic Kjeldahl Distillation Unit, VELP Scientific Inc., Usmate, Italy) . The SCFAs were determined as described by Palmiquist and Conrad using gas chromatography (GC, model 5890, Hewlett Packard, Little Falls, MI, USA) equipped with a capillary column (30 m length x 0.25 mm inner diameter, 1 m phase thickness, Supelco Nukol; Sigma-Aldrich, Mississauga, Canada), and flame ionization detector (FID). 2.7. Statistical Analysis All the obtained data were analyzed statistically using one-way analysis of variance (ANOVA) with SPSS 11.0 statistical software. Differences among means were determined using the Duncan test. Data were analyzed using the following model: Yij = U + Ai + Eij where U is the overall mean, Ai is the effect of dietary treatments; and Eij is the random error. For the growth performance and the nutrient digestibility variables, the statistical repetitions were 7 cages per dietary treatment including 3 rabbits each, as the rabbits were housed in groups (7 cages per treatment, 3 rabbits per cage). For the blood plasma and cecal variables and the digestibility trial, the statistical repetitions were the individual rabbit. Statistical significance was accepted at p <= 0.05. 3. Results 3.1. Phytochemicals of the Experimental Feed Additives The phytochemicals compounds in AKE and PKE determined by GC/Mass analysis are presented in Table 2 and Table 3, respectively. The experimental AKE and PKE contained 15 and 26 phytochemicals with different concentrations and functional groups. Both extracts contained 2(3h)-Furanone, 5-Heptyldihydro in abundance; however, the AKE contained almost double the concentration of this component higher than the PKE. 1,1-Dimethyl-2 Phenylethy L Butyrate, 1,3-Dioxolane, and 4-Methyl-2-Phenyl- were the most components detected in AKE. For the PKE extract, Cyclohexanol, 10-Methylundecan-4-olide, Propanoic Acid, Phenylmethyl Ester, and Butanoic acid, 3-methyl-, butyl ester were the highest detected components. 3.2. Animal Growth Performance Effects of AKE, PKE, or their mixture on rabbit growth performance, feed intake, and feed conversion ratio are presented in Table 4. All the experimental treatments increased (p < 0.05) the total final BW, total weight gain, and ADG, and enhanced the feed conversion ratio (FCR) compared to the control group; however, both PEK and the Mix groups had similar outstanding experimental growth performance indicators higher (p < 0.05) than the AKE and control groups. 3.3. Nutrient Digestibility and Nitrogen Balance Results of the apparent nutrient digestibility and nitrogen balance of rabbits rated with AKE, PKE, and their mixture compared to control rabbits are shown in Table 5. The mixture treatment presented the highest (p < 0.05) DM, OM, CP, EE, and NFC digestibilities compared to other treatments. No differences were detected among the experimental groups in fiber digestibility. 3.4. Blood Plasma Antioxidant and Immunological Indicators Results presented in Table 6 showed the effects of the experimental extracts of the fruit kernels on blood plasma antioxidant indicators and SRBCs of growing rabbits. All rabbits treated with the experimental extracts had reductions (p < 0.001) in blood plasma concentrations of MDA, and increases in TAC (p < 0.001), superoxide dismutase (p = 0.017), and catalase (p = 0.019) levels compared to rabbits fed the control diet. All rabbits treated with the fruits kernel extracts had similar higher increases (p < 0.05) in antibody titers against SRBCs compared to the control at weeks 11, 12, and 13 weeks of age. 3.5. Cecal Measurements, Fermentation Characteristics, and Microbial Count Results in Table 7 showed that all the experimental fruit kernel extracts positively affected (p < 0.05) the cecal length, and empty and free weights compared with the control group. Cecal pH was reduced (p = 0.0009) by all the experimental extracts, while the Mix treatment resulted in the lowest (p = 0.01) cecal ammonia concentrations among the experimental treatments. Significant increases (p < 0.05) in concentrations of cecal total and individual SCFAs were observed for all rabbits treated with the fruit kernel extracts compared to those fed the control group. Cecal total bacteria, coliform, and anaerobic counts were diminished (p < 0.05), while L. acidiophilus and L. cellobiosus were enhanced (p < 0.05) with the experimental extracts compared to the control. 4. Discussion The experimental extracts of AKE and PKE were chosen due to their impressive range of biologically active phytochemicals . In the current study, most of the phytochemicals identified either in AKE or PKE were found to have various health benefits and beneficial applications such as antioxidant, anti-microbial, and anti-inflammatory activities . The most obvious commonality between the AKE and PKE extracts is the substantial quantity of a fatty ester named 2(3h)-Furanone, 5-Heptyldihydro. It is noticed that the AKE contained almost double the concentration of this component higher than the PKE. Moreover, PKE contained a wider range of phytochemical components than AKE and considerable concentrations of Cyclohexanol, 10-Methylundecan-4-olide, Propanoic Acid, Phenylmethyl Ester, and Butanoic acid, 3-methyl-, butyl ester were identified, while other bioactive components were identified in abundance in AKE different from PKE. These findings indicated that AKE and PKE may have different bioactive activities from each other and, thus, their mixture might have unique biological effects that differ from the individual extract. Soltan et al. reported that the binary and tertiary combinations of different plant secondary metabolites from different plant sources directly enhance the bioactivities of these mixtures through their antioxidant and antimicrobial effects. The similar outstanding indicators of growth performance (final BW, total weight gain, and ADG), and enhanced FCR found for PEK and the Mix groups compared to other groups might be due to providing certain bioactive compounds in these groups that improve nutrient digestion and absorption. The phytochemicals that exist in the fruit kernels may enhance the gut-intestinal microbial ecosystem, thus helping to improve digestive efficiency and nutrient absorption in rabbits . Therefore, the enhancements in all nutrient digestibility by all the experimental fruit kernel extracts could be a result of the combination of antibacterial phytochemicals with energy-yielding nutrients (fatty acid esters) found in these extracts. Such combinations of these components may support bacterial symbiosis and the growth of cecal fermentative species, resulting in increased nutrient digestion . A similar relationship between nutrient digestibility and rabbit health was described by Bovera et al. . Despite the restricted number of experimental rabbits (this was the major limitation in the current study), the positive effects of these extracts on nutrient apparent digestibility would be accompanied by enhancements in animal growth performance and health status. Some plant phytochemicals, even if existing at very low levels in a mixture, can interact with each other acting as indifferent, antagonistic, additive, or synergistic agents . It seems that the Mix treatment provided the animals with synergetic bioactive compounds that improve nutrient digestion and nutrient absorption since the Mix group had the highest nutrient digestibility values among all the experimental groups. This suggestion can be proved by the superiority of the Mix group in the body N retention compared to other treatments. Moreover, these results indicate efficient protein digestion, absorption in the total alimentary tract, and high utilization of dietary protein for the growth of rabbits that were treated with Mix extract, as all animals received the same diet with similar feed intakes. Decreasing cecal ammonia concentration in rabbits treated with Mix extract may partly confirm the efficiency of protein utilization. Based on the existing study results, it can be suggested that the Mix extract may enhance ammonia utilization; however, the specific mechanisms by which the Mix treatment reduced cecal ammonia are not clear and, thus, more studies are needed to assess these mechanisms. Generally, ammonia constitutes the largest proportion and causes the greatest harm among all kinds of gases in livestock industries . In this context, the extracts of fruit kernels may provide an advantage for further developing the ammonia emissions reduction approach from rabbits. Recently, there is growing attention on using alternative natural antioxidants from plant extracts in livestock production . In the current study, the treatments of all fruit extracts resulted in notable enhancements of the blood antioxidant indicators. Fruit kernels have been already employed as a source of plant antioxidants, including apricot, peach, sweet cherry, nectarine, and plum oils . The oral administration of AKE and PKE or Mix resulted in the current findings' considerable reductions in MDA levels and increases in blood serum antioxidant properties (TAC, superoxide dismutase, and catalase). This may be a result of the phytochemicals naturally found in the kernel extracts. Earlier studies showed a notable decrease in MDA level and an increase in the antioxidant defense level following the administration of the AKE and PKE extracts containing other components than what we detected in the current study. For example, Karadimou et al. reported that PKE contains significant amounts of phenolic compounds and unsaturated fatty acids that are used as active natural antioxidant dietary supplements. Others found that the main active components of PKE which play an important role in the regulation of a variety of physical and biological functions were oleic and linoleic acid which aid in preventing the negative effects of free radicals on the body . Kalia et al. related the antioxidant activity of AKE to b-carotene, catechins, neochlorogenic acid, caffeic acid, and flavonoids. These differences might due to the differences in the method of the determination of the phytochemicals rather than the differences in the extraction methods and the plant varieties . Regardless of these differences in phytochemicals responsible for the antioxidant activity of the experimental extracts, the increases in superoxide dismutase can play a key role in protecting body cells from free radicals and oxidative damage . Similarly, the endogenous defense mechanisms, including increases in the TAC, and catalase activities may reduce oxidative damage . Generally, such enhancements of antioxidant status may partly explain the improvements in the growth performance of rabbits treated with the experimental fruit kernels. The oral administration of AKE, PKE, and Mix extracts increased the antibody titers of growing rabbits against SRBCs. These findings imply that these extracts may enhance the immunological responses of rabbits. There is a wealth of literature reporting a conjugation of enhancements of immunity response with improvements in blood antioxidant status. Adding coconut oil and watercress oil to the diet of growing rabbits improved their immunity synchronized with a decrease in pathogenic cecal bacteria and an improvement in blood antioxidant status . Bendich reported that the presence of carotenoids in AKE can protect phagocytic cells from antioxidative deficiencies, enhance T and B lymphocyte proliferative responses, and alleviate the production of necessary interleukins, resulting in improvement in antibody titer against SRBCs. Similarly, Abdelnour et al. observed that supplementing rabbits with thyme essential oil improved blood antioxidant status while boosting the formation of immunological bodies such as IgM and IgG. The changes in cecal ammonia concentration and SCFAs profile by the experimental extracts indicated that these extracts can modify the cecal microbial community. The enhancement of total SCFAs as well as acetate, butyrate, and propionate concentrations by the treatments of fruit kernel extracts could partly support their superiority in activating specific populations of cecal microorganisms that are involved in fiber digestion, as SCFAs are the main endproducts of the fiber cecal microbial activity, and enhancement of gut cell development . In particular, the increase of butyric acid which known for its positive effect on enterocytes and, in general, on intestinal health . Such enhancements of SCFAs may partly explain the improvement in cecal weight and length combined with the pH decrease observed for all kernel extracts treatments compared to the control . It is worth noting that decreasing the SCFAs is the typical mode of action of antibiotic growth promoters such as monensin . The active components naturally found in the experimental extracts might also play a role in the characteristics of the cecal fermentation profile since the major phytochemicals identified either in AKE or PKE were found to have antimicrobial activities . The reductions in the total bacterial coliform and anaerobic cecal microbial counts may confirm the antimicrobial activity of these extracts, however, the increases in the total counts of L. acidiophilus and L. cellobiosus may have been a result of a combined effect of low cecal pH and kernel extracts antimicrobial activity against specific microbes that providing suitable conditions for enhancing the growth of other acidic tolerant microorganisms like L. acidiophilus and L. cellobiosus . Therefore, it can suggest that these extracts somehow exert similar selectivity against the growth of specific cecal microbial strains while promoting certain other cecal microorganisms. However, these extracts presented nutritional advantages for the growing rabbits, but further. These results indicated the advantages of using these extracts for growing rabbits; however, oral administration may not be the optimal practical method for using such extracts on a commercial scale. This is another limitation of the current study, that has to be considered in our further studies. 5. Conclusions The results of the current study revealed various-faceted biological activities of the phytochemical compounds that naturally exist in the experimental kernel extracts. Oral administration of AKE, PKE, and their mixture for growing rabbits resulted in positive effects on their growth performance, digestibility, nitrogen balance, cecal fermentation, and antioxidant and immune status. The animals treated with the mixture treatment of AKE and PKE had superiority in nutrient digestion and N retention compared to other treatments, thus, synergetic bioactive effects might exist among the compounds of AKE and PKE. Prospective experiments are needed to understand the mode of action of the biologically active chemical compounds in the mixture of these extracts. Acknowledgments The authors are grateful to the members of the laboratory of Animal Nutrition of the Livestock Research Department, Arid Lands Cultivation Research Institute, City of Scientific Research and Technological Applications. Author Contributions Conceptualization, M.B., and Y.A.S.; methodology, M.B.; software, M.B.; validation, M.B., A.S.M. and Y.A.S.; formal analysis, M.B.; investigation, M.B., A.S.M.; resources, A.S.M. and M.B.; data curation, M.B.; writing--original draft preparation, M.B.; writing--review and editing, Y.A.S.; visualization, M.B., A.S.M. and Y.A.S. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by the Institutional Review Board (or Ethics Committee) of the Pharmaceutical and Fermentation Industries Development Center of the City of Scientific Research and Technological Applications (SRTA-City)--(protocol code IACUC76-1A-0123). Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. animals-13-00868-t001_Table 1 Table 1 Composition (%) and chemical analysis of the basal diet. Item Basal Diet Ingredients (%) Berseem clover hay 28.0 Barley 17.3 Corn yellow 17.9 Wheat bran 12.0 Soybean meal 20.0 Molasses 3.00 Di-Ca-Ph 1.00 Sodium chloride 0.30 Vitamin premix 1 0.30 Lysine 0.10 Methionine 0.10 Chemical composition Organic matter(%) 89.7 Crude protein (%) 17.2 Crude fibre (%) 13.1 Neutral detergent fibre (%) 37.5 Acid detergent fiber (%) 21.4 Ether extract (%) 3.45 Non-fiber carbohydrate (%) 55.9 Hemicellulose (%) 16.2 Calcium (%) * 0.83 Available phosphorus (%) * 0.31 1 Contains (unit/ kg diet): Vitamin A, 12,000 IU; vitamin D3, 2000 IU; vitamin E, 11 IU; vitamin K, 2 mg; d-Ca pantothenate, 10 mg; folic acid, 1 mg; choline chloride, 250 mg; manganous oxide, 60 mg; ferrous sulfate, 30 mg; zinc oxide, 50; copper sulfate, 10 mg; ethylenediamine dihydrocodeine, 1 mg; cobalt sulfate heptahydrate, 0.1 mg sodium selenite.* Calculated. animals-13-00868-t002_Table 2 Table 2 Chemical constituents identified by gas chromatography/mass spectrometry analysis in apricot extract (AKE). Peaks Compounds RT (min) Peak Area (%) Molecular Formula Structure 1 D-Limonene 5.39 1.94 C10H16 2 Propanoic acid, 2 methyl-, 3-methylbutyl ester 5.93 4.44 C9H18O2 3 1,6-Octadien-3-Ol, 3,7-Dimethyl- 7.00 3.74 C10H18O 4 Butanoic acid, 3-methyl-, 3-methylbutyl ester 7.09 7.11 C10H20O2 5 Acetic acid, phenylmethyl ester 8.53 6.41 C9H10O2 6 1,6-Octadien-3-Ol, 3,7-Dimethyl-, Acetate 10.72 1.07 C12H20O2 7 1,3-Dioxolane, 4-Methyl-2-Phenyl 11.20 10.76 C10H12O2 8 Butanoic Acid, Phenylmethyl Ester 13.21 2.21 C11H14O2 9 Butanoic acid, 2-methyl-, phenylmethyl ester 14.21 1.94 C12H16O2 10 2-Buten-1-one, 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)- 14.76 0.91 C13H20O 11 2(3h)-Furanone, 5-Hexyldihydro- 16.21 10.32 C10H18O2 12 3-Buten-2-one, 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)- 16.50 0.66 C13H20O 13 1,1-Dimethyl-2 Phenylethy L Butyrate # 16.70 14.36 C14H20O2 14 2-Phenoxyethyl isobutyrate 17.40 9.10 C12H16O3 15 2(3h)-Furanone, 5-Heptyldihydro- 18.75 25.01 C11H20O2 RT = retention time. animals-13-00868-t003_Table 3 Table 3 Chemical constituents identified by gas chromatography and mass spectrometry analysis in peach kernel extract (PKE). Peaks Compounds RT (min) Peak Area (%) Molecular Formula Structure 1 3-Hexen-1-ol, acetate 4.58 1.45 C8H14O2 2 Acetic acid, hexyl ester 4.99 1.68 C8H16O2 3 Morpholine, 4-(1-butenyl)- 5.12 0.62 C8H15NO 4 1-methyl-4-(1-methylethenyl)-, (S)- D-Limonene 5.39 0.54 C10H16 5 Butanoic acid, 3-methyl-, butyl ester 5.72 7.87 C9H18O2 6 Propanoic acid, 2-methyl-, 3-methylbutyl ester 5.93 0.28 C9H18O2 7 1,6-Octadien-3-Ol, 3,7-Dimethyl- 7.00 1.38 C10H18O 8 Isoamylisovalerate 7.09 1.91 C10H20O2 9 Acetic Acid, Phenylmethyl Ester 8.57 1.99 C9H10O2 10 Acetic acid, decyl ester 9.74 0.6 C12H24O2 11 Propanoic Acid, Phenylmethyl Ester 10.95 8.64 C10H12O2 12 1,3-Dioxolane, 4-methyl-2-phenyl- 11.21 7.94 C10H12O2 13 Cyclohexanol, 5-methyl-2-(1-methylethyl)-, acetate 11.77 14.10 C12H22O2 14 Butanoic Acid, Phenylmethyl Ester 13.21 0.83 C11H14O2 15 Pentanoic acid, phenylmethyl ester 14.21 0.63 C12H16O2 16 2-Buten-1-one, 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-, (E)- 14.29 0.83 C13H20O 17 2-Cyclopenten-1-One, 3-Methyl-2-(2-Pentenyl)-,(Z)- 14.40 2.71 C11H16O 18 (Z)-1-(2,6,6-Trimethyl-1-Cyclo Hexen-1-Yl)-2 Buten-1-One 14.76 1.49 C13H20O 19 2(3H)-Furanone, 5 hexyldihydro- 16.20 7.63 C10H18O2 20 1,1-Dimethyl-2 Phenylethy L Butyrate 16.69 4.03 C14H20O2 21 2-Phenoxyethyl isobutyrate 17.40 2.40 C12H16O3 22 2(3H)-Furanone, 5-Heptyldihydro- 18.71 14.99 C11H20O2 23 10-Methylundecan-4-olide 21.09 9.24 C12H22O2 24 e-Dodecalactone 21.70 1.47 C12H22O2 25 9-Octadecenoic acid, methyl ester, (E)- 29.57 0.97 C19H36O2 26 9,19-Cyclolanost-24-en-3-ol, (3a) 42.80 3.24 C30H50O RT = retention time. animals-13-00868-t004_Table 4 Table 4 Growth performance, feed intake and feed conversion ratio of New Zealand White growing rabbits (36-92 days of age) treated with apricot kernel extract (AKE), peach kernel extract (PKE), or their mixture (1:1). Items Treatments SEM p-Value Kernel Extract Control AKE PKE Mixture Initial body weight (g/rabbit) 725 741 745 735 26.5 0.977 Final body weight (g/rabbit) 2195 c 2304 b 2437 a 2433 a 25.6 0.001 Total weight gain (g/rabbit) 1470 c 1562 b 1692 a 1697 a 29.8 0.001 Average daily gain (g/day) 26.2 c 27.9 b 30.2 a 30.3 a 0.61 0.001 Feed intake (g/experimental period) 5571 5561 5556 5578 17.9 0.275 Daily feed intake (g/day) 99.4 99.3 99.2 99.6 0.36 0.277 Feed conversion ratio 3.79 a 3.56 b 3.28 c 3.29 c 0.06 0.001 SEM = standard error of the mean. a,b,c Means with a different superscript in the same row are significantly different (p < 0.05). animals-13-00868-t005_Table 5 Table 5 Nutrient digestibility and nitrogen balance of New Zealand White growing rabbits treated with apricot kernel extract (AKE), peach kernel extract (PKE), or their mixture (1:1). Item Treatments SEM p-Value Kernel Extracts Control AKE PKE Mixture Nutrient digestibility Dry matter 60.0 c 64.3 b 64.4 b 68.8 a 0.622 0.001 Organic matter 62.5 c 64.7 bc 67.6 b 70.8 a 0.647 0.001 Crude protein 69.0 b 70.5 b 70.9 b 73.5 a 0.605 0.008 Ether extract 52.1 d 59.9 c 62.4 b 67.1 a 0.872 0.001 Crude fiber 57.6 58.1 59.2 61.0 1.39 0.293 Nitrogen free extract 65.5 b 66.9 b 69.1 ab 71.8 a 0.97 0.012 Nitrogen (N) balance N intake (g/day) 2.77 2.77 2.76 2.77 0.03 0.100 Fecal N excretion (g/day) 1.12 1.093 0.97 0.87 0.02 0.670 Urinary N excretion (g/day) 0.53 0.46 0.48 0.48 0.03 0.315 Body N retention (g/day) 1.12 c 1.21 b 1.31 b 1.42 a 0.04 0.001 N retained (% N intake) 40.4 b 43.8 b 47.5 a 51.3 a 1.03 0.006 SEM = standard error of the mean. a,b,c,d Means with a different superscript in the same row are significantly different (p < 0.05). Intakes of nitrogen, fecal nitrogen, and urinary nitrogen were not affected either by the individual or the mixture of the fruit kernel extracts. The group treated with the mixture presented the highest (p = 0.001) body retention of nitrogen while both PKE and the mixture treatments had similar higher (p = 0.006) nitrogen retained (as % nitrogen intake) than the other treatments. animals-13-00868-t006_Table 6 Table 6 Blood plasma antioxidant indicators and antibody titers against sheep red blood cell counts (SRBCs) of New Zealand White growing rabbits treated with apricot kernel extract (AKE), peach kernel extract AKE, or their mixture (1:1). Item Treatments SEM p-Value Kernel Extracts Control AKE PKE Mixture Antioxidant indicators Malondialdehyde (mmol/L) 12.9 a 10.6 b 9.9 b 9.97 b 0.265 <0.001 Total antioxidant capacity (mmol/L) 0.60 b 1.18 a 1.16 a 1.24 a 0.057 <0.001 Superoxide dismutase (U/L) 27.0 b 35.7 a 36.1 a 36.6 a 3.497 0.017 Catalase [U/g] 490 b 595 a 591 a 607 a 28.9 0.019 Antibody titers against SRBCs SRBCs at 11 weeks of age 0.73 b 0.89 a 0.88 a 0.86 a 0.025 0.010 SRBCs at 12 weeks of age 0.74 b 0.85 a 0.86 a 0.82 a 0.03 0.044 SRBCs at 13 weeks of age 0.71 b 0.79 a 0.88 a 0.81 a 0.04 0.020 SEM = standard error of the mean. a,b Means in a row not sharing the same superscript differ significantly (p < 0.05). animals-13-00868-t007_Table 7 Table 7 Cecal measurements, fermentation characteristics, and microbial count of New Zealand White growing rabbits treated with apricot kernel extract (AKE), peach kernel extract (PKE), or their mixture (1:1). Item Treatments SEM p-Value Kernel Extracts Control AKE PKE Mixture Cecal measurements Cecum length (cm) 40.5 b 49.2 a 49.7 a 48.9 a 0.202 0.0001 Full cecum weight (g) 100 b 121 a 120 a 121 a 1.162 0.0007 Empty cecum weight (g) 24.7 b 27.9 a 28.1 a 27.9 a 0.377 0.018 Cecal fermentation pH 6.43 a 5.59 b 5.44 b 5.49 b 0.082 0.0009 Ammonia (mmol/L) 13.4 a 11.1 b 11.0 b 9.17 c 0.212 0.001 Total short-chain fatty acids (mmol/L) 51.6 b 69.3 a 69.0 a 70.1 a 3.62 0.05 Butyric acid (mmol/L) 9.1 b 10.3 a 10.2 a 10.5 a 0.165 0.017 Acetic acid (mmol/L) 50.4 b 63.0 a 63.1 a 65.1 a 0.367 0.0001 Propionic acid (mmol/L) 5.12 b 6.27 a 6.23 a 6.4 a 0.09 0.0001 Cecal microbial count (x105) (CFU/mL) Total bacterial 4.85 a 3.03 b 3.01 b 2.98 b 0.14 0.0001 Total coliform 3.41 a 2.18 b 2.19 b 2.11 b 0.125 0.005 Total anaerobic 1.91 a 1.22 b 1.21 b 0.91 b 0.27 0.0015 L. acidiophilus 1.85 b 8.95 a 8.99 a 9.01 a 0.132 0.0179 L. cellobiosus 1.41 b 9.98 a 9.10 a 9.26 a 0.08 0.015 SEM = standard error of the mean. a,b,c Means within a row having different superscripts are significantly different (p < 0.05). 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PMC10000094

Recognizing wildlife based on camera trap images is challenging due to the complexity of the wild environment. Deep learning is an optional approach to solve this problem. However, the backgrounds of images captured from the same infrared camera trap are rather similar, and shortcut learning of recognition models occurs, resulting in reduced generality and poor recognition model performance. Therefore, this paper proposes a data augmentation strategy that integrates image synthesis (IS) and regional background suppression (RBS) to enrich the background scene and suppress the existing background information. This strategy alleviates the model's focus on the background, guiding it to focus on the wildlife in order to improve the model's generality, resulting in better recognition performance. Furthermore, to offer a lightweight recognition model for deep learning-based real-time wildlife monitoring on edge devices, we develop a model compression strategy that combines adaptive pruning and knowledge distillation. Specifically, a student model is built using a genetic algorithm-based pruning technique and adaptive batch normalization (GA-ABN). A mean square error (MSE) loss-based knowledge distillation method is then used to fine-tune the student model so as to generate a lightweight recognition model. The produced lightweight model can reduce the computational effort of wildlife recognition with only a 4.73% loss in accuracy. Extensive experiments have demonstrated the advantages of our method, which is beneficial for real-time wildlife monitoring with edge intelligence. automatic wildlife recognition shortcut learning data augmentation model compression Fundamental Research Funds for the Central Universities2021ZY70 Beijing Municipal Natural Science Foundation6214040 This work was jointly supported by the Fundamental Research Funds for the Central Universities (2021ZY70) and the Beijing Municipal Natural Science Foundation (6214040). pmc1. Introduction Accurate wildlife density and abundance monitoring assists in analysis of the causes of biodiversity loss and assessment of the impacts of conservation measures . According to the International Union for Conservation of Nature (IUCN), up to 17,000 species are considered as "data deficient" . Therefore, there is an urgent need for effective large-scale wildlife monitoring systems with great spatiotemporal resolution. Camera traps have become an essential tool for wildlife monitoring in the recent decades, collecting huge amounts of data every day . Because manual annotation of such huge amounts of data is time-consuming, automatic wildlife recognition is an appealing method for analyzing these data . Deep learning methods have recently emerged as the dominant method for automatically recognizing wildlife. Xie et al. introduced the SE-ResNeXt model for recognizing 26 wildlife species in the Snapshot Serengeti dataset, with the highest Top-1 and Top-5 accuracy levels of 95.3% and 98.8%, respectively. Silva et al. utilized ResNet50 to classify different species of "bush pigs", with a best accuracy of 98.33%. Nguyen et al. designed Lite AlexNet to identify the three most common species in South Central Victoria, Australia, with an accuracy of 90.4%. Tan et al. compared three mainstream detection models, YOLOv5, FCOS, and Cascade R-CNN, on the Northeast Tiger and Leopard National Park wildlife image dataset (NTLNP dataset). YOLOv5, FCOS, and Cascade R-CNN all obtained high average precision values: >97.9% at mAP_0.5 and >81.2% at mAP_0.5: 0.95. These studies indicate that deep convolutional neural networks (CNNs) can perform well in wildlife recognition. Although existing automatic wildlife recognition methods have achieved higher and higher accuracy, these models' capacity to generalize across diverse datasets is not as strong as it could be. Geirhos et al. suggested that the aforementioned issue might be attributed to shortcut learning, in which these models tend to learn simple decision rules during training. These learned decision rules can only perform well on datasets that are independent and identically distributed (i.i.d.). In a non-independent and identically distributed (non-IID) dataset, however, performance deteriorated. Because camera traps are typically deployed in fixed locations, wildlife monitoring images appear to have similar backgrounds over time. This demonstrates a strong coupling link between wildlife and their backgrounds, providing shortcuts for a deep learning model to recognize wildlife via the backgrounds. These models may fail to recognize the same species with different backgrounds given the shortcut-learned decision rules. To increase the accuracy and generalization capabilities of wildlife recognition models, shortcut learning must be avoided. Many strategies for mitigating shortcut learning have been investigated. Szegedy et al. proved that data distribution has a direct impact on deep neural network learning and generated adversarial samples by adding perturbations to the data to avoid shortcut learning. Cubuk et al. improved the generalization of object recognition models by augmenting data with information, geometric distortion, and color distortion. Arjovsky et al. used causality to distinguish the false correlation and the interest region in the sample and then proposed invariant risk minimization (IRM), a novel learning framework that can estimate nonlinear, invariant, causal predictors across different training environments, allowing for out-of-distribution generalization. Finn et al. utilized meta-learning to train a model on multiple learning tasks, resulting in high generalization performance with only a modest number of new training samples. Overall, data augmentation is a a simple and effective technique for avoiding shortcut learning. Furthermore, it is beneficial to conduct recognition directly on a camera trap to improve the effectiveness of wildlife monitoring . Due to the limited computing capability and memory of camera traps, a lightweight recognition model is required. Model compression is a commonly used method to generate a lightweight model . Knowledge distillation transfers the knowledge gained by a large teacher network to a small student network without losing validity, allowing model compression to be realized. The structure of the student model is crucial to the success of compression and the performance of the compressed model. Wen et al. suggested a structural sparse learning approach for obtaining the student network from a large CNN, which accelerated AlexNet by 5.1 and 3.1 times on CPU and GPU, respectively, with only a 1% drop in accuracy. Rather than compressing the teacher network, Du et al. selected a shallow reference model as the student network, which was then combined with a random forest model to generate more precise probability values for each class. Crowley et al. separated the normal convolution of the teacher model into different groups of point-wise convolutions to construct several student models with test errors ranging from 5.0% to 7.87%. Model pruning is another popular model compression method that can significantly reduce the number of parameters by removing certain parts of the model . It is regarded as an effective method for achieving a student network. There are two types of pruning, i.e., unstructured pruning and structured pruning. Unstructured pruning approaches remove weights on a case-by-case basis, e.g., HashedNet , which uses a hash function to randomly group network weights, then allows weights in the same group to share parameters to minimize model complexity. Structured pruning methods remove weights in groups, such as a channel or a layer, and are often more efficient than unstructured pruning methods. Li et al. removed convolution kernels with low effect on network accuracy and then retrained the pruned model to boost accuracy. Luo et al. developed a pruning technique for ThiNet based on a greedy strategy. By considering network pruning to be an optimization issue, the statistical information obtained from the next layer's input-output relationship is utilized to determine how to prune the present layer. ThiNet can reduce the parameters and FLOPs of ResNet-50 by more than half, whereas top-5 can only reduce them by 1%. He et al. utilized a channel pruning approach to reduce the channel information in the input feature map before retaining it in the output feature map by adjusting the weights. Under a scenario with five-fold acceleration, the approach only degrades the accuracy of a VGG16 network by 0.3%. To achieve a high compression ratio, Jin et al. proposed a hybrid pruning strategy that integrated kernel pruning and weight pruning. Aghli et al. first employed the weight pruning method to create a student model, then knowledge distillation was achieved by minimizing the cosine similarity between the layers of the teacher and student networks. When compared to the state-of-the-art methods, higher compression rates can be achieved with comparable accuracy. All in all, it appears that the combination method of model pruning and knowledge distillation is more suited for generating a lightweight student network with acceptable accuracy. In this paper, we propose a lightweight automatic wildlife recognition model design method that avoids shortcut learning. To the best of our knowledge, this is the first work that focuses on the shortcut learning of camera trap image recognition. First, two data augmentation strategies--image synthesis (IS) and regional background suppression (RBS)--are introduced in order to prevent the wildlife recognition model from shortcut learning and improve its performance. The Resnet50-based wildlife recognition model is then pruned with the genetic algorithm and adaptive BN (GA-ABN) to construct the student model. Finally, utilizing the Resnet50-based wildlife recognition model as a teacher model, knowledge distillation is employed to fine-tune the student model, yielding a lightweight automatic wildlife recognition model. The technological framework for the design of the lightweight automatic wildlife recognition model is depicted in Figure 1. To summarize, the contribution of this work is two-fold:(1) We introduce a novel mixed data augmentation method that combines IS and RBS to mitigate shortcut learning in wildlife recognition. (2) We propose an effective model compression strategy based on GA-ABN for adaptively reducing the redundant parameters of a large wildlife recognition model while maintaining accuracy. 2. Materials and Methods 2.1. Wildlife-6 Dataset From 2010 to 2019, we utilized infrared camera traps to collect wildlife monitoring images in Saihanwula National Nature Reserve in Inner Mongolia . We selected images of six common species: red deer, goral, roe deer, lynx, badger, and wild boar . After discarding images that were falsely triggered or damaged, the LabelMe software was used to annotate the remaining images with bounding boxes and categories. The annotated dataset is denoted as Wildlife-6. Details of Wildlife-6 are shown in Table 1. 2.2. Mixed Data Augmentation In this section, a mixed data augmentation method combining IS and RBS is proposed to diversify the background. 2.2.1. Image Synthesis Based on Random Pasting We present an image synthesis method for generating new training samples by randomly pasting wildlife instances into background images of camera traps. Figure 2 shows the image synthesis procedure. There are two main stages: (1) target segmentation and (2) random pasting. In the target segmentation stage, wildlife instances are extracted using the weakly supervised semantic segmentation model Inter-pixel Relation Network (IRNet) . First, we trained the ResNet50-based wildlife recognition model and generated the class activation mapping (CAM). The confidence region of CAM was then obtained using the DenseCFR method, which was based on the thresholding of the foreground and background. The random walk method was applied to select pairs of points in the confidence region, which were then input into the IRNet for training. During training, IRNet learns to produce class boundary maps to determine class boundaries and compute class centroids. The optimal class centroids are achieved by iteration. Finally, the random walk algorithm is used to compute the attention scores of the pixel points in the CAM, as well as the domain propagation of the instance image based on the semantic affinity between adjacent pixels, to obtain the entire instance region, i.e., the wildlife instance. Furthermore, in the random pasting stage, the extracted wildlife instances are randomly rotated, resized, and then pasted to a random background image to disrupt their inherent directional properties and reduce the object's dependency on the entrenched scene. Figure 3 depicts synthetic sample instances. 2.2.2. Regional Background Suppression We propose a target-guided background suppression method to guide the network to focus on the foreground (wildlife). Unlike the cutout method, our method only randomly suppresses the background regions. The background suppression is divided into the following stages. To begin, separate the image into foreground (wildlife) and background. To properly extract the foreground, we label the wildlife in the image with a bounding box, and the region outside the bounding box is regarded as background. Then, given a random starting point and height-width ratio, we generate a rectangular mask mb with all values equal to 0. With the bounding box region mf, it is critical to guarantee that mfmb=. In this case, the random mask suppresses background features, forcing the network to focus on foreground (wildlife) features. The generated images with background occlusion may be utilized not only to model occlusion phenomena in the wild, but also to help the model focus on more foreground (wildlife) information. 2.3. Lightweight Method for Automatic Wildlife Recognition Model 2.3.1. Pruning Method Based on Genetic Algorithm and Adaptive Batch Normalization A structural prune method based on GA-ABN is presented to construct the student model. First, the genetic algorithm(GA) is applied to obtain an optimal pruning strategy. To begin, a set of sub-networks are created as the initial population using a random sampling method. A fitness function is introduced to evaluate each sub-network in the initial population and is shown in Equation (1). (1) fj=njpj/p0 where nj is the validation accuracy of the j-th sub-network, and the parameters pj and p0 represent the number of the j-th sub-network and the initial network, respectively. The fitness score of each sub-network is then determined in the selection stage. The top 20 sub-networks with the highest fitness scores are chosen. During the crossover mutation step, a new population with 100 new sub-networks is generated based on the 20 previously selected sub-networks. The selection and mutation steps are repeated until the total fitness of the new population remains constant. Finally, an optimal pruned network can be achieved. Furthermore, because the commonly used global BN may result in sub-optimal performance , we updated the BN to an adaptive BN to overcome this issue. For mini-batch samples of size N, the training data xi, the statistics of m, and s2 used in the adaptive BN are computed with Equations (2) and (3). (2) mB=1Ni=1Nxi (3) sB2=1N-1i=1Nxi-mB2 2.3.2. Fine-Tuning Based on Knowledge Distillation with MSE Loss Knowledge distillation is introduced to improve the accuracy of the student model. As shown in Figure 4, we jointly train the teacher model and the student model with knowledge distillation loss, resulting in the student model's distribution being comparable to that of the teacher model. The knowledge distillation with MSE loss is introduced to fine-tune the pruned model . The MSE loss is applied to compute the difference between the output distributions of the teacher and student models. For comparison, two types of KL divergence-based knowledge distillation are included: knowledge distillation with soft KL (KD-SKL) and knowledge distillation with hard KL (KD-HKL). In KL divergence-based knowledge distillation, the teacher model imposes a supervision signal on the student model. KD-SKL adopts probability scores as a supervision signal, whereas KD-HKL uses one-hot pseudo labels, resulting in two different KL divergence losses. Following knowledge distillation, the fine-tuned student model may be viewed as a lightweight wildlife recognition model with relatively high performance. 3. Experiments and Results 3.1. Experiments Setup Because ResNet-50 provides excellent performance in wildlife image recognition , we built the ResNet50-based wildlife recognition model as the teacher model. During training, the dataset was divided into training and test sets in a 7:3 ratio. All images were downsized to 448x448 before being fed to the model. Table 2 shows the software and hardware used in all experiments. All of the models' main training settings are the same, as indicated in Table 3. 3.2. Evaluation Metrics The following metrics are introduced to evaluate the performance of the models:(1) Classification performance The accuracy calculated by Equation (4) is used to evaluate the classification performance. (4) ACCcls=i=1cTPii=1cTPi+FPi where TPi (true positive) represents the number of correctly classified images of the ith class and FPi (false positive) represents the number of misclassified images of the ith class. C is the total number of categories. (2) Performance of mitigating shortcut learning A heatmap can indicate where a model focuses on during classification . We propose the foreground ratio of the heatmap (FRoH) to evaluate the performance of mitigating shortcut learning. The FRoH is defined by Equation (5):(5) FRoH=HboxHtotal where Htotal is the sum of the thermal values of all pixels in the heatmap, and Hbox is the sum of the thermal values of the pixels in the labeled bounding box. Figure 5 shows an intuitive schematic diagram of FRoH. FRoH indicates how much the model focuses on the targets (wildlife). The higher the FRoH value, the more the model is focused on the wildlife, implying that the model classifies images primarily on wildlife features rather than background and that shortcut learning is mitigated. (3) Performance of model compression The number of parameters, number of calculations, and FPS (frames per second) are introduced to quantify the performance of model compression. Equations (6) and (7) specify the number of parameters and number of calculations of the convolutional layer, respectively. (6) params=coutxK2xcin (7) FLOPs=2HWcinK2+1cout where Cout and Cin represent the number of output and input channels, respectively. H and W represent the height and width of the input feature map, respectively. K is the size of the convolution kernel. 3.3. Experiments Results of Mitigating Shortcut Learning 3.3.1. Comparison Experiments with Different Data Augmentation Methods Using the ResNet50-based wildlife recognition model as a baseline, five different data augmentation methods were compared, including repeat sampling, cutout , IS, RBS and the mixed method. The results shown in Table 4 indicate that our proposed method achieves the best performance, with ACCcls and FRoH values of 91.23% and 44.71%, respectively. Surprisingly, the results using repeat sampling are somewhat lower than the baseline. This is because simple random repeat sampling has no effect on the overall distribution of the dataset. The strong coupling between wildlife and background remains, resulting in no improvement in recognition accuracy. The cutout method has a higher FRoH score than the baseline, implying that the cutout method can decrease the coupling between wildlife and background to some extent. However, the cutout method has a slightly lower ACCcls value than that of the baseline. Because the cutout method's optional suppression regions cover the whole image, including the wildlife region, the wildlife feature may be lost. Both IS and RBS perform better than the baseline. IS outperforms RBS because it actively diversifies the background, resulting in more abundant variety than simply suppressing a portion of the background. As expected, the mixed data augmentation method outperforms IS and RBS. 3.3.2. Visualization Analysis To conduct a more in-depth analysis of the performance improvement brought by the mixed data augmentation method, we calculated a heatmap using gradient-weighted class activation mapping (Grad-CAM) . Heatmaps of example images with or without mixed data augmentation method are illustrated in Figure 6. It can be observed that with mixed data augmentation, the trained wildlife recognition model can precisely focus on the wildlife . However, in the absence of mixed data augmentation, the trained wildlife recognition model is easily disturbed by the background or can only acquire incomplete wildlife features . Figure 7 shows the recognition results for example images of gorals, badgers, red deer, and roe deer. Without the mixed data augmentation, the wildlife recognition model predicted the wrong categories of wildlife , which can be ascribed to an excessive focus on the background. The wildlife recognition model with the mixed data augmentation method is completely concerned with all wildlife, resulting in accurate predictions . 3.3.3. Class-Wise Accuracy Confusion matrices were computed to investigate the effect of the proposed mixed data augmentation strategy on the recognition performance of different species, as shown in Figure 8. When comparing Figure 8a,b, the mixed data augmentation strategy improved the recognition accuracy of all species. Lynxes and badgers, in particular, improved by 7.3% and 6.1%, respectively. The limited sample size and background type of these two species, together with their small size, made it easier for the model to accomplish recognition by background, and so enrichment of background variety resulted in a larger improvement. 3.3.4. Performance on Other Dataset We investigated the mixed data augmentation strategy further on the NACTI dataset. The NACTI dataset contains around 3.7 million camera trap monitoring images from five locations across the United States. We chose eleven species for comparative experiments. As with the Wildlife-6 dataset, the bounding boxes containing wildlife were annotated and used in the following experiments. Table 5 shows the results on the NACTI dataset. The ACCcls and FRoH values were both enhanced after utilizing the mixed data augmentation method, with the FRoH being increased to a greater extent. The improvement in ACCcls is rather minimal due to the excellent accuracy of the baseline and the more diverse backgrounds in the NACTI dataset. In summary, the mixed data augmentation strategy can assist the wildlife recognition model in focusing more on the wildlife, resulting in higher classification accuracy and generalization across datasets. In other words, the shortcut learning can be mitigated. 3.4. Experiments Results of the Lightweight Automatic Recognition Model 3.4.1. Comparison with Different Pruning Method With the ResNet50-based wildlife recognition model as the teacher model, two pruning methods, the GA-ABN and random sampling algorithms, were compared. The compression ratio of FLOPs was set to 50 +- 5%. The fitness variations of the sub-networks generated by two pruning methods during the searching process are depicted in Figure 9. It can be shown that the fitness (i.e., accuracy) of the random sampling method improved significantly at first, but then almost equalized in the later search period. This is due to the lack of an optimization mechanism. Taking advantage of GA's evolution mechanism, our method can achieve higher and higher fitness as time passes, implying a better pruning strategy. Meanwhile, the final fitness of our method is greater than that of the pruning method using random sampling. We further fine-tuned the optimal pruned model obtained by two pruning methods. As shown in Table 6, the accuracy of the ResNet50-based model is 91.23%, the number of parameters is 23.52 M, the FLOPs is 16.48 G and the FPS is 0.85. The number of parameters of the two pruned models were reduced by almost half. Furthermore, the accuracy values of the two pruned models were decreased as expected, and the pruned model by GA-ABN achieved greater accuracy with fewer parameters than the pruned model by random sampling. Similar to the numbers of parameters, the FLOPs of both pruned models declined by almost half, resulting in higher FPS. The FPS values of both pruned models increased by nearly 62 times as compared with the ResNet50 model. Although the FLOP and FPS of our method are similar with those of random sampling, our method has a higher accuracy than random sampling. Furthermore, the compression ratio of FLOPs was set to 25 +- 5% to verify the generalization of the proposed pruning method. As shown in Table 7, although FLOPs and FPS are the same, the accuracy of the pruned model achieved by our method is still higher than that of the pruned model using random sampling. Altogether, the pruning method using GA-ABN can effectively reduce the number of computations in the model, speed up the model operation, and maintain relatively high accuracy under different pruning ratios, showing that the GA-ABN pruning method has excellent compression performance. 3.4.2. Fine-Tuning Based on Different Knowledge Distillation Methods Three fine-tuning strategies based on KD-MSE, KD-SKL, and KD-HKL were compared with the pruned ResNet50 model using GA-ABN as the baseline model (P-ResNet50). As shown in Table 8, all three fine-tuning strategies outperformed the P-ResNet50 model in terms of accuracy. Furthermore, the accuracy of KD-MSE was 1.88%, 0.66%, and 1.39% higher than those of P-ResNet50, KD-SKL, and KD-HKL, respectively, indicating that KD-MSE is the most effective fine-tuning method. The advantage of KD-MSE originates from the fact that it enables the student network to inherit the output distribution of the teacher network via an explicit MSE constraint. KD-HKL, in particular, performs worse than KD-SKL, which we attribute to possible noise introduced by a hard supervision signal during training. 4. Discussion Wildlife camera trap image recognition based on deep learning offers enormous potential, but it is also challenging due to the complexity of the wild environment . Our work investigates a mixed data augmentation technique for mitigating shortcut learning of deep learning models caused by similar backgrounds in certain images from the same camera trap. Intelligent recognition implemented directly on edge devices can improve the effectiveness of wildlife monitoring , we further propose a novel model compression strategy that integrates pruning and knowledge distillation to develop lightweight wildlife recognition models applicable to camera traps. 4.1. Overcoming Shortcut Learning with Data Augmentation Overcoming shortcut learning can enhance model generalization performance. However, to the best of the authors' knowledge, no work has focused on shortcut learning in camera trap image recognition. Because dataset distribution has a direct influence on deep neural network learning, data distribution variation has the potential to alleviate shortcut learning . Several data augmentation strategies, which can vary the data distribution, have been employed to alleviate shortcut learning . Diversifying the background in camera trap images can help distinguish the foreground (wildlife) and background and prevent the recognition model from shortcut learning. Given that repeat sampling has no effect on data distribution, it is therefore not surprising that the recognition performance has not been improved. The cutout method modifies the data distribution using random masks and improves the FRoH score compared to the baseline, but it may also suppress the foreground (wildlife) feature, which lowers the ACCcls in comparison to the baseline. IS and RBS both retain foreground features while modifying the data distribution, achieve higher FRoHs than the baseline, guide the model to focus more on the foreground, and avoid shortcut learning very well. As expected, a mixed data augmentation approach that combines IS and RBS performs the best. Using the Wildlife-6 dataset, our recognition accuracy (91.23%) is higher than that of the literature (90.2%), demonstrating that our technique is effective. 4.2. Lightweight Wildlife Recognition Model Because camera traps have limited computational capacity and memory, lightweight models are required for deep learning-based automatic wildlife recognition in camera traps . Model compression strategies based on model pruning and knowledge distillation are the dominant approaches for developing lightweight models . In knowledge distillation methods, selecting a suitable student model is crucial. Model pruning can produce beneficial tiny networks, but it can also reduce accuracy . A potential strategy appears to be model pruning to generate student models and knowledge distillation for fine-tuning . The key to structured pruning is to search for redundant channels or layers, which is an optimization problem. GA can effectively avoid falling into local optima and has been utilized in model pruning . Experiments show that the proposed GA-ABN can search for and achieve sub-networks with higher final fitness scores more quickly when compared to random sampling algorithms. The GA-ABN method can achieve a higher accuracy than random sampling algorithms. The greater the compression rate, the more obvious the benefit. With a compression rate of 50 +- 5%, the FPS of our method reaches 53.49, about 63 times that of ResNet50, which is sufficient for provision of real-time results (30 FPS or more) . Furthermore, we introduced three kinds of knowledge distillation methods, KD-MSE, KD-SKL and KD-HKL to fine-tune the pruned model to improve accuracy. MSE loss can make the student model learn the output distribution of the teacher model more intuitively , which makes the KD-MSE achieved the highest accuracy improvement among three methods. Finally, a lightweight wildlife recognition model with high accuracy but a massively reduced number of parameters and a significantly lower calculation can be achieved. The proposed lightweight wildlife recognition model design method has the potential to enable the application of edge intelligence. Future research will investigate the lightweight model's performance on embedded devices. 5. Conclusions This study proposed a lightweight wildlife recognition model design method that combines a mixed data augmentation strategy, as well as a model compression method with integrated pruning and knowledge distillation. The mixed data augmentation method, combined with IS and RBS, was introduced to modify the data distribution of the dataset, guiding the model to focus more on the wildlife feature. The ACCcls and FRoH were both improved after using the mixed data augmentation method, demonstrating that shortcut learning was mitigated. Furthermore, the lightweight model was constructed using an integrated method that first employed the structural pruning technique based on GA-ABN, then fine-tuned the pruned model with KD-MSE. Using GA-ABN pruning and a compression rate of 50 +- 5%, the number of model parameters reduced by 57.4%, FLOPs decreased by 46.1%, FPS improved to 62.9 times, and accuracy decreased by just 4.73%. After fine-tuning with KD-MSE, the accuracy of the model improved by 2.12%. Overall, this study provides a novel method for developing lightweight wildlife recognition models, which can result in lightweight models with relatively high accuracy and significantly decreased computing cost. Our method has important practical implications for utilizing deep learning models for wildlife monitoring on edge intelligent devices. This can help to support long-term wildlife monitoring, biodiversity resource assessments, and ecological conservation. In the future, we intend to deploy our lightweight model to camera traps and augment them with wireless communication capabilities to create a real-time wildlife monitoring system. The performance of proposed system will be evaluated in the wild. Author Contributions Conceptualization, J.X. and X.L.; methodology, Y.Z. and X.L.; software, Y.Z. and X.L.; validation, Y.Z. and X.L.; formal analysis, Y.Z. and X.L.; investigation, X.L.; data curation, Y.Z.; writing--original draft preparation, Y.Z. and X.L.; writing--review and editing, J.X., Y.Z. and J.Z.; supervision, J.X.; project administration, J.X. and J.Z.; funding acquisition, J.X. and J.Z.; All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The datasets used in this study are available from the corresponding author on reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The technological framework for the design of the lightweight automatic wildlife recognition model. Figure 2 Image synthesis method. Figure 3 Synthetic samples. Figure 4 Fine-tuning based on knowledge distillation. (a) indicates mean square error (MSE) distillation loss and (b) indicates hard soft kullback-leibler (KL) distillation loss. Figure 5 Schematic diagram of FRoH. Figure 6 Heatmaps of typical images. The first row contains the input images, the second row contains the heatmaps of images without the mixed augmentation method, and the third row contains the heatmaps of images with mixed augmentation method. Figure 7 Recognition results with or without mixed augmentation method. From up to down, there are the images of goral (label 0), badger (label 1), red deer (label 2), roe deer (label 3). The first column is the input images, second column are the heatmaps of images without mixed augmentation method, the third column is the heatmaps of images with mixed augmentation method. The top left corner of each heatmap presents the predicted label. Figure 8 Confusion matrix. (a) indicates class wise accuracies of ResNet-50 and (b) indicates class wise accuracies of our method (+Mixed(+RBS+IS)). Figure 9 Fitness variations of the sub-networks during the search under 50 +- 5% compression ratio. The solid line shows the sliding average of the fitness (N = 3). animals-13-00838-t001_Table 1 Table 1 Details of Wildlife-6. Species Number of Images Red deer 1094 Goral 761 Roe deer 1310 Lynx 377 Badger 190 Wild boar 906 Total 4638 animals-13-00838-t002_Table 2 Table 2 Setup of the experiments. Test Environment Type System Ubuntu16.04 Graphics card Nvidia 1080 ti (11 GB/Nvidia) CPU Intel Core i5-9400F @ 2.9GHz RAM 32 GB Framework Pytorch1.4.1 Programming language Python3.6 animals-13-00838-t003_Table 3 Table 3 Training settings. Item Value or Method Optimizer SGD Momentum 0.9 Initial learning rate 0.0001 Learning rate decay cosine decay Batch size 64 Iterative scheme early stop, patience = 50 SGD = stochastic gradient descent. animals-13-00838-t004_Table 4 Table 4 Results of different data augmentation methods. Methods Acccls FRoH ResNet-50 89.76% 43.40% + Repeat sampling 88.23% 43.36% + Cutout 89.03% 43.83% +RBS 90.13% 43.57% + IS 90.72% 43.95% +Mixed (+RBS + IS) 91.23% 44.71% RBS = regional background suppression; IS = image synthesis. animals-13-00838-t005_Table 5 Table 5 Results of the NACTI dataset. Methods Acccls FRoH ResNet-50 99.21% 46.48% +Mixed (+RBS + IS) 99.24% 49.35% RBS = regional background suppression; IS = image synthesis. animals-13-00838-t006_Table 6 Table 6 Performances of ResNet50 and the pruned models with compression ratio of 50 +- 5%. ResNet50 Random Sampling Our Method Accuracy 91.23% 84.90% 86.50% Parameters 23.52 M 10.65 M 10.01 M FLOPs 16.48 G 8.85 G 8.89 G FPS 0.85 53.2 53.49 FLOPs = floating-point operations per second; FPS = frames per second. animals-13-00838-t007_Table 7 Table 7 Performances of the pruned models with compression ratio of 25 +- 5%. Random Sampling Our Method Accuracy 83.80% 86.20% Parameters 5.65 M 5.65 M FLOPs 4.73 G 4.73 G FPS 56.62 56.62 FLOPs = floating-point operations per second; FPS = frames per second. animals-13-00838-t008_Table 8 Table 8 Result of different fine-tuning framework on our dataset. Method Accuracy P-ResNet50 86.50% KD-SKL 87.72% KD-HKL 86.99% KD-MSE 88.38% KD-SKL = knowledge distillation with soft kullback-leibler; KD-HKL = knowledge distillation with hard kullbackleibler; KD-MSE = knowledge distillation with mean square error. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000095

Iron flocculation is widely used to concentrate viruses in water, followed by Fe-virus flocculate formation, collection, and elution. In the elution stage, an oxalic or ascorbic acid re-suspension buffer dissolved iron hydroxide. After the concentration of viral hemorrhagic septicemia virus (VHSV) in seawater (1 x 101 to 1 x 105 viral genome copies or plaque-forming unit (PFU)/mL), the recovery yield of the viral genome using quantitative real-time PCR (qRT-PCR) and viral infectivity using the plaque assay were investigated to evaluate the validity of the two re-suspension buffers to concentrate VHSV. The mean viral genome recovery yield with oxalic and ascorbic acid was 71.2 +- 12.3% and 81.4 +- 9.5%, respectively. The mean viral infective recovery yields based on the PFU were significantly different between the two buffers at 23.8 +- 22.7% (oxalic acid) and 4.4 +- 2.7% (ascorbic acid). Notably, although oxalic acid maintains viral infectivity over 60% at a viral concentration above 105 PFU/mL, the infective VHSVs were not sufficiently recovered at a low viral concentration (102 PFU/mL, <10%). To support this result, concentrated VHSV was inoculated in Epithelioma papulosum cyprini (EPC) cells to confirm cell viability, viral gene expression, and extracellular viral titer. All results demonstrated that oxalic acid buffer was superior to ascorbic acid buffer in preserving viral infectivity. iron flocculation oxalic acid ascorbic acid viral hemorrhagic septicemia virus viral genome recovery viral infective recovery Pukyong National UniversityThis work was supported by a research grant from Pukyong National University (2022). pmc1. Introduction Environmental DNA (eDNA), which is genetic material extracted from environmental samples, including water, can be applied as a pathogen monitoring tool . Despite the applicability of eDNA for disease surveillance in aquaculture, the low concentration of viral particles released from diseased aquatic animals in seawater makes it difficult to directly use water samples, in their natural state, in general, using molecular methods (e.g., polymerase chain reaction) without a concentration process. Among the several concentration methods, such as polyethylene glycol precipitation , ultracentrifugation , tangential flow filtration , and the negatively charged membrane method , viral particle coagulation has been applied to recover viral particles from water . In the coagulation method, various filters and metal substances, such as aluminum, sodium alginate, and sodium silicate, are used as coagulants to increase the efficiency of viral recovery yields by combining viruses with membrane filters . Notably, the iron flocculation method, based on ferric chloride as a coagulant, is widely used because of the high recovery yields of viral particles . Iron flocculation consists of three steps: Fe-virus colloid formation, collection, and elution. Since viral particles can be released under different water conditions (freshwater or seawater), several factors, including Fe concentration, pH, salinity, stirring speed, alkalinity, collection filter type, and re-suspension buffer, should be considered . Notably, the elution step involving the dissolution of iron hydroxide by a re-suspension buffer is not essential to detect viruses by molecular assays ; the re-suspension buffer employed in the step is closely associated with the recovery of virus infectivity . Thus, based on the purpose of this study, it is necessary to evaluate the viral recovery yield under experimental conditions. Viral hemorrhagic septicemia virus (VHSV), an enveloped negative-sense single-stranded RNA virus (family Rhabdoviridae, genus Novirhabdovirus), is the most serious pathogen causing mass mortality in both freshwater and marine fish, resulting in significant economic loss . VHSV can be horizontally transmitted through water containing the virus released from the infected fish . In Korea, since the first outbreak of viral hemorrhagic septicemia (VHS) in olive flounder (Paralichthys olivaceus) in Pohang in 2001 , it has been reported annually as an endemic disease . Notably, in aquaculture systems, infective viral particles in seawater emitted from diseased fish could be a source of reinfection and disease outbreaks. In a previous study , even though viral genomic recovery yields of VHSV (genotype IVa) were 23.0% using a negatively charged membrane, recovery yields of viral infectivity were not determined. Thus, since the analysis of infective viral particles is a crucial factor in determining the distributional transmission and interaction with susceptible species to aquatic viral disease, both quantitative genomic analysis and analysis of infective viral particles in seawater are needed for surveillance. Therefore, to apply iron flocculation as a diagnostic procedure for disease surveillance, viral genomic and infectious recovery yields should be determined. In this study, the viral recovery yields of VHSV were compared between two re-suspension buffers (oxalic acid and ascorbic acid) in the iron flocculation process. Viral genomic recovery was determined based on a quantitative real-time PCR (qRT-PCR) assay. Viral infective recovery was analyzed using a plaque assay on EPC cells. 2. Materials and Methods 2.1. VHSV Culture and Titration 2.1.1. Virus Culture Epithelioma papulosum cyprini (ECACC, Cat No. 93120820; EPC) cells were cultured in Leibovitz's medium (L-15; Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1x antibiotic-antimycotic solution (GenDEPOT, Katy, TX, USA) at 25 degC. VHSV 21OfPh isolate (genotype IVa) from diseased olive flounder was propagated in an L-15 medium containing 5% FBS at 15 degC for 5 days. 2.1.2. Plaque Assay Following the appearance of the cytopathic effect (CPE), the viral titer of the supernatant was determined using a plaque assay. Briefly, EPC cells (approximately 4 x 106 cells/well) seeded in 6-well plates were inoculated with 200 mL of 10-fold serially diluted supernatants for 2 h at 15 degC. After the washing of the cells with phosphate-buffered saline (PBS; Gibco, Grand Island, NY, USA), a semi-solid overlay medium consisting of 2% (w/v) SeaPlaque TM GTG TM agarose (Lonza, Rockland, WA, USA) was added. Following the appearance of visible plaques (4-5 days post inoculation) which could be visually and sufficiently counted as individual plaques, cells were fixed and stained with 25% formaldehyde and 0.5% crystal violet for 4 h. Then, the semi-solid medium was carefully removed, followed by washing the residue with tap water, and the number of plaques was counted. 2.1.3. Quantitative Real-Time PCR Quantification of the viral cDNA copies using primers and TaqMan probes targeting a fragment of the VHSV nucleoprotein (N) gene was performed by qRT-PCR . Briefly, qRT-PCR was performed with a reagent mixture containing 10 mL of HS Prime Q-Master Mix (2X, Real-time PCR for TaqMan Probe; GenetBio, Daejeon, Korea), 800 nM of each primer (forward and reverse; Table 1), 200 nM of the probe, 0.4 mL of 50X ROX dye (GenetBio, Daejeon, Korea), 6 mL of nuclease-free water, and 0.8 mL of cDNA using the StepOne real-time PCR system (Applied Biosystems, Waltham, MA, USA). The standard curve was generated using recombinant plasmid DNA harboring the VHSV nucleoprotein (N) gene (82 bp) cloned based on the pGEM T-vector (Promega GmbH, Mannheim, Germany). To determine the cut-off value for quantitative analysis, 95% of the limit of detection (LOD95%) was set as described by Uhlig et al. . Following serial two-fold dilutions of plasmid DNA (from 40 copies mL-1 to 0.3125 copies mL-1), qRT-PCR was conducted with 12 replicates. LOD95%, which indicates 95% of a positive result at the lowest number of viral DNA copies and confidence intervals, was decided through probit regression analysis using MedCalc(r) (version 20.015). 2.2. Concentration of VHSV in Seawater by Iron Flocculation 2.2.1. Iron Flocculation Process To compare VHSV particle recovery between viral genomic and infective particles by iron flocculation, VHSV particles in seawater were concentrated with minor modifications as described previously . First, before the pre-filtration process, seawater was stored at room temperature (approximately 20 degC) for 1 h to precipitate debris, and pre-filtered using a vacuum pump (Gast, Benton Harbor, MI, USA) with a glass microfiber filter (GF/A; pore size, 1.6 mm; Whatman, Maidstone, UK). If water flux was declined by membrane fouling, the membrane filter was replaced with a new filter. Then, 50 mL of iron chloride solution [4.83 g of iron (III) chloride hexahydrate (FeCl3*6H2O) per 100 mL of distilled water] was added to 500 mL of serially diluted VHSV-spiked seawater (1 x 101 to 1 x 105 viral genome copies/mL, 1 x 101 to 1 x 105 PFU/mL) and gently mixed using a magnetic stir bar and stir plate (<120 rpm) for 1 h at 20 degC to allow Fe-VHSV flocculate formation. The Fe-VHSV flocculate was collected on a polycarbonate (PC) membrane filter (pore size, 1.0 mm; Whatman, Maidstone, UK) holder with a polyethylene sulfone (PES) membrane filter (pore size, 0.8mm; Whatman, Maidstone, UK) using a peristaltic pump (<12 psi; Eyela, Japan). Following collection of the Fe-VHSV flocculates, the membrane was transferred to a 5 mL round-bottom tube, and 1 mL of re-suspension buffer (oxalic or ascorbic acid buffer; pH 6.0 +- 0.2, as previously described by Kim et al. ) was added. Viral re-suspension was performed for 2 h using a Bio RS-24 Mini-Rotator (30 rpm; 170 Biosan, Riga, Latvia) in a dark room at 4 degC. 2.2.2. Comparison of Viral Genome Copies and Infective Viral Particle Recovery To compare the viral genomic recovery yields using different re-suspension buffers in the iron flocculation process, total RNA from the concentrate of VHSV-spiked seawater (200 mL) was extracted using the yesRTM Total RNA Extraction Mini Kit (GenesGen, Busan, Korea). The extracted RNA exhibited an absorbance ratio between 2.0 and 2.2 when measured at A260/280 nm with a NanoVue Plus Spectrophotometer (GE Healthcare, Chicago, IL, USA) used for the cDNA template. RNA was reverse-transcribed using the PrimeScriptTM 1st cDNA Synthesis Kit (Takara, Kusatsu, Japan) according to the manufacturer's instructions. The number of viral cDNA copies in the concentrate was determined using qRT-PCR, as described in Section 2.1.3. Moreover, the viral infective recovery yields under different re-suspension buffers were compared using the plaque assay described in Section 2.1.2. The recovery yield of viral genome copies was calculated based on the total viral cDNA copies (genome copies/mL) of VHSV-spiked seawater and concentration products (recovered viral cDNA copies per mL x volume of concentration product/spiked viral cDNA copies per mL x volume of seawater). The recovery yield of infective particles was calculated based on the total spiked infective particles (PFU/mL) in seawater and concentration products (recovered PFU per mL x volume of concentration product/spiked PFU per mL x volume of seawater). The experiments were performed in triplicate, and the results of the recovery rate (%) were presented as the mean +- standard deviation (SD). The positive results of qRT-PCR were determined using a cut-off value of LOD95%. Statistical analyses were conducted using a two-way analysis of variance (ANOVA) with GraphPad Prism software (version 9.5.0), and statistical significance was set at p < 0.05. 2.3. Assessment of Infectivity Recovery for Viral Concentrates Eluted with Different Buffers In Experiment 1 (Exp. 1), to clarify the infectivity recovery yield under different re-suspension buffers, the viability and morphology of EPC cells inoculated with the concentrated VHSV-spiked seawater were compared. The EPC cells (approximately 5 x 105 cells/well) seeded into 24-well plates were inoculated with 100-fold diluted concentrates (originally, VHSV-spiked seawater at 1 x 101 to 1 x 105 PFU/mL) at 15 degC. After 2 h of viral adsorption, followed by washing with PBS, VHSV-concentrate-inoculated cells were incubated with fresh L-15 medium containing 5% FBS for 48 h. Cell viability was determined using the Cellrix(r) Viability Assay Kit (Medifab, Seoul, Korea) according to the manufacturer's instructions. Briefly, EPC cells were reacted with the reagent at 15 degC for 3 h, and absorbance was measured at 450 nm using an Infinite(r) 200 microplate reader (Tecan, Mannedorf, Switzerland). Cell viability compared to naive EPC cells was calculated using a two-way ANOVA with GraphPad Prism software (ver. 9.5.0). Statistical significance was set at p < 0.05. In Experiment 2 (Exp. 2), to compare the viral gene expression of the concentrate eluted by different re-suspension buffers, the expression levels of N and glycoprotein (G) genes were investigated in concentrate-inoculated EPC cells. After inoculation with the concentrate under the same experimental conditions as in Exp. 1, EPC cells were incubated for 48 h with fresh L-15 medium containing 5% FBS. The relative expression levels of the VHSV N and G genes were determined by qRT-PCR. Briefly, total RNA was extracted from concentrate-inoculated cells using the yesRTM Total RNA Extraction Mini Kit (GenesGen, Busan, Korea), followed by cDNA synthesis using the PrimeScriptTM 1st cDNA Synthesis Kit (Takara, Kusatsu, Japan). For gene expression analysis, qRT-PCR was carried out using the StepOne real-time PCR system (Applied Biosystems, Waltham, MA, USA) with the Prime Q-Master Mix (2X, real-time PCR with SYBR Green I; GenetBio, Daejeon, Korea). The reaction was performed in a final volume of 20 mL, containing 10 mL of 2X Prime Q-Master Mix, 1 mL of each primer, 7 mL of distilled water, and 1 mL of cDNA. The primers and qRT-PCR conditions used are presented in Table 1. The relative quantitation values of the amplified genes in triplicate samples were measured using the 2-DDCt method as described by Rao et al. . Statistical analysis was conducted using a two-way ANOVA with GraphPad Prism software (version 9.5.0), and significance was supported by a p-value < 0.05. In Experiment 3 (Exp. 3), the viral cDNA copies of the extracellular virus from concentrate-inoculated EPC were compared under the same experimental conditions in Exp.1. Following incubation of concentrate-inoculated EPC cells for 48 h, the supernatant was collected, and viral cDNA copies in each triplicate well were determined by qRT-PCR, as described in Section 2.1.3. Statistical analyses were conducted using a two-way ANOVA with GraphPad Prism software version 9.5.0. Statistical significance was set at p < 0.05. 3. Results 3.1. Determination of the LOD95% Value for qRT-PCR To support the reliability of qRT-PCR by differentiating false-positive from non-specific reactions, the LOD95% value was determined in 12 replicates with low viral copies of pDNA harboring N gene fragments. From the standard curve , the correlation coefficient (R2) of the plotted points (viral pDNA copies from 3.17 x 101 to 3.17 x 108 copies/mL) was 0.9975. The LOD95% value was 9.57 pDNA copies/mL . Therefore, the cut-off value for qRT-PCR was set at 9.57 copies/mL. 3.2. Genomic and Infective Recovery Yields Using Iron Flocculation from VHSV-Spiked Seawater Viral recovery yields were found to be higher as more VHSVs were present in the seawater. Based on the viral cDNA copies (spiked VHSV copies at 1 x 105-1 x 101 viral cDNA copies/mL of seawater), the mean +- SD viral genomic recovery yields by ascorbic acid (81.4 +- 9.5%) were superior to oxalic acid (71.1 +- 12.3%) (Table 2). Meanwhile, based on the PFU (spiked VHSV copies at 1 x 105-1 x 101 PFU/mL of seawater), the mean (+- SD) viral infective recovery yields by oxalic acid and ascorbic acid were significantly different at 23.8 +- 22.7% and 4.4 +- 2.7%, respectively (Table 3). Notably, even though VHSVs were spiked at a concentration of 1 x 101 to 1 x 102 PFU/mL in seawater, infectious particles were not identified in concentrates eluted with ascorbic acid. 3.3. Assessment of Viral Infectivity Recovery at Different Re-Suspension Buffers Based on a comparison of cell viability and morphology (Exp. 1), the relative viability of oxalic-acid-resuspended concentrates inoculated on the EPC cells was significantly decreased compared to that of ascorbic-acid-resuspended concentrates . Moreover, the typical CPE caused by VHSV infection, including retracted and rounded refractile cells, were observed to be more pronounced in oxalic acid groups at viral concentrations ranging from 1 x 103 to 1 x 105 PFU/mL . Regarding viral gene expression (Exp. 2), the expression levels of both the VHSV G and N genes were significantly upregulated in the oxalic acid group than in the ascorbic acid group in a concentration-dependent manner . Furthermore, viral cDNA copies of the extracellular virus (Exp. 3) showed a significant difference (approximately a 12 to 531-fold difference) between the oxalic and ascorbic acid groups at spiked concentrations of 1 x 102 to 1 x 105 PFU/mL . 4. Discussion With a low concentration of virus particles in the water below the detection limit, a general molecular assay can be difficult to apply without a concentration process. Among the virus concentration methods, iron flocculation is broadly applied to concentrate aquatic animal viruses in water, such as red sea bream iridovirus (RSIV) , tilapia tilapinevirus (TiLV) , cyprinid herpesvirus-2 (CyHV-2) , and white spot syndrome virus (WSSV) . Notably, several conditions in the iron flocculation process are associated with the viral recovery yield and preservation of infectivity. Herein, to investigate the applicability of surveillance of VHSV in seawater, the viral genome and infective recovery yields were assessed in vitro using different re-suspension buffers (oxalic and ascorbic acid) in the iron flocculation process. There are a variety of VHSV genotypes (I, II, III, and IV). To identify their genotypes and genetic variants, nucleotide sequence analysis is needed. qRT-PCR developed by Garver et al. has been recommended as a validated method sufficient for VHSV surveillance in all life stages of susceptible species . Based on the LOD95% and the reliability of positive results of at least 95%, a cut-off value of qRT-PCR described by Garver et al. , 9.57 pDNA copies/mL were served . Accordingly, quantitative results below the cut-off were not served as valid data in this study. In terms of viral genome recovery yield, viral cDNA copies eluted by oxalic and ascorbic acid were recovered over 70% without significant differences (Table 2). In our previous study , the viral genomic recovery yields of WSSV as a DNA virus also showed no significantly different results between re-suspension buffers. Thus, re-suspension buffer containing oxalic and ascorbic acid for eluting concentrates in iron flocculation might not be associated with genomic recovery yields. Viral infectivity is an important factor as one of the criteria for assessing actual viral transmission in disease surveillance or prediction. Of note, between oxalic- and ascorbic-acid-eluted concentrate, the recovery of viral infectivity in oxalic acid remarkably appeared above VHSV-spiked concentration greater than 1 x 105 PFU/mL (>60%; Table 2) and was different from findings in the ascorbic acid group (<10%). Notably, at a low viral concentration (<1 x 102 PFU/mL), the infective VHSVs were slightly recovered by oxalic acid (<10%). In contrast, infective VHSVs were not recovered in ascorbic acid. As the proteins or lipids in serum can protect the surface of viral particles from damage, several previous studies have used serum sources (e.g., FBS) during viral concentration or storage to preserve viral infectivity . However, in the iron flocculation experiment in this study, the infective recovery yields were not improved by the addition of FBS (exhibited less than 3.9% of recovery yield at 0.1 and 1% FBS addition in either seawater or re-suspension buffer; data not shown). Since the recovery yield of the iron flocculation method is influenced by the formation of the Fe-virus flocculate and its dissolution during the re-suspension process , the macromolecular substances present in FBS might interfere with the formation of the flocculate (spiked in seawater) and re-suspension process (spiked in oxalic acid buffer). The EPC cell viability in the oxalic acid group was significantly lower than that in the ascorbic acid group . Moreover, the levels of viral gene expression and extracellular viral cDNA copies in oxalic acid were significantly higher than those in ascorbic acid, supporting the infectivity preservation of concentrates eluted by oxalic acid in the iron flocculation process . pH is one of the most important parameters that influence the preservation of viral infectivity, as strong acidic (approximately <pH 3) or basic (>pH 12) conditions could damage viral particles. Meanwhile, a previous study exhibited that VHSV could maintain infectivity between pH 6 and 9 for 24 h . Based on these findings, the pH levels (approximately 6.0) of the re-suspension buffers used in this study were confirmed to be sufficient for viral re-suspension without affecting VHSV infectivity. In iron flocculation, ascorbic acid advances the dissolution of iron hydroxide by reducing Fe3+ to Fe2+ to allow chelation with EDTA . In more detail, when reducing the Fe3+ to Fe2+, ascorbic acid enhances the production of radicals by the Fe2+/EDTA/H2O2 reaction complex. These radicals are generated by the one-electron oxidation of the respective reduced compounds, which promotes an additional oxyradical-mediated chain reaction . Even though ascorbic acid, known as vitamin C, likely seems to be associated with improved EPC cell viability, oxygen-free radicals produced by it might reduce the ability to preserve infectivity during the elution step in iron flocculation, consistent with a previous study . Oxalic acid advances the dissolution of iron hydroxide by binding directly to iron hydroxide to release trivalent Fe3+, which prevents re-precipitation and allows chelation with EDTA during iron flocculation . Moreover, oxalic acid is used as a reductant to remove surface-adsorbed 55Fe from phytoplankton cells and other particles, reacting with EDTA by chelation . Thus, as EDTA could inactivate the virus, oxalic acid reacting with EDTA might prevent virus inactivation during re-suspension. 5. Conclusions In conclusion, this study evaluated the applicability of iron flocculation to the concentration of VHSV-IVa particles in seawater by comparing two re-suspension buffers (oxalic and ascorbic acid buffers). The viral genomic recovery yields were not significantly different between re-suspension buffers. However, although oxalic acid conserves helped to recover VHSV-IVa infectivity better than ascorbic acid at viral concentrations above 104 PFU/mL (oxalic acid, >35%; ascorbic acid, <10%), the infective VHSVs were not sufficiently recovered at a low viral concentration (<1 x 102 PFU/mL; oxalic acid, <10%; ascorbic acid, no recovery). This result was supported by an in vitro assessment of EPC cells inoculated with concentrates in terms of PFU recovery yield, cell viability, viral gene (G and N) expression, and extracellular virus titer. Nevertheless, for disease surveillance and prediction in aquaculture, it is essential to evaluate the validity of Fe flocculation in natural environmental samples. Author Contributions Conceptualization, N.-G.R. and K.-I.K.; methodology, N.-G.R., E.-J.B. and M.-J.K.; formal analysis, N.-G.R., E.-J.B. and M.-J.K.; writing--original draft preparation, N.-G.R.; writing--review and editing, K.-I.K.; project administration, K.-I.K.; funding acquisition, K.-I.K. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available upon request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Quantification of viral cDNA was performed by VHSV specific qRT-PCR using primers and a TaqMan probe, and the cut-off value was set based on LOD95%. (A) Standard curve indicating a linear relationship between Ct (cycle number of threshold) values and the serial ten-fold pDNA (3.17 x 101 to 3.17 x 108 copies/mL) containing the N gene fragment. (B) The probability of detection (POD) curve was indicated with a solid line, and a 95% confidence interval was highlighted as the gray zone. LOD95% (9.57 viral pDNA copies/mL) was denoted using a dotted line. Figure 2 Comparison of cell viability and morphology in EPC cells inoculated by VHSV concentrates. VHSV-spiked seawater (1 x 101 to 1 x 105 PFU/mL) was concentrated using the oxalic or ascorbic acid buffer, and after 48 h post-infection in EPC cells, the cell viability and morphology were analyzed. Statistical analysis was performed via a two-way ANOVA (*, p < 0.05) using GraphPad Prism software version 9.5.0. (A) Relative cell viability (means +- standard deviation, SD). (B) The morphology of EPC cells 48 h post-infection. Figure 3 Comparison of the preserved infectivity of concentrates resuspended in oxalic and ascorbic acid buffers. Concentrates from VHSV spiked seawater (1 x 101 to 1 x 105 PFU/mL) were inoculated in EPC cells, and 48 h post-infection, viral gene expression levels in EPC cells and total viral cDNA copy number in the supernatant were quantified using qRT-PCR (means +- SD). A significant difference was found via two-way ANOVA (*, p < 0.05) using GraphPad Prism version 9.5.0. (A) G gene expression. (B) N gene expression. (C) Extracellular viral titer (viral cDNA copies/mL). animals-13-00943-t001_Table 1 Table 1 Primers uses in this study. Purpose Primer Primer Sequence (5'-3') Condition Product Size (bp) Reference Quantification VHSV (N gene) TaqMan real-time F-ATGAGGCAGGTGTCGGAGG R-TGTAGTAGGACTCTCCCAGCATCC 50 degC, 2 min; 95 degC, 10 min (95 degC, 15 s; 60 degC, 1 min) x 40 82 Garver et al. Probe 5'-FAM-TACGCCATCATGATGAGT-BHQ1-3' Gene expression EPC cell b-actin F-GCTATGTGGCTCTTGACTTCGA R-CCGTCAGGCAGCTCATAGCT 94 degC, 10 min (94 degC, 20 s; 58 degC, 1 min) x 35 85 Yang et al. VHSV Glycoprotein F-AACTGTCTCCAAAGAAGTGTGT R-GCCATCAAGGAGATAATGTG 95 degC, 30 s (95 degC, 15 s; 60 degC, 15 s; 72 degC, 15 s) x 40 94 Zhang et al. Nucleoprotein F-TTGGAGAACTGCAACACTTCAC R-CGGTCAGGATGAAGGCGTAG 82 animals-13-00943-t002_Table 2 Table 2 VHSV genomic recovery yield from artificial virus-spiked seawater by qRT-PCR. Re-Suspension Buffer Spiked Viral cDNA Copy (Viral cDNA Copies/mL) Recovered Viral cDNA Copy (Viral cDNA Copies/mL) Genomic Recovery Yield (%) a Oxalic acid 1.0 x 105 4.3 x 107 +- 7.4 x 106 87.7 +- 14.8 1.0 x 104 3.4 x 106 +- 1.5 x 105 68.3 +- 30.0 1.0 x 103 3.5 x 105 +- 5.8 x 104 70.6 +- 11.4 1.0 x 102 2.9 x 104 +- 1.6 x 104 58.0 +- 32.0 1.0 x 101 N.D. b - Mean of recovery yield 71.2 +- 12.3 Ascorbic acid 1.0 x 105 4.7 x 107 +- 4.2 x 106 95.2 +- 8.4 1.0 x 104 3.8 x 106 +- 4.7 x 105 76.4 +- 9.4 1.0 x 103 4.0 x 105 +- 8.1 x 104 79.5 +- 16.3 1.0 x 102 3.7 x 104 +- 3.8 x 103 74.4 +- 7.6 1.0 x 101 N.D. b - Mean of recovery yield 81.4 +- 9.5 a Recovery (%) = (Recovered viral cDNA copies per mL x vol/spiked viral cDNA copies per mL x vol) x 100. b ND, not detected or undetermined based on the LOD95% for qRT-PCR. animals-13-00943-t003_Table 3 Table 3 VHSV infective particle recovery yield from artificial virus-spiked seawater by plaque assay. Re-Suspension Buffer Spiked PFU (PFU/mL) Recovered PFU (PFU/mL) PFU Recovery Yield (%) a Oxalic acid 1.0 x 105 3.2 x 107 +- 2.0 x 106 64.0 +- 4.0 1.0 x 104 1.8 x 106 +- 1.3 x 105 35.5 +- 2.5 1.0 x 103 4.1 x 104 +- 6.1 x 103 8.1 +- 1.2 1.0 x 102 2.7 x 103 +- 2.9 x 102 5.5 +- 0.6 1.0 x 101 3.0 x 102 +- 1.1 x 102 6.0 +- 2.2 Mean of recovery yield 23.8 +- 22.7 Ascorbic acid 1.0 x 105 3.6 x 106 +- 3.9 x 105 7.2 +- 0.8 1.0 x 104 2.6 x 105 +- 1.6 x 104 5.2 +- 0.3 1.0 x 103 4.3 x 103 +- 1.0 x 103 0.9 +- 0.2 1.0 x 102 N.D. b - 1.0 x 101 N.D. b - Mean of recovery yield 4.4 +- 2.7 a Recovery (%) = (Recovered PFU per mL x vol/spiked PFU per mL x vol) x 100. b ND, undetermined based on the plaque assay. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000096

The birth process is a crucial event for piglet survival. Along with increasing litter sizes, not only has the duration of parturition increased, but placental blood flow per piglet has reduced and placental area per piglet has become smaller, making these piglets more susceptible for hypoxia. Diminishing the risk of piglet hypoxia by either reducing the total duration of parturition or increasing fetal oxygenation may reduce the incidence of stillbirth and early post-partum mortality. This review discusses options to do so by nutritionally supporting the sow in the final pre-partum period, after discussing the role of uterine contractions and placental blood flow. Providing sufficient energy seems to be a logical first step, but also other nutrients needed for uterine contractions, such as calcium, or enhancing uterine blood flow by using nitrate seem promising. These nutrient requirements may depend on litter size. sow parturition placental blood flow uterine contractions energy requirements This research received no external funding. pmc1. Introduction The parturition process is challenging for both the sow and her piglets. For the sow, parturition is an energy demanding, stressful, and painful event . For piglets, it is also a stressful event, and the odds of dying are highest during parturition and the first days of life . The parturition process in sows has been studied mainly from a behavioral or endocrine point of view . Only a few studies, however, have investigated peri-partum uterine contractions and placental or umbilical blood flow and the changing metabolic status and nutritional requirements of the sow during the peri-partum period . Along with increases in litter size, the challenges to the perinatal piglet have increased, which are either related to in utero circumstances such as a decrease in uterine blood flow per piglet , a decrease in piglet and placental size , or an increase in farrowing duration . The survival rate for piglets mainly depends on fetal oxygenation, which in turn is related to farrowing duration , the duration and intensity of uterine contractions , and placental blood flow and therefore oxygen flow . The maternal diet needs to provide the nutrients for uterine contractions and for sufficient placental blood flow, and its role has been investigated in recent studies . This review focuses on possible interventions in the maternal diet that may facilitate the parturition process, affecting uterine contractions and/or placental blood flow in the perinatal period. 2. Uterine Contractions The parturition process in the sow can be divided into three stages: (1) increase in myometrial activity and dilation of the cervix (approximately 6-12 h), (2) expulsion of the piglets with the sow lying down and in abdominal straining (approximately 5-8 h), and (3) expulsion of the placenta (approximately 4 h, which may already start during stage 2) . Several reviews discuss the complex endocrine changes in the peripartum period , so here, we only highlight the major changes. The increase in myometrial contractions in stage 1 results from a cascade of endocrine events. Fetal cortisol induces a release of endometrial PGF2a, which induces luteal regression and thereby results in a decline in progesterone. PGF2a also stimulates the release of relaxin by the corpora lutea, producing oxytocin and uterine smooth muscle contractions . Exogenous prostaglandin injection to induce luteal regression and thereby induce parturition does not influence the parturition process itself. It is mainly a tool to optimize parturition management when given, at most, two days before parturition . The placenta produces estrogens, and the changed progesterone/estrogen ratio increases the expression of oxytocin receptors on the myometrium, causing an increase in number as the ratio between progesterone and estrogen changes . The changed progesterone/estrogen ratio stimulates myometrial contractions starting at 4-9 h before the expulsion of the first piglet. These contractions last for 2-3 min each and occur at regular intervals . Contractions keep increasing in frequency and amplitude, and straining efforts of the sows start to appear the last few hours before expulsion of the first piglet . As soon as the first fetus enters the cervix, stage 1 of parturition is considered to be completed . Then, the Ferguson reflex is activated, releasing oxytocin from the pituitary. The increased oxytocin levels stimulate abdominal muscle straining to expel fetuses . The frequency of uterine contractions is highest when piglets and the placentae are being expelled , but large variations occur among sows in frequency, duration, and amplitude and for an individual sow from one hour to the next of the expulsion phase . During this phase, on average, uterine contractions last for 1-2 min and occur at a frequency of 18 per hour . Maffeo et al. gained insight into the frequency and amplitude of contractions during different timepoints of the parturition process using two implanted strain gauges (one in each horn) during spontaneous births. The frequency, amplitude, and duration of contractions 12, 5, and 1 h before the birth of the first piglet, during piglet expulsions, and during placenta expulsion are shown in Figure 1. Exogenous oxytocin injections can maintain and reinforce spontaneous parturition by increasing the frequency of contractions 13-fold and their intensity 2-fold when compared to spontaneous contractions. This can result in a reduced duration of farrowing, but could also impair the normal physiology of contractions , which may result in a reduction in piglet vitality at birth and an increase in incidence of stillbirth . This is likely due to damage of the umbilical cord or a decrease in placental blood flow . During the expulsion phase, tubo-cervical contractions move the fetuses towards the cervix. In addition, cervico-tubal contractions occur, which are likely meant to shorten the uterine horns and to prevent accumulation of fetuses at the caudal ends of the uterine horns and/or to keep fetuses at a fixed place to keep the umbilical cord functional before expulsion . Contractions are initiated at the two ends of the horns and convey (either as a tubo-cervical or cervico-tubal contraction) to the proximal end of the horns , but may rebound in the opposite direction when reaching the end of the horn . Empty parts of the horn also contract . Cervico-tubal contractions end when the horn is empty of piglets, indicating that the presence of piglets close to the cervix initiate these contractions . It is estimated that four to five uterine contractions, with an average duration of 11.5 s and an intensity of 9.4 mm Hg, are needed to expel one fetus . As soon as the horns are completely empty, contractions are only tubo-cervical and appear very frequent and regular for placentae expulsion . It is unknown whether or not there is synchrony in the timing of contractions between the two horns, but this seems likely, since muscle fibers fuse at the common uterine body , and the birth order of fetuses from both uterine horns appears to happen fully at random from one horn or the other . It is also unclear whether crowding of piglets occurs during contractions. It might be that crowding does occur when fetuses are stuck or when a stillborn piglet causes a delay in the birth process. The birth interval after which a stillborn piglet is born is approximately twice as long as that of a liveborn piglet (28 vs. 15 min) . It is unknown whether the increase in birth interval is a cause or consequence of the increased birth interval . 3. Uterine Blood Flow Most studies evaluating the duration of farrowing in sows only consider stage 2 of parturition, the time during which fetuses are expelled , as this stage determines the level of asphyxiation of piglets and can easily be observed. Asphyxiation mostly occurs due to strong uterine contractions combined with placental space limitation and/or reduced placental-uterine connection, which together reduce or obstruct placental blood flow . As an initial response to reduced blood oxygen levels, fetal heartrate drops and fetal movements increase, which in turn promote myometrial contractions, making a positive feedback system to reduce the duration of farrowing . In fetuses with a prolonged inadequate oxygen supply, blood CO2 concentrations will rise, and hypoxia starts to occur. To reduce fetal oxygen consumption, not only will fetal limb and body movements reduce , but also fetal heartrate falls (bradycardia) and metabolic rate reduces . When fetal blood O2 concentration drops below a certain threshold level, adenosine triphosphate (ATP) production shifts to anaerobic glycolysis, and fetal lactate levels increase . This anaerobe metabolism is faster than aerobe metabolism, but can only provide energy for a short period of time (up to 2 min) . Lactate also lowers blood pH, which can affect functioning of the central nervous or cardiovascular system . Lactate levels at birth have been related with chances of dying during lactation. For example, English and Wilkinson showed that piglets that died pre-weaning had higher blood lactate concentrations at birth than survivors (383 vs. 303 mg lactate/mL blood; p < 0.01, respectively). Furthermore, Langendijk et al. found a higher pre-weaning mortality when blood lactate concentrations in umbilical cord blood was increased (8.5% and 10.9% for 4.45-6.40 mmol/L and >6.40 mmol/L, respectively). Thus, the level of asphyxia at birth appears to be related to the chances for pre-weaning survival. It is not known whether the number, duration, and amplitude of contractions, or the duration of stage 1 of parturition, is related to litter size. It is also not known whether the durations of stage 1 and 2 of parturition are related. It is known that the duration of stage 2 of parturition is related to litter size; it indeed takes more time to deliver more piglets . Combining the data of 15 studies that measured the duration of stage 2 of parturition in the last 18 years shows an estimated increase of 27 min in duration of stage 2 of parturition per extra piglet . The deviation from the predicted line for farrowing duration based on litter size is sometimes quite large, which may be caused by differences in e.g., breed, housing, or management (i.e., use of birth assistance and exogenous hormones). Summarizing, the total duration of parturition (stage 1, 2, and 3), in which a sow experiences frequent and powerful uterine contractions, can take up to 24 h in the hyper-prolific sow . Research on duration of parturition mainly focuses on phase 2 of parturition, i.e., the period during which the piglets are born, since this phase is the most easy to observe. It is unknown what the impact is of phase 1 on the sow, her piglets, and how related phase 1 and 2 of parturition are with each other. Most of the studies investigating the intensity, number, and duration of uterine contractions for the different phases of parturition in the sow were done three to four decades ago . It is unknown whether the intensity, number, and duration of uterine contractions relate to the current litter sizes and other aspects of the current highly prolific sow. 4. Placental and Umbilical Cord Functionality The placenta is responsible for nutrient and oxygen exchange between the sow and her fetuses. The fetus has a diffuse placenta in which many closely spaced chorionic villi are distributed over the entire outer surface of the chorion , which ensures transport and diffusion of nutrients from the maternal to the fetal blood. Additionally, specific structures called areolae on the placenta absorb the products secreted by the endometrial glands (e.g., growth hormones, hormones, transport proteins lymphokines, cytokines) . The surface area of the chorio-allantoic membrane mainly increases in size between day 35 to 70 of gestation, with little change between day 70 to 100 of gestation . Vascularization of the allantoic membrane starts at approximately day 15 post-insemination, i.e., 2 days after contact between the trophoblast and maternal epithelium , and increases until mid-gestation, after which vascularity remain relatively constant . By that time, blood vessels occupy about 3-4% of the chorio-allantoic membrane, but with large variation among individual fetuses, among litters, and between breeds . Blood capillaries from the chorionic villi merge and eventually form larger vessels that enter the umbilical cord . In addition to vascularization, nutrient supply to the fetus is also affected by uterine blood flow, which increases as gestation progresses . Although it seems likely, it is not known, whether vascularization of the placenta and placental blood flow are related. Blood flow and placental area per piglet both seem negatively correlated with litter size, which likely explains why average piglet birth weight decreases as litter size increases . No studies were found showing a clear relationship between litter size and placental vascularization. Wilson et al. found differences between breeds in placental vascularization at the fetal-maternal interface. Vascular density was higher in Meishan placentas compared to Yorkshire placentas, although placental size was larger in Yorkshire sows. Whether placental blood flow differs between breeds has not been evaluated. Placental characteristics and the incidence of pre-weaning mortality appear to be related, although these relationships might be confounded with piglet birth weight. Both placental surface (-20.4%) and placental weight (-14.8%) were lower in piglets that died before weaning compared to surviving conspecifics, which was most likely caused by a lower birth weight of piglets that died before weaning . Baxter et al. found no difference in vascularization score of placentas of piglets that survived or died before weaning. The umbilical cord connects the fetus to the placenta, and it contains one vein that carries oxygen and nutrient-rich blood to the fetus and two smaller arteries that transport deoxygenated blood from the fetus back to the placenta . These vessels are surrounded by Wharton's jelly, a gelatinous connective tissue consisting mainly of hyaluronic acid, in which collagenous and reticular fibers form a loose meshwork . An intact and functional umbilical cord is of crucial importance for fetal oxygen and nutrient supply. Umbilical cord length of piglets was found to be 35 cm on average (ranging between 17 to 50 cm) and was positively correlated with piglet weight . Umbilical cord length is not correlated to the position of a piglet in the uterus, but its elasticity (up to 37.5% of its length) allows it to stretch as a piglet is transported through the uterine horn at parturition, making it possible for piglets at the end of the uterus to be born with intact umbilical cords. The tension required to break an umbilical cord varies from 545 to 2000 g . The percentage of piglets born with a broken umbilical cord lies between 21 to 71% , and Rootwelt et al. showed that broken umbilical cords occur most in the second and last third of piglets born (2.3 times more often compared to the first third of piglets born). When or where an umbilical cord breaks has, to our knowledge, not been studied in pigs. It can be hypothesized that the umbilical cord breaks at a weak spot or occurs randomly over the full length of the umbilical cord, potentially caused by a weak spot in the Wharton's jelly or the first place where umbilical cord blood flow has stopped. It is also unclear which placental or other sow and/or piglet characteristics might be related with umbilical cord length, thickness, strength, or breaking point. It seems likely that larger piglets, which have a larger placenta , also have a thicker umbilical cord that may also be less prone to breaking. Curtis et al. suggested that stillborn piglets (that weighed less than live-born litter mates) have a smaller umbilical cord that is more likely to break, suggesting a relationship with piglet birth weight and umbilical cord thickness/strength. However, Langendijk and Plush found a similar weight distribution in live and stillborn piglets, suggesting that the hypothesis of Curtis et al. might not be true. Piglets born alive but with a broken umbilical cord (as observed at the moment of birth) showed a lower vitality score and had an higher risk for post-partum death compared to piglets born with an intact umbilical cord . A recent review estimated the association between incidence of stillbirth and a broken umbilical cord before expulsion to be 50% or more . In addition, even when the cord does not break, extensive stretching might lead to vasoconstriction and limited blood flow, increasing the risk for stillbirth . In summary, in larger litters, placental blood flow per piglet is reduced and placental area per piglet is smaller , which likely explains why average piglet birth weight is lower and partially explains why incidence of pre-weaning mortality increases. An intact and functional umbilical cord is key for fetal oxygen and nutrient supply and therefore survival. Studies on how, where, or when an umbilical cord breaks and which sow and/or piglet characteristics are related to its breaking are limited. A better understanding of the complex interactions between placental/umbilical cord blood flow, contractions, breaking of the umbilical cord, and other characteristics of the modern sow might provide insights in how perinatal piglet losses can be reduced. In conclusion, placental and/or umbilical cord blood flow might be under pressure in large litters, which might be related to stillbirth and pre-weaning mortality. 5. The Potential of Maternal Nutrition to Reduce Farrowing Duration Relationships between placental characteristics, uterine contractions, placental and umbilical cord blood flow, and piglet losses are summarized in Figure 3. Additionally, in this figure, the potential effects of maternal nutrients on these events are included. Providing the right nutrients to the sow and her fetuses may not only enhance placental development and fetal growth but could also affect uterine blood flow in the perinatal period and/or affect the duration of farrowing. Studies evaluating nutritional solutions aiming to reduce farrowing duration by enhancing uterine contractions or affect placental characteristics and therefore affecting piglet losses during or shortly after parturition will be discussed in the next paragraph. Nutritional interventions in the perinatal period aiming to decrease stillbirth and to increase piglet vitality right after birth should stimulate uterine contractions (frequency or intensity), increase placental nutrient and/or oxygen supply to the fetus, and/or provide the sow with the energy to prevent fatigue. To prevent constipation and metritis, mastitis and agalactia (MMA) feed allowance in some European countries is lowered to 2.0-3.0 kg/sow/day, beginning 2-3 days before the expected farrowing date. It can be questioned whether this lower energy and nutrient intake and the type of nutrients supplied sufficiently facilitates energy and nutrient requirements during parturition. Consequently, feeding strategies in the perinatal period might need to be reconsidered. 5.1. Energy The total duration of farrowing (stage 1, 2, and 3) can take up to 24 h in the hyper-prolific sow and is positively related to litter size . When we expect our sows to give birth to larger litters, we should provide them with the right nutrients to perform this activity. Focus on energy requirements seems to be a logical first step, since farrowing is likely a highly energy-demanding activity . It seems likely that modern sows do experience a limitation in available energy around farrowing. Van Kempen et al. suggested that sow exhaustion during farrowing caused by energy depletion could impair the number and intensity of uterine contractions, thereby increasing the duration of farrowing and consequently increasing stillbirth rate. That sow exhaustion occurs was also suggested by Mosnier et al. , who found a higher sow plasma lactate concentration at day 1 post-partum (approximately 1.4 mmol/L) compared to day 4 (approximately 0.9 mmol/L). The higher concentration of lactate in sow blood is likely due to increased metabolic activity and uterine contractions of sows during farrowing (also seen by an increase in body temperature ). A recent study, in which lactate levels were determined more frequently around parturition (every 6 h pre-farrowing and every 3 h post-partum), showed that sow blood lactate levels were indeed increased during parturition, but were already increased at 9 and 3 h before the expulsion of the first piglet , which is likely related to higher activity during nest-building behavior and by increased uterine contractions during phase 1 of parturition. Three hours after farrowing, lactate levels started to decrease again , indicating that sows shifted back to their aerobe metabolism. The energy requirement for the farrowing process is expected to be comparable to moderate to heavy exercise . Recent estimates of energy requirements estimated during the transition period (10 days pre-farrowing to 10 days post-farrowing) included maintenance, heat loss, mammary growth, fetal growth, and colostrum/milk production, but not requirements for the farrowing process itself, resulting in the lowest estimated energy requirements at the day of farrowing . Other recent evaluations of amino acid and energy requirements also did not include parturition requirements . Feyera et al. were the first to give an estimate of energy requirements of farrowing, which was based on an evaluation of different feed amounts and therefore daily energy intake around farrowing, aiming for the shortest farrowing duration and lowest number of interventions during farrowing. The estimated energy requirement for farrowing was 16 MJ ME (approximately 30% of the total energy requirements on the day of farrowing). In Figure 4, the calculated energy requirements of sows provided by Theil et al. and Feyera et al. are combined for the last day of gestation, the day of farrowing, and day 1 and 2 of lactation. These estimates included energy requirements for maintenance purposes, heat loss due to reproduction costs and diet induced thermogenesis , colostrum/milk production, fetal growth, mammary growth, growth of uterine tissue, nest-building behavior, and energy requirements for farrowing. In addition to the study by Feyera et al. , Che et al. evaluated effects of energy intake on the day of farrowing on farrowing duration. Strategies of how the energy intake was increased differed between these two studies. Che et al. increased energy level of the diet by increasing fat levels (soybean oil) and increasing daily feed supply with 0.2 kg/sow/day (from day 90 of gestation until farrowing), while Feyera et al. increased feeding levels (from 1.8 to 5.0 kg/sow/day from day 108 of gestation until 24 h after farrowing). Both studies also differed in average litter size and average farrowing duration. Because farrowing duration increases with increasing litter size , it can be assumed that energy requirements for farrowing also increase with litter size. For estimating the energy requirements per piglet born or per 60 min of farrowing, it was assumed that the energy requirements were met when farrowing duration was shortest. For the treatments with the shortest farrowing duration, number of piglets born and average farrowing durations are shown in Table 1. Calculations on average energy requirement per piglet and per 60 min for both studies turned out to be quite close. Calculations suggest that optimal daily energy intake on the day of farrowing depends on the average litter (2.44 MJ ME/piglet born) and/or farrowing duration (8.66 MJ ME/hour of farrowing duration). Since only two studies were available, the findings should be confirmed in additional experiments. 5.2. Glucose as a Source of Energy during Farrowing ATP (adenosine triphosphate), derived primarily from glucose by glucogenesis, is the main energy source for uterine contractions . Blood glucose levels rise during farrowing, which can be explained by the increased glucolysis under the influence of adrenalin and cortisol . Sow blood glucose levels originate from carbohydrates in the diet and/or glycogen reserves. A negative correlation was found between sow arterial glucose level, measured 1 h after the birth of the first piglet and farrowing duration, suggesting that a low energy status of the sow indeed increased farrowing duration. Feyera et al. showed that farrowing duration linearly increased with time when the last meal was more than 3.13 +- 0.34 h before the onset of farrowing (defined as the birth of the first piglet). Theil et al. showed that sows lack glucogenic energy on the day of farrowing and start using the glycerol part of triglicerides as an energy source (rather than nonesterified fatty acids in a normal catabolic state), making triglycerides and glucose the only two energy sources for the uterus during farrowing . Although duration of farrowing has been related to the energy status of the sow, other dietary factors (e.g., type of energy, mineral levels, other supplements) might play a role as well. These factors will be discussed below. 5.3. Other Carbohydrates The role of dietary fibers in sow nutrition around farrowing are mostly studied in relation to the prevention of constipation and therefore easy passage of piglets through the birth canal . Effects of dietary fibers on the duration of farrowing have been reviewed extensively and will thus not be discussed here. No information is available on possible effects of dietary fibers on uterine contractions. However, dietary fibers can be a source of energy from the gastrointestinal tract up to several hours after a meal . The type of carbohydrates consumed appears to be an important factor for exercise performance in athletes . The glycemic index (GI) of carbohydrates is a tool to predict blood glucose, insulin, and therefore energy supply of diets High GI foods (e.g., sugars and starches) provide a high and relatively short peak in blood glucose. Low GI feed ingredients (e.g., pectins) could provide lower but longer levels of blood glucose. Combining different types of carbohydrates, providing fast-, medium-, and slow-release glucose, might increase the period after feeding in which sufficient glucose is available to supply the energy needed for the farrowing process. It can be concluded that a better understanding of perinatal energy requirements (and the composition of these energy sources) is needed to optimize the farrowing process and consequently reduce piglet losses. 5.4. Calcium and Magnesium Calcium is an essential mineral for muscle contractions and therefore also essential for myometrial contractions during farrowing . Studies evaluating calcium requirements in the peri-partum period are limited. Geisenhauser et al. evaluated effects of a single-dose calcium supplementation on top of feed (400 mmol Ca, source calcium lactate) on the day of farrowing and found a significant reduction (-34% on sow level) in the incidence of dystocia (defined as birth interval > 60 min) and decreased time for placenta expulsion (4-19 min faster). Le Cozler et al. evaluated plasma calcium levels in gilts before, during, and after farrowing and observed no change in plasma calcium levels during parturition (measuring 2 h before the birth of the first piglet to 7 h after). This is in contrast to a recent study of Nielsen et al. , who also evaluated plasma calcium profiles around parturition (measuring 33 h before the birth of the first piglet until 24 h after farrowing) and found a drop in calcium levels 9 and 3 h before the expulsion of the first piglet, but no changes in sow blood calcium levels during the first 24 h post-farrowing. These ambiguous results in perinatal plasma calcium profiles might be related with differences in litter size (12.2 vs. 24.6 total born for Le Cozler et al. and Nielsen et al. , respectively) and/or farrowing duration (175 vs. 486 min for Le Cozler et al. and Nielsen et al. , respectively), suggesting that hyper-prolific sows might have higher calcium requirements in the perinatal period. The benefits of calcium supplementation to sows before farrowing on incidence of stillbirth and piglet vitality remain unclear but might be related to the plane of feeding in this period and the calcium source and concentration in the diet. Magnesium promotes the relaxation of smooth muscle cells and inhibits contractions of the uterine myometrium. Magnesium sulphate is used in human medicine to prevent pre-term labor and pre-term birth , which suggests that magnesium supplementation before farrowing might have a negative effect on myometrial contractions. However, Le Cozler et al. observed a drop in magnesium levels in sow blood 1 h after the first piglet was born, which was likely due to the role of magnesium in dephosphorylation of ATP to provide energy for muscle contractions, as ATP must be bound to a magnesium ion to be biologically active. The synergistic and antagonistic role of magnesium with calcium might explain why calcium levels were constant and magnesium levels dropped during parturition . In pig husbandry, magnesium is used in sow diets as an effective laxative to prevent constipation . However, as with calcium, research on magnesium supplementation for sows in the perinatal period is limited. Plush et al. showed an increase in stillbirth incidence (+0.3 stillborn piglets/litter, p = 0.01) when sows were supplemented with magnesium sulphate (2.85 kg/mton feed, receiving 2.5 kg of feed/sow/day) from 5 days pre-farrowing until 3 days post-farrowing. It can be speculated that magnesium induced relaxation of the myometrium, which consequently increased the duration of farrowing, but this was not evaluated. Vitamin D is essential for intestinal calcium absorption, plays a central role in calcium homeostasis, and directly impacts muscle contractions . Vitamin D is usually added to sow diets in the form of cholecalciferol (vitamin D3), which is transported to the liver and hydroxylated to 25-hydroxycholecalciferol [25(OH)D3], or by directly feeding the 25(OH)D3 . Although requirements for sows are known for gestation and lactation , vitamin D requirements specifically during parturition are not. Some studies evaluated maternal vitamin D supplementation on offspring status in muscle fibers and therefore lean development and growth performance in later life . However, we found no studies that evaluated maternal vitamin D supplementation and the effects on parturition characteristics and piglet losses. It can be concluded that although both calcium and magnesium play an important role in myometrial contractions, research on supplementing sows with one or both of these minerals in the perinatal period is very limited. Consequently, calcium and magnesium requirements of the sow in the perinatal period and potential factors influencing these requirements (e.g., litter size) are currently unknown. 5.5. Vasoactive Components Dietary arginine (as recently reviewed by ) and nitrate supplementation to the sow both aim to influence placental vascularization and/or placental-fetal blood flow. Although converted differently, both arginine (oxidized in a reaction catalyzed by the NO synthase family ) and nitrate (non-enzymatically converted via the NO3-NO2-NO pathway ) are precursors for nitric oxide (NO). NO is an endothelium-derived relaxing factor, causing vascular vasodilation , which plays an important role in regulating placental-fetal blood flow and consequently nutrient and oxygen transfer from mother to fetuses . This higher blood, and therefore nutrient and oxygen, flow may lead to an increased piglet birth weight and/or oxygenation during parturition, which is hypothesized to lead to a lower incidence of stillbirth, increased vitality, and therefore a decreased incidence of pre-weaning mortality. Arginine is mostly supplemented in the first stage of gestation to increase placental angiogenesis , with several studies showing a beneficial effect on embryo survival, fetal development, placental weight, piglet weight, and number born alive (as reviewed by ). Fewer studies have used arginine supplementation up to or close to the moment of parturition. Neither placental weight (when supplementing 1% of L-arginine from day 22 until day 114 of gestation ) nor piglet birth weight and stillbirth rate (when supplementing 25.5 g/d from day 77 of gestation until term ) were affected in these studies. Van den Bosch et al. evaluated effects of dietary nitrate supplementation starting 7 days before farrowing and found a linear dosage effect on piglet vitality (by scoring individual piglet vitality ) and piglet birth weights and a tendency for a lower pre-weaning mortality rate, which may have been driven by an increased placenta size and/or vasodilation. The use of NO precursors to enhance either placental vascularization and/or blood flow may benefit piglet vitality and survival. 6. Conclusions Parturition is not only a stressful, painful, and energy-demanding event for sows, but it also affects the perinatal survival of her offspring. Along with increases in litter size, farrowing duration has increased and uterine blood flow per piglet, placental development, and piglet weight have decreased, which has increased the challenges to the perinatal piglet. Potential maternal nutritional factors that stimulate uterine contractions and/or increase uterine blood flow (by providing adequate energy and/or minerals or by the use of NO precursors) may reduce the duration of parturition and/or increase perinatal piglet survival. However, knowledge on the exact nutritional requirements before, during, and after parturition and the impact that meeting these requirements may have on piglet characteristics and perinatal survival is limited. Current feeding strategies in the perinatal period might not support the modern hyper-prolific sow adequately in energy and other nutritional requirements. Author Contributions M.v.d.B. was the main author of this publication. H.v.d.B., N.S. and B.K. contributed to content and reviewed this publication. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data presented in this review is available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Myometrial contractions in the sow 12, 5, and 1 h pre-partum during spontaneous parturition (* indicate the birth of a piglet) and during the placentae expulsion, measured by two stain gauges implanted in each uterine horn as adapted from Maffeo et al. . Figure 2 Relationship between average litter size and average duration of parturition (stage 2, expulsion of piglets) based on averages of 15 studies conducted over the last 18 years . Figure 3 Relationships between farrowing and placental characteristics on piglet vitality, incidence of stillbirth and pre-weaning mortality, and potential roles of maternal nutrients on these relationships. Dotted lines indicate a negative effect. Grey boxes will be discussed in current paragraph. Figure 4 Energy requirements of sows on the last day of gestation, the day of farrowing, and day 1 and 2 of lactation, adapted from , including estimated energy requirements for additional heat loss, nest building behaviour and farrowing . ME = metabolizable energy. animals-13-00910-t001_Table 1 Table 1 Calculation on estimated energy requirements on the day of farrowing per piglet and per 60 min of farrowing based on Che et al. and Feyera et al. . Study Che et al. Feyera et al. Average Average litter size 14.8 20.4 Average farrowing duration (min) 238.4 359.8 Optimal daily energy intake determined based on the shortest duration of farrowing (MJ ME/sow/day) 33.8 53.0 Energy requirement on the day of farrowing per piglet (MJ ME) 2.28 2.60 2.44 Energy requirement on the day of farrowing per 60 min of farrowing (MJ ME) 8.49 8.83 8.66 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000097

This review aims to discuss the effects of dietary fiber sources with various levels on stereotypic behaviors in sows. There are a variety of dietary fiber sources that are supplemented to feeds for sows. However, dietary fiber sources have different physio-chemical properties, leading to controversial results in feed motivation, nutrient digestibility, and behaviors in sows fed fiber-rich diets. Findings from previous studies indicated that soluble fiber delays nutrient absorption and decreases physical activity after feeding. In addition to this, it increases volatile fatty acid production, provides energy, and prolongs the feeling of satiety. It also prevents certain stereotypies and thus is paramount to sow welfare. fiber stereotypies animal welfare sow gestation This research received no external funding. pmc1. Introduction Animal welfare has been becoming an important area of concern for livestock producers over the last three decades. Public concern over the methods used to raise food-producing animals has increased, and these concerns are leading to voluntary and mandated changes in the methods used to produce livestock . The primary aim of these changes in production methods is to improve the welfare of livestock. Accurately determining the welfare of animals is difficult and should employ a multidimensional approach . Multiple factors (e.g., animal health, immune system competence, housing, behavior, growth performance, reproduction, environmental conditions, and physiological characteristics) should be taken into consideration for the assessment of animal welfare . Most people intuitively assume that hunger and welfare are negatively correlated. Hunger is a relatively easy concept to understand, but a difficult sensation to measure in animals. There are many factors (mixing of pigs different origin, nutrient deficient, high stocking density, boredom, temperature variation, stress, discomfort, or pain) that contribute to animal welfare, and the presence of hunger could not necessarily mean that welfare is compromised. Further research and advances in animal welfare assessment are necessary to shed greater light on the relationship between hunger and welfare. It is possible to measure hunger directly through operant conditioning tests and indirectly through stereotypical behaviors and postures of the sows . Operant conditioning tests are likely to give the most accurate results, but they are not easily available and amenable to production conditions. Therefore, in the current discussion, stereotypical behaviors and the sow posture will be considered as markers of hunger. Stereotypic behaviors are actions that are frequently repeated, have no apparent purpose, and appear to be useless to the animal. According to , stereotypical actions can include, but are not limited to, biting on bars, sham or vacuum chewing (chewing motions unrelated to eating), and nosing or licking the floor or feeder in the absence of food (Table 1). In addition to stereotypic behaviors, the proportion of time spent standing and/or active as opposed to lying (Table 1) is used to indicate hunger because sows do not appear satiated . We acknowledge that using simplified ways to assess a complicated physiological process such as eating motivation might be misleading . However, in production, basic approaches are now the only viable tools available. Stereotypic behaviors are more common in sows, and they are closely related to the restricted feeding levels used in commercial production , which supports the idea that stereotypies might be used as a sign of hunger and feeding motivation . The level of feeding and associated satiety experienced by sows appears to be inversely related to the duration of sow stereotypies . Interestingly, stereotypic behavior can be expressed in restrictively-fed gilts housed individually or in groups , indicating the importance of feeding level to animal welfare. 2. Approaches to Mitigate Hunger and Stereotypic Behaviors 2.1. Higher Feeding Levels An obvious approach to ameliorating hunger and associated stereotypes is to offer pregnant sows a much higher quantity of feed. Increasing the amount of nutrient-dense diets used in commercial production effectively satiates sows and reduces stereotypic behavior. Unfortunately, this approach also supports excessive maternal weight gain during pregnancy, which suppresses feed intake during the subsequent lactation and compromises sow longevity . Furthermore, increased feeding of nutrient-dense diets increases production costs and results in sows with a large body size that do not easily fit in existing accommodations . It is unlikely that this approach alone will alleviate hunger in gestating sows. Since it is well known that an increase in feed intake plays an important role in decreasing stereotypical behavior, many researchers have concentrated on providing diets that are relatively low in nutrient density and bulk density in order to decrease stereotypical behavior . Consequently, low nutrient-dense diets can be fed to sows at relatively higher levels without excessive gains in sow body weight. Typically, low-density diets contain a high proportion of fibrous feed ingredients. Examples of fibrous feed ingredients commonly used to dilute the nutrient density of diets include sugar beet pulp, soybean hulls, wheat bran, oat hulls, alfalfa meal, alfalfa hay, and wheat bran. One must carefully choose the fibrous ingredient for use in sow diets because they differ dramatically in nutrient composition and digestibility. 2.2. Feeding a High-Fiber Diet 2.2.1. Dietary Fiber Dietary fiber has been used to describe the plant-derived component of feed and foods, which was isolated or synthetic non-digestible carbohydrates and resistant to digestion by mammalian enzymes . Dietary fiber could be categorized by source, chemical characteristics, resistance to digestion, and beneficial physiological effects. However, the simple way to classify with dietary fiber is to be divided into two primary classes: soluble dietary fiber and insoluble dietary fiber (Table 2) . Simply stated, they are classified based on their ability to dissolve in water. Soluble dietary fiber typically includes compounds such as mix-linkage glucans, arabinoxylan, gums, and oligosaccharides including fructooligosaccharide, pectins, and b-glucans (Table 3). On the other hand, insoluble dietary fiber contains cellulose, hemicelluloses, lignin, resistant starches, and polyphenols (Table 3). Both soluble and insoluble dietary fiber are found in feed ingredients for pigs (Table 2). However, the solubility of dietary fiber in feedstuff varies depending on botanical origin of the plants, processing methods, and harvest conditions . Dietary fiber has many different functions and activities as it passes through the gastrointestinal tract. For example, soluble dietary fiber could delay gastric emptying and may slow nutrient uptake, while insoluble dietary fiber stimulates the production of beneficial bacteria in the colon, increases the passage rate, and alters the digestive enzyme activity . Based on these functions of dietary fiber (Table 3), they could be thought to impact on the satiation for sows because of their properties of adding bulking and viscosity in the gut. The use of dietary fiber for sow is a reasonable method to reduce hunger and stereotypies. While fiber is a carbohydrate, it is not easily digestible. This means it could provide feelings of fullness after feeding without spiking blood sugar or providing extra calories . In addition, dietary fiber mitigates the symptom of constipation, thereby alleviating stress because pregnant sows are often subject to feeding restriction. Although the addition of dietary fiber plays an important role in alleviating sow hunger and reducing abnormal behavior, it may be affected by the type of fiber and the inclusion level in the diet . animals-13-00879-t002_Table 2 Table 2 Fiber composition of feed ingredients used in pig diets a. Ingredients Type of Fiber, % Detergent Method, % Soluble Insoluble ADF NDF ADL Barely 5.4 9.7 5.8 18.3 2.3 Corn 0.9 6.0 2.9 9.1 0.3 Corn DDGS 3.0 14.1 12.0 30.5 2.6 Oat, whole 3.6 9.8 13.7 25.3 - Oat hulls 4.9 65.7 32.1 65.9 5.4 Rye 3.7 8.4 4.6 12.3 0.8 Sorghum 0.6 5.1 4.9 10.6 0.4 Sorghum hulls 10.0 45.0 41.6 59.4 - Sugar beet pulp 25.2 18.0 23.5 44.9 - Wheat 2.3 6.8 3.6 10.6 1.0 Wheat bran 2.5 23.8 11.0 32.3 - a Reprinted/adapted with permission from Ref. . 2012, NRC.; Reprinted/adapted with permission from Ref. . 2015, Jha and Berrocoso; Reprinted/adapted with permission from Ref. . 2016, Jimenez-Moreno et al. animals-13-00879-t003_Table 3 Table 3 Characteristics of dietary fiber and relationships to gut effects a. Dietary fiber Solubility GIT Effect b Cellulose Insoluble Substrate for microbial fermentation Changes digesta mixing Alteration of digestive enzyme activity Stimulates passage rate Lignin Insoluble Resistant starches Insoluble Hemicellulose Insoluble Polyphenols Insoluble Mix-linkage glucans Soluble Changes in mixing and diffusion Slows gastric emptying and glucose absorption Increases viscosity in the upper GIT Arabinoxylan Soluble b-glucan Soluble Pectins Soluble Gums Soluble Non-digestible oligosaccharides Soluble Fructooligosaccharide Soluble a Reprinted/adapted with permission from Ref. . 2000, Jimenez-Escrig and Sanchez-Muniz; Reprinted/adapted with permission from Ref. . 2007, Maljaars et al.; Reprinted/adapted with permission from Ref. . 2010, Gunness and Gidley; Reprinted/adapted with permission from Ref. . 1994, Low et al.; Reprinted/adapted with permission from Ref. . 2015, Zhang et al.; Reprinted/adapted with permission from Ref. . 2016, Gunness et al.; Reprinted/adapted with permission from Ref. . 2019, Williams et al. b Gastrointestinal tract effect. 2.2.2. Sources of Dietary Fiber When fibrous ingredients are incorporated into the diet of sows, the carbohydrate composition would be changed from a high-starch diet to a less-starch diet (more non-starch polysaccharides). In general, dietary fiber has a lower nutritional value than other ingredients, but the ingestion of high-fiber diets has a beneficial effect on animal welfare. Minimizing stereotypies in sows during the gestation period requires providing sufficient feed to satisfy feed motivation. The bulk offered by a diet rich in fibrous ingredients could reduce the abnormal behaviors in sows without excessive fatness and reduce reproductive performance. Different sources and types of dietary fiber have been used to evaluate how those fiber sources mitigate symptoms of feed motivation and stereotypies in sows. However, the results have been controversial . Generally, the inclusion of dietary fiber in the diet may reduce appetite and voluntary feed intake. It is not clear, however, whether a lowered palatability or gut fill is the major reason for reduced feed intake. Krogh et al. reported that feeding high-fiber diets based on sugar beet pulp (12%) or alfalfa meal (17%) to sows during the lactation period had no differences in feed intake, weight gain, and backfat thickness. Similarly, no differences were observed in feed intake and weight gain when sows were fed the diets with 5% of resistant starch and fermented soybean fiber . However, feeding sows with a konjac flour (2%) during gestation increased the subsequent lactation feed intake of sows compared to a control diet. Additionally, increased feed intake was observed in sows fed the diet with wheat straw (12%) and sugar beet pulp (16%) compared to the control diet . Moreover, sows fed sugar beet pulp (20%) increased their feed intake compared to those fed control diets . Weng tested diets containing 20% wheat bran, soya hull, and rice hull for sows. In that study, sows fed the diet with rice hull showed higher weight gain during gestation and consumed more feed during lactation. Similarly, Feyera et al. tested four different fiber sources (mixed fiber, palm kernel expellers, sugar beet pulp, and soy hull) for sows. In this study, sows fed mixed fiber and palm kernel expellers had higher average daily feed intake compared to sows fed sugar beet pulp, and soy hull (Table 4). These results indicated that wheat straw, sugar beet pulp (16% or 20%), konjac flour, rice hull, palm kernel expellers, and mixed fiber showed relatively higher feed intakes compared to other fiber sources. The most likely explanation may be that dietary fiber sources could have considerably different physiological functions between feedstuff, and this may affect the feed intake and weight gain of sows. Inclusion of fibrous feed ingredients in the diet of gestating sows does not necessarily depress nutrient digestibility. In vivo digestibility of fibrous feed ingredients is usually determined in young growing pigs. However, it is clear that sows have a greater capacity to extract energy from fibrous feedstuffs compared with growing pigs . So, one must carefully extrapolate the digestibility data determined in growing pigs to sows using regression equations or by predicting the energy content of feeds from chemical analysis . Generally, the nutritional value of ingredients with high levels of soluble fiber is greater than ingredients with elevated levels of insoluble fiber because soluble fiber is more completely fermented in the gastrointestinal tract of sows . According to Renteria-Flores , who fed pregnant sows diets that contained high levels of soluble, insoluble, or a combination of soluble and insoluble fiber, the diet high in soluble fiber from oat bran was superior in energy digestibility and similar in nitrogen digestibility compared to the low-fiber control diet. However, high levels of insoluble fiber from wheat straw and high levels of both soluble and insoluble fiber from sugar beet pulp depressed the digestibility of energy and nitrogen. Similarly, Feyera et al. supplemented dietary fiber sources such as mixed fiber, palm kernel expellers, sugar beet pulp, and soy hull in the gestational diet for sows. Sows had a similar amount of dietary fiber supplementations during the gestation period. The feed supplemented with soy hull and sugar beet pulp was highest in energy digestibility, while palm kernel expellers and mixed fiber showed lowest in protein and non-starch polysaccharides digestibility. However, sows fed the diet supplemented with 2% of konjac flour had higher neutral detergent fiber and crude protein digestibility compared to the control diet, whereas there were no differences in the digestibility of gross energy, acid detergent fiber, crude fiber, and dry matter ( , Table 4). The degree of nutrient digestibility is dependent on the source of dietary fiber. In this context, feeding soluble dietary fiber sources to sows increases nutrient digestibility more than sows fed insoluble dietary fiber sources. Ingestion of soluble dietary fiber increases digesta viscosity and thereby slow down the digesta passage rate, allowing more time to nutrient absorption. animals-13-00879-t004_Table 4 Table 4 Results of studies evaluating the effect of dietary fiber sources on feed intake, nutrient digestibility, and behaviors of sows. Feeding Periods Fiber Sources Ingredient Concentrations Main Results References Gestation Control vs. oat bran 34% No effect on N digestibility and feed intake, but increased energy digestibility Renteria-Flores et al. Control vs. wheat straw 12% Decreased N and energy digestibility, increased feed intake Control vs. sugar beet pulp 16% Decreased N and energy digestibility, increased feed intake Gestation Control vs. konjac flour 2% Increased CP and NDF digestibility and lactation feed intake a Sun et al. Lactation Control vs. alfalfa meal 17% No effect on feed intake, weight gain, and backfat thickness Krogh et al. Control vs. sugar beet pulp 12% Gestation Control vs. sugar beet pulp 20% Increased feed intake Shang et al. Control vs. wheat bran 30% No effect on feed intake Lactation Wheat bran vs. soya hull vs. rice hull 20% in all diets Rice hull showed higher BW gain during gestation and feed intake during lactation than other fiber sources Weng Gestation MF vs. SBP vs. PKE vs. SH b Supplemented in gestation diet in same level SH and SBP showed the highest energy digestibility and lowest for PKE Feyera et al. PKE showed the lowest protein digestibility, MF showed the lowest NSP digestibility c Increased ADFI in MF and PKE compared to SBP and SH Gestation Control vs. resistant starch 11% Lowest time of fighting frequency Sapkota et al. Control vs. sugar beet pulp 27% Increased percentage of standing behavior Control vs. soybean hulls 19% Highest percentage of resting behavior Gestation and lactation Control vs. resistant starch 5% Decreased the time on standing behavior, no difference in feed intake and BW Huang et al. Control vs. fermented soybean fiber 5% No differences in behaviors (lysing, sitting, licking, chewing, drinking), no difference in feed intake and BW Gestation MIDD-SY vs. DDGS-GM d 30% of wheat middlings and 15% of soybean hulls vs. 30% of distillers dried grains and 30% of corn germ meal Sows fed MIDD-SY increased percentage of eating behavior and decreased percentage of sitting behavior Lopez et al. a CP: crude protein, NDF: neutral detergent fiber. b Mixed fiber: MF, sugar beet pulp: SBP, palm kernel expellers: PKE, and soy hulls: SH. c NSP: non-starch polysaccharides. d MIDD-SY: 30% wheat middlings and 15% soybean hulls, DDGS-GM: 30% distillers dried grains with solubles and 30% corn germ meal. Stereotypic behaviors could be a major problem in individual gestation stalls due to animal welfare concerns and public perception (sustainability of production systems or acceptance of practices involving animals). Dietary fiber increases chewing, which limits intake by promoting the secretion of saliva and gastric juice, resulting in an expansion of the stomach and increased satiety. Sapkota et al. recorded sows' behaviors in nine different time points (starting at 0830 h). They included dietary fiber sources (resistant starch: 11%; sugar beet pulp: 27%; soybean hulls: 19%) in diets for sows and they found that the percentage of sows standing (71%) was highest in sows fed sugar beet pulp, while the percentage of resting was greatest for soybean hulls. However, bar chewing and nosing behaviors were not affected by fiber sources. The inclusion of 30% of wheat middlings and 15% of soybean hulls in sow's diet results in an increased percentage of eating behavior (11.7 vs. 7.3), while percentage of sitting behavior was reduced in sows fed diets containing 30% distillers dried grains and 30% corn germ meal. However, lying, standing, drinking, sham-chewing, walking, and oral-nasal-facial behaviors were not affected by fiber sources . While most stereotypic behaviors were not affected by dietary fiber sources, increasing resting behavior and reducing standing behavior was observed when sows were fed soybean hulls (19%) and resistant starch (5%), which may be indicative of decreased motivation of feeding and increased postprandial satiety. The soluble and insoluble ratios of dietary fiber sources (wheat middlings (30:1), soybean hulls (5 to 15:1), sugar beet pulp (5:1)) would be an important parameter to determine the physio-chemical characteristics of fiber . The sugar beet pulp or soybean hulls are a moderately fermentable fiber, which contains both insoluble and soluble fiber components (high in pectin, cellulose, and hemicellulose) in a desirable ratio compared to other dietary fiber sources. Most studies above showed that supplementation with soybean hulls in the diet for sows reduced stereotypic behaviors (increased resting and decreased standing behaviors). This suggests that low solubility of fiber sources may be less effective in suppressing huger, resulting increased standing and eating behaviors in sows. Discrepancies among the studies reported above may be due to dietary fiber source, fiber inclusion levels, or soluble and insoluble content (ratio) affecting outcome measures. However, the inclusion of dietary fiber in the diet for sows may reduce stereotypic behaviors because feeding fiber increases chewing, resulting in an expansion of the stomach and increased satiety due to accelerating the secretion of saliva and gastric juice. Based on the studies above, sugar beet pulp or soybean hulls could be potential candidates for dietary fiber sources to alleviate abnormal behaviors in sows. The positive effects of sugar beet pulp or soybean hulls on sow behavior may relate to the character of fiber present. Both sugar beet pulp and soybean hulls are high in soluble fiber which is highly fermented by sows. Fermentation of dietary fiber increases the production of short chain fatty acids (SCFA) which are readily absorbed through the intestinal wall into the blood. These SCFAs are available as an energy source during the interprandial period when glucose supply from the gut is diminishing. Because fermentation of dietary fiber requires more time relative to starch digestion, the SCFAs are available for a sustained period of time after each meal. De Leeuw et al. demonstrated that a diet containing 45% sugar beet pulp improved the stability of plasma glucose and insulin in sows during the interprandial period from 3 to 12 h after a meal compared to sows fed a starch-based diet. Improved stability of plasma glucose and insulin levels was associated with a significant decline in postural changes of sows during the period from 5 to 12 h after a meal. Decreased postural changes presumably indicate that sows are more content and less restless. In a later study, De Leeuw et al. demonstrated that fermentation of dietary fiber in the hindgut is more important than the increased gut fill associated with high-fiber diets in improving the stability of plasma glucose and insulin. Similarly, Brouns et al. observed decreased peak postprandial concentrations of glucose and insulin and a prolonged elevation of serum insulin in pigs fed a diet high in sugar beet pulp. These data suggest that fermentable fibers are more effective in improving the welfare of gestating sows compared with non-fermentable fiber sources. 2.2.3. Levels of Dietary Fiber A logical approach to controlling stereotypic behaviors and excessive body weight gains during pregnancy is to provide sows with a high-fiber diet . In agreement with the literature, by increasing levels of dietary fiber in the diets for sows, a corresponding reduction in nutrient digestibility and feed intake could be observed . A higher intake of dietary fiber increases the luminal viscosity and water-binding capacity of digesta (soluble fiber), as well as passage rate in the small intestine (insoluble fiber). These physio-chemical properties of fiber may affect the feed intake and stereotypic behaviors of sows (Table 5). The ingestion of high-fiber diets could decrease the feed intake because of the increased bulkiness of the diet. Moreover, in addition, increasing dietary fiber inclusion leads to a decrease in dietary energy intake, causing sows to increase feed intake to keep up with their energy requirements. However, results could be varied depending on dietary fiber sources and concentrations in the diet. Le Gall et al. tested different levels of dietary fiber (low: 13%, medium: 21%, high: 31%, very high: 38%, total dietary fiber (TDF)) for sows. The diets were formulated with various dietary fiber sources including wheat, maize, barley, soybean meal, and a combination of fibrous ingredients (sugar beet pulp, wheat bran, maize bran, and soybean hulls). They found that increasing supplementation of dietary fiber linearly reduced feed intake. However, Sun et al. found a linear increase in feed intake when sows were fed diets based on different levels of konjac flour (0, 0.6, 1.2, or 2.2%) during the lactation period. Loisel et al. provided low-fiber (13%, TDF) and high-fiber (23%, TDF) diets to sows during the gestation period. In this study, however, a high-fiber diet fed to sows was expected to increase feed intake compared to a low-fiber diet, but no difference was observed in feed intake. Similarly, there was no difference in voluntary feed intake when sows were fed a high-fiber diet (15%, crude fiber) compared to the control diet during the lactation period (3%, crude fiber) . Studies on these sows suggested that the fiber concentration below 15% of crude fiber may not affect feed intake. However, if the TDF concentration in the diet exceeds 23%, it is considered that the feed intake may be reduced for sows. Ingestion of high-fiber diets has the potential to adversely affect energy and nutrient utilization due to the regulation of the digestive process and glycemic response . Depending on the fiber source and consumed amount, different effects on nutrient digestibility could be promoted. Rijnen et al. reported that group-housed sows were able to utilize energy from sugar beet pulp silage, which is high in soluble fiber, as efficiently as energy from starch. An important observation is that diets containing very high levels of fibrous feed ingredients can be just as digestible as high-starch diets for gestating sows. However, the study by Oelke et al. showed that increasing the amount of total dietary fiber from soybean hull (0, 12, or 24%, TDF) in sow diets linearly decreased in the digestible energy and apparent total tract digestibility of dry matter, gross energy, crude protein, non-fibrous carbohydrates, and organic matter. Similarly, Calvert et al. reported that using 50% and 95% alfalfa meal in the gestational diet decreased the digestibility of dry matter, protein, energy, and fiber components (neutral detergent fiber, acid detergent fiber, and cellulose) compared to 5% alfalfa treatment. In addition, the digestibility of dry matter, organic matter, nitrogen, carbohydrates, and energy decreased linearly with increasing fiber levels (from 13% to 38%, TDF), while the digestibility of crude fiber and acidic detergent fiber increased linearly . This improvement is associated with the development of the digestive tract in sows, allowing increased degradation of dietary fiber fractions. Holt et al. also reported that dry matter, energy, and nitrogen digestibility was decreased by feeding a high-fiber diet (15%, crude fiber) to sows. Based on the studies, nutrient digestibility has been reported to be negatively affected by the feeding of high-fiber diets to sows. The addition of fibrous feedstuffs (over 15%, TDF) in the diet for sows could reduce nutrient digestibility due to the increased digesta viscosity (soluble) or decreased digesta transit time (insoluble), which slow down the diffusion of the substrates and lead to less mixing time for digestive enzymes. Few studies have compared the effects of different fiber sources and inclusion levels on the stereotypic behavior of sows. A diet containing 50% sugar beet pulp was more effective in reducing stereotypic behaviors and aggression in gilts than a diet containing 50% of mixed fiber sources (grass meal, wheat bran, oat hulls). Both high-fiber diets increased the time spent eating and resting while they reduced the time spent standing. These behavior patterns all suggest improved satiation of the sows . Bergeron et al. reported that sows fed very high-fiber diets (23%, crude fiber) spent less percentage of time in chain manipulation, vacuum chewing, nose rubbing, and object biting than sows fed a control diet (5%, crude fiber). Additionally, sows fed very high-fiber diets spent less time standing than sows fed the control diet. However, there were no differences in sitting and standing time between treatments. Holt et al. suggested that feeding a high-fiber diet contained with soybean hulls (15%, crude fiber) increased the percentage of time for sitting and feeding activity, while it decreased the percentage of time lysing for sows during the gestation period compared to sows fed a control diet (3%, crude fiber). The increased feeding activity is associated with increased time spent standing and these behaviors seem to be driven by a physiological need for energy and gut fill (feed motivation). Guillemet et al. tested a control diet (3%, crude fiber) and high-fiber diet (12%, crude fiber) which was formulated with wheat, barley, soybean meal, and mixed fiber. However, no differences were observed in nesting, resting, sitting, standing, ventral, and lateral recumbency behaviors in sows during parturition. Bernardino et al. evaluated sows' behaviors in two feeding periods (one hour before and one hour after feeding) with low-fiber (3%, crude fiber) and high-fiber diets (13%, crude fiber) during the gestation period. They did not find any differences in feeding motivation and any behaviors for duration or frequency (sleep, lysing, standing, sham chewing, rooting floor, licking floor, fence and gate interaction, interacting with mats, and vocalization) between the treatments. However, sows fed the low-fiber diet showed longer and frequent sham-chewing and licking floor behavior before feeding compared to after feeding treatment. DeDecker et al. used two different floor spaces (1.7 m2 and 2.3 m3) and diets (control: 3 and 9%, acidic detergent fiber (ADF) and neutral detergent fiber (NDF) and fiber: 17 and 28%, ADF and NDF) for sows during gestation periods. They concluded that the sows that were fed the fiber diet and raised at 1.7m2 performed lower oral-nasal-facial and sham-chewing behaviors compared to the sows that were fed the control diet and raised at 1.7m2. Consequently, definitive recommendations on the best fiber source or optimal levels of fiber to eliminate behavioral vices are difficult. However, levels of dietary fiber in diets between 12 and 15% (crude fiber) for sows may not affect the stereotypies. animals-13-00879-t005_Table 5 Table 5 Results of studies evaluating the effect of various levels of dietary fiber on feed intake, nutrient digestibility, and behaviors of sows. Feeding Period Fiber Sources or Treatments Ingredient or Fiber Concentrations Main Results References Gestation Low fiber vs. high fiber a 13 and 23%, TDF levels No effect on feed intake, BW, and backfat thickness Loisel et al. Gestation Alfalfa meal 5, 50, or 95% Decreased dry matter, fiber components, protein and energy digestibility with increasing alfalfa meal Calvert et al. Gestation Control vs. SBP vs. MFS b 5, 13%, or 14% SBP and MFS increased time spent eating and resting and decreased spent standing Danielsen and Vestergaard Gilts Sugar beet pulp silage 0, 10, 20, or 30% Increased energy digestibility Rijnen et al. Non gestation and lactation Low vs. medium vs. high vs. very high fiber c 13, 21, 31, or 38%, TDF levels Linear decrease nutrients (DM, OM, N, CHO, and energy) digestibility and daily food intake, but linear increase in CF and ADF digestibility Le Gall et al. 79 to 113 days of gestation Soybean hull 0, 12, or 24% Linear decrease nutrients digestibility Oelke et al. No effect on feed intake and BW Gestation Control vs. high fiber vs.very high fiber d 5, 18, or 23%, CF Sows fed very high-fiber decreased time performing stereotypies Bergeron et al. Post weaing to lactation Control vs. soybean hulls 3% or 15%, CF Increased percentage of time sitting and feeding activity and decreased percentage of time lying during gestation period and nutrient digestibility, no effect on feed intake during lactation period Holt et al. Gestation and lactation Control vs. high fiber e 3% or 12%, CF No effect on activities and postures in sows Guillemet et al. Gestation Control vs. high fiber f 3 and 9% vs. 17 and 28%, ADF and NDF g Decreased oral-nasal-facial and sham-chewing behaviors DeDecker et al. Gestation and lactation Konjac flour 0, 0.6, 1.2, 2.2% Linear decrease non-feeding oral behavior, linear increase in feed intake during lactation period Sun et al. Gestation and lactation Low fiber vs. high fiber h 3% or 13%, CF No effect on feed motivation test and any behaviors for duration or frequency Bernardino et al. a Low fiber was based on barley and wheat, high fiber was based on barley, wheat, soybean hulls, wheat bran, sunflower meal, and sugar beet pulp. b SBP: sugar beet pulp, MFS: mixture of green grass meal, wheat bran, and oat hulls. c Dets were based on wheat, maize, barley, soybean meal and a combination of fibrous ingredients (sugar beet pulp, wheat bran, maize bran, and soybean hulls). d High-fiber and very high-fiber diets were formulated with oat hulls and alfalfa meal. e Control diet was based on wheat, barley, and soybean meal, high fiber was based on wheat, barely, and fiber rich feedstuffs (sugar beet pulp, soybean hulls, wheat bran). f Control diet was based on soybean meal and high-fiber diets was based on wheat middlings and soybean hulls. g ADF: acidic detergent fiber, NDF: neutral detergent fiber. h Soybean hulls was the main fiber source. In most studies that demonstrated a significant effect of dietary fiber on stereotypic behaviors, fibrous ingredients were included at high levels (over 20%, TDF). These high inclusion rates for fibrous ingredients significantly decrease the bulk density of feed which creates nearly insurmountable problems with conveyance of mash feed through augers and storage bins and difficulty in thoroughly mixing diets. The high inclusion rates for fibrous ingredients make it difficult to produce high quality pellets for pelletized feed. These handling problems make many diets proven to be effective in modifying sow behavior impractical for commercial production. Holt et al. addressed this practical problem by formulating a high-fiber, mash diet based on corn, soybean meal, and 40% soybean hulls, which readily flowed through commercial feed delivery systems. Sows receiving the high-fiber diet were allotted 20% more feed (2.42 vs. 2.03 kg/day) than control sows each day in an attempt to compensate for the energy-diluting effects of soybean hulls. The high-fiber diet had no effect on reducing the occurrence of stereotypic feeding behaviors in sows for 3 h around feeding time. They used sows that were in their second or greater pregnancy and experienced at least one gestation period housed individually in stalls. These sows may have been unresponsive to the effects of dietary fiber because their feeding behaviors were already sensitized by previous housing in stalls. These older sows may have developed a consistent repertoire of behaviors, including stereotypic behaviors, that were elicited by feeding. Consequently, regardless of diet composition, sows expressed these behaviors as a result of feeding. Lawrence and Terlouw referred to this phenomenon a "channeling" of behaviors. Similarly, van der Peet-Schwering et al. noted that stereotypic behaviors increase as sows age and that high-fiber diets are less effective in reducing stereotypies in older sows. Differences between the reported works and previous studies may be related with quality of fiber, inclusion of fiber level (adding oil is necessary if we used a greater amount of fiber that affect palatability and feed intake), multiparous sows (age and experience of farrowing), format of diet (mash or pellet), and floor space for sows. The abovementioned studies reported on diverse types of dietary fiber that mentioned "high fiber or very high fiber", which contained varying inclusion levels of fiber sources. The scientific literature is clear that specific formulations of high-fiber diets can decrease the stereotypic behavior of gestating sows. Based on the studies, the inclusion of high levels of sugar beet pulp (13-30%) or soybean hulls (13%) seems to provide a consistent reduction in stereotypic behaviors and increases the satiety of sows . 3. Other Approaches to Decrease Stereotypies Feeding management practices may influence the expression of stereotypic behaviors. We theorized that offering sows two meals per day instead of one might provide more feeding opportunities for sows throughout the day which would decrease their need to exhibit stereotypic behaviors. However, Holt et al. found that total time sows expressed stereotypic behaviors each day increased when the daily feed was divided in two meals compared to a single daily meal. The channeling of feeding-associated behaviors may explain why twice daily feeding was not beneficial. Manu et al. offered one meal, two meals, and three meals per day for sows to evaluate how feeding frequency affects cortisol response and behaviors (food anticipatory activity, total feed activity, and indication of hunger). They suggested that no difference was found between the sows fed two or three meals because all sows had a similar energy intake. However, the counts of food anticipatory activity, total feeding activity, and indication of hunger were greater for sows fed three meals compared to sows fed one or two meals. These results indicated that feeding frequency did not provide sufficient gut fill, which may not reduce the stereotypic behaviors in sows. In support of this theory, Poulopoulou et al. reported that feeding sows three meals per day increased feed intake during lactation period, while a reduced degree of shoulder lesions was observed in sows fed three meals per day compared to sows fed two meals per day. Therefore, frequent feeding, regardless of the fiber content of the diet, is not likely to decrease the occurrence of stereotypic behaviors in sows that already display these abnormal behaviors. However, more frequent feeding may effectively reduce the development of stereotypic behaviors in young sows. Feeding motivation is the product of internal physiological cues and external stimuli . Consequently, one may be able to enhance satiation of the sow and decrease the occurrence of stereotypic behaviors with a combination of high-fiber diets and some specific metabolic modifier. Brouns et al. reported diets high in fermentable fiber from sugar beet pulp blunted the postprandial response in insulin and glucose and increased circulating levels of short-chain fatty acids. Presumably, these metabolic changes were present in sows that exhibited an enhanced level of satiety after consuming high-fiber meals. Possibly, some intermediary metabolite normally responsible for the control of feed intake could be manipulated by diet composition to mimic conditions of a large meal. If so, sows could receive limited amounts of a diet moderately high in fiber and achieve the same level of satiation as sows fed large quantities of diets very high in fiber. This approach, if successful, would minimize the practical problems of very high-fiber diets while achieving the reduction in stereotypic behaviors realized when feeding diets very high in fiber. 4. Conclusions There are proven methods of controlling the hunger and associated stereotypic behaviors in pregnant sows. However, these methods have important limitations, which prevent them from being rapidly adapted to commercial production systems. More detailed research into the mechanisms responsible for the decline in stereotypic behaviors caused by high-fiber diets will provide solutions to these practical challenges. In the interim, sows should receive diets that contain over 13% crude fiber and contain ingredients with high concentrations of fermentable fiber if a goal is to minimize expression of stereotypic behaviors. The beneficial effects of high-fiber diets will only be realized if the sow's nutrient requirements for maintenance, growth, and reproduction are met. Feeding diets in meal rather than pelleted form may be more effective in reducing stereotypies realizing that feed handling problems are magnified. Author Contributions J.-C.J. provided the conceptualization of the theme and topics for the manuscript; S.D., G.-I.L., J.-C.J. and Y.-Y.K. contributed to writing the original draft, and reviewing and editing the final submitted version of the manuscript. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. animals-13-00879-t001_Table 1 Table 1 Definition of behaviors for behavioral observation of pregnant sows a. Behaviors Definition Sham chewing Continuous chewing without the presence of visible food in the oral cavity Rooting the floor or feeder Snout touches the ground followed by head Standing Body supported by the four limbs Lying ventrally Lying with the belly on the ground with all the limbs under the body Lying laterally Lying sideways, with all the limbs extended laterally Licking the floor or feeder The tongue touches the floor and is followed by movements with the head Interacting fence or gate Biting or nibbling the fence wire or gate Interacting with mats Snout or tongue touches mats followed by head movements Bites Bite on any parts of the body (tail, vulva, ear, body) Facing Face to face, with a fixed view to the other animal Pushing Pushing another animal using the head or the muzzle Vocalization Sound emission emitted by the animal Belly nosing Snout movements to stimulate the milk flow a Reprinted/adapted with permission from Ref. . 2004, Zonderland et al.; Reprinted/adapted with permission from Ref. . 2020, Tatemoto et al. 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PMC10000098

The aim of this study was to compare the cargos of miRNA in exosomes isolated from the milk of healthy (H) cows, cows at risk of mastitis (ARM), and cows with subclinical mastitis (SCM). Based on the number of somatic cells and the percentage of polymorphonuclear cells, 10 cows were assigned to group H, 11 to group ARM, and 11 to group SCM. After isolating exosomes in milk by isoelectric precipitation and ultracentrifugation, the extracted RNA was sequenced to 50 bp long single reads, and these were mapped against Btau_5.0.1. The resulting 225 miRNAs were uploaded to the miRNet suite, and target genes for Bos taurus were identified based on the miRTarBase and miRanda databases. The list of differentially expressed target genes resulting from the comparisons of the three groups was enriched using the Function Explorer of the Kyoto Encyclopedia of Genes and Genomes. A total of 38, 18, and 12 miRNAs were differentially expressed (DE, p < 0.05) in the comparisons of H vs. ARM, ARM vs. SCM, and H vs. SCM, respectively. Only 1 DE miRNA was shared among the three groups (bta-mir-221), 1 DE miRNA in the H vs. SCM comparison, 9 DE miRNAs in the ARM vs. SCM comparison, and 21 DE miRNAs in the H vs. ARM comparison. A comparison of the enriched pathways of target genes from the H, SCM, and ARM samples showed that 19 pathways were differentially expressed in the three groups, while 56 were expressed in the H vs. SCM comparison and 57 in the H vs. ARM comparison. Analyzing milk exosome miRNA cargos can be considered as a promising approach to study the complex molecular machinery set in motion in response to mastitis in dairy cows. milk exosomes miRNA mastitis cow Ministry of Agriculture, ItalyDM 36296 del 19/12/2018 BEST MILK-MIPAAF, DM 36296 del 19/12/2018. Ministry of Agriculture, Italy. pmc1. Introduction The milk of cows is a source of nutrients and other compounds . Moreover, breastfeeding modulates the development of the immune system , and this activity is also regulated by microRNAs (miRNAs) . Milk is a rich source of extracellular vesicles (EVs), which are involved in cell communication . Milk-derived EVs contain mRNA, miRNA, ribosomal RNA (rRNA), long noncoding RNA (lncRNA), transfer RNA (tRNA), and DNA in addition to lipids, metabolites, and proteins . MiRNA in milk is a class of single-stranded noncoding RNAs about 22 nucleotides in length that induce post-transcriptional silencing of genes by binding to the 3' untranslated region of target genes . In this way, miRNAs control multiple cellular functions, from cell differentiation to tissue development and immune regulation, and consequently many aspects of health and disease . Mastitis is a common disease of dairy cows that causes high economic losses and often requires the administration of antimicrobial drugs , with potential implications for antimicrobial resistance , a topic under the scrutiny of the World Health Organization (WHO, accessed on 14 December 2022). Somatic cell count (SCC) in milk is the most common routine tool used at the farm level to screen cows for mastitis, and 200,000/mL SCC is the European cutoff to identify an inflammatory response to infection , while the cutoff is >150,000/mL SCC for New Zealand and >250,000/mL for Australia . Cows with high SCC levels can be later subjected to microbial culture or molecular analysis to confirm and diagnose the type of infection and stage of disease. Recently, it has become possible to routinely calculate the percentage of polymorphonuclear neutrophils (PMN) and lymphocytes (LYM) within the SCC , also defined as differential somatic cell count (DSCC), which is an estimate of the percentage of polymorphonuclear cells and allows a more accurate classification of intramammary infection in dairy cows . Interestingly, analysis of SCC and DSCC in milk is also related to milk quality, in particular to coagulation characteristics for cheese production . The identification of early biomarkers of mastitis is important and forms the basis for defining and applying farm protocols to reduce the spread of infectious agents, implement hygiene measures, and thus limit the use of antimicrobial drugs. Moreover, signaling pathways and cellular functions activated in response to an inflammatory response or disease can be assessed by the study of miRNA contained in milk exosomes, which have gained attention as markers of cow health and mastitis . In fact, the availability of next-generation sequencing technology has stimulated studies that use genomics as a diagnostic tool for mastitis while enabling the molecular response of the host to microbial invasion . These findings may also provide new insights for understanding the variable prevalence of mastitis among dairy cow breeds and the high individual susceptibility . It has been reported that miRNAs contained in milk exosomes are conserved among mammalian species, at least among humans, pigs, cows, and pandas . The miRNAs in milk exosomes are acid-resistant and protected from degradation in the intestine . Their transfer from mother to infant has been documented ; notably, miRNAs in milk exosomes of cow can be incorporated into human cells such as intestinal cells and macrophages . Bovine exosomes isolated from milk have been used as vehicles for drug delivery for therapeutic purposes , confirming their uptake in the gut. These findings open the perspective of considering milk exosome loading as a newer aspect of milk safety and quality . The aim of this study was to measure miRNA cargoes in exosomes isolated from milk samples of lactating cows classified as healthy or at risk or affected by mastitis based on the European cutoff of 200,000/mL SSC and the proportion of PMN. The regulatory network of differentially expressed miRNA between these groups of cows was also investigated to reveal possible signaling pathways and cellular functions activated in response to an inflammatory response or disease. Moreover, to evaluate whether the presence of an inflammatory process can influence the expression of miRNAs and milk quality, in the present study milk was collected from the all the four quarters of each cow. 2. Materials and Methods 2.1. Animals and Housing Sixty cows were recruited for the study from a herd on a commercial farm (46.1134059, 13.2817455; N 46deg6'48.261'', E 13deg16'54.283''; Italy). Cows were housed in loose housing with cubicles, fed the same total mixed ration, and not treated with antibiotics during the previous 20 days. The milking parlor (parallel 12 + 12) was located next to the bedding area. All protocols, procedures, and care of the animals were in accordance with the Italian legislation on animal care (DL n.116, 27/1/1992), and the study was approved by the Ethics Committee of the University of Udine (OBPA Prot. N. 9/2020). Before starting the sampling, milk from these cows was analyzed for SCC and DSCC values obtained from the official register of the breeders' association (Associazione Allevatori del Friuli Venezia Giulia, Codroipo, Italy; www.aafvg.it, accessed on 14 December 2022). SCC and DSCC were measured in milk samples using a Fossomatic 7DC (FOSS Electric A/S, Hillerod, Denmark; according to ISO 13366-2/IDF 148-2:2006). The 60 cows in the herd were divided into 3 groups of 20 cows each according to the classification proposed by Zecconi et al. : healthy (group H, SCC/mL < 200,000 and DSCC <= 69.3%); at risk (group ARM, SCC/mL < 200,000 and DSCC > 69.3%); with subclinical mastitis (group SCM, SCC/mL > 200,000 and DSCC > 69.3%). Milk samples were collected again during the subsequent monthly official record of the breeders' association. Based on the analysis of SCC and DSCC of the second sampling, the 60 cows were reclassified as follows: 34 cows were in group H (11 primiparous and 23 multiparous; 10 Days in Milk (DIM) < 70 and 24 DIM > 70), 13 cows were in group ARM (6 primiparous and 7 multiparous; 2 DIM < 70 and 11 DIM > 70), and 13 cows were in group SCM (2 primiparous and 11 multiparous; 3 DIM < 70 and 10 DIM > 70). 2.2. Extracellular Vesicles Isolation and RNA Extraction For EVs isolation, the teats were cleaned with disposable wipes and disinfected (ethanol, 70%) before milking; then, the first flow of milk from each quarter was discarded, and a pooled sample of about 100 mL of milk for each cow was collected in sterile Falcon tubes, which were kept on ice during the collecting period and stored at -80 degC until analysis. EVs were isolated using a modified isoelectrical precipitation method . Briefly, frozen milk samples were thawed at room temperature and centrifuged at 2000x g at 4 degC for 10 min to separate fat. After elimination of the supernatant fat, the samples were centrifuged at 12,000x g at 4 degC for 40 min to remove cell debris. The supernatant was diluted 1:1 with distilled water and heated to 37 degC for 10 min in a water bath after adding HCl 6N to reach pH 4.6 for casein precipitation. Samples were centrifuged at 5000x g at room temperature for 20 min, and the supernatant was transferred into clean tubes and frozen at -80 degC overnight. After thawing, the samples were filtered with Millipore membrane filters of 1.00, 0.45, 0.20 mm (Merck Life Science, Milan, Italy), and the filtrate was centrifuged at 100,000x g at 4 degC for 1 h. An aliquot of the pellet was resuspended in 0.1 M PBS pH 7.4 for the exosome characterization analysis. The Nanosight (Malvern Panalytical, Malvern, UK) under light scatter mode was used to visualize size distribution of the isolated milk exosomes in the 50-250 nm range. Transmission electron microscopy (TEM) with the immunogold labeling method was used to directly detect exosomes based on their size and specific surface proteins. Nickel TEM grids (Electron Microscopy Sciences, Hatfield, PA, USA), 400 mesh, with a formvar/carbon film, were floated on a drop of the pellet suspension fixed in 4% paraformaldehyde. Immunogold labeling was performed as previously described using anti-HSC70 (ab19136, Abcam, Cambridge, MA, USA) and CD63 (ab193349, Abcam, Cambridge, MA, USA) primary antibodies coupled with a 10 nm gold particle (EY Lab. Inc., San Mateo, CA, USA). The grids were then washed with several drops of water and stained with methyl cellulose-uranyl acetate (4% uranyl acetate and 2% methyl cellulose in a ratio of 1:9) before being subjected to microscopic analysis. Grids were analyzed on Philips CM10 and images recorded at 80 kV. The results of TEM confirmed the presence of round-shaped exosomes with diameters of about 80 nm. Another aliquot of the pellet was suspended in 300 mL of mirVanaTM miRNA Isolation Kit lysis buffer (Ambion, ThermoFisher Scientific, Milan, Italy) and frozen at -80 degC. Extraction of miRNAs from the isolated EVs was performed with the commercial kit mirVanaTM (ThermoFisher Scientific, Milan, Italy). The lysate was then mixed with 30 mL of homogenate additive and incubated on ice for 10 min and extracted once with 300 mL of acid phenol:chloroform. Samples were then centrifuged, and the aqueous (upper) phase was collected in a new tube. After washing in 100% absolute ethanol at room temperature, the lysate-ethanol mixture was placed in a filter cartridge and centrifuged until all the lysate solution had passed through the filter. The filter cartridge was then washed repeatedly until 40 mL of RNase-free water preheated to 95 degC was added. MiRNAs were collected by spinning for 25 s at 10,000x g. 2.3. miRNA Sequencing Ten samples of the H group, 11 samples of the ARM group, and 11 samples of the SCM group were sequenced. RNA purity, integrity, and concentration were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, US). FASTQ raw sequence files were subsequently quality-checked with FASTQC software . Then, sequences with a low quality score Q < 20 or including adaptor dimers were trimmed using Cutadapt 4.2 software . Samples were selected on the basis of an RNA Integrity Number (RIN) of >7 and an rRNA 28 s/18 s ratio of >1.8. Libraries were prepared with Illumina(r) TruSeq(r) Small RNA Library Prep protocol (Illumina Inc., San Diego, CA, USA); 1 mg of total RNA for each library (minimum concentration of 200 ng/mL) was sequenced to 50 bp long single reads (average of 64 million reads per sample) using 8 lanes in 6-plex on an Illumina HiScanSQ (Illumina Inc., San Diego, CA, USA). Sequences were mapped against Btau_5.0.1 using Bioconductor Rsubread . The raw data in FASTQ format were uploaded to NCBI Sequence Read Archive (Bioproject ID PRJNA902552). 2.4. Data Mining and Bioinformatic and Statistical Analysis The resulting miRNA sequences were firstly divided into those annotated in Bos taurus (no. 184) and those in other vertebrate taxa (no. 622), whilst sequences annotated to other phyla were not included in the downstream analysis. To discover which miRNAs annotated in various vertebrate organisms overlapped in Bos taurus, sequences were searched in the miRbase . The final number of miRNAs considered for Bos taurus was 225. These miRNAs were uploaded on the miRNet suite and normalized with the DESeq2 method for the comparisons of H vs. SCM, ARM vs. SCM, and H vs. ARM. The target genes of these mRNAs for Bos taurus were found based on miRTarBase version 8.0 and miRanda database . The list of differentially expressed (DE) target genes (p < 0.05 after Benjamini and Hochberg correction for multiple testing) resulting from each comparison were enriched to the Function Explorer on the Kyoto Encyclopedia of Genes and Genomes accessed on 14 December 2022) database to find the statistically relevant pathways linked to the DE miRNAs and genes. Only enrichments with a p-value of <0.05 after Benjamini and Hochberg correction are reported. 3. Results The characterization of the isolated milk exosomes was obtained by Nanoparticle Tracking Analysis (NTA), and the results are displayed as averaged finite track-length adjusted (FTLA) concentrations . Under electron microscopy, the exosomes were round with a diameter of approximately 80 nm . Moreover, the exosomes were positive for known exosomal markers CD63 and HSC70 via immunogold labeling. The total number of reads from RNAseq obtained from 32 samples was 63,219,487, but after the quality check, 28,199,543 were annotated as RNAfam, and 18,507,870 were detected as miRNAs. A value of 656,318 counts per million (CPM) was calculated for miRNA followed by 196,430 CPM for RNA , and the contribution of other RNA types was minimal. In the miRNet workflow, DESeq2 was applied to assess the DE of miRNAs in the different health states (H_ARM, ARM_SCM, and H_SCM). A total of 38 (16 upregulated and 22 downregulated), 18 (5 upregulated and 13 downregulated), and 12 (3 upregulated and 9 downregulated) miRNAs were significantly DE in the H_ARM, ARM_SCM, and H_SCM comparisons (Supplemental Table S1). The Venn diagram showed that 1 DE miRNA was shared between the three groups (bta-mir-221), 1 DE miRNA in the H_SCM comparison (bta-mir-1247-5p), 9 DE miRNAs in the ARM_SCM comparison (bta-mir-142-5p; bta-mir-128; bta-mir-671; bta-mir-19a; bta-mir-146b; bta-mir-222; bta-mir-142-3p; bta-mir-15b; and bta-mir-205), and 21 DE miRNAs in the H_ARM comparison (bta-mir-2892; bta-mir-96; bta-mir-1343-3p; bta-mir-194; bta-mir-1281; bta-mir-2885; bta-mir-30c; bta-mir-1307; bta-mir-2415-3p; bta-mir-365-3p; bta-mir-542-5p; bta-mir-502deg; bta-mir-7857-3p; bta-mir-30b-5p; bta-mir-2431-3p; bta-mir-328; bta-mir-210; bta-mir-186; bta-mir-31; bta-mir-1839; and bta-mir-425-3p). The target genes of the DE miRNA were 2195 for the H_ARM comparison, 713 for the H_SCM comparison, and 1313 for the ARM_SCM comparison (Supplementary Table S2). Based on these genes, analysis of pathways revealed 57, 47, and 56 pathways significantly affected in the H_ARM, ARM_SCM, and H_SCM comparisons, respectively (p < 0.05 after HB correction for multiple testing) (Supplementary Table S3). A comparison of these enriched pathways from healthy, subclinical mastitic, and mastitis-prone cows showed that 19 pathways were differentially expressed in the three groups , while 16 were expressed only in the H_SCM group, 12 in the H_ARM comparison, and 18 in the ARM_SCM comparison. Based on the degree and betweenness results, the top miRNAs were highlighted for each network (Supplementary Table S4). In the H_ARM network, bta-mir-2415-3p, bta-mir-2431-3p and bta-mir-6517, bta-mir-339a, bta-mir-1343-3p, bta-mir-2407, bta-mir-328, and bta-mir-1306 are reported, and these miRNAs were downregulated in the healthy cows compared with the at-risk cows. In the H_SCM pairwise comparison, the miRNAs bta-mir-339a, bta-mir-1247-5p and bta-mir-1306, bta-mir-345-5p, bta-mir-320a, and bta-mir-2388-3p were downregulated in the healthy cows compared to the cows with subclinical mastitis. The significantly enriched KEGG signaling pathways are also shown in Figure 4, highlighting the NF-KB signaling pathway, T cell receptor signaling pathway, TNF signaling pathway, adherens junction, and leukocyte transendothelial migration for H_ARM. For H_SCM, the NF-KB signaling pathway was not enriched and was replaced by the NOD-like receptor signaling pathway. Figure 4A,B depict the connections between the miRNAs and target genes according to the selected KEGG pathways. They show the relevance of one miRNA in relation to the other miRNAs in regulating the relevant genes and pathways for immune functions. 4. Discussion The analysis of miRNAs was performed in milk samples collected from a single milking and revealed 225 unique miRNAs associated with Bos taurus or showing sequence homology. The total number of miRNAs in the isolated milk exosomes was higher than that reported by Saenz-de-Juano et al. and lower than that reported by Sun et al. , Cai et al. , and Ma et al. , and is likely dependent on library construction, reference Bos taurus genome and sequence depth, and isolation techniques. In the present study, the mammary glands health status of the lactating cows was assessed by total (SCC) and differential counts (DSCC) of somatic cells in milk . This method is routinely used by the Italian Breeders Association to classify lactating cows as healthy, affected by subclinical or chronic mastitis, or at risk of mastitis, based on validated cutoffs . The leukocyte counts and miRNA cargos of exosomes in milk were measured in milk samples from healthy and mastitis-affected cows collected during a single milking. In this context, Saenz-de-Juano et al. reported that the miRNA in exosomes and SCC did not vary on consecutive days and thus could reflect the condition of the individual cow at the level of the mammary gland. The results in Figure 2 and in the Supplementary Table S1 clearly show that the number of DE miRNAs from healthy cows (H) was higher compared with the ARM groups (21 miRNAs) than with the SCM groups (1 miRNA). Among the different studies on the cargos of isolated milk EVs, only four investigated the effects of mastitis on the presence and expression of miRNAs, three of them referred to Staphylococcus aureus infection, and only one compared subclinical with clinical mastitis. In particular, Ma et al. collected milk samples from the four quarters of cows infected with this bacterial species, and Cai et al. collected milk from the udder after infusion of Staphylococcus aureus colonies. Sun et al. also infected the mammary gland with this pathogen, but only in two quarters, and used the corresponding quarters of individual cows as negative controls. In the fourth study , milk from cows with subclinical mastitis was collected from the infected quarters and compared with milk from the quarters with low SCC. The DE miRNAs in healthy and diseased cows were not the same in all four studies, except bta-miR-142-5p, which was always upregulated, not only in the cows infected with Staphylococcus aureus but also in the milk exosomes isolated from the milk of cows with subclinical mastitis. Our results showed that this miRNA was not DE compared with healthy cows (H_ARM, H_SCM), whereas it was downregulated in cows in the early phase of inflammation (ARM) compared with cows with subclinical mastitis (SCM) (p < 0.01). The upregulation of bta-miR-142-5p in cows with subclinical mastitis (SCC > 200,000/mL) compared with at-risk cows (SCC < 200,000/mL) is consistent with the results of Saenz-de-Juano et al. and with the results of other studies in which cows were at the onset of mastitis after infection with Staphylococcus aureus. However, only one target gene for bta-miR-142-5p was found in cattle in the miRbase database. Interestingly, bta-miR-1247, the only upregulated miRNA in the H_SCM comparison, has been related to inflammation in bovine endometrial stromal cells treated with lipopolysaccharide affecting the MAPK pathway, which was significantly found in an H_ARM comparison . Among the downregulated miRNAs in H_SCM and H_ARM comparisons, bta-mir-339a and bta-mir-320 were negatively associated with lactogenic differentiation in bovine mammary epithelial cell culture . Bta-mir-22 and mir-345 microRNAs have been recently identified as differentially expressed in bovine macrophages in response to M. bovis expression . Their differential expression is suggested to regulate host gene expression to enhance pathogen survival. Mir-345 is also a methylation-sensitive microRNA involved in cell proliferation. In addition to being downregulated in macrophages in response to M. tuberculosis, it is hypermethylated in T cells from TB-infected cattle . A few other miRNAs found in the present study agreed with previous results, namely, bta-miR-142-3p, bta-miR-221, and bta-miR-103 with Cai et al. and bta-miR-22-3p with Saenz-de-Juano et al. . In addition, bta-miR-146a, bta-miR-146b, bta-miR-223, bta-miR-2285b, bta-miR-378-b, and bta-miR1246 were common between the studies, but the overlap did not extend to all of these studies. However, bta-miR-146a was found to alleviate intestinal colitis in a mouse colitis model by activating NF-kB , and it was found to be differentially expressed in bovine mammary glands affected by mastitis . The experimental conditions in these investigations differed from those in the present study, suggesting that there is a great deal of individual variability in terms of infection-specific change, as previously suggested by Saenz-de-Juano et al. . Indeed, the immunological response and milk concentrations of biomarkers also differed according to the type of mastitis . Nevertheless, genetic variants and factors other than infection influence the cargos of EVs in the milk of lactating cows. Stressors such as group relocation and ambient temperature have been reported to modulate miRNA levels in milk EVs from dairy cows . Since miRNAs are also involved in mammary gland plasticity and the synthesis of milk components, genetic background is another factor to be considered . Notably, bta-mir-320 and bta-mir-345 show 100% homology with human sequences, whereas bta-mir-146a is 93.8%. Since it is known that miRNAs can be absorbed in the human intestine, the study of miRNAs in milk exosomes may be a new area to investigate in relation to human health. Significantly enriched pathways (Supplementary Material Table S3) underscored a different pattern between subclinical and at-risk cows compared with healthy cows. In the H_SCM comparison, at least nine pathways were directly or indirectly involved in the cellular immune response (leukocyte transendothelial migration; prolactin signaling pathway; Rap1 signaling pathway; signaling pathways regulating pluripotency of stem cells; NOD-like receptor signaling pathway; regulation of actin cytoskeleton; VEGF signaling pathway; thyroid hormone signaling pathway; and thyroid hormone synthesis). From a biochemical point of view, these data support the evidence for the increase in leukocyte count in subclinical mastitis. In the H_ARM group, however, the pattern was different. In the ARM samples, an increase in the percentage of PMN and lymphocytes (DSCC) was observed, but the total leukocyte count (SCC) was not yet above the cutoff generally considered as inflammation (200,000 cells/mL). Among the signaling pathways of this comparison, at least 11 related to immune cell function and modulation of inflammation (phosphatidylinositol signaling system; lysosome; Fc epsilon RI signaling pathway; Jak-STAT signaling pathway; chemokine signaling pathway; platelet activation; NF-kappa B signaling pathway; inflammatory mediator regulation of TRP channels; inositol phosphate metabolism; sphingolipid signaling pathway; and HIF-1 signaling pathway). Moreover, the target genes of bta-mir-339a were enriched for 10 significant pathways, including the MAPK signaling pathway , as for bta-mir-1247 in the present study. The different patterns observed in the two comparisons can be explained by the different health status of the mammary gland. In the H_SCM comparison, we compared healthy with subclinical mastitis conditions, and in SCM there were clear signs of inflammation with an increase in leukocyte count above 200,000 cells/mL . Therefore, one can expect a prevalence of signaling pathways involved in cell immune responses. The H_ARM comparison includes samples in the initial stages of inflammation (ARM). In these animals, overt inflammation had not yet started, and SCC was <200,000 cells/mL, while PMN content was increased. ARM status can develop in two ways: spontaneous healing or subclinical mastitis. The regulation of the immune and inflammatory response is crucial for the final outcome. Therefore, we can assume that at this stage genes regulating the activity of cells, including epithelial cells that are also able to secrete inflammatory mediators and antibacterial substances , may play a role leading to one of the two possible outcomes in terms of the success of the defense response and the virulence of the invading pathogens. These results are supported by assessing the degree (number of connections of nodes) and the betweenness (a measure of the number of shortest paths through a node; this filter retains genes connecting clusters) from the miRNet analysis, which allows us to highlight the most relevant miRNAs for the H_ARM and H_SCM networks . The interconnections of these miRNAs with selected significantly enriched KEGG pathways highlighted in Figure 4 (T cell receptor signaling pathway, TNF signaling pathway, leukocyte transendothelial migration, adherens junction, and NF-KB signaling pathway) were based only on the target sequences of the genes. The H_ARM comparison was the most interconnected and showed that a complex network exists between the miRNAs and the target genes within the selected signaling pathways. In contrast, in the H_SCM comparison, the DE miRNAs formed separate gene networks, indicating a more focused regulatory role of miRNAs within each pathway. However, these miRNAs do not correspond to those reported in the limited studies of exosomes in bovine mastitis . Searches of the miRbase database and the literature revealed limited information on the regulatory activity of these miRNAs in the inflammatory process and immune response. The expression of bta-miR-2431-3p, bta-miR-2415-3p, bta-mir-2407, bta-mir-345-5p, and bta-mir-328 was reported in an in vitro study of viral infection of a bovine kidney cell line, but these miRNAs were not significantly affected by treatment . In another study of DE miRNAs in bovine testicular and ovarian tissues , bta-miR-6517, bta-miR-1343-3p, and bta-mir-2388-3p were highlighted, but their biological significance was not elucidated. A sequencing and annotation study reported the expression of bta-mir-671 and bta-miR-1306 , but their regulatory roles were not investigated. The limitations of the present study are that the extracellular vesicles were isolated from whole milk and were not related to a specific pathogen but were referred to an inflammatory process. Moreover, potential confounding factors, such as parity and stage of lactation, were not evaluated. Nevertheless, the origin of the extracellular vesicles is another aspect that deserves attention, and further studies are needed to determine the role of miRNAs in the inflammatory response of the mammary gland and their potential effects on human health to reach more comprehensive conclusions. 5. Conclusions Extracellular vesicles and their miRNA cargos in milk are considered a promising approach to study the complex molecular machinery set in motion in response to mastitis in dairy cows and could influence milk quality. However, the question of whether miRNAs are influenced only by mastitis or also depend on various experimental conditions and environmental factors deserves further investigation. Acknowledgments We thank Maurizio Francescutti of the Associazione Regionale Allevatori della Friuli Venezia Giulia for the assistance. Supplementary Materials The following supporting information can be downloaded at: Table S1: miRNA differentially expressed (p < 0.05 with HB correction) in milk exosomes sampled from healthy (H) cows, cows with subclinical mastitis (SCM), and cows at risk of mastitis (ARM); Table S2: Target genes of the miRNA differentially expressed (p < 0.05 after HB correction for multiple testing) in milk exosomes sampled from healthy (H) cows, cows with subclinical mastitis (SCM), and cows at risk of mastitis (ARM); Table S3: Pathway analysis of the target genes of miRNA differentially expressed (p < 0.05 after HB correction for multiple testing) in milk exosomes sampled from healthy (H) cows, cows with subclinical mastitis (SCM), and cows at risk of mastitis (ARM). Target genes are reported in Supplementary Table S3; Table S4: Number of connections (degree) and betweenness values of miRNA differentially expressed (p < 0.05 with HB correction) in milk exosomes sampled from healthy (H) cows, cows with subclinical mastitis (SCM), and cows at risk of mastitis (ARM). Supplementary Figure S1: Bovine milk exosomes isolation and validation. (A) Averaged FTLA concentration/size with the size of the exosome peaks annotated. (B,C) Exosome morphology recorded by transmission electron microscopy and immunogold labeling (10 nm gold particles) of CD63 marker (B) and HSC70 marker (C). Scale bar: 200 nm. Click here for additional data file. Author Contributions B.S. designed the experiment and contributed to milk sampling, bioinformatic and biostatistics computation, and writing and reviewing the manuscript; M.C. (Michela Cintio) performed sampling, exosome isolation, RNA extraction, and data analysis; S.S. assisted milk sampling, biostatistics computation, and writing and reviewing the manuscript; E.S. was involved in RNA extraction and bioinformatic and biostatistics computation; D.L. sequenced data and performed bioinformatic analysis; A.Z. assisted results interpretation and writing and reviewing the manuscript; M.C. (Monica Colitti) contributed to design of experiments, exosome identification, and biostatistics computation. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was approved by the Ethics Committee of the University of Udine (OBPA Prot. N. 9/2020). Informed Consent Statement Informed consent was obtained from the owner of the animals. Data Availability Statement Raw data FASTQ can be downloaded from NCBI Sequence Read Archive accessed on 1 December 2022), Bioproject ID PRJNA902552). Further inquiries can be directed to the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Mean small RNA and microRNA (miRNA) abundance in reads per million (RPM) of the milk samples collected from healthy and mastitic cows. Figure 2 Venn diagram showing the number of unique or shared differentially expressed miRNAs in milk exosomes sampled from healthy (H) cows, cows with subclinical mastitis (SCM), and cows at risk of mastitis (ARM). H_ARM, H_SCM, and ARM_SCM refer to the pairwise comparisons between groups (Supplementary Table S1). Venn diagram was created by a web tool available at accessed on 14 December 2022. Figure 3 Venn diagram showing the number of unique or shared KEGG pathways of the target genes significantly enriched in the comparisons between healthy (H) cows, cows with subclinical mastitis (SCM), and cows at risk of mastitis (ARM). H_ARM, H_SCM, and ARM_SCM refer to the pairwise comparisons between groups (Supplementary Table S3). Venn diagram was created by a web tool available at accessed on 14 December 2022. Figure 4 Network analysis of miRNA differentially expressed between healthy (H) cows, cows at risk of mastitis (ARM), and cows with subclinical mastitis (SCM). Figures were generated by miRNet suite . (A) H_ARM comparison: in red are the genes of the NF-KB signaling pathway; in green the genes of the T cell receptor signaling pathway; in yellow the genes of the TNF signaling pathway; in purple the genes of the leukocyte transendothelial migration; and in black the genes of the adherens junction. (B) H_SCM comparison: in red are the genes of the NOD-like signaling pathway; in green the genes of the T cell receptor signaling pathway; in yellow the genes of the TNF signaling pathway; in purple the genes of the leukocyte transendothelial migration; in black the genes of the adherens junction. (Supplementary Table S4 reports the miRNAs in the networks). Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000099

A recent study revealed that organically raised Bronze turkeys showed a high prevalence of green liver discoloration. This alteration is commonly associated with the Turkey Osteomyelitis Complex and potentially caused by opportunistic bacteria. Therefore, 360 organically fattened Bronze turkeys were examined post-mortem throughout two fattening trials with two examinations each to determine possible infectious risk factors and reduce disease prevalence. Clinical and pathoanatomical examinations were performed on every hen. Histopathological, bacteriological, parasitological, and virological examinations were performed on at least six hens without and, if applicable, six hens with green livers on each examination date. Overall, 9.0% of all hens had a green liver without a correlation with bacterial or parasitological findings but multiple health impairments. The discoloration correlated significantly with the detection of immunosuppressive turkey hemorrhagic enteritis virus at the early stage and macro- and histological joint/bone lesions at the late fattening stage, indicating the presence of two different predisposing pathogeneses. Flocks not being vaccinated against hemorrhagic enteritis but having a virus-positive sample showed the highest prevalence of green liver discoloration and developed worse in various parameters. In conclusion, an adequate vaccination schedule and the prevention of field infections may lead to a decreased risk of performance reduction and improved animal health. Turkey Osteomyelitis Complex green liver hemorrhagic enteritis virus aseptic bone necrosis Federal Ministry of Food and Agriculture (BMEL)2819OE059 The project was supported by funds from the Federal Ministry of Food and Agriculture (BMEL) based on a decision of the parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food (BLE) under the Federal Programme for Ecological Farming and Other Forms of Sustainable Agriculture [2819OE059]. pmc1. Introduction The organic farming associations and Council Regulation (EU) No 2018/848 of 20 May 2018 on organic production and labeling of organic products place special requirements on the organic rearing of turkeys in Germany . This serves to improve animal welfare as well as climate and environmental protection. During extensive statistical surveys from 2007 to 2017, the health status of conventionally and organically raised turkeys in Germany was recorded . The latest study revealed that organically raised turkeys (Kelly Broad Breasted Bronze [BBB]) showed an approximately nine times higher prevalence of green livers (GL) at processing compared to conventionally raised B.U.T. 6 (British United Turkeys). The prevalences of GL for organically raised Bronze toms and hens were 34.8% and 33.2%, versus 3.8% and 4.4% for conventionally raised B.U.T. 6 toms and hens, respectively . The pathogenesis of GL is mainly attributable to the accumulation of inter- and intrahepatic endogenous biliverdin . This bile pigment represents a degradation product of erythrocytes. It is particularly relevant in birds due to the absence of the enzyme biliverdin reductase, which converts biliverdin to bilirubin . Besides bacterial infection, other possible causes for the accumulation of biliverdin are increased hemolysis due to mechanical trauma, intoxication, hepatitis, necrosis, or obstructive outflow disorders . Turkey osteomyelitis complex (TOC) is a disease predominantly described in conventionally fattened turkeys. It is commonly associated with GL and bone alterations, leading to economic losses due to the discarding of complete or partial carcasses . The descriptions of this disease complex have many similarities with the findings from the predecessor study. A statistically significant correlation (p <= 0.05, r = 0.49) between prevalences of GL and joint pathologies was found at processing . However, previous studies indicate that the alterations of TOC are associated with opportunistic bacteria like Escherichia (E.) coli or Staphylococcus (S.) aureus . A further immunosuppressive effect of infection with the turkey hemorrhagic enteritis virus (HEV), leading to a higher prevalence of pathological findings associated with coliform bacteria, has been suggested . Transient immunosuppression and related hemorrhagic enteritis are mediated by the release of proinflammatory cytokines as well as necrosis and apoptosis of primary target cells, B lymphocytes, and macrophages . HEV belongs to the family Adenoviridae, genus Siadenovirus, and species Turkey siadenovirus A. It includes two strains, the avirulent turkey HEV and the virulent turkey adenovirus 3 . It is a non-enveloped, linear, double-stranded DNA virus with an approximate genome length of 26.3 kb and is characterized by an open reading frame (ORF). The ORF encodes for the glycoside hydrolyzing enzyme sialidase . Besides different bacteria, several other viruses and diseases in turkeys are known to affect the liver. These include, among others, avian hepatitis E virus, turkey viral hepatitis, aviadenoviruses, highly pathogenic avian influenza A viruses, and reoviruses . This study was designed to identify associations between GL and infectious risk factors in organically raised turkeys. It was carried out to gain knowledge about the potentially infectious causative agents of GL disease in organic turkey farming. Further, it was conducted to implement strategies to avoid circumstances leading to discoloration and the associated TOC at the time of processing. The results may help to assess the importance of preventing HEV infection during an early stage of fattening to avoid economic losses and improve health and welfare. 2. Materials and Methods The examinations did not require notification or approval, as in accordance with the German Animal Welfare Act (SS 7, paragraph 2, sentence 3). 2.1. Animals and Time Points of Examination A total of five organic turkey farms fattening Bronze turkey hens of a Cartier genetic (including rearing farms) were selected for this investigation. In part, the selection was based on the selection criteria of high prevalence of GL at processing from the previous study . This study included two consecutive trials, with the first examination trial extending over six months, starting in September 2020 and ending in March 2021. The second trial started in February 2021 and finished with the final examination in August 2021. As a consequence of avian influenza control measures in one farm's area (flock No. 41, second examination) and quarantine measures in the scope of the SARS-CoV-2 pandemic (flock No. 31, second examination), two examinations were canceled. Due to the avian influenza epidemic in Germany in 2020/2021, flocks No. 21 (second examination trial) and No. 22 (first examination trial) were not granted any access to the outdoor enclosure. In Flock No. 11 (second examination trial), access to the outdoor enclosure became available during the end of the fattening phase. The turkeys of flock No. 41 were kept outside during the most part of the fattening period with variable access to woodland and a small stable (in the second trial, no access was allowed from week 11 onward). Recorded data of the farm workers of each farm considered in this investigation included the vaccination program, flock diseases and treatments, and daily mortality (Table S1). With respect to health protection measures during the SARS-CoV-2 pandemic, no personal attendance at the slaughterhouses was possible. Clinical and detailed post-mortem examinations were performed on 20 turkeys per flock between the 70th and 75th days of fattening (the first or early examination time) and the 120th to 127th days of fattening (the second or late examination time), respectively. In addition, an inspection of the turkeys was performed on the first days of rearing. The sample size was calculated based on an average prevalence of GL of 27.7% (Bronze- and B.U.T. 6/TP7/TP9-hens) in organically raised hens, with the lower limits of the 95% confidence intervals of 18.5% . Using this lower limit with a maximum herd size of 2.500 turkeys [the maximum permissible number of fattening turkeys in one housing unit according to Regulation (EU) No. 2020/464 of 26 March 2020] and calculation according to Cannon and Roe, a sample size of n = 16 was required to demonstrate the presence of GL in organically raised hens . Sample size was limited to 20 randomly drawn individuals per investigation time and flock. This can be explained by the fact that the sample, on the one hand, had to comprise a sufficient number of carriers, while on the other hand, all animals had to be purchased and thus removed from the food chain. This study includes only female turkeys to maximize validity, as the prevalence of GL showed a notably greater variance in the Bronze hen flocks (1.7% to 75.0%) than in the tom flocks (1.7% to 55.0%). Therefore, influences on GL development could potentially be demonstrated . Hens were then taken to the Clinic for Birds and Reptiles at the University of Leipzig and provided with food and water overnight. The next day, all turkeys were euthanized after being stunned (Large Poultry Stunner, Friedr. Dick GmbH & Co., KG, Deizisau, Germany) by subsequent exsanguination through a unilateral neck cut severing the carotid artery and jugular vein. All turkey hens were euthanized for routine diagnostic necropsy. 2.2. Data Assessment Necropsy was conducted following the protocol by Schmidt et al. including the 10-cut procedure protocol implemented by the Food Safety and Inspection Service . Pathoanatomical examinations were performed on 360 turkeys. Depending on the occurrence of GL, six hens without and additionally up to six further hens with discoloration were chosen for histopathological, bacteriological, parasitological, and virological examinations. Further examinations were performed on 67 hens at the early and 63 hens at the late fattening stages (in total, 130 hens) (Table S1). Swabs for microbiological examination were taken from the liver, heart, kidney, spleen, lungs, small intestine, shoulder joint, hip joint, knee joint, ankle joint, and the epiphysis of the proximal tibiotarsus. In the case of an ambiguous parasitological result, the diagnosis was confirmed by PCR of the 18S rDNA fragment specific for Eimeria of turkeys and/or for Tetratrichomonas (T.) gallinarum and Histomonas (H.) meleagridis . The chymus of the jejunoileum was sequentially diluted 1:10 using phosphate-buffered saline (PBS) for quantifying coliform bacteria as colony-forming units (CFU) (starting with 0.5 g in 4.5 mL PBS). Different volumes of the first two dilutions were cultivated for 24 h at 37 degC on a selective Gassner agar (Sifin, Berlin, Germany), which allows differentiation of lactose fermentation. A Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometer (Bruker, microflex LT mass spectrometer, Bruker Daltonik GmbH Leipzig, Germany) was used to identify bacteria forming colonies on Gassner agar. Analyses for Brachyspira (B.) sp. were performed randomly at six time points, including at least one examination of each flock. The chymus of the cecum was cultured anaerobically on selective horse blood agar plates designed to detect avian brachyspira as described by Harms et al. . Samples of liver, kidney, spleen, lungs, heart, duodenum, ileum, cecum, and bursa cloacalis were fixed in 4.5% neutral buffered formalin (Formaldehyde-Solution 37%, Merck, Darmstadt, Germany) for at least 24 h. Bone fragments from the femoral head, proximal tibiotarsus, distal tibiotarsus, and proximal tarsometatarsus were decalcified (OSTEOMOLL, Merck KGaA, Darmstadt, Germany) for at least 24 h prior to further processing. Formalin-fixed samples were dehydrated, routinely embedded in paraffin wax, and sectioned at 4 mm. All sections were stained with hematoxylin and eosin for histopathological examination. Samples for molecular biological examinations were pooled separately for hens with and (if applicable) without GL, despite the assumption that a whole flock would be affected by virus infection. In each case, DNA was extracted from 10 to 15 mg of each pooled sample using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) or IndiSpin Pathogen Kit (INDICAL BIOSCIENCE GmbH, Leipzig, Germany). RNA was isolated from 15 to 30 mg of each pooled sample using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). Samples included the trachea and a joint swab for detection of mycoplasma . Samples of liver and duodenal tissue as well as a joint swab were collected for amplification of avian orthoreoviruses . Liver tissue samples were taken to detect the avian hepatitis E virus . For the detection of adenoviruses, spleen and duodenum homogenates were examined . In the case of HEV detection, positive samples were further characterized to determine the virulence and, thus, relevance of the virus. For this purpose, the partial ORF1, E3, and fib knob domains were amplified . For amplification of the partial ORF1, E3, and fib knob domains, 2 mL of DNA was mixed with 5 mL of 5X Q5 Reaction Buffer (New England BioLabs, Ipswich, MA, USA), 0.5 mL of dNTPs (final concentration 200 mM; Thermo Fisher Scientific, Waltham, Massachusetts, USA), 1.25 mL of each oligonucleotide primer (final concentration 0.5 mM), and 0.25 mL of Q5 Hot Start High-Fidelity DNA Polymerase (final concentration 0.02 U/mL) (New England BioLabs, Ipswich, MA, USA). The PCR protocol started with activation of the polymerase for 30 s at 98 degC, followed by 40 cycles of denaturation for 10 s at 98 degC, annealing for 15 s at 60 degC, and elongation for 60 s at 72 degC. The reaction ended with a final elongation step lasting 2 min at 72 degC. Subsequently, the PCR product was analyzed by agarose gel electrophoresis. Positive PCR products were purified using the GeneJET PCR Purification Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and subsequently sent to Microsynth Seqlab (Gottingen, Germany) for Sanger sequencing. Nucleotide sequences were analyzed and edited using the GENtle program (Magnus Manske, University of Cologne, Germany), and comparison was performed using the Basic Local Alignment Search Tool of the National Center for Biotechnology Information (NCBI; accessed on 16 May 2022). Phylogenetic analyses and the construction of phylogenetic trees were carried out using the software MEGA X . The evolutionary history was illustrated using the maximum likelihood method and the JTT (Jones-Taylor-Thornton) matrix-based model . For this analysis, the Neighbor-Join and BioNJ algorithms were applied to a matrix of pairwise distances estimated using the JTT model, and then the topology with superior log likelihood value was selected. All sequences of HEV were deposited in NCBI GenBank, with NCBI accession numbers (acc. No.) OM994418, OM994422 to OM994426 for the E3 and fib knob domain genes, and OP171872, OP171876 to OP171880 representing the partial ORF1 gene (Tables S2 and S3). 2.3. Statistical Analysis Statistical analysis was performed with IBM SPSS Statistics for Windows Version 28.0 (IBM SPSS, Armonk, NY, USA) . Tests were implemented to examine infectious agents based on the appearance of GL. Calculations were performed separately for both examination dates to differentiate between the different ages of the hens. A Chi-square test was used to investigate the relationship between two nominal or categorical variables. Fisher's exact test was utilized instead of the Chi-square test when cell frequencies were below five for 2 x 2 tables. The means of two independent groups were compared using Student's t-tests for independent samples with a 95% confidence level. The model assumptions of normality and homogeneity of variance were examined by Shapiro-Wilk and Levene's test. If the data did not follow a normal distribution, the non-parametric Mann-Whitney U test was performed to compare the median values of two samples. A one-way analysis of variance, including a Tukey post-hoc test, was implemented to compare the means of multiple samples. The assumption of normal distribution and homogeneity of variances was determined using the tests described above. A Games-Howell post-hoc test was performed if the data failed to meet the assumption of homogeneity of variances. The non-parametric alternative for the one-way analysis of variance was the Kruskal-Wallis test followed by a post-hoc test. A 95% confidence interval was calculated for the prevalence rates of clinical and pathological findings. For all calculations, results were considered significant if the double-sided p-value was equal to or less than 0.05. The p-value is supplemented by the specific effect size for the statistical test (d = Cohend's d, ph = phi coefficient, r = Pearson correlation coefficient). 3. Results and Discussion 3.1. Gross Pathological and Histopathological Findings In total, 31 of 343 (9.04%; 95% CI [0.06, 0.12]) Bronze hens (of which 17 turkeys were toms and excluded from further examination) showed GL discoloration. Divided by age, 16 of 184 (8.76%; 95% CI [0.05-0.13]) were affected at the early fattening stage, and 15 of 159 (9.43%; 95% CI: [0.06-0.15]) at the late fattening stage. The prevalence at the individual examination times varied between 0.0 and 68.4%. The most common finding was a focal GL discoloration limited to the caudal margin of the liver. Multifocal or diffuse green coloration appeared rarely. In a previous study, the mean prevalence of GL in organically farmed turkeys was 33.15% (processing batches from nine flocks with 60 Bronze hens each) . Therefore, the mean prevalence for GL is significantly lower in the current study. In addition, examinations in the current study were performed on Bronze hens of a Cartier genetic. This is due to the decreased proportion of organically fattened Kelly BBB turkeys in Germany, which were investigated in the previous study in favor of other turkey genetics. Because both genetics were kept under the same fattening conditions, a comparison of the two studies seems legitimate. The marked discrepancy in GL prevalence between the two studies, however, may be in part due to the different genetics of the turkeys examined. The histopathological examination of the livers revealed the accumulation of bile pigment in 4.62% (95% CI [0.02, 0.09]) of the examined livers and solely at the late stage of fattening. Contrarily, inflammatory reactions consisting of slightly multifocal infiltrates consisting of heterophils, lymphocytes, and plasma cells were seen at both examination times, with a prevalence of 9.23% (95% CI [0.05, 0.15]). There was a significant correlation between GL and the presence of inflammatory cells at both fattening stages (early fattening stage: p <= 0.05, ph = 0.34; late fattening stage: p <= 0.05, ph = 0.30), as well as with the accumulation of bile pigment (p <= 0.001, ph = 0.58) at the late fattening stage (Table 1). Therefore, damage to the hepatic tissue concerning GL can be assumed. The bodyweight (BW) of hens between their 70th and 75th days of fattening varied from 1.83 kg to 5.41 kg (mean = 3.53 kg, SD = 0.57). Between 120 and 127 days, it ranged from 4.27 kg to 11.91 kg (median = 8.94, variance = 1.60). At the late examination date, differences in BW of more than 2.0 kg between individuals were found in all flocks. Hens with GL had a significantly (p <= 0.05, d = 0.56) lower mean BW compared to hens without GL in the early fattening period. The discrepancy increased up to 1.45 kg (95% CI [0.36, 2.54]) at the later stage of fattening for hens with GL as opposed to those without (p <= 0.001, d = 1.19) (Table 1). The reduced body weight in the presence of GL indicates impaired physical development and emphasizes that GL is an indicator of a superordinate pathologic condition. Over both examination dates, the mean of the relative liver weight was statistically significantly (early fattening stage: p <= 0.05, d = 0.36; late fattening stage: p <= 0.01, d = 0.28) higher in hens with GL (Table 2). High relative liver weights as a consequence of liver pathology in cases of GL were already described in the literature . Splenomegaly, defined as follicular hyperplasia, was documented in 5.54% (95% CI [0.035, 0.083]) of hens. Most were seen at the early examination date and predominantly belonged to flock No. 31 in the second trial. However, there was a significant correlation between GL and splenomegaly at the late fattening stage (p <= 0.001, ph = 0.44). The hens showed a statistically significant (p <= 0.01, r = 0.35) higher median spleen weight at this stage (Table 1 and Table 2). Gastrointestinal lesions were seen in 81.54% (95% CI [0.74, 0.88]) of the turkey hens. The most common finding was a slight to mild mucosal and submucosal inflammation consisting of heterophils and lymphocytes. They were occasionally accompanied by intestinal villi thickening and merging. Inflammatory reactions frequently affect more than one intestinal segment. Concerning the targeted organ of this study, a significant relation was proven between the occurrence of GL and catarrhal duodenitis at the early fattening period (p <= 0.001, ph = 0.47) (Table 2). A total of 34.69% (95% CI [0.29, 0.40]) of the hens showed skin injuries predominantly located at the snood, and there was no significant correlation for GL. Bilateral footpad dermatitis (FPD) of differing severity was present in every hen. The evaluation method for the footpads is based on a scheme implemented by Mayne and Hocking et al. . All lesions were restricted to the upper skin layers, and no alterations of the subcutaneous tissue were found. There was no statistically significant correlation between the severity of FPD and GL (Table 1). Macroscopic alterations of the joints were solely seen in the late fattening phase, with a prevalence of 6.92% (95% CI [0.04, 0.12]). There was a statistically significant correlation between macroscopic bone alterations and GL in the late fattening phase (p <= 0.001, ph = 0.42) (Table 1). Aseptic bone necrosis was characterized by cell debris and heterophilic and plasmacellular infiltrates accompanied by proliferation of connective tissue and fibrin. It was localized within the epiphyseal growth plate at the proximal tibiotarsus in three cases (2.31%, 95% CI [0.007, 0.06]) and at the distal tibiotarsus in another case (0.77%, 95% CI [0.001, 0.035]). Of those findings, three were seen at the later stage of fattening. Therefore, a significant (p <= 0.05, ph = 0.40) correlation with GL is given at this stage (Table 2). TOC is defined by green livers associated with lesions of the musculoskeletal system . Although this study found a correlation between GL and joint/bone lesions, swollen joints did not necessarily indicate GL discoloration. Regarding histological examinations, the relation between aseptic bone necrosis and GL discoloration corresponds to the literature describing TOC as an immune-associated process possibly leading to further lesions . However, a bacterial infection is not necessarily involved or no longer detectable. This could explain the finding of a single hen with systemic S. aureus infection and simultaneous GL discoloration . Due to the small sample size (five flocks within two trials), no statistical calculations regarding husbandry and management-associated factors were performed. However, an impact of the housing situation in addition to climatic conditions on several health parameters is likely. Regarding the overall fattening period, total losses ranged from 1.38% to 10.32% (mean = 5.32%, SD = 2.69). A higher median mortality existed in flocks where GL appeared during necropsy. Vaccination against hemorrhagic enteritis virus with DINDORAL SPF (Merial GmbH, Hallbergmoos, Germany) was implemented in flocks No. 21 and 22 in the first examination trial and flock No. 41 in the second examination trial. The time point of vaccination varied between the 21st and the 34th day of fattening (Table S1). 3.2. Parasitological Findings The prevalences of cecal parasites were 45.49% (95% CI [0.37, 0.54]) for T. gallinarum, 26.92% (95% CI [0.20, 0.35]) for Eimeria (E.) meleagridis, 23.08% (95% CI [0.17, 0.31]) for H. meleagridis, and 3.08% (95% CI [0.01, 0.07]) for Heterakis (H.) gallinarum. No significant relationship between GL and the detection of E. meleagridis or H. gallinarum could be proven. In contrast, hens with GL significantly more often harbored T. gallinarum (p <= 0.05, ph = 0.27) at the late fattening stage and H. meleagridis at the early fattening stage (p <= 0.001, ph = 0.52) (Table 2). The parasite T. gallinarum usually induces latent infections of the ceca without clinically manifest outbreaks. Similar to H. meleagridis, the documented association with GL may indicate a general weakening of the affected hens and a higher susceptibility for secondary infections . 3.3. Bacteriological Findings Extraintestinal bacterial growth was rarely seen in the hens examined. This included one hen at 72 days of fattening with detection of E. coli in the spleen, shoulder joint, and knee joint. Despite the presence of osteomyelitis in the distal tibiotarsus, this hen showed no GL. Another hen at 120 days of fattening had a systemic infection from the cultivation of S. aureus in her liver, spleen, knee joint, and duodenum. Concurrently, this hen had GL discoloration and inflammatory responses in the liver and spleen on histology. The detection of Mycoplasma sp. succeeded with a prevalence of 9.23% (95% CI [0.05, 0.15]) and predominantly at the late fattening stage. Concerning GL, there was no significant relationship to the detection of mycoplasma. Even though joint/bone lesions predominantly occurred at a later age as well, there was no significant correlation between macroscopic and/or histologic bone lesions and the presence of mycoplasma (Table 2). Most isolates showed high sequence identities with uncultured Mycoplasma sp. from geese of ambiguous relevance for turkeys. Swabs taken from the duodenum revealed E. coli in 29.23% (95% CI [0.22, 0.37]) and Candida (C.) albicans in 24.62% (95% CI [0.18, 0.33]) of hens. There was no significant correlation between the detection of C. albicans or E. coli and GL. The median of the results for quantitative analyses of the commensal E. coli in the jejunoileum from each examination time varied between 180 and 110.000 CFU/mL. There was no statistically significant correlation between E. coli counts and GL. The most diverse bacterial culture was present in the ceca. Slow-growing thermophilic and microaerobic Campylobacter sp. were detected with a total prevalence of 84.62% (95% CI [0.78, 0.90]). Furthermore, Clostridium (C.) perfringens was commonly isolated, with a prevalence of 52.31% (95% CI [0.44, 0.61]). Brachyspira were found with a prevalence of 13.04% (n = 46; 95% CI [0.03, 0.23]). This includes B. innocens in 50.00%, B. pilosicoli in 16.67%, and brachyspira, which could not be further typified in the remaining positive samples. There was no significant correlation between the detection of Campylobacter sp., C. perfringens, or Brachyspira spp. and GL discoloration (Table 2). It must be noted that four examination trials had problems with clostridial infection during the fattening phase, followed by antimicrobial treatment. Samples were taken at specific time points in which the infection may have resolved despite the persistence of inflammation. Therefore, limited detection of bacterial agents may be explicable. 3.4. Virological Findings In six out of 27 pooled samples from three flocks, HEV was detected irrespective of vaccination status. In addition, turkey adenovirus 4 (TAdV-4) from two flocks in the late fattening period and turkey adenovirus 5 (TAdV-5) from two flocks in the early fattening period were detected. Avian orthoreoviruses or avian hepatitis E virus RNA were not detected in any pooled sample. Hens from flocks with a positive finding of HEV in the pooled sample revealed GL significantly more often (p <= 0.001, ph = 0.35) than those without virus detection. Furthermore, hens from positive tested flocks more frequently showed a predominantly slight or moderate catarrhal duodenitis (p <= 0.001, ph = 0.47) at the early fattening stage (Table 1 and Table 2). In contrast, there was no significant association between the finding of GL and the detection of TAdV-4 or TAdV-5 in the pooled sample at this stage. At the late examination date, hens with HEV-positive samples (7.91 kg, variance = 1.03) showed a significantly (p <= 0.001, r = 0.59) lower median BW compared to hens without virus detection (9.20 kg, variance = 1.14). Intranuclear inclusion bodies and splenic lymphocytic depletion are characteristics of a pathologically manifest infection with HEV . These alterations were found in five hens in the early fattening period in the second examination trial from flock No. 31. These hens were not vaccinated against HEV but tested virus positive, and one of these hens concurrently had a GL. One key finding of HEV is the eponymous hemorrhagic enteritis, which is primarily localized in the duodenum . In this study, hens with HEV detection only exhibited slight catarrhal duodenitis, indicating a certain effect on intestinal health, nonetheless. Immunization against HEV is usually part of the routine vaccination regimen for turkeys in Germany. Therefore, we compared the obtained HEV samples with the avirulent turkey HEV strain Virginia (Dindoral(r) SPF, Merial GmbH, Hallbergmoos, Germany; NCBI acc. No.: AY849321) used for vaccination in Germany . The HEV samples showed high sequence identity (98.98-100.00%) at nucleotide levels at partial ORF1, E3, and fiber knob compared to the listed sequence of the avirulent turkey HEV strain Virginia. This is emphasized by the high sequence identity (98.62-100.00%) at amino acid levels (Tables S2 and S3). The phylogenetic analysis revealed the presence of two main clusters. Isolates from flock 31 cluster close to the Virginia avirulent vaccine strain (Dindoral(r) SPF) as well as the Virginia virulent strain isolated from turkeys with clinical HE. Isolates from flocks 21 and 22 cluster next to the commercial Oralvax HE vaccine strain listed at NCBI . Based on the present phylogenetic analysis, it can be assumed that all isolates cluster close to other field and vaccine strains described in the literature . Turkey adenoviruses are known to be widespread in Germany, with particularly avirulent vaccine strains circulating between different turkey flocks. However, knowledge of types and disease conditions is lacking . Possible reasons for the contact with the virus could be insufficient cleaning between herds during the rearing or fattening phase or indirect transmission of the virus through employees, insects, rodents, or equipment. Clinical manifestations of HEV infection in flock 31 can be explained by the genetic drift of the circulating HE field and the vaccine strains through which strains gain virulence. However, infection with a HE-isolate does not necessarily lead to a clinical outbreak but can still increase susceptibility to opportunistic pathogens . Therefore, it is necessary to use vaccines that are adapted to the currently circulating strains . Examined turkey flocks were assigned to five subgroups to clarify the potential impact of HEV-vaccination and HEV- or TAdV-5 infection in the early fattening period (flock virus-positive at the first examination time point). The groups were as follows: (a) hens unvaccinated against HEV with detection of TAdV-5; (b) hens unvaccinated against HEV with detection of HEV; (c) HEV-vaccinated hens with detection of HEV; (d) HEV-vaccinated hens without AdV-detection; and (e) hens unvaccinated against HEV without AdV-detection (Table 3). The highest mean mortality rate was seen in the subgroup of HEV-unvaccinated, positive hens. This group also showed the highest prevalence of GL in the early and late fattening periods. Most of the histological bone lesions occurred in groups with either HEV- or TAdV-5-detection for both examination times. At the early fattening period, unvaccinated hens with HEV detection showed the highest prevalence of catarrhal duodenitis. Most of the histological bone lesions were also found during the late fattening period in this group. The mean BW of the hens chosen for further examination did not differ significantly between the five subgroups at the early fattening stage. The same calculations were performed for the relative spleen and liver weights. Although there were no statistically significant differences regarding the relative liver weight, the highest BW proportion at the early fattening stage was found within the group of HEV-unvaccinated and HEV-positive hens. In addition, the mean relative spleen weight at this stage differed significantly between the five groups (p <= 0.001, e2 = 0.32). A Tukey post-hoc analysis revealed a significantly higher relative spleen weight (p <= 0.05) for unvaccinated hens with HEV detection compared to the other groups. At the early fattening stage, hens of the HEV-unvaccinated but infected subgroup most often harbored C. perfringens (100.00% of the hens) and H. meleagridis (58.82% of the hens, 95% CI: [0.36-0.79]). Regarding the detection of HEV, a virally mediated long-term adverse impact can be implied based on the reduced fattening performance at the end of the fattening stage. Overall development of flocks with HEV detection that had not been vaccinated against HEV was worse compared to those that were either HEV-infected but vaccinated or unvaccinated against HEV and AdV-negative. Furthermore, a poor health condition can be presumed due to high prevalences of GL in HEV-unvaccinated but HEV-positive flocks, indicating liver damage; high relative spleen weights, indicating an immunogenic reaction; the presence of inclusion bodies and splenic lymphatic depletion as a pathological side effect of natural HEV infection; and finally, high mortalities. Confirmation is given when assessing the development of HEV-positive and HEV-unvaccinated hens with a high prevalence of GL in addition to joint/bone lesions at the late fattening period. Since the isolates originate from unvaccinated hens infected with a presumably mutated vaccine or field strain, this emphasizes the role of HEV as a predisposing factor for GL and the importance of an adequate vaccination. 4. Conclusions The causative etiology of GL cannot be restricted to one single pathogen. Statistical calculations partially need to be interpreted carefully due to the comparatively low prevalence of GL. Despite the fact that only one hen had a simultaneous S. aureus infection, an initial bacterial etiology for the other hens could not be excluded in this study. Furthermore, antimicrobial treatment during the fattening phase could be an explanation for the limited detection of bacterial agents. Bacterial or viral infections determine the general health condition and should be managed adequately to avoid potentially higher GL prevalences. Discoloration should be considered an indicator of a negative impact on individual health status and animal welfare that also entails economic damage. This can be assumed due to decreased weight gain, high relative liver weights, the presence of inflammatory cells in the liver, increased prevalence for catarrhal duodenitis and joint/bone lesions, and a tendency for higher flock mortality in the presence of GL. Furthermore, HEV as an immunosuppressive agent correlated significantly with GL, facilitated secondary infections, increased flock mortality, and led to impaired physical development. This is particularly evident when the hens were not vaccinated against this viral pathogen, which emphasizes the need for proper management of this virus. This study indicates two different pathogeneses for GL with a correlation with immunosuppressive HEV at the early and bone/joint lesions at the late fattening stage. However, the results of this study may be coincidental because other important factors, such as husbandry practices or climate, were not evaluated. Further standardized investigations to determine and evaluate possible infectious risk factors are necessary. This study expanded the knowledge about green liver discoloration in fattening turkeys, nevertheless. Acknowledgments The authors thank the diagnostics team of the Institute of Virology as well as Eric Engel, Katrin Erfurt, and Jana Schomburg (Institute of Virology, Faculty of Veterinary Medicine Leipzig) for their excellent technical assistance. Supplementary Materials The following supporting information can be downloaded at: Table S1: Anamnestic data of Bronze turkey hens of a Cartier genetic from five organic turkey farms (two trials each); Table S2: Nucleotide (nt) and amino acid (aa) sequence identities of detected hemorrhagic enteritis virus at the early fattening period in organic fattened turkeys compared to virulence genes E3 (nt 21247-22116 [aa AAX51187.2]) and Fiber (nt 22519-23883 [aa AAX51189.1]) of avirulent turkey hemorrhagic enteritis virus strain Virginia (NCBI acc. No.: AY849321.1) in %; Table S3: Nucleotide (nt) and amino acid (aa) sequence identities of detected hemorrhagic enteritis virus at the early fattening period in organic fattened turkeys compared to virulence gene ORF 1 (nt 313-1953 [aa AAX51169.2]) of avirulent turkey hemorrhagic enteritis virus strain Virginia (NCBI acc. No.: AY849321.1) in %; Table S4: List of samples used for comparative analysis in this study with NCBI accession numbers (acc. no). The table includes several (virulent) field samples as well as commercial vaccine strains against hemorrhagic enteritis (HE). Sorted by authors and publication journals . Click here for additional data file. Author Contributions V.S., M.-E.K.-J. and K.C. conceived and supervised this study; The experimental procedures and statistical analysis were performed by L.C. and I.S.; C.G.B. and J.H. conducted the quantification of coliform bacteria and the detection of avian brachyspira; K.H., T.W.V. and M.H. conducted all virological examinations, including the interpretation and visualization of the results. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The examinations required no notification or approval in accordance with the Animal Protection Act (SS 7, paragraph 2, sentence 3). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The data presented in this review is available on request from the corresponding author. Conflicts of Interest The authors report that there are no competing interest to declare. Figure 1 Phylogenetic tree based on concentrated sequences of partial ORF1, E3, and Fiber knob genes (original tree). The evolutionary history was illustrated using the maximum likelihood method and the JTT matrix-based model . The tree with the highest log-likelihood (-1554.26) is indicated. The percentage of trees in which the associated taxa are clustered is indicated next to the branches. For this analysis, the Neighbor-Join and BioNJ algorithms were applied to a matrix of pairwise distances estimated using the JTT model and the topology with the superior log likelihood value. This analysis included 22 amino acid sequences. Six sequences belonging to this study are marked with a black triangle. Vaccine strains are marked with an empty square. The phylogenetic analysis was based on the amino acids of six sequences from this study in comparison to 16 reference sequences from NCBI . The final data set included a total of 496 positions. The evolutionary analyses were performed in MEGA X . animals-13-00918-t001_Table 1 Table 1 Comparison of different values for all 343 female Bronze turkey individuals with and without green liver discoloration in the early (70 to 75 days of fattening) and late (120 to 127 days of fattening) fattening stages, respectively. The presented data includes means (x ), medians (x ), standard deviations (SD), p-values, and (if applicable) effect sizes (ES) within the same age group. Value 70 to 75 Days of Fattening 120 to 127 Days of Fattening Green Liver No Green Liver Green Liver No Green Liver n = 16 n = 168 n = 15 n = 144 (x )/(x ) +- SD (x )/(x ) +- SD p-Value/ES (x )/(x ) +- SD (x )/(x ) +- SD p-Value/ES Bodyweight 3.19 +- 0.47 3.57 +- 0.57 0.01/d = 0.56 7.51 +- 1.95 8.97 +- 1.10 <=0.001/d = 1.19 Skin injuries (%) 0.00 17.26 0.08 66.67 55.56 0.41 Splenomegaly (%) 6.25 7.74 1.00 26.67 0.69 <=0.001/ph = 0.44 Macroscopic joint lesions (%) 0.00 0.00 40.00 3.47 <=0.001/ph = 0.42 HEV (%) 93.75 32.74 <=0.001/ph = 0.35 33.33 23.61 0.53 FPD Score 4.00 +- 0.27 4.00 +- 0.12 0.90 4.00 +- 0.29 4.00 +- 0.64 0.48 HEV: Hemorrhagic Enteritis Virus, FPD: Foot pad dermatitis. Data include means and medians (in italic). animals-13-00918-t002_Table 2 Table 2 Comparison of different values of the further examined 130 female Bronze turkey individuals with and without green liver discoloration in the early (70 to 75 days of fattening) and late (120 to 127 days of fattening) fattening stages, respectively. The presented data includes means (x ), medians (x ), standard deviations (SD), p-values, and (if applicable) effect sizes (ES) within the same age group. Value 70 to 75 Days of Fattening 120 to 127 Days of Fattening Green Liver No Green Liver Green Liver No Green Liver n = 9 n = 58 n = 15 n = 48 (x )/(x ) +- SD (x )/(x ) +- SD p-Value/ES (x )/(x ) +- SD (x )/(x ) +- SD p-Value/ES Rel. weight spleen (%) 0.11 +- 0.00 0.08 +- 0.00 0.12 0.09 +- 0.00 0.06 +- 0.00 0.004/ r = 0.35 Rel. weight liver (%) 2.25 +- 0.55 1.91 +- 0.33 0.01/ d = 0.36 1.67 +- 0.51 1.26 +- 0.15 0.004/ d = 0.28 Inflammatory cells liver 22.22 1.72 0.045/ ph = 0.34 33.33 8.33 0.03/ ph = 0.30 Macroscopic joint lesions (%) 0.00 0.00 40.00 3.47 <=0.001/ ph = 0.42 Bile pigment liver 0.00 0.00 - 40.00 0.00 <=0.001/ ph = 0.58 Lymphocytic depletion spleen 11.11 10.34 1.00 0.00 0.00 - Aseptic bone necrosis 0.00 1.72 1.00 20.00 0.00 0.01/ ph = 0.40 Catarrhal duodenitis 66.67 12.07 <=0.001/ ph = 0.47 26.67 10.42 0.20 Catarrhal jejunitis 22.22 46.55 0.28 33.33 37.50 0.77 Catarrhal typhlitis 22.22 62.07 0.03/ ph = 0.27 73.33 81.25 0.49 Qual. Escherichia coli duodenum 11.11 27.59 0.43 46.67 29.17 0.21 Quan. Escherichia coli duodenum (CFU) 4000 +- 1.8 x 108 3950 +- 5.6 x 1011 0.41 2.800 +- 1.1 x 1011 1350 +- 2.5 x 1010 0.68 Candida albicans duodenum 66.67 32.76 0.07 6.67 12.50 1.00 Staphylococcus aureus extraintestinal 0.00 0.00 - 6.67 0.00 0.24 Escherichia coli extraintestinal 0.00 6.70 1.00 6.67 2.08 0.42 Campylobacter sp. 77.78 68.97 0.71 100.00 100.00 - Clostridium perfringens 88.89 62.07 0.15 40.00 37.50 1.00 Brachyspira sp.1 16.67 0.00 0.27 0.00 27.78 0.28 Mycoplasma sp. 0.00 3.45 1.00 6.67 18.75 0.43 Tetratrichomonas gallinarum 77.78 43.10 0.08 66.67 35.43 0.03/ ph = 0.27 Histomonas meleagridis 77.78 13.79 <=0.001/ ph = 0.52 13.33 27.08 0.49 Heterakis gallinarum 0.00 0.00 - 0.00 8.33 0.56 Eimeria meleagridis 0.00 29.31 0.10 26.67 29.17 1.00 rel. = relative; qual. = qualitative; quan. = quantitative. 1 Investigations were performed randomly at six examination points. Data include means and medians (in italic). animals-13-00918-t003_Table 3 Table 3 Presentation of different values for flocks or selected hens of (a) hens unvaccinated against HEV with detection of TAdV-5, (b) hens unvaccinated against HEV with detection of HEV, (c) HEV-vaccinated hens with the detection of HEV, (d) HEV-vaccinated hens without AdV-detection, and (e) hens unvaccinated against HEV without AdV-detection. The presented data includes means (x ), except for the relative liver weight, which is given as medians (x ). Group a b c d e Value--Flocks n 37 39 31 20 57 Mortality (%) 5.54 9.15 3.04 7.08 3.57 Green livers 1 (%) 0.00 35.90 3.20 0.00 1.80 Green livers 2 (%) 12.80 20.00 5.00 10.00 5.00 Value--Selected hens n 12 17 13 6 19 BW 1 (kg) 3.33 3.57 3.69 3.42 3.42 BW 2 (kg) 7.11 8.03 10.29 7.96 9.41 Rel. weight spleen 1 (%) 0.07 0.12 0.08 0.09 0.09 Rel. weigh liver 1 (%) 2.04 2.07 1.84 1.73 1.95 Bone lesions 1,3 (%) 16.70 5.90 30.80 0.00 10.50 Bone lesions 2,3 (%) 5.90 20.00 14.30 12.50 0.00 Catarrhal duodenitis 1 (%) 0.00 64.71 7.69 16.67 0.00 Clostridium perfringens1 (%) 50.00 100.00 53.85 50.00 57.89 Histomonas meleagridis1 (%) 25.00 58.82 0.00 0.00 10.53 1 first examination (70th-75th day of fattening); 2 second examination (120th-127th day of fattening); 3 histological bone lesions; Data include means and medians (in italic). 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PMC10000100

Growth and histological parameters were evaluated in Atlantic salmon (74 g) that were fed alternative phospholipid (PL) sources in freshwater (FW) up to 158 g and were transferred to a common seawater (SW) tank with crowding stress after being fed the same commercial diet up to 787 g. There were six test diets in the FW phase: three diets with different doses of krill meal (4%, 8%, and 12%), a diet with soy lecithin, a diet with marine PL (from fishmeal), and a control diet. The fish were fed a common commercial feed in the SW phase. The 12% KM diet was compared against the 2.7% fluid soy lecithin and 4.2% marine PL diets, which were formulated to provide the same level of added 1.3% PL in the diet similar to base diets with 10% fishmeal in the FW period. A trend for increased weight gain with high variability was associated with an increased KM dose in the FW period but not during the whole trial, whereas the 2.7% soy lecithin diet tended to decrease growth during the whole trial. A trend for decreased hepatosomatic index (HSI) was associated with an increased KM dose during transfer but not during the whole trial. The soy lecithin and marine PL diets showed similar HSI in relation to the control diet during the whole trial. No major differences were observed in liver histology between the control, 12% KM, soy lecithin, and marine PL diets during transfer. However, a minor positive trend in gill health (lamella inflammation and hyperplasia histology scores) was associated with the 12% KM and control diets versus the soy lecithin and marine PL diets during transfer. krill phospholipid salmon smolt feed gills This research received no external funding. pmc1. Introduction Farmed salmon are typically transferred from early phase production in tanks on land to seawater cages that constitutes a challenging environment, where fish can experience significant mortality before reaching harvest size. For example, mortality in Atlantic salmon ranged from 15 to 16% from 2017 to 2021 in Norway, with approximately 35% of sea cage mortality occurring in the first 0-3 months at sea for the 2010-11 salmon generations in the Norwegian-farmed Atlantic salmon . This mortality in the early sea cage phase leads to significant economic loss . Thus, research on optimal nutrition to produce robust smolts for improved survival and growth after transfer to the sea cage is of interest to the aquaculture industry . Fish meal (FM) and fish oil (FO) dominated early commercial salmon feed formulations and provided essential nutrients, but usage of these marine ingredients has declined over time as they are limited resources at generally higher prices compared to alternative ingredients where sustainability measures are also considered . Antarctic krill meal (KM; Euphausia superba) is a commercially known ingredient in salmon feeds, with potential benefits toward enhancing growth and health in salmonids . The krill fishery in the Antarctic Southern Ocean is considered highly regulated and sustainable . KM provides a range of nutrients including proteins (similar amino acid profile to FM); water soluble nitrogenous components (free amino acids, peptides, nucleotides, and trimethylamine N-oxide), which can act as potential feed attractants; astaxanthin; marine omega-3 fatty acids (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)); and phospholipids (PLs) . Substantial evidence exists showing that dietary PL can improve growth, survival, and health (reduced intestinal steatosis and deformities) in the larval and early juvenile stages of the fish . In addition, KM and krill oil (KO) reduced fat accumulation in the hepatocytes in comparison to soybean lecithin as the PL sources in the diet of seabream larvae . In addition, there was an indication that seabream juveniles that were fed a diet with 9% KM had lower hepatocyte vacuolization (fat storage) versus a control diet without KM that was higher in fishmeal , and a non-significant trend for lower hepatocyte vacuolization was indicated for seabream larvae that were fed a diet with krill oil versus soybean lecithin as the PL source . PLs from different sources can have different properties. KM has approximately 40% PL consisting of the total lipid with phosphatidylcholine (PC) at >80% of the total PL and ca. 18% EPA + DHA of the total lipid . In comparison, fluid soy lecithin can have approximately 46% PL of product (does not include glycolipids and complex sugars) with ca. 35% PC of the total PL and ca. 55% 18:2n-6 of the total FA as the major FA with no EPA + DHA . KM has been documented in the diet of seawater salmon , however, only KO has been documented in the diet of freshwater salmon during the pre-transfer to the seawater phase . The objective of the present study was to document the effect of the KM dose as a source of PL and compare it against other PL sources in the feed of freshwater Atlantic salmon during the pre-transfer phase followed by the early seawater phase by evaluating the growth and histological health parameters. A four-level graded dose response for KM up to 12% of the diet along with a comparison of alternative PL sources (soy lecithin and marine PL from fishmeal) formulated to provide the same level of added 1.3% PL in the diet as 12% KM was evaluated in freshwater diets for salmon during the pre-transfer phase. Fish identified by pit tag with this pre-transfer freshwater feeding history were then transferred to a common seawater tank with crowding stress after transfer and a drop in water temperature at transfer (crowding and water temperature drop can be experienced at transfer commercially) and then were fed the same commercial feed. Gill and liver histology were also compared for salmon that were fed the alternative PL source diets at the end of the freshwater pre-transfer period. 2. Materials and Methods 2.1. Feed Formulation and Composition Three different sources of PL were tested in pre-transfer freshwater feeds: (i) krill meal (QrillTM Aqua; Aker BioMarine Antarctic ASA) at four levels for dose response (4%, 8%, and 12% of diet), (ii) fluid soy lecithin as a vegetable PL source, and (iii) marine phospholipid-rich oil sourced from North Atlantic fish species from Triple 9 (TripleNine, Trafikhavnskaj 9, DK-6700 Esbjerg, Denmark)). , and a control diet. The trial diets are referred to as Control, KM4, KM8, KM12, VegPL, and MarPL, respectively. Trial feeds were formulated using a commercial formulation program with external oil mix calculations and produced by extrusion at Cargill Innovation Center (Dirdal, Norway) for ca. 74 g fish with lipid nutrients and then adjusted for purposes of the trial. The 4-mm pre-transfer freshwater trial feeds were formulated and analyzed to have similar digestible energy (22.1-23.6 MJ/kg gross energy), protein (46-49% range), and fat (22-24% range) (Table 1) and with similar calculated 1.1% EPA + DHA in diet, 15-16% saturated in total FA, and 1.3 n-6/n-3 fatty acid (FA) ratio across trial feeds. Protein was analyzed by the Dumas principle using the Elementar Rapid Max N system. Fat was analyzed by low-field nuclear magnetic resonance scan using the NMR Analyzer Bruker minispec mq10 system (Cargill Innovation Center, Dirdal, Norway). Gross energy was analyzed by the Leco gross energy bomb calorimetry system (Cargill Innovation Center, Dirdal, Norway). Moisture was predicted by the NIR FOSS DS2500 system (Cargill Innovation Center, Dirdal, Norway) by using the feed model at Cargill. A similar 1.3% PL in diet across pre-transfer freshwater diets was calculated from the addition of 12% krill meal, fluid soy lecithin, and marine PL test ingredients to base formulations with the same 10% fishmeal level across the diets. There was variation in the other ingredients (added oil, plant ingredients, and micronutrients) needed for balancing or reaching nutrient targets. The choline level was formulated to be the same for control and VegPL diet with MarPL and KM12 providing additional choline to these diets in the form of phosphatidylcholine (PC). However, formulated choline levels for control diet and fluid soy lecithin diets were in excess of the NRC 2011 requirements for salmonids and in excess of the lowest choline level used by Hansen and coworkers with no growth differences observed (1340 to 4020 mg choline/kg diet dose response trial for 456 g initial weight salmon). Lipid accumulation in the gut was reduced for salmon (456 g initial weight) at increased choline levels . The formulation and composition of feeds are given in Table 1. 2.2. Fish Trial Conditions The experiment was performed according to the guidelines and protocols approved by the European Union (EU Council 86/609; D.L. 27.01.1992, no. 116) and by the National Guidelines for Animal Care and Welfare published by the Norwegian Ministry of Education and Research. Atlantic salmon (Salmo salar) with an initial weight of ca. 67 g were used for the trial. The fish were pit-tagged and randomly distributed into 24 freshwater flow-through tanks (1 m diameter and 0.45 m3 volume) to have 40 fish per tank at the start of trial diet feeding. These fish after 15 days of tank acclimation were 74 +- 12 g (average +- SD for all 960 fish in 24 tanks at the start of trial feeding) and then were fed the freshwater pre-transfer trial diets (Table 1) over a 53-day period. Water temperature averaged 14.3 degC (13.3-15.3 degC range) with 107% average oxygen saturation at the inlet and 90% oxygen saturation at the outlet during the freshwater acclimation and trial diet feeding period. Fish were fed the six trial diets to four replicate tanks during the 53-day freshwater pre-transfer period using an automatic belt feeder with continuous feeding for 20 h per day in excess of satiation level. Feed intake was calculated on a weekly basis by collecting and weighing uneaten pellets as well as by weighing the amount fed. There was a 12 h light: 12 h dark photoperiod regime from Day 0 at freshwater tank acclimation to Day 33 after which a 24 h light regime was used to initiate smoltification. After this freshwater pre-transfer feeding period, fish from all the tanks (17-20 fish per tank from the 24 freshwater tanks) were transferred to a larger common seawater flow-through tank (5 m diameter and 21.6 m3 volume with 28.5 ppt salinity, and no acclimation time from 0 ppt freshwater to 28.5 ppt seawater) with a water temperature drop at transfer (ca. 14 to 9 degC) and crowding stress after transfer (lowered water level to ca. 20 cm for one hour with supplemental oxygen for all 459 fish of ca. 167 g within a ca. 0 to 30 h period after transfer) in the common seawater tank after all 17-20 fish per tank from the 24 freshwater tanks fish were transferred over and then were fed a common commercial extruded salmon diet (EWOS AS) for a further 98 days. Daily water temperature was lower during the seawater phase averaging 9.4 degC (8.5-11.1 degC range). 2.3. Fish Growth The 40 fish per tank were weighed individually with pit-tag identification on acclimation to the freshwater tanks (Day 0), at the start of trial diet feeding (Day 15), at intermediate weighing (Day 33), and after 53 days of trial feeding in the freshwater (Day 68). The fish weight gain in the freshwater pre-transfer period from Day 15 (start of freshwater trial diet feeding) to 68 were compared statistically between diets. A total of 17-20 fish from each of the 24 freshwater tanks were transferred to the common seawater tank on Day 68 with fish weighing performed on Days 35, 73, and 98 after transfer to seawater. There were 9 to 17 fish representing the original tanks in the freshwater period with 50 to 58 fish representing each of the test diets from the freshwater period at final weighing in seawater at 98 days after transfer to the common seawater tank. The fish weight gain over the whole trial period in freshwater and seawater from Day 15 to 166 days were statistically compared between diets. 2.4. Hepatosomatic Index Hepatosomatic index (HSI) is the liver weight percent of the whole body weight. HSI was measured on 10 fish randomly sampled per tank (four tank replicates per diet) to study 40 fish per diet at the end of the freshwater pre-transfer period when fed test diets and 40 fish per diet (identified by pit-tag) at the end of the seawater phase when fed the common commercial diet. 2.5. Histology Gill and liver histology were performed on the fish involved in the dietary phospholipid source comparison (KM12, VegPL, and MarPL) and on fish fed the Control diet at the end of the freshwater pre-transfer period. Liver (half tissue section) and gill (left gill arch 2) tissues were randomly sampled from five fish per tank to give a total of 20 liver and 20 gill tissues per diet group for histological analysis. The tissues were fixed in formalin (4% formaldehyde) and stored at room temperature until sent to Pharmaq Analytiq AS (Harbitzallee 2A, 0275 Oslo, Norway) for histological analysis. 2.6. Statistical Analysis The weight gain for the different periods was modelled by computing the weight gain of each tagged individual and then using a hierarchical generalized additive model (GAM) with the spline function to describe the possibly non-linear dose-response. A random effect of tank was added to the model to account for the multiple individual observations per experimental unit. The total feed intake over the periods of interest was modelled with a single level GAM with a spline function describing the dose-response function. Hepatosomatic index (HSI) was modelled by a hierarchical GAM model using a spline function to describe the dose-response function, mean-centered round weight of the fish as a covariate, and a random effect of tank to account for the multiple individual observations per tank. From this model the expected liver weight was solved for an average-sized sampled fish and expressed as HSI by dividing the expected liver weight with the mean round weight of the sample. Gill and liver histology scores are ordinal variables for which common arithmetic operations, such as sum or mean, are not defined and therefore scores require an ordinal model returning the score probability for evaluation. A hierarchical GAM for ordinal data was set up by using a spline function to describe the dose-response function, and a random effect of tank was included to account for multiple individuals observed per tank. The models for weight gain, feed intake, and HSI assumed the error distribution is the normal distribution, and the model for gill and liver scores assumed the model is ordinal and the errors followed the ordered categorical family. All data processing and statistical modelling was conducted with the R language . The GAMs were estimated with the "gam" function of the R language add-on package "mgcv" . The outcomes from the fitted statistical models are presented graphically by showing the mean response and the 95% credible intervals. The mean (median) response and the 95% credible intervals were computed with the help of a parametric bootstrap (with 10,000 random draws per parameter) by taking the 25%, 50%, and 97.5% quantiles of the computed response vector. In the case of a categorical predictor variable (for comparing the different PL sources), the graphs show the mean and an error bar of the 95% credible interval. In the case of a continuous predictor (for the dose-response of krill meal inclusion), the mean response is shown as a median dose-response curve and the 95% credible interval is shown as a confidence band around the mean curve. This way both the magnitude of any potential effect (biological significance) and the uncertainty of any effect estimate (statistical significance) can be shown in the same graph for all the results independent of the response following the normal, binomial, or ordered categorical distribution. 3. Results 3.1. Growth Performance Atlantic salmon of 74 g (overall tank average) were fed the six test diets up to 158 g (overall tank average), growing 2.1-times the initial fish weight to the end of the freshwater pre-transfer period. There was no clear trend for increased feed intake with KM dose in the FW pre-transfer phase . A trend for increased feed intake was indicated for the Control and KM12 diets compared to the MarPL and VegPL diets in the PL source comparison for the FW pre-transfer phase . There was overall high variability for the feed intake comparisons. A trend for increased fish weight gain with high variability was indicated with increased KM dose in the FW phase . There was similar weight gain during the whole trial with feeding the KM dose in the FW pre-transfer phase followed by feeding the same commercial diet in a common tank for the SW phase . Fish fed the KM12 diet had increased weight gain compared to the VegPL diet with the MarPL and Control diets having intermediate weight gains in the PL source comparison for the FW pre-transfer phase . Weight gain was similar for the fish that were fed KM12, MarPL, and Control diets, with a trend for higher indicated weight gain than the VegPL group during the whole trial, with feeding the KM dose in the FW pre-transfer phase followed by feeding the same commercial diet in a common tank for the SW phase . 3.2. Hepatosomatic Index A trend for decreased hepatosomatic index (HSI; liver% of fish weight) was indicated for the fish that were fed increased KM dose from 0 to 12% of diet at the end of the freshwater pre-transfer feeding phase . There was no decrease in HSI with feeding KM dose at the end of the whole trial after the FW pre-transfer phase followed by feeding the same commercial diet in a common tank for the SW phase . A lower HSI was indicated for the fish that were fed the KM12 diet compared with the fish that were fed the MarPL, VegPL, and Control diets at the end of the freshwater pre-transfer feeding phase with a similar minor HSI trend observed over the whole trial . 3.3. Histology 3.3.1. Gill Histology An increased probability for very mild to mild gill lamella inflammation and hyperplasia score was indicated for the salmon that were fed the VegPL and MarPL diets compared to the Control and 12% KM diets at the end of the freshwater pre-transfer phase after 53 d of feeding the trial diets . Other following gill histology responses were evaluated with no major differences between the diets: vascular lesions, filament inflammation, necrosis of respiratory epithelium, necrosis affecting deeper tissues, fusion of lamella ,and other lesions noted as present or absent. 3.3.2. Liver Histology No major differences were observed in liver histology between the control, 12% KM, soy lecithin, and marine PL diets at the end of the FW pre-transfer phase after 53 d of feeding the trial diets (data not shown). The following liver histology responses were evaluated: total amount of abnormal tissue, inflammation, necrosis, inflammation in liver tissue or capsule (peritonitis), peribiliary or perivascular inflammation, neoplasia, fibrosis, lipid deposition, other degenerative changes, vascular lesions, and other lesions noted as absent or present. 4. Discussion The present study evaluated the effect of different phospholipid sources fed over 53 d in the freshwater pre-transfer phase followed by feeding the same commercial diet over 98 d in a common seawater tank on growth performance and health parameters of Atlantic salmon. KM was evaluated in dose response (4%, 8%, and 12.0% of diet), and diets with 2.7% fluid soy lecithin (VegPL) and 4.2% MarPL as alternative PL sources were formulated to provide the same level of added 1.3% PL in diet as 12% KM. All the test diets contained 10% fishmeal in the FW phase. A trend was indicated for increased fish weight gain (high variability) with increased KM dose in the FW pre-transfer phase but a carry-over effect on growth was not observed for the same salmon fed the same commercial diet after seawater transfer. Salmon (104 g initial weight) that were fed krill meal at 7.5 and 15% of diet for higher fishmeal diets (40-52% of diet range) than the current trial had increased growth after transfer to sea cage . Fishmeal provides PL, so higher fishmeal diets may reduce the need for KM as a PL source . However, KM also provides amino acids (protein), water-soluble nitrogenous components (potential feed attractants), astaxanthin, and EPA + DHA, hence, it is more than a PL source. KM feeding may need to continue after sea water transfer to have a positive effect on growth at the end of the trial, noting the positive effects of KM on salmon growth observed in other but not all trials, which can depend on life stage and challenges, diet composition, KM refining (de-shelling etc.), and inclusion level . A trend for decreased fish weight gain was indicated for the VegPL diet in the FW phase and over the whole trial compared with the control diet, whereas the MarPL diet showed more similar growth to the control diet over the whole trial, noting that only one PL level tested for MarPL and fluid soy lecithin matched that provided by KM12, so optimal dose was not evaluated. The choline level was formulated to be the same for the control and VegPL diets with KM12 and MarPL providing additional choline to these diets in the form of phosphatidylcholine (PC). Formulated choline levels for the control diet and fluid VegPL diets were in excess of the NRC 2011 requirements for salmonids and in excess of the lowest choline level used by Hansen et al. in 2020 with no growth differences observed (1340 to 4020 mg choline/kg diet dose response trial for 456g initial weight salmon) . Lipid accumulation in the gut was reduced for these salmon (456 g initial weight) at increased choline levels . Effects of increased choline with KM inclusion cannot be ruled out and further research would be needed to separate choline from PL effects for these smaller pre-transfer salmon (74 to 158 g fish weight) that were fed lower fat pre-transfer diets (22-24% fat) than during the seawater growth with choline requirements for reducing the lipid accumulation in the intestine, potentially dependent on dietary fat level . Higher growth was generally observed for PL provided by KO over soy lecithin at various PL doses for the first-feeding stage of salmon, but this growth trend was not consistent at various PL doses over the whole trial from the first-feeding to smolt . PL from KO was indicated to be more effective than fluid soy lecithin for reducing intestinal steatosis in smaller salmon (2.5 g salmon, but no steatosis observed across diets for 10-20 g salmon) and low level of vertebral deformities . Marine PL sources (FM and KO) were also compared against soy lecithin at a similar ca. 3.5% PL of diet level for the first-feeding Atlantic salmon (0.14 g initial weight) with these PL sources, giving similar growth to ca. 2.4 g final fish weight with no conclusive mortality or intestinal histology differences between PL sources but these parameters were generally improved for the PL source diets with higher PL compared to the control diets with lower PL. An uncertain observation of higher average growth was indicated for the marine PL sources over soy lecithin at intermediate weighing for salmon at ca. 0.6 g . Effects of PL cannot be isolated from KM but the increased growth for KM12 over the VegPL diet in the pre-transfer phase may be due to PL, choline, water soluble nitrogenous components, etc., noting that there was also an indicated trend for decreased growth of VegPL versus the control diet in the pre-transfer phase. Addition of KM did not give a clear increase in feed intake compared to the control diet and there was an indicated trend of decreased feed intake for the MarPL and VegPL diets, but strong conclusions cannot be made due to the high variability. Feed intake can only be measured on a tank basis, so it was not possible to estimate feed intake of fish with different pre-transfer freshwater feeding histories in a common tank that were fed the same diet in the seawater phase. A trend for decreased hepatosomatic index (HSI) was indicated with increased KM inclusion and for the 12% KM diet versus the other PL sources added to provide the same PL level in the pre-transfer phase, but the effect of KM on decreasing HSI was not carried over into the seawater phase with fish that were fed the same diet in a common tank . There was no difference in the liver lipid droplet accumulation based on histology (normal scores only) for salmon that were fed the diets containing different PL sources at the end of the freshwater pre-transfer period. The lower HSI in KM12 could be due to the positive effects from krill PL (and choline) on the lipid transport and deposition in organs, with this effect of feeding 12% KM to Atlantic salmon documented by with less pale livers and reduced liver fat. The authors further supported this observation with a significantly higher expression of the cadherin 13 (Chd) gene in the 12% KM group associated with circulating levels of the adipocyte-secreted protein adiponectin that has potential anti-inflammatory effects and plays an important role in metabolic regulation and is associated with the fatty liver index in humans . However, Chd expression was not studied in the current study, and hence, further studies are warranted to explore the association between Chd expression, his, and absolute fat accumulation in the liver in salmon. Increased choline, which KM provided in this trial, was shown to reduce fat accumulation in the intestine of Atlantic salmon . Choline supplementation was also indicated to reduce HSI in Atlantic salmon, but this was not reflected in lower liver fat or histological vacuolization, noting that there are variable trends of dietary choline deficiency on the liver fat level of fish reported in the literature . PL from KO was indicated to be more effective than fluid soy lecithin for reducing intestinal steatosis in smaller salmon (2.5 g salmon but no steatosis observed across diets for 10-20 g salmon). Further studies are required to associate higher liver fat with welfare in salmon. Gills are one of the most vital organs of fish, due to their function in respiration, osmoregulation, excretion of nitrogenous waste, pH regulation, and hormone production . Gill health has become one of the most significant health and welfare challenges in the salmon aquaculture industry in Norway, Scotland, and Ireland . The gill disorders are generally complex and multifactorial and are related to both biological factors, such as parasites and pathogens, handling stress, treatments, or due to the environmental factors, such as temperature, salinity, algal blooms, etc. Hence, the gill diseases are challenging to prevent and control and lead to high mortality, reduced production performance, and impaired fish welfare, cumulating in huge economic losses . There were no differences reported for histological parameters investigated except in the presence of ectopic epithelial cells containing mucus in the lamina propria in the hindgut (potential inflammatory marker) of salmon (grown from 2.3 to 3.9 kg in sea cages) that were fed 15% fishmeal diet but not for 12% KM of diet in a 5% fishmeal diet, which may suggest anti-inflammatory effects of KM . KM provides astaxanthin (166 mg/kg in the KM used for the present study) to the diet as a natural antioxidant with potential anti-inflammatory properties . KM and MarPL also provide EPA + DHA attached to PL, which may affect bioavailability of EPA + DHA for use in cell membranes and inflammatory response but this is not documented in fish. In the current study, there was decreased probability for very mild to mild gill lamella inflammation and hyperplasia scores indicated in salmon that were fed 12% KM compared to the soy lecithin and marine PL diets but gill histology for salmon that were fed the 12% KM diet was similar to the control diet without KM . 5. Conclusions Overall, increased KM tended to increase growth (high variability), whereas the VegPL diet tended to decrease growth compared to the control diet in the FW pre-transfer phase. The positive growth trend indicated for KM fed pre-transfer was not carried over into the seawater phase for fish fed the same diet. A minor positive trend in gill health (lamella inflammation and hyperplasia histology scores) was indicated for the 12% KM and Control diets compared with the VegPL and MarPL diets in the FW pre-transfer phase. Hepatosomatic index tended to decrease with KM fed in the pre-transfer phase, noting that all livers evaluated by histology were considered normal for lipid droplet accumulation. Only one VegPL and MarPL dose was tested, so dose effect of these PL sources and comparison with krill oil to better isolate the PL effect from other nutrients in KM as well as a post-transfer feeding comparison of these PL sources could be areas to research further in transfer diets for salmon. Acknowledgments The authors would like to thank Lena Burri for her feedback to the manuscript. Supplementary Materials The following supporting information can be downloaded at: Table S1: Fish weight, fish weight gain, feed intake and FCR for the 53~d of trial diet feeding in freshwater pre-transfer period; Table S2: Fish weight and fish weight gain for common seawater tank posttransfer period with fish fed the same commercial diet over 98~d. Click here for additional data file. Author Contributions R.K. designed and executed the study. She was also involved in the analysis and interpretation of the results and writing of the manuscript. J.V. was involved in running the trial and data analysis. D.N. was involved in design, interpretation of results, and writing of manuscript. K.R. did the statistical analysis of the study. K.K. was involved in analyzing and interpretation of results and writing of manuscript. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The experiment was performed according to the guidelines and protocols approved by the European Union (EU Council 86/609; D.L. 27.01.1992, no. 116) and by the National Guidelines for Animal Care and Welfare published by the Norwegian Ministry of Education and Research. Data Availability Statement The data is available in the present manuscript and Supplementary File. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Modelled feed intake in relation to increasing KM inclusion after 53 d of feeding in the freshwater pre-transfer phase. Dots denote observed tank mean feed intake with error bars giving 95% credible intervals. Figure 2 Modelled feed intake for diets containing different phospholipid sources after 53 d of feeding in the freshwater pre-transfer phase. Dots denote observed tank mean feed intake with error bars giving 95% credible intervals. Figure 3 Modelled weight gain with increasing KM inclusion after 53 d of feeding in the freshwater pre-transfer phase plotted with 95% credible intervals as the grey band. Dots denote observed tank mean weight gain. Figure 4 Modelled weight gain with increasing KM inclusion over 53 d in the freshwater pre-transfer phase followed by feeding the same commercial diet over 98 d in the common seawater tank plotted with 95% credible intervals as the grey band. Dots denote observed tank mean weight gains. Figure 5 Modelled weight gain for feeding diets with different phospholipid sources after 53 d of feeding in the freshwater pre-transfer phase plotted with 95% credible intervals. Dots denote observed tank mean weight gains. Figure 6 Modelled weight gain after feeding diets with different phospholipid sources for the whole trial, with 53 d in the freshwater pre-transfer phase on test diets followed by feeding the same commercial diet over 98 d in the common seawater tank. The bars are plotted with 95% credible intervals. Dots denote observed tank mean weight gains. Figure 7 Modelled hepatosomatic index (HSI) with increasing KM inclusion after 53 d of feeding in the freshwater pre-transfer phase plotted with 95% credible intervals as the grey band. HSI is derived from the model for a 160 g average-sized sampled fish. Figure 8 Modelled hepatosomatic index (HSI) for different phospholipid sources over 53 d in the freshwater pre-transfer phase followed by feeding the same commercial diet over 98 d in a common seawater tank plotted with 95% credible intervals as the grey band. HSI is derived from the model for a 792 g average-sized sampled fish. Figure 9 Modelled hepatosomatic index (HSI) for different phospholipid sources after 53 d of feeding in the freshwater pre-transfer phase plotted with 95% credible intervals. HSI is derived from the model for a 157 g average-sized sampled fish. Figure 10 Modelled hepatosomatic index (HSI) for different phospholipid sources over 53 d in the freshwater pre-transfer phase followed by feeding the same commercial diet over 98 d in a common seawater tank plotted with 95% credible intervals. HSI is derived from the model for a 792 g average-sized sampled fish. Figure 11 Modelled probability for gill histology score of 1 described as very mild to mild as the most severe observation over a 0 score (no lesions/normal tissue) for (a) inflammation of lamella and (b) hyperplasia in gills of the fish that were fed diets containing different phospholipid sources after 53 d of feeding in the freshwater pre-transfer phase. Error bars give 95% credible intervals. animals-13-00835-t001_Table 1 Table 1 Formulation and expected composition of test diets for the feeding trial in freshwater. Control KM4 KM8 KM12 MARPL VEGPL Ingredient (% diet) Krill meal 4.0 8.0 12.0 Marine PL 4.2 Fluid soy lecithin 2.7 Fish oil 6.9 5.5 4.1 2.7 1.8 6.9 Plant oil 10.4 11.0 11.6 12.1 11.3 7.5 Fish meal (LT94) 10.0 10.0 10.0 10.0 10.0 10.0 Plant ingredients 68.8 65.9 62.9 59.9 68.8 68.6 Microingredients * 4.0 3.6 3.4 3.3 3.9 4.3 Total 100.0 100.0 100.0 100.0 100.0 100.0 Composition Protein (%; Dumas) 48.9 47.1 47.7 48.1 45.9 46.1 Fat (%; LfNMR) 24.0 24.4 24.3 24.2 22.7 21.8 Moisture (%; NIR) 6.0 6.5 6.1 5.9 6.3 8.2 Gross energy (MJ/kg; bomb calorimeter) 23.6 23.1 23.2 23.1| 22.1 22.7 Formulated PL% of diet from test ingredient 0 0.4 0.9 1.3 1.3 1.3 * Vitamins, minerals, and amino acids; * Formulated choline levels in excess of NRC 2011 requirements for salmonids (Section 2). Abbreviations: KM = krill meal; LfNMR = low-field nuclear magnetic resonance; NIR = near-infrared spectroscopy; PL = phospholipid. 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PMC10000101

Boar taint is an unpleasant odour and flavour released during heat treatment of pork from uncastrated male pigs. The two main compounds responsible for boar taint are androstenone and skatole. Androstenone is a steroid hormone formed in the testis during sexual maturity. Skatole is a product of microbial degradation of the amino acid tryptophan in the hindgut of pigs. Both of these compounds are lipophilic, which means that they can be deposited in adipose tissue. Several studies have reported heritability estimates for their deposition from medium (skatole) to high magnitudes (androstenone). In addition to efforts to influence boar taint through genetic selection, much attention has also been paid to reducing its incidence using various feeding strategies. From this point of view, research has focused especially on the reduction in skatole content by supplementation of feed additives into the nutrition of entire male pigs. Promising results have been achieved using hydrolysable tannins in the diet. To date, most studies have investigated the effects of tannins on the production and accumulation of skatole in adipose tissue, intestinal microbiota, growth rate, carcasses and pork quality. Thus, the objective of this study was, in addition to determining the effects of tannins on androstenone and skatole accumulation, to assess the effects of tannins on the sensory traits of meat from entire males. The experiment was performed on 80 young boars--progeny of several hybrid sire lines. Animals were randomly assigned to one control and four experimental groups (each numbering 16). The control group (T0) received a standard diet without any tannin supplementation. Experimental groups were supplemented with 1% (T1), 2% (T2), 3% (T3) or 4% (T4) SCWE (sweet chestnut wood extract) rich in hydrolysable tannins (Farmatan). Pigs received this supplement for 40 days prior to slaughter. Subsequently, the pigs were slaughtered, and sensory analysis was applied to evaluate the odour, flavour, tenderness and juiciness of the pork. The results showed a significant effect of tannins on skatole accumulation in adipose tissue (p = 0.052-0.055). The odour and flavour of the pork were not affected by tannins. However, juiciness and tenderness were reduced by higher tannin supplementation (T3-T4) compared to the controls (p < 0.05), but these results were sex-dependent (in favour of men compared to women). Generally, women rated tenderness and juiciness worse than men regardless of the type of diet. sensory traits boars diet hydrolysable tannins consumers Ministry of Agriculture of the Czech RepublicQK1910217 QK1910400 Czech University of Life Sciences PragueSV21-5-21360 Institute of Animal Physiology and Genetics AS CR, v.v.i. Libechov67985904 This work was supported by the Ministry of Agriculture of the Czech Republic (NAZV projects QK1910217 and QK1910400), by the Czech University of Life Sciences Prague (SGS project SV21-5-21360), and by the Institute of Animal Physiology and Genetics AS CR, v.v.i. Libechov (RVO 67985904). pmc1. Introduction Animal welfare has become a very important factor in livestock breeding, including that of pigs. A general ban on surgical castration of entire male pigs was expected in all EU member states by the end of 2018. Due to various circumstances, it was postponed until after 2021. One feasible alternative to painful surgical castration is to fatten all males. It is well known that boars grow faster and more efficiently, and they have higher lean meat content in carcass than surgical castrates. On the other hand, fattening boars increases the risk of and results in a higher incidence of boar taint and thus the dissatisfaction of consumers with such meat. The incidence of boar taint is mainly attributed to two substances, androstenone and skatole , especially after heat treatment of pork. Androstenone (a-androst-16-en-3-one) is a steroid hormone synthesized in the Leydig cells of the testis of uncastrated boars. This compound has an odour similar to urine or sweat, is perceived by approximately two-thirds of the human population and has been shown to be different according to country/region. Deposition of androstenone in fat tissue has high heritability estimates (0.55-0.88), which indicates that it is affected mainly by genetics . Skatole (3-methyl-indole) is a metabolite derived from microbial catabolism of the amino acid tryptophan in the hindgut of pigs. Its deposition is influenced mainly by environmental factors (h2 = 0.23-0.55) , especially nutrition, the system of feeding and the housing conditions . Since both of these compounds are lipophilic, they can accumulate in adipose tissue and therefore may have a negative effect on sensory attributes and result in the rejection of such meat by consumers. Since the odour of skatole is negatively perceived by practically the entire human population, reduction efforts have focused mainly on various feeding strategies in fattening all males. Promising results have been achieved by supplementation of diets with a variety of feed additives, such as chicory root or inulin , raw potato starch , sugar beet pulp , Jerusalem artichoke , oligofructose and fructooligosaccharides and tannins . Tannins are plant metabolites with great diversity, resulting in different physiological effects according to their form, animal species and amount of supplementation . Sweet chestnut (Castanea sativa Mill.) wood extract, consisting mainly of hydrolysable tannins, has antimicrobial and antiviral properties. Therefore, products containing this substance are used in animal nutrition, especially in piglets, as supportive treatment for diarrhoea after weaning . Several studies have demonstrated a reduction in total protein digestibility, as well as inhibition of microbial activity, in the colons of pigs after supplementation of the pig diet with hydrolysable tannins . Lower disponibility of tryptophan and cell debris in the hindgut may lead to reduced intestinal production of skatole . These findings are interesting from the entire male production point of view. Apart from a few studies dealing with Iberian pigs fed natural sources , other research on the effects of hydrolysable tannins (HTs) has been mainly aimed at growth and carcass parameters, meat quality and oxidative stability or the intestinal skatole production and microbiota composition in the large intestines of boars , but almost no research has addressed the effect of HTs on the sensory traits of entire male meat. The main objective of the present study was to assess the impact of hydrolysable tannins on parameters of eating quality, considering the possible effect of the sex of consumers, as well as to investigate the relationships between sensory evaluation and the content of skatole and androstenone in adipose tissue. 2. Materials and Methods 2.1. Animals and Diet Eighty young boars were used in the experiment. They were the progeny of Landrace sows and Yorkshire x Pietrain boars. Two weeks before the experiment, the pigs were housed in pairs/pens at the experimental test station of the Research Institute for Animal Production (RIAP) Nitra. Subsequently, the boars were randomly distributed within litters to one control and four experimental groups (each containing 16 animals). Control pigs (T0) received a diet without any supplementation. Experimental groups received the same diet as the control group but supplemented with 10 (T1), 20 (T2), 30 (T3) or 40 (T4) g/kg sweet chestnut wood extract (SCWE) rich in hydrolysable tannins (Table 1). The producer of the Farmatan product is Tanin Sevnica d.d., Sevnica, Slovenia, and the supplier was Product Feed a.s., Luzianky, Slovakia. The content of tannins in this product is 73 +- 2% (the value declared by the producer). The analysis of feed was performed according to the Folin-Ciocalteu method . The total phenolic content is expressed as gallic acid equivalents and is 45.1%. Supplementation of the diet with tannins started when the boars reached an average live weight of 80 kg (after a 2-week transitional period) and lasted for 40 days prior to slaughter. Access of the animals to drinking water and feed (automatic feeders--Schauer s.r.o., Nitra, Slovakia) was ad libitum. 2.2. Slaughter and Sample Collection Entire males were slaughtered in one batch at the experimental slaughterhouse of RIAP Nitra. The average live weight of the pigs was 122.28 kg +- 5.63 kg. Standard slaughter conditions were used: electrical stunning (90-100 V, 0.9-1.0 A, 50 Hz) followed by exsanguination. Evisceration was completed approximately 20 min post mortem. Chilling of the carcasses (air temperature 2-4 degC, velocity 0.5-1.0 m.s-) started approximately 60 min after slaughter and was continued overnight. After 24 h of chilling of the carcasses, musculus longissimus thoracis (LT) samples (1.0 cm thick) with subcutaneous fat were removed from the right side of the carcass (at the level of the last rib) and stored at -20 degC until sensory evaluation. 2.3. Sample Preparation One day before sensory evaluation, the raw LT sample was thawed overnight at 4 degC. Subsequently, each LT slice was trimmed to have 5 mm of subcutaneous fat. Each LT slice was placed in a grill and cooked for 4 min at 180 degC. The average measured internal temperature of the samples was 80 degC. After grilling, each steak was cut into four strips and immediately served to different panellists. 2.4. Sensory Evaluation Panellists were recruited from the staff of RIAP Nitra. All of them were consumers who liked and ate pork regularly (2-3 times weekly). Before the sensory evaluation of samples, consumers were tested for their sensitivity to androstenone according to the modified method of Weiler et al. . On the basis of this method, 20 panellists were selected (12 men and 8 women aged 32 to 60 years old). In total, 320 samples were evaluated in eight sessions (each of 40). In each session, eight panellists evaluated five samples (one from each dietary group). Each panellist participated in several sessions. Meat samples were randomly served to the consumers, and the attributes classified were odour, flavour, tenderness and juiciness. The scale applied in the sensory test was structured into 5 points, with 1 being the worst and 5 the best evaluation (Table 2). The panellists were asked to score for odour first, followed in order by flavour, tenderness and juiciness. 2.5. Skatole and Androstenone Determination Fat samples (100 g from a part of the belly) from entire males were removed 24 h after slaughter. One part of each sample was individually packed in a microtene bag, marked and frozen (-20 degC) until panel testing. The second part of each fat sample was transported to the authorized private laboratory of EKOLAB, s.r.o. (Kosice, Slovakia), to analyse the androstenone and skatole concentrations according to the methods of Ampuero Kragten et al. and Bekaert et al. . Briefly, adipose tissue samples were melted in a microwave for 4 min. Liquid fat was transferred to centrifuge tubes (2 mL), and 1.75 mL of extraction solvent methanol:hexane (9:1) were added. The extract was ultrasonically cleaned in a bath at 50 degC for 5 min and centrifuged for 5 min at 10,000x g. After cooling, approximately 2.0 mL of extract were then injected into a gas chromatograph equipped with a mass spectrometry (MS) detector (Shimadzu GCMS-TQ8030, Shimadzu Corp., Kyoto, Japan) at an injection temperature of 260 degC. The limits for detection were 0.02 mg/g for androstenone and 0.01 mg/g for skatole. 2.6. Statistical Analysis The observed data were evaluated by 1-way analysis of variance (ANOVA) with fixed effects using the following model: yij= m + ai + eij with N (0,s2) for androstenone and skatole For multiple comparisons of treatment means, Bonferroni's test was used . Dunnett's test was not used to compare the control and treatment groups since, from the analyses of variance of both traits, it could be concluded that differences between all groups by the F test were nonsignificant. The observations of sensory traits were evaluated by 2-way ANOVA with fixed effects using the following statistical model: yijk = m + ai + (ab)jj + eijk with N (0,s2) Pearson's correlation coefficients of androstenone and skatole with sensory traits were calculated. 3. Results 3.1. Effect of Tannins on Androstenone and Skatole Levels of boar taint compounds in adipose tissue with regard to tannin supplementation are shown in Figure 1. The samples that were high in both androstenone and skatole levels (greater than thresholds of 1.0 and 0.2 mg/kg, respectively) in the control group numbered 5, whereas in the supplemented groups, they numbered 1, 2, 3 and 0 for the T1, T2, T3 and T4 groups, representing 31.25%, 6.25%, 12.5%, 18.75% and 0%, respectively. The total number of samples high in androstenone, skatole or both compounds was 10 (62.5%). After tannin supplementation, these numbers were reduced to 7%, 6%, 8% and/or 7% (43.75, 37.5, 50.0 and/or 43.75%). Deposition of androstenone in adipose tissues was not affected by supplementation of the EM diet with tannins (Table 3). A numerical reduction, however nonsignificant (p = 0.052-0.055), in skatole concentration in fat tissue was found after administration of 2%, 3% and 4% tannins compared to the control group. Correlations between observed traits are presented in Table 4. Generally, all the relationships of concentrations of androstenone or skatole in adipose tissue with sensory traits were small and negative. Higher correlations were observed between skatole and eating quality parameters, with the exception of juiciness, which was more correlated with androstenone concentration. 3.2. Effect of Tannins on Eating Quality of Pork Supplementation of the pig diet with tannins did not show any impact on the panellists' scores for meat odour and flavour (Table 5 and Table 6). However, significant differences among the treatment groups were found for tenderness and juiciness (Table 7 and Table 8). Men scored both traits significantly better in meat from the control EM group than in meat from the T3-T4 or T4 group (3.05 vs. 2.74 and 2.81; 3.28 vs. 2.75, p < 0.05). Moreover, significant differences in the evaluation of these two traits were observed between the sexes of the panellists. Women were more critical than men regarding both tenderness and juiciness traits regardless of diet group (2.70 vs. 3.00, p < 0.05, and 2.65 vs. 2.99, p < 0.01, respectively). 4. Discussion Generally, tannins are considered to be antinutrients, as they create compounds with other nutrients, such as proteins, minerals, or digestive enzymes, and therefore are capable of reducing feed palatability, feed intake and nitrogen digestibility . However, pigs, wild as well as domestic, seem to be relatively resilient to the intake of feedstuffs with a high content of tannins without any negative consequences on performance or health status . This resistance is associated with elevated synthesis of proline-rich proteins (PRPs) in the saliva, which bind tannins from feedstuffs and prevent intoxication of organisms with diets rich in hydrolysable tannins . Moreover, recent studies have suggested that tannins have antimicrobial, anticancer, and antioxidant properties and can improve feed efficiency and reduce bacterial proteolytic reactions in piglets, thus protecting them from severe diarrhoea during weaning . It is well known that wild pigs, as well as some special native breeds of domestic pigs (Iberian, Cinta, etc.), can eat foodstuffs rich in tannins (content: 4-7%); therefore, a dose of 40 g per kg of feed mixture was selected as the highest dosage in the present study. It is well known that the level of skatole in fatty tissue is influenced by many processes, such as formation, absorption, metabolism and deposition. The main role is associated with the activities of digestive enzymes in the CYP450 family, such as 2E1, 2A19, 1A1, and 1A2, in the liver . Generally, data relating the effect of tannins on skatole production and accumulation in pig adipose tissue are limited. Some studies have shown that tannins can inhibit proliferation and apoptosis in the caecum. This inhibition results in decreased skatole production in the large intestine due to the lower availability of cell debris from lower apoptosis and tryptophan . In the present study, skatole accumulation in adipose tissue tended to decrease with increasing tannin supplementation. Similar numerical decreases were observed in other studies after 2-3% or 3% tannin supplementation, but surprisingly, lower supplementation (1.0 or 1.5%) resulted in higher skatole accumulation than in the control group . Candek-Potokar et al. suggest that this result could be associated with lower activity of CYP2E1 and CYP2A19 enzymes, two major enzymes of the phase 1 metabolism of skatole in the liver. It should be mentioned that some of the above studies used products with lower contents (only 50%) of hydrolysable tannins than our study. Regarding the effect of tannins on androstenone, the present study showed that supplementation with these additives did not have any impact, even though the two highest doses had higher (although nonsignificant) androstenone concentrations in fat tissue than the control group. This result is partially in contrast with that of other studies in which numerical reductions were found after supplementation of diet with 1-3, 3, and 3% hydrolysable tannin extracts. However, the pigs in the latter study had low levels of androstenone overall. Generally, the results regarding the effects of tannin supplementation on skatole and androstenone levels in back fat were not consistent and were highly dependent on the dose of tannin supplementation, often reporting curvilinear dependence. Therefore, research to determine the optimal dose of tannin supplementation for reducing boar taint is still needed. Moreover, the effects of hydrolysable tannins on androstenone are still unclear, and if any are confirmed, further studies will be needed to clarify the mechanism of action. As previously mentioned, very few studies have been published thus far relating the effects of hydrolysable tannins on pigs. Those studies focused mainly on intestinal skatole production, growth rate, carcass and meat quality, and the intestinal microbiota , but almost none focused on the sensory properties of pork. Thus, our results related to the effects of tannins on eating quality are difficult to compare with those of other studies. Only one study in pigs and one in sheep investigated the impact of tannin supplementation on the sensory traits of meat. In the present study, the odour and flavour of boar meat were not affected by tannin supplementation. Panellists scored these two parameters in the control and supplemented groups at almost the same level, and the differences were not significant. Bee et al. reported different findings. Panel members detected a stronger boar taint odour but not flavour in the meat of entire males supplemented with 1.5% tannins compared to the controls and group with 3% tannins. In lambs, Priolo et al. observed lower sheep meat odour in meat from animals supplemented with tannins compared to those not supplemented. Contrary to odour and flavour, the other two sensory traits--tenderness and juiciness--in the present study showed significant differences. Panellists, but only men, scored (p < 0.05) tenderness better in the control group than in the two supplemented groups (T3-T4), and juiciness in the control group was evaluated better than in T4. This outcome is contrary to the results of a study that reported no significant effect of tannin supplementation on juiciness and/or tenderness. Regarding differences between the sexes of panellists in the present study, women scored tenderness and juiciness worse than men in both the control and supplemented groups. It seems that higher supplementary tannin doses (3-4%) in our study significantly reduced juiciness (T4) and tenderness (T3-T4) compared with the control group. However, this result may be sex-dependent. 5. Conclusions Supplementation with tannins had no effect on androstenone accumulation in adipose tissue. In contrast, the concentration of skatole in fat tissue tended to decrease with increasing tannin supplementation. The odour and flavour of pork were not affected by tannin supplementation, but its juiciness and tenderness were lower after supplementation of the entire male diet with higher concentrations of tannins. The effect of the sex of consumers on sensory evaluation showed significant differences for the tenderness and juiciness of pork in favour of men. It seems that tannin concentrations greater than 3 in the diet negatively affect some sensory characteristics. These findings might be useful in solving appropriate feeding strategies for entire males to reduce boar taint, as well as to maintain good eating quality of their meat. Acknowledgments The authors thank the company EKOLAB, s.r.o. (Kosice, Slovakia), for its analyses of androstenone and skatole and Jana Zelenakova for her technical support. Author Contributions Conceptualization, I.B. and R.S.; Data curation, I.B.; Formal analysis, J.C. and O.B.; Methodology, P.F.; Statistics, P.F.; Writing--original draft, I.B. and M.S.; Writing--review and editing, R.S. and J.C. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was performed in accordance with Act on Animal Veterinary Care No. 39/2007 of the Slovak Republic and was approved by the Animal Care Committee of the Research Institute for Animal Production (RIAP) in Nitra, Slovakia. The approval code is 12/4557/2021, and the approval date is 9 September 2021. Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available upon request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Diversity of androstenone (AND) and skatole (SKA) regarding tannin treatments. animals-13-00893-t001_Table 1 Table 1 Ingredients and chemical composition of diets. Diet T0 T1 T2 T3 T4 Ingredients, g/kg Wheat 150 150 150 150 150 Barley 360 360 360 360 360 Corn 150 150 150 150 150 Wheat bran 80 70 60 50 40 Soybean meal 110 110 110 110 110 Rapeseed meal 100 100 100 100 100 Hydrolysable tannins 1 0 10 20 30 40 Mineral suppl. 25 25 25 25 25 Premix 10 10 10 10 10 Ground limestone 10 10 10 10 10 Fodder salt 5 5 5 5 5 Analysis, g/kg DM 2 898.4 896.3 898.8 899.0 895.8 Crude protein 168.4 167.6 167.8 164.7 165.2 Crude fibre 50.8 51.6 49.9 51.4 50.5 Crude fat 27.2 26.5 25.5 26.4 26.7 Crude ash 43.7 45.8 44.9 43.3 44.7 ME 3, MJ/kg 13.9 13.8 13.8 13.7 13.8 1 SCWE--sweet chestnut wood extract, 2 DM--dry matter, 3 ME--metabolisable energy. animals-13-00893-t002_Table 2 Table 2 Scale for sensory evaluation of meat. Trait Scale 5 4 3 2 1 Odour very distinctive, typical of roasted meat, without any foreign smell distinctive, typical smell of roasted meat less distinct, faintly typical aroma of roasted meat, with a faint foreign smell atypical aroma of roasted meat with a stronger foreign smell bland, impure scent with a strong foreign smell Flavour very distinctive, typical of roasted meat, without any extraneous flavour distinctive, typical taste without noticeable foreign flavour less distinct, faintly typical flavour of roasted meat, with a faint foreign flavour bland and atypical taste of roast meat with a noticeable foreign flavour bland with a foreign flavour, unpleasant to disgusting Juiciness meat very juicy meat juicy meat less juicy meat almost dry meat dry Tenderness meat slightly fibrous, very tender and soft meat still tender, stringy, fragile and soft meat thicker, fibrous, less fragile and quite firm meat almost coarsely fibrous with stiff fibres, hard meat coarsely fibrous, firm, very hard animals-13-00893-t003_Table 3 Table 3 Effect of tannin supplementation on androstenone and skatole accumulation in fatty tissue. Trait Treatment SEM p Value T0 T1 T2 T3 T4 Androstenone, mg/g 0.79 0.43 0.62 0.80 0.84 0.064 n.s. Skatole, mg/g 0.28 a 0.22 0.17 b 0.16 b 0.15 b 0.042 0.052-0.055 T0--control group, T1--1% tannin supplementation, T2--2% tannin supplementation, T3--3% tannin supplementation, T4--4% supplementation, a,b Different superscripts within rows indicate significant differences at p < 0.05; n.s.--non significant. animals-13-00893-t004_Table 4 Table 4 Pearson's correlation coefficients between traits. Trait Skatole Odour Flavour Juiciness Tenderness Androsterone 0.32 -0.02 -0.10 -0.19 -0.13 Skatole - -0.12 -0.13 -0.03 -0.17 animals-13-00893-t005_Table 5 Table 5 Effect of tannin supplementation on sensory evaluation of odour. T0-T4 T0 T1 T2 T3 T4 SEM p Value Men 3.28 3.30 2.96 3.37 3.62 3.16 0.07 n.s. Women 3.31 3.27 3.60 3.20 2.90 3.60 0.14 n.s. Total 3.30 3.28 3.28 3.29 3.26 3.38 0.17 n.s. n.s.--non significant. animals-13-00893-t006_Table 6 Table 6 Effect of tannin supplementation on sensory evaluation of flavour. T0-T4 T0 T1 T2 T3 T4 SEM p Value Men 3.32 3.39 3.13 3.43 3.43 3.21 0.06 n.s. Women 3.25 3.37 3.60 3.13 3.13 3.02 0.11 n.s. Total 3.28 3.38 3.37 3.28 3.28 3.11 0.13 n.s. n.s.--non significant. animals-13-00893-t007_Table 7 Table 7 Effect of tannin supplementation on sensory evaluation of tenderness. T0-T4 T0 T1 T2 T3 T4 SEM Men 3.00 1 3.05 a 3.40 3.00 2.74 b 2.81 b 0.06 Women 2.70 2 2.87 3.00 2.20 2.63 2.81 0.11 Total 2.85 2.96 3.20 2.60 2.68 2.81 0.12 a,b Different superscripts within rows indicate significant differences at p < 0.05; 1,2 Different superscripts within columns indicate significant differences at p < 0.05. animals-13-00893-t008_Table 8 Table 8 Effect of tannin supplementation on sensory evaluation of juiciness. T0-T4 T0 T1 T2 T3 T4 SEM Men 2.99 ++ 3.28 a 3.07 3.06 2.82 2.75 b 0.05 Women 2.65 ? 2.53 2.60 2.67 2.88 2.58 0.10 Total 2.82 2.91 2.83 2.86 2.85 2.66 0.12 a,b Different superscripts within rows indicate significant differences at p < 0.05; ++, ? Different superscripts within columns indicate significant differences at p < 0.01. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000102

Background: Scan-related anxiety ("scanxiety") is distressing to people living with and beyond cancer. We conducted a scoping review to promote conceptual clarity, identify research practices and gaps, and guide intervention strategies for adults with a current or prior cancer diagnosis. Methods: Following a systematic search, we screened 6820 titles and abstracts, evaluated 152 full-text articles, and selected 36 articles. Definitions, study designs, measurement methods, correlates, and consequences of scanxiety were extracted and summarized. Results: The reviewed articles included individuals living with current cancer (n = 17) and those in the post-treatment phase (n = 19), across a breadth of cancer types and disease stages. In five articles, authors explicitly defined scanxiety. Multiple components of scanxiety were described, including those related to scan procedures (e.g., claustrophobia, physical discomfort) and scan results (e.g., implications for disease status and treatment), suggesting varied intervention approaches may be needed. Twenty-two articles used quantitative methods, nine used qualitative methods, and five used mixed methods. In 17 articles, symptom measures specifically referenced cancer scans; 24 included general measures without reference to scans. Scanxiety tended to be higher among those with lower education levels, less time since diagnosis, and greater baseline anxiety levels (three articles each). Although scanxiety often decreased immediately pre- to post-scan (six articles), participants reported the waiting period between scan and results to be particularly stressful (six articles). Consequences of scanxiety included poorer quality of life and somatic symptoms. Scanxiety promoted follow-up care for some patients yet hindered it for others. Conclusions: Scanxiety is multi-faceted, heightened during the pre-scan and scan-to-results waiting periods, and associated with clinically meaningful outcomes. We discuss how these findings can inform future research directions and intervention approaches. Scanxiety cancer imaging scan stress anxiety National Cancer InstituteK99/R00 CA245488 R35 CA197730 P30 CA008748 Work on this scoping review was supported by the National Cancer Institute (K99/R00 CA245488; R35 CA197730; P30 CA008748). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. pmc1. Introduction "Scanxiety" (defined here as distress and/or anxiety occurring before, during, and after cancer-related imaging/scans ) is widely recognized as upsetting in patient reports, popular press, and patient education materials . Given that scans yield highly personal and consequential results regarding disease status, treatment response, and recurrence, it is not surprising that awaiting these results can be anxiety-provoking for patients during and following cancer treatment. Indeed, stress and anxiety are often ranked as highly concerning for patients awaiting scan results , and people living with cancer report that scanxiety is a notable and challenging part of their cancer experience . Accordingly, summarizing the emerging empirical research on scanxiety among people with cancer has the potential to advance the science of cancer survivorship, as well as to inform potential intervention approaches that may ease this difficult time period that arises repeatedly for cancer survivors. In a prior scoping review, Bui and colleagues summarized the quantitative measurement methods, prevalence, and severity of scanxiety among those with and without cancer. They concluded that the literature lacked a consistent definition of scanxiety . There remains a need to clarify scanxiety's definition and components in order to inform measurement and intervention strategies. A useful approach for doing so is to integrate qualitative research on patients' experiences. Furthermore, because those with cancer repeatedly experience uncertainty about disease status while undergoing follow-up scans to establish stage and treatment plans, evaluate progression and treatment response, and detect recurrence, they may have unique scanxiety experiences and needs compared to those without cancer who undergo screening procedures. Accordingly, there is a need to advance the conceptualization of scanxiety by including qualitative findings, to review scanxiety literature specifically in those diagnosed with cancer, and to inform broad research directions in this area. In this systematic scoping review, we synthesized findings from empirical articles on scanxiety among adults living with (e.g., those with current disease or in active treatment) and beyond (e.g., those deemed cured, post-treatment, or in remission) cancer. The objectives of this review were to describe the definitions, measurement methods, symptom levels, and correlates of scanxiety, and to identify research gaps and promising strategies for intervention. To achieve these objectives, we conducted a scoping review with a systematic search strategy. Given the current lack of conceptual clarity and to take an inclusive approach, we considered a range of psychological symptoms around a scan (including distress, stress, and anxiety) to reflect scanxiety. In this review, we describe the multi-faceted nature of scanxiety, outline measures that specifically focus on anxiety with respect to scans versus those that measure more general anxiety, and summarize research conducted to date regarding scanxiety's correlates and consequences. We then discuss how this information can inform future research directions and opportunities for intervention strategies during this stressful yet under-addressed period in clinical care. 2. Methods Because our goals were to examine and clarify definitions of scanxiety and its components, the methods used to study it, and current knowledge gaps, we selected a scoping review method (vs. a systematic review method to address a more specific research question, ). We used a previously-established scoping review methodology to guide our study methods and applied the Preferred Reporting Items for Systematic Reviews and Meta-Analyses for Scoping Reviews . A protocol describing the objectives, inclusion criteria, and methods was posted on Open Science Framework (osf.io/wm79v) in March 2021 . As described below, the approach was updated to include an interim search and updated data extraction procedures, due to the time elapsed after the updated search as well as institution changes among multiple co-authors. For similar reasons, the data extraction phase included one rater (rather than two as initially planned) to chart the data for each included article. References were compiled through a systematic search conducted by a librarian with expertise in clinical medicine (J.S.). Based upon initial search terms generated by the corresponding author (H.M.D.V.: cancer; scan; imaging; CT; MRI; recurrence; anxiety; distress; stress; scanxiety), the librarian constructed an extensive search strategy with expanded terms. The three main search clusters were cancer survivors, check-ups involving imaging, and patient anxiety. Medical Subject Headings, Emtree, PsycINFO and Cochrane terms were combined within each cluster using 'OR'; these clusters were then combined using 'AND'. The search strategy is available in the Supplementary Materials. The databases searched were MEDLINE, Embase, PsycINFO, CINAHL, Cochrane and Web of Science. The initial search was conducted in July 2020 for articles published in English between 1 January 1980 and 31 July 2020, and an updated search was conducted in March 2021. In order to include interim articles, the first author also conducted a limited search in PubMed up to July 2022. Reference management, abstract/title and full-text screening decisions, and data extraction utilized the Covidence reference management system. Prior to initial article screening, references were downloaded into Endnote and then transferred to the Covidence review management system, which was used to identify and remove duplicates. The remaining titles and abstracts were then screened for inclusion using initial eligibility criteria that were clarified through consensus meetings. Titles and abstracts were indicated for inclusion if they reported data from adults undergoing cancer-related imaging tests, who also had a measure of psychological symptoms or recalled symptoms experienced around the time of the scan. Using these criteria, abstracts and titles were screened independently by two team members (N.G., M.L., and/or H.M.D.V.) for possible inclusion. Disagreements for initial screening were resolved via tiebreaker vote from the third member. For records that met the initial criteria, full texts were reviewed by two study authors (H.M.D.V. and L.C.H.) and evaluated for final inclusion. The initial eligibility criteria above were refined to guide the full-text review phase and revised iteratively via consensus discussions (Table 1). Inclusion and exclusion decisions (along with exclusion reasons) for the full-text review phase were recorded using Covidence. Disagreements were resolved via consensus discussion between the two raters. The first author developed a data abstraction template and utilized this tool to extract and chart the data from the included articles. The following information was extracted by a single rater from the included articles as available: study design and methodology; demographic and cancer-related characteristics of participants; study definition of scanxiety; methods, instrument, and timing of scanxiety assessment; levels of scanxiety symptoms from quantitative measures; predictors and correlates of scanxiety; characteristics of the scan; and descriptions and effects of interventions. For qualitative articles, we also extracted themes and descriptions of scanxiety. Based on the goals of this scoping review, critical appraisal tools were not used. To synthesize results, we grouped articles into those that used quantitative, qualitative, and mixed methods. To gain insight into research gaps, we summarized measurement characteristics (e.g., whether the study used a previously-established or newly-created measure; whether the assessment referenced scanxiety or scans specifically or instead included general measures of anxiety/stress) and the timing of assessments with respect to the scan. In addition, we grouped articles according to period in the cancer continuum (during vs. post-treatment) and summarized the cancer types and stages of the study samples' participants. To gain insight on promising intervention strategies, we reviewed articles' results for correlates of scanxiety to identify modifiable characteristics, and applied expertise in clinical psychology to suggest how these patterns could inform intervention development. 3. Results The systematic search of multiple databases returned 10,319 references, as summarized by the PRISMA flow diagram in Figure 1. After removing duplicates, 6,820 titles and abstracts were screened. Of these, 152 full-text articles were evaluated for inclusion, including eight identified in the updated search. At the full-text review stage, articles were most frequently excluded due to a focus on individuals without current or prior cancer (such as those undergoing initial cancer screening) or due to not incorporating scans into the study design, data collection, or results. Ultimately, 36 full-text articles were included in the review. 3.1. Study Types and Designs There were 22 quantitative articles, summarizing 8 cross-sectional , 12 longitudinal , and 2 intervention trial study designs . There were 9 qualitative and 5 mixed methods articles . 3.2. Definitions and Elements of "Scanxiety" In five articles, authors explicitly defined scanxiety (Table 2; ). Several articles credited Bruce Feiler for first using and describing the term in his 2011 Time magazine article, who wrote that "scans are like revolving doors, emotional roulette wheels ..." and "[scans] engender 'scanxiety' as they approach" . A common component of these definitions included a time course, suggesting that distress or anxiety can occur before, during, and after imaging procedures. One article explicitly emphasized that scanxiety is "normal" . Among articles that did not define scanxiety, several did include a definition of a closely-related concept (e.g., fear of recurrence), or referenced that aspects of the scan experience can be stressful (e.g., "anxieties can be heightened around the time of a scan..." ). Several articles' narratives described specific elements of the scan experience that may be anxiety-provoking for patients. Most commonly, articles noted the uncertainty and fear associated with what the results would show, and the implications of scan results for their disease status and treatment . Authors also described anxiety-provoking aspects of the imaging procedures, such as specialized or unfamiliar technology/equipment , discomfort , and claustrophobia arising from being in an enclosed space . Concerns about exposure to radiation from the procedures were also noted . Given the variety of concerns described, there are likely individual differences in what part of the scan experience is considered most stressful. These components suggest that scanxiety is multi-faceted, and people with cancer may have multiple and differing concerns relating to scans . Quantitative results in several articles provided further detail on aspects of scanxiety. In a quantitative study of patients undergoing low dose PET/CT with 18-F-fluor-2-deoxi-D-glucose (18F-FDG), patients rated pre-selected reasons for their scanxiety . Before a scan, 79% rated that they were most anxious about the results; others reported that they were most anxious about the scan procedure (12%), their illness (3%), or other areas (6%). This pattern did not change much afterwards in the post-scan assessment, with 87% reporting being most anxious about the scan results . Other articles focused on the scan procedures (per the specific study goals), but did not appear to ask explicitly about anxiety related to the scan results. These articles illustrate additional concerns that patients may have around scans. For example, researchers focused on which experiences during MRI and PET/CT procedures were most stressful to esophageal cancer patients . Following a series of scans, participants reported that body position in the scanner (52%) and waiting time before scanning (19%) were most stressful during PET/CT. The noise of the scanner (26%), scan time (22%) and body position (22%) were noted as stressful during the MRI. Comparing the ratings for MRI versus PET/CT procedures showed that anxiety did not differ between them . In another quantitative study of cancer survivors in the United States who participated in the Health Information National Trends Survey (HINTS), 73% of respondents reported at least some worry about the effects of radiation from surveillance scans (i.e., medical imaging radiation; MIR), and 16% reported "a lot" of worry . Qualitative findings also indicated a range of components of the scan experience that were stressful. For example, in focus groups, post-treatment breast cancer survivors described that undergoing scans reminded them of their initial diagnosis and promoted fears of recurrence . Similarly, individual interviews with survivors of several cancer types suggested that scans served as a reminder of the "fragility of survivor status" and resulted in "iatrogenic uncertainty" . Claustrophobia , exposure to radiation , pain and discomfort associated with the procedures , and receiving information from electronic reports were also mentioned as sources of anxiety in qualitative interviews. Among people living with advanced cancer, results from open-ended questions and semi-structured interviews suggested that "most anxiety centered around the scan result and its implications" and that the procedures themselves were a less significant driver of anxiety . Taken together, these findings suggest that multiple aspects of scans may be anxiety-provoking for patients . 3.3. Populations Examined Below, we summarize descriptive statistics of cancer-related characteristics from the articles' samples. The number of articles summarized for these characteristics often exceeds the total number of included articles (n = 36) because some included individuals across multiple cancer types, stages, or other characteristics. In 23 articles, the sample comprised individuals with a single cancer type . In 13 articles, individuals with multiple cancer types were included in the sample . In two articles, individuals' cancer type was not specified . Across articles, samples most commonly included people with breast (n = 18; ), lung (n = 10; ), or gastrointestinal (n = 9; ) cancer. Most articles included people with solid tumors, while fewer (n = 5; ) included those with hematological malignancies (lymphoma or leukemia). Seventeen articles included individuals living with cancer (i.e., those with current cancer, typically in active treatment ), and 19 included individuals in the post-treatment period . Three did not specify participants' status along the cancer continuum . People with non-metastatic cancer were included in 22 articles , and those with metastatic cancer were included in 11 articles . Specifically, 9 , 14 , 12 , 16 , and 11 articles included people with Stage 0, I, II, III, and IV disease, respectively. Thirteen articles did not specify the stage of participants' cancer . The reasons that participants were undergoing scans varied. Often, people who were undergoing scans for different reasons were included in the same studies. The most commonly listed purposes of scans were routine post-treatment monitoring to detect disease recurrence (n = 21; ) and routine monitoring to detect disease progression or treatment response (n = 15; ). Other articles included participants undergoing scans for post-diagnosis staging or treatment planning (n = 6; . Only one article included individuals who were undergoing investigative scans for new symptoms . The scans' purpose was not specified in two articles . Articles included a wide variety of scan types (Table 3). 3.4. Measurement Methods Measurement of psychological symptoms included diverse concepts, such as distress, anxiety, stress, fear of cancer recurrence, and emotional well-being. Some articles included assessments that were specifically worded with reference to a scan, while others included a measure of general symptoms without a specific focus. For the purpose of consistency and brevity, and to reflect that these concepts were measured around or associated with scan procedures, we refer to all of these psychological symptoms as "scanxiety" in this paper. Quantitative measures were used in 27 articles, including the five mixed methods articles . Of these, 10 articles used more than one quantitative measure . The most frequently used quantitative assessment tools were single-item measures that were developed for the purpose of the given study. Such measures were used in eight articles . Across articles, there were 28 unique assessment tools; see Table 4. There were 14 scan-specific measures (used across 17 articles) that were worded specifically with respect to scans, and 14 general measures (used across 24 studies) that assessed psychological symptoms without a specific reference to scans. Among the articles that used quantitative measures, 20 provided information about the timing of survey administration with respect to the scan or scan results discussion . In seven articles, the timing of survey administration was unspecified or was not timed with respect to the scan . In four articles, scanxiety was assessed only before a scan procedure or scan results discussion . In five articles, scanxiety was assessed only after a scan or scan results discussion . In 11 articles, scanxiety was assessed both before and after a scan procedure or scan results discussion . Fourteen articles used qualitative methods, including the five mixed methods articles that also incorporated quantitative measures above . Of these, 13 articles used semi-structured interviews (n = 4 in focus groups , n = 9 individually ) and one used open text box responses . In nine articles, the interview topic guides or open text questions explicitly asked about scanxiety or emotions with respect to scans . In the other five articles using qualitative methods, responses and themes regarding scanxiety emerged when asking other questions about cancer survivorship, follow-up care, and quality of life, and were thus reported in the results . 3.5. Consequences of Scanxiety Of the articles reviewed, eight quantitatively investigated and five qualitatively summarized the effects of scanxiety. These articles highlighted effects on quality of life, somatic symptoms, receipt of follow-up care, and other healthcare experiences. 3.5.1. Quality of Life Several articles highlighted links between scanxiety and poorer quality of life. Patients with metastatic cancer who reported scanxiety had poorer quality of life on the EORTC-QLQ-30 than those who did not . In another article, advanced lung cancer patients who experienced higher scanxiety reported poorer quality of life on the FACT-L than those with lower scanxiety . By examining the subscales of the FACT-L, the authors concluded that this relationship was primarily driven by an association between scanxiety and emotional well-being. Consistent with these findings, metastatic cancer patients' anxiety as assessed before a scan results discussion was associated with poorer psychological well-being on the McGill quality of life questionnaire . 3.5.2. Somatic Symptoms Findings from included articles suggested that scanxiety is linked to somatic symptoms. For example, advanced cancer patients reported that they experienced trouble sleeping (32%), feelings of dread (29%), poor concentration (26%), irritability (25%), and restlessness/agitation or tension (24%) due to scanxiety . Other somatic symptoms including pain, low appetite, and racing heart, among others . In a study of women undergoing mammograms following breast cancer treatment, participants with higher fear of cancer recurrence experienced poorer sleep both immediately before and one week after their mammograms compared to those with lower fear of cancer recurrence . Similarly, advanced cancer patients described difficulty sleeping, fatigue, irritability, poorer concentration, and lower motivation for daily activities in qualitative interviews . 3.5.3. Receipt and Experiences of Follow-Up Care There were complex relationships between scanxiety and receipt of follow-up care, and the direction of the effects differed by article. For example, in a qualitative study of post-treatment lymphoma survivors, some reported that fear of recurrence motivated them to complete follow-up care, while others said it was a barrier . These different patterns were also observed in quantitative studies. Among people with metastatic cancer, 16% reported that they had delayed follow-up care due to scanxiety . A similar pattern was observed in post-treatment breast cancer survivors, such that those with higher levels of anticipatory anxiety were less likely to undergo mammography in the following year than those with lower levels of anticipatory anxiety . Specifically, compared to women without anticipatory anxiety, those with median levels of anticipatory anxiety were 32% less likely to undergo a mammogram in the next year. Women with higher anticipatory anxiety also reported more negative responses to pain during the mammogram, which partially explained the relationship between anxiety and mammogram adherence in a mediation model. Findings from a latent class analysis of adult survivors of childhood cancers indicated a different pattern . Those in the "worried" latent class were more likely to report completing mammography, as well as other aspects of follow-up care including ECG and bone densitometry, within the recommended time frame than those in other latent classes. Finally, in a study involving a series of three scans, baseline anxiety scores did not differ between those who went on to complete all of their scans in the series versus those who did not . Qualitative findings added further context to these relationships. Following breast cancer treatment, some women noted that concern about recurrence was a motivator for completing follow-up surveillance care, while others reported that fear of recurrence was a barrier to follow-up care . Another article highlighted that mammography was perceived as reassuring, and thus "worth the discomfort it may cause", suggesting that anxiety was not perceived as a barrier to completing follow-up mammograms . In survivors with various cancer diagnoses and cancer statuses, it was noted that follow-up tests (e.g., mammograms) were reassuring for some, while at least one participant noted that they did not experience this relief and considered discontinuing follow-up . The effects of scanxiety on other healthcare experiences were examined in two articles. Among advanced cancer patients, those with higher anxiety prior to a scan results discussion were less likely to report their recently-discussed scan results accurately (compared to their oncologists' reports) than those with lower anxiety . In lung cancer patients, anxiety was not related to motion artifacts during the scan, which can affect the quality of the imaging for interpretation . 3.6. Correlates of Scanxiety Of the articles reviewed, 21 quantitatively evaluated factors that were associated with scanxiety , and 12 described qualitative themes regarding such factors . Findings are synthesized below, grouped by factors that reflect sociodemographic characteristics, cancer-related characteristics, scan-related factors, timing of assessment, clinic- and system-related factors, and psychological and behavioral factors. 3.6.1. Sociodemographic Characteristics In two articles, women reported higher scanxiety or were more likely to endorse scanxiety compared to men . In Abreu and colleagues' study, there were no gender differences in scanxiety prior to the scan procedure, but men reported higher scanxiety than women afterward . Gender was not associated with scanxiety in four articles , although there was a trend toward higher scanxiety in women in two of these articles . Age was not associated with scanxiety in most articles . In a study of advanced cancer patients, younger participants were more likely to endorse experiencing scanxiety than older participants . Similarly, in people undergoing active surveillance or primary intervention for kidney cancer, those who were younger had poorer mental health scores than those who were older . Few articles examined the relationship between scanxiety and race or ethnicity. In advanced lung cancer patients, there was no significant relationship between race or ethnicity and scanxiety . Among cancer survivors in the HINTS study, Black individuals and those who were foreign-born were more worried about exposure to medical imaging radiation than white and US-born individuals, respectively, in unadjusted analyses, but these relationships did not remain in adjusted models . While education level was not associated with anxiety in three articles , several articles suggested that scanxiety may be worse among those with lower education levels. In the HINTS study, cancer survivors with lower educational attainment reported more worry about medical imaging radiation than those with higher educational attainment in unadjusted analyses, but this relationship was weakened in adjusted models . Similarly, people with lower education had higher scanxiety when undergoing imaging than those with higher education . In a study of advanced cancer patients, those with lower education levels were more likely to endorse scanxiety than those with higher education levels, but interestingly, health literacy was not associated with scanxiety . In this study, participants who lived in more remote locations were also more likely to report experiencing scanxiety than those who lived in more urban locations . Other sociodemographic factors were not associated with anxiety or stress around the time of scans, including smoking status , income , and relationship/marital status . Medical imaging radiation worry was not associated with health insurance status, but those with lower income levels were more likely to report medical imaging radiation worry than those with higher income levels . 3.6.2. Cancer-Related Characteristics Several articles included individuals with and without cancer and examined whether scanxiety varied with cancer history. Using daily data collection before and after mammograms, Porter and colleagues observed that breast cancer survivors had a slightly greater increase in daily stress from baseline to mammogram day compared to control women without a cancer history . On the other hand, patients undergoing imaging tests for cancer had lower anxiety levels on the day of the exam than those who underwent imaging for other health conditions . In a three-week daily diary study, participants with cancer reported significantly higher fear of cancer recurrence across the study period and greater peak levels of fear of cancer recurrence on the day of their mammogram compared to their spouses . Another study indicated a different pattern, in which 71% of patients and 81% of their caregivers reported scanxiety symptoms on a Greek version of the IES-R, though the score cutoff and statistical significance of this comparison was not reported in the conference abstract . In two quantitative articles, time since diagnosis was not related to levels of scanxiety . Other articles suggested a complex relationship. Among advanced cancer patients, those who were diagnosed less than a year ago endorsed similar rates of experiencing scanxiety (vs. not experiencing scanxiety) compared to those who were diagnosed more than a year ago . However, in the subset of participants who endorsed scanxiety, those who were diagnosed less than a year ago rated their peak anxiety as higher than those with a longer time since diagnosis . In a cross-sectional study, patients with a recent diagnosis within six months or less had higher levels of scanxiety than those who had been living longer with cancer . However, in the same study, participants also reported that scanxiety did not dissipate over time . In the HINTS study, those who had completed treatment within the past 10 years reported higher worry about medical imaging radiation than those who had completed their treatment more than 10 years ago . Qualitative findings similarly highlighted that scanxiety may decrease over time for some people with cancer, but not for others. For example, some patients with metastatic lung cancer reported that their anxiety diminished as their condition stabilized, while others reported that scanxiety is "always there" . Among veterans with bladder cancer, some noted that anxiety lessened over time with repeated procedures, and with meeting "experienced" patients who had been living with cancer for a longer period of time . Few articles investigated differences in scanxiety based on cancer stage, cancer type, treatment type, or health status. Advanced lung cancer patients' scanxiety did not vary according to their cancer histology, stage, or the presence of an actionable tumor mutation . Patients with progressive lung cancer did not experience significantly higher levels of scanxiety than those whose disease was stable or improving . On the other hand, cancer survivors with poorer self-rated health had higher levels of medical imaging radiation worry than those who rated their health better . Larger proportions of those with breast and lung cancer reported higher medical imaging radiation worry compared to other cancer types, and those who received radiation treatment were more likely to report worry than those who did not . Among those with advanced cancer, those with breast cancer were more likely to report experiencing scanxiety around a recent scan compared to those with other cancers . Clinical trial participation was not associated with scanxiety . 3.6.3. Scan-Related Characteristics The number of prior scans or frequency of scans was not reported in most articles we reviewed. In two articles, anxiety level was not significantly different for those who were undergoing the 18F-FDG PET/CT scan procedure for the first time compared to those who had completed it previously . Similarly, among patients who were undergoing a series of multiple scan types, including MRI and CT, anxiety was not statistically different between the first and second MRIs . Nevertheless, in qualitative interviews with advanced cancer patients, increasing familiarity with the procedures was noted to be linked to lower scanxiety . Findings were inconsistent for whether scanxiety varied by scan type. For example, among lung cancer patients undergoing CT and MRI scans, state anxiety scores did not differ significantly by scan type . However, in the semi-structured interview component of this mixed methods study, participants described that MRI scans prompted more anxiety, claustrophobia, and discomfort than CT scans . In Goense and colleagues' study, participants underwent both MRI and PET/CT scans; they concluded that anxiety did not vary significantly by scan type . Follow-up regimen type was also not associated with differences in mental well-being between kidney cancer patients who received active surveillance including regular imaging (CT, MRI, and ultrasound) and those who received primary intervention . On the other hand, three studies found group differences in anxiety according to scan type, with anxiety being higher for MRI (vs. CT) , for endoscopic ultrasonography (vs. cervical ultrasonography, CT, or PET) , and for rigid (vs. flexible) cystoscopy . Several articles examined characteristics of the scan experience. For example, one study indicated that patients' experiences during the scan (e.g., discomfort, difficulty) were associated with higher anxiety directly following the procedure . Similarly, focus groups with breast cancer survivors suggested that pain and discomfort during mammogram procedures were a source of anxiety . The reason that the scan was being conducted was not related to anxiety . Interestingly, a study of advanced cancer patients did not indicate a significant association between type of result received (stable or better vs. progressive) and retrospective anxiety reports . Patients' expectations about the nature of the results were also not associated with pre-scan distress levels . Among advanced lung cancer patients attending an appointment, those who reviewed a recent scan during the visit did not have significantly higher scanxiety severity compared to those who did not review a scan at the visit . 3.6.4. Timing of Assessment Articles with longitudinal designs examined the effect of time by comparing scanxiety at different points in the scan experience. In two articles, anxiety increased from baseline to pre-mammogram . In six articles, anxiety decreased from pre- to post-scan procedure . In one article, participants experienced higher anxiety following the scan compared with their pre-scan anxiety . In at least two articles , participants had received their results by the post-scan time point. However, the timing of results was not always clear with respect to the study assessments. 3.6.5. Other Clinic or System-Level Factors In qualitative articles, the waiting period between the scan procedure and receiving scan results was often described as the most distressing period . Participants described that anxiety was present until the results of the scan were known . In separate articles of post-treatment breast cancer and lymphoma survivors, both research teams noted that the days between the scan and receiving results were particularly stressful and reminded participants of the time period of when they were first evaluated for and diagnosed with cancer . Wait times may be shortened when receiving results electronically (e.g., via patient portals), which was perceived positively by some patients . Qualitative findings also suggested that other stressors arose with this option for some patients, such as the potential to feel worse after a "bad result" or to need reassurance from an oncologist . Although this waiting period was commonly noted as anxiety-provoking in qualitative articles, few quantitative articles examined this factor. Advanced cancer patients who waited more than two days for their scan results were more likely to retrospectively report experiencing scanxiety . With respect to results delivery, advanced cancer patients who were notified about their results by their preferred provider had greater decreases in their distress following this discussion than those who received results from a provider that differed from their preference . However, participants' distress before imaging and after results was not associated with whether their preferences were met for the time frame or method for delivering results . Among lymphoma patients, those who reported a worse patient-doctor relationship had higher anxiety than those who reported a better relationship, although this assessment was not timed around scans specifically . On the other hand, worry about medical imaging radiation was not associated with physician trust in a heterogeneous sample of cancer survivors . In another article, advanced cancer patients rated clinic-level factors as helpful around the time of the scan or scan results , although the statistical relationships between these factors and anxiety were not tested specifically. Several factors reported to be helpful around the time of scans were having experienced (81%) and friendly (88%) staff conducting the scan procedures, knowing what to expect about the procedures (82%), and undergoing the scan at a familiar location (i.e., close to home or at the center they received treatment, 71%). Around the time of receiving scan results, several factors reported to be helpful were the availability of scan results at the time of their oncologist appointment (91%), receipt of results from their oncologist in clinic (90%), and discussing the treatment plan in the same visit (81%) . 3.6.6. Psychological and Behavioral Characteristics Some articles indicated that scanxiety was more common among those with certain baseline psychological factors. Those with higher general anxiety symptoms and fear of cancer progression were more likely to endorse experiencing scanxiety . General anxiety symptoms were also associated with patients' ratings of peak anxiety around the time of the scan and distress on the day of imaging . In a study of people with bladder cancer, those with higher baseline anxiety also had higher anxiety following the cystoscopy procedure . On the other hand, this relationship was not observed between elevated general anxiety and worry about medical imaging radiation . The likelihood of reporting scanxiety in the context of a recent scan did not differ for those with and without clinical depression . In a daily diary study, patients with higher threat sensitivity (i.e., a tendency to have stronger responses to threatening cues or situations) had higher fear of cancer recurrence on the day of their mammogram and slower rates of recovery (decreases in fear of cancer recurrence) following the mammogram than those with lower threat sensitivity . Other articles examined how health behaviors and coping strategies were related to scanxiety. For example, people who searched for information prior to their scan did not have higher levels of anxiety compared to those who did not . In bladder cancer patients, low perceived control over the procedures was noted, and the ability to watch the procedure in real time was noted to help improve feelings of control . Empowerment or self-efficacy in managing their health by undergoing the procedure was noted as a positive factor . However, in a trial testing a self-management approach following treatment for endometrial cancer, women randomized to patient-initiated follow-up (self-referral if alarming symptoms occurred) had smaller decreases in fear of recurrence compared to control participants receiving regularly-scheduled hospital-based follow-up . Articles also summarized ways of coping; patients reported using a wide range of strategies, including distraction, relaxation, positive self-talk, and self-management of care to tolerate scans and manage scanxiety. Among post-treatment breast cancer survivors, greater confidence in one's ability to cope with potential recurrence was associated with lower fear of recurrence around surveillance . Following a PET-CT scan, state anxiety scores were lower among cancer patients who were assigned to listen to music immediately before a PET-CT scan compared to those assigned to a control condition . Several articles examined how social relationships were associated with scanxiety. Although emotional and instrumental social support were not associated with pre-scan distress, patients who reported greater social isolation had higher distress on the day of their imaging compared to their socially connected counterparts . In a dyadic design using daily diary methods, partner responsiveness (i.e., the degree to which one perceives their partner to respond with genuine interest and care) and capitalization attempts (i.e., the process of sharing good news with one's partner) were not related to fear of cancer recurrence before a mammogram . Contrary to hypotheses, patients whose partners were more responsive had higher fear of cancer recurrence on the day of the mammogram than those with whose partners were less responsive . 4. Discussion In this scoping review, we identified and synthesized findings from 36 articles on scanxiety among adults diagnosed with current or prior cancer. The articles included a notable breadth of individuals across cancer types, disease status, and stage, suggesting that scanxiety occurs across the cancer continuum. Our synthesis indicated that there are various components of the scan experience that prompt anxiety, which may differ across individuals. These components appear to cluster around aspects of the scan procedure itself and around the uncertainty associated with high-stakes results. The waiting period between the scan procedure and receipt of the results was described as particularly stressful, although few quantitative articles captured this period of time or described how results were delivered. Our review also summarized the measures and methods used in scanxiety research. The assessment tools used to measure scanxiety varied widely, with 28 unique assessment tools, and often relied on newly-created single items for study-specific purposes. While this leads to a rich literature to examine scanxiety from different angles, this variation limits conclusions about levels or consistent factors relating to scanxiety. Strengths of our approach include the large literature base screened and reviewed, the inclusion of both quantitative and qualitative research designs, and the focus on cancer survivorship. Although comprehensive searches were conducted in July 2020 and March 2021, an adjusted search approach limited to Pubmed was conducted for the final period of this review (March 2021 to July 2022), a limitation. Below, we discuss how the findings of this review could be used to inform future research directions and to generate hypotheses and approaches for interventions (Table 5). 4.1. Current Research Findings, Methods, and Gaps Conceptual clarity varied across articles. Few articles provided a specific definition of scanxiety. This may have been because research questions on scanxiety were often secondary to other study aims. With limited conceptual clarity, it is not surprising that many different survey measures were used, with little harmonization across articles. To advance the science of scanxiety, there is a need to examine how it relates to closely-related constructs that may be exacerbated around scans, such as fear of recurrence or progression, anticipatory anxiety, and state anxiety. Determining if these constructs are largely the same, strongly correlated, or composed of different components would help to harmonize measurement strategies for indexing anxiety around scans. Because precise measurement methods are especially needed for assessing the impact of interventions, a strong interim approach might be to utilize measures that are worded specifically in reference to a scan (Table 2), such as the Impact of Events Scale (six items) and the modified Distress Thermometer. Relatedly, neighboring literature on anxiety while awaiting other types of tests (e.g., blood tests) or routine follow-up examinations should be evaluated to determine if experiences are similar during these waiting periods. For example, many patients receive key prognostic information about their disease from blood tests such as carginoembryonic antigen (CEA), prostate-specific antigen (PSA), or cancer antigen 125 (CA125) levels. As technology becomes more sophisticated, additional blood tests (e.g., cell-free DNA, or "liquid biopsy" for mutational analysis of tumors) bring with them the potential for additional distress to patients. The extent to which similar processes of heightened anxiety and uncertainty occur around these types of tests is needed. Based on the definitions and elements of scanxiety from our review, we suggest that this experience may not be limited to scans, but instead may occur in the context of other types of follow-up tests that involve uncomfortable procedures, waiting for results, and uncertainty around implications for disease status. During the article screening process, we identified articles that focused on different types of tests or follow-up without mention of imaging, but we did not review these articles based on our research objectives and inclusion criteria. Because the search terms were designed to focus on imaging-related articles, our review of literature on other testing methods would not have been comprehensive or representative. However, we identified additional articles that could be relevant to the concepts raised here, an area for future work. Findings might also inform considerations for administering existing measures or developing new ones. For example, measures or terminology that apply to anticipatory anxiety around multiple types of tests may be needed. Scanxiety is currently used to describe these processes specifically around scans, and we are not aware of broader terms that encompass anxiety around new or alternative tests that result in similar prognostic information. The characteristics of the samples and designs in our review also point to areas in which research can be extended. For example, most of the current scanxiety literature focuses on those with solid tumors, and fewer articles included individuals with hematological malignancies. In addition, articles typically focused on individuals who were undergoing routine, scheduled scans to detect progression, treatment response, or recurrence. Only one study included individuals who were undergoing investigative scans due to new symptoms, but it seems that anxiety would likely be even higher in this context. Third, scanxiety among family members/care partners has been under-studied. Research is needed to determine their anxiety levels and needs for psychosocial support, and interactions between these factors and the patient's experience around the time of scans. Broader literature suggests that family members'/care partners' general anxiety is significant and can be higher than patients' anxiety , so it is possible that periods around scans are even more stressful for them. Another future direction is to determine how delivery of scan results may impact scanxiety, especially in the context of newer formats such as direct release through patient portals . Given that a common focus of scanxiety was on what the scan results showed, additional research that examines how results are delivered and what they showed would be useful. These details about results delivery were rarely included in the articles reviewed, with several exceptions . Finally, research addressing how scanxiety affects day-to-day life, clinical experiences, and follow-up care receipt is limited. Because existing articles highlighted that scanxiety drives some patients to seek greater follow-up care and others to seek less, there is a need for research to test "for whom" and "how" scanxiety affects follow-up care. Both patterns can be problematic but likely require different management strategies. Our review also highlights areas for methodological improvements in scanxiety research. For example, psychometrics of quantitative measures were rarely provided. Overall, the description of timing of measurements with respect to the scan procedure and the results delivery could also be improved. Longitudinal assessments often included pre- and post-scan time points, but it was often unclear when scan results were delivered with respect to the post-scan assessment. To the extent possible in a given study design, it would be useful to report the nature of the test results as well as whether, when, and how results were delivered. Prospective designs that captured the period between the scan procedure and receipt of the results were especially limited. Future research on this time period is needed, given that it was identified as the most stressful by patients in qualitative articles. In addition, methods that allow for repeated measures around these times, such as daily diary designs or ecological momentary assessment, could help to elucidate fluctuating patterns of anxiety over time with respect to these events. While these methods are acceptable and feasible in young survivors of childhood cancer around the time of scans , evaluating their acceptability and feasibility during these time periods in older survivors and those with active disease would be useful. Based on our experience with the screening phase of this project, description of the components of clinical follow-up procedures could be improved in future articles. Some of the screened abstracts and articles had a focus on routine follow-up, but did not describe what procedures were included in these visits, or whether they involved scans or other types of tests. We excluded these articles if scans were not mentioned. However, operationalizing the types of tests or procedures in regular follow-up could potentially expand the pool of scanxiety articles available to draw upon. Accordingly, it is possible that the group of available articles is actually larger than what was reviewed here. In addition, in the broader cancer literature on anxiety and stress, it is possible that symptoms are assessed at the time of tests or follow-up visits while awaiting results, and thus affected by scanxiety. Future work on anxiety could be advanced by including questions about whether participants are awaiting upcoming tests, scans, or follow-up care to help interpret fluctuations in anxiety. 4.2. Promising Approaches to Intervention To develop and test interventions for scanxiety, a key step is to learn which individuals are most at risk for scanxiety. While more research is needed and findings were not always consistent, our review indicated that women and those with lower education levels, shorter time since diagnosis, and greater levels of baseline anxiety may be more likely to experience scanxiety, while associations with other factors were less consistent. If these patterns hold true in future research, this information could be used to emphasize the inclusion of these individuals in the intervention development process, tailor strategies to their needs, and prioritize them for intervention delivery. For example, screening for baseline characteristics (e.g., general anxiety or fear of recurrence) could be used to inform which patients may be likely to need or benefit from additional support around the time of scans. Future research supporting these patterns could also inform intervention components. For example, it is possible that helping patients manage high levels of general anxiety could have a positive impact on their scan-specific anxiety. However, we did not analyze these patterns using meta-analysis, and the measures and time points examined were quite heterogeneous. Given these limitations, our synthesis can be used to generate hypotheses rather than to draw firm conclusions on whether these factors are related to higher scanxiety. Further research is needed to determine which patients may be most prone to increases in anxiety around the time of scans. Few articles examined modifiable or theory-driven individual-level factors to inform intervention targets that mitigate scanxiety, another area for future work. Nevertheless, the existing literature can be used to identify promising intervention approaches. For example, a study of post-treatment breast cancer survivors investigated whether factors from the cognitive-behavioral model were related to anxiety. Women who were more confident in their ability to cope with potential recurrence experienced lower fear of cancer recurrence around their surveillance mammograms than those with lower coping self-efficacy . These findings suggest that interventions to strengthen peoples' coping skills for how they might handle the potential "bad news" of recurrence might be useful, as opposed to reassuring oneself that risk is low (which was associated with greater anxiety in the study). Because the waiting period before receiving results is often uncontrollable and described as the most stressful period, strategies to pass the time during this difficult window might be especially useful. For example, advanced cancer patients described using strategies such as distraction and pleasant activities to cope with scanxiety . Similarly, engaging in enjoyable flow activities that fully occupy one's mind could make the waiting period more tolerable . When developing and testing interventions, researchers should consider that anxiety may center around the scan procedures themselves, around the uncertainty associated with what the results may show, or both. It is likely that different types of intervention strategies may be needed to address these various components effectively. For example, a patient who is primarily concerned about the procedure due to claustrophobia may benefit from cognitive behavioral therapy, including exposure exercises. This approach would likely be ineffective and inappropriate for someone whose primary concern is the uncertainty of results. Short interventions around the time of scans, including real-time strategies employed in the waiting area or during scan time, may be beneficial and acceptable for those with procedural anxiety. Just-in-time micro-interventions represent a promising approach that may be well suited for these moments . On the other hand, a common focus of scanxiety is the ongoing uncertainty associated with the possibility of recurrence or progression, or anxiety-provoking reminders of initial diagnosis. Interventions that are employed outside of the scan procedure itself, perhaps in the lead-up to the scan or emphasized during the waiting period between the scan and results receipt, may be better suited to address anxiety arising from uncertainty about the results. In this case, people may be expected to benefit from interventions that promote meaningful engagement in activities, mindful awareness, coping with fear of progression or recurrence, or acceptance-based strategies. As research on the most beneficial strategies to support those with scanxiety emerges, a helpful approach could be to offer several different evidence-based stress management tools , such that patients may practice those that resonate most with them. This review also suggests that addressing clinic- and system-level factors could help mitigate scanxiety. For example, findings from qualitative articles repeatedly suggested that wait times, especially the waiting period between the scan procedure and receipt of the results, was particularly anxiety-provoking for patients. Shortening wait times to results could shorten periods of heightened anxiety for patients. Strategies that improve patients' ability to know what to expect, such as implementing a set routine for results delivery or pre-scheduling discussion appointments, may also be useful. While the direct release of results via patient portals may provide a way to shorten wait times, data also indicates that some patients experience heightened anxiety or confusion around technical terms when viewing these results independently; thus, they may benefit from additional support, education, or informed decision-making to determine if this is the right approach for them . Other patients may benefit from addressing aspects of the scan procedure itself. Improving the scan experience (e.g., by making adjustments that reduce pain or discomfort) could help to alleviate procedural anxiety. Finally, clinicians may benefit from considering which patients could struggle most with scanxiety. Shared decision-making with such patients (e.g., considering a longer interval to the next scan) as well as communicating about how to act on (e.g., anticipated treatment decisions based on results) and cope with (e.g., by voicing unending support for the patient no matter what the results, offering additional psychosocial supports as available) the potential results are other promising directions for future research and clinical practice. 5. Conclusions Our review--which involved screening and reviewing a large literature base, included quantitative and qualitative research designs, and focused on cancer survivorship--provides insight on the nature of scanxiety, research practices and gaps, and promising strategies for interventions. In summary, scanxiety occurs across the cancer continuum. It may focus on procedural or uncertainty-related components of the scan experience. Future work to determine the ideal measurement strategies and timing of assessments would advance the current understanding of scanxiety and intervention approaches to help manage it. Efforts to harmonize measures, examine the waiting period between scans and scan results, and identify intervention targets are particularly needed. Emerging literature indicates that some individuals may be more prone to elevated anxiety around the time of scans, and suggests promising approaches at the individual and clinic/system levels to help manage scanxiety. Given that scan experiences are common and repeated across the cancer continuum, strategies to support patients around these times have the potential to improve quality of life and other outcomes for those living with and beyond cancer. Supplementary Materials The following supporting information can be downloaded at: File S1: OVID Search Strategy. Click here for additional data file. Author Contributions Conceptualization: H.M.D.-V. and L.C.H.; Methodology: H.M.D.-V., L.C.H., and J.S.; Data Curation: H.M.D.-V.; Project Administration: H.M.D.-V., L.C.H., N.G., M.L.; Interpretation: H.M.D.-V., L.C.H., N.G., J.S., M.L., A.S.E., H.G.P.; Writing--Original Draft Preparation: H.M.D.-V.; Writing--Review and Editing: H.M.D.-V., L.C.H., N.G., J.S., M.L., A.S.E., H.G.P.; Resources: H.M.D.-V. and H.G.P. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest H.M.D.V. discloses a financial relationship (immediate family member) with Dechra. A.S.E. discloses royalties from reviewing UpToDate topics on gastrointestinal cancers, medical oncology, and palliative care. Figure 1 PRISMA diagram. Figure 2 Dimensions of scanxiety, clustering around scan procedures and scan results. cancers-15-01381-t001_Table 1 Table 1 Eligibility criteria for full text screening. Inclusion: Participants: Adults (ages 18 and older) with current or prior cancer diagnosis Concept: Undergoing imaging tests for detecting disease progression or recurrence Context: Completed a measure of psychological symptoms while awaiting scans or scan results or provided retrospective recollection of such symptoms. Types of studies: Observational studies, case studies, qualitative studies, conference abstracts, and intervention studies (single-arm and randomized controlled trials) Exclusion: Study sample composed primarily of children (mean sample age under 18 years), due to likely differences in scan experiences and assessment methods People undergoing initial diagnostic cancer screening (i.e., no current or prior cancer diagnosis) Scans or scan result discussions not considered in study design, measurement, or results Review articles or study protocols Full text unavailable in English cancers-15-01381-t002_Table 2 Table 2 Definitions of scanxiety listed in included articles. "Ten years ago, Feiler coined the term, 'scanxiety' to describe the emotional distress that patients experience immediately before or after medical imaging ." "Scanxiety represents a complex array of negative and stressful emotions linked with cancer scans, and the uncertainties and fears that may accompany them ." "Scanxiety" or scan-associated anxiety describes the distress before, during, or after a cancer-related scan and was a term first coined by a patient writing for the Time magazine in 2011. ... There are no agreed criteria that define scanxiety. Unlike anxiety disorders, which are characterised by excessive fear and anxiety, scanxiety is often considered a normal reaction to a scan. Scanxiety is a transitory emotional state, which is consistent with the concept of state anxiety . Though scanxiety may not be pathological in the same manner as an anxiety disorder, scanxiety may be a negative experience that impairs quality of life ." "Distress leading up to, during and after an imaging scan has been termed 'scanxiety' ." "'Scanxiety' refers to the often-debilitating anxiety patients with cancer experience in the period surrounding imaging studies for their cancer ." cancers-15-01381-t003_Table 3 Table 3 Summary of included articles. Sample Characteristics Scan Characteristics Scanxiety Measures Citation N Cancer Type Cancer Stage Cancer Status Scan Type Scan Purpose Assessment Tool Scan-Specific vs. General Measure Design; Assessment Time Points Quantitative Studies Abreu et al., 2017 232 Multiple Not specified Not specified F-FDG PET/CT Staging/early post-diagnosis Detect progression Detect recurrence Likert item (anxiety, 10-point) Scan-specific Longitudinal: Immediately pre- and post-scan procedure Bauml et al., 2016 103 NSCLC III, IV Living with cancer Examples given: CT, MRI, PET Detect progression Impact of Events Scale-6 item version (IES-6) Scan-specific Cross-sectional: Waiting room prior to medical oncology appointment (scans discussed for 73% of patients) Bjelic-Radisic et al., 2017 284 Breast 0-IV Post-treatment Multiple aspects of follow-up: Mammography, breast ultrasound, abdominal ultrasound, chest x-ray, bone scintigraphy Detect recurrence Breast cancer psychosocial assessment screening scale (BC-PASS)Likert items (distress about follow-up components, 1-4) Likert items (anxiety and stress before follow-up visits; 0-10 and 1-4) General (BC-PASS) Scan-specific (Likert items) Cross-sectional: Not specified with respect to scan Cox et al., 2013 613; varied by analysis Multiple; prior childhood cancers Not specified Post-treatment Mammogram, ECG (ultrasound or multi-gated acquisition scan), bone density scan (DEXA or CT) Detect recurrence Other: Detect late effects Three summed items on health fears (future health, cancer recurrence, finding a problem at follow-up visits; 1-5) General Longitudinal: Not specified with respect to scan Derry et al., 2019 94 Multiple; solid tumors III, IV Living with cancer Not specified Detect progression Single item (anxiety about diagnosis, not at all to completely) General Cross-sectional: Prior to scan results discussion appointment Goense et al., 2018 27 Esophageal Not specified Living with cancer MRI and PET/CT to predict clinical response to neoadjuvant tx Staging/early post-diagnosis information Likert rating (anxiety, 1-5) Scan-specific Cross-sectional: Directly following the last of several MRI and PET/CT scans Grilo et al., 2017 81 Multiple Not specified Not specified 18F-FDG PET/CT Staging/early post-diagnosis Detect progression Detect recurrence State Trait Anxiety Inventory (STAI)-state General Longitudinal: Before and after scan Hay et al., 2018 452 Multiple Not specified Living with cancer Post-treatment Not specified; item asked about radiation from medical imaging tests such as x-rays, mammograms, radioactive dye, etc. Not specified Single item from HINTS on Medical Imaging Radiation (MIR) worry (not at all to a lot) Scan-specific Cross-sectional: Not specified with respect to scan Jeppesen et al., 2018 214 Endometrial I Post-treatment Ultrasound; other imaging as needed Detect recurrence Fear of Cancer Recurrence Inventory (FCRI) General Intervention trial: Not specified with respect to scan Koinis et al., 2022 218 Multiple IV Living with cancer Not specified Detect progression Modified Greek version of Impact of Events Scale (IES)-Revised Scan-specific Cross-sectional: Within one week after scan Krajewski et al., 2017 100 Bladder Not specified; non-muscle- invasive Living with cancer Cystoscopy Detect progression Hospital Anxiety and Depression Scale (HADS) General Longitudinal: Directly before procedure and within 7-10 days after Lo Re et al., 2016 260 (41% were oncology patients) Multiple Not specified Not specified MRI, breast imaging (mammogram, ultrasound, breast MRI), x-ray, CT, ultrasound Not specified STAI-state STAI-trait General Cross-sectional: Before scan procedure (presumably in waiting area) Martinez-Lorca and Martinez-Lorca, 2022 108 Multiple Not specified Living with cancer 18F-FDG-PET-TAC Early post-diagnosis/staging Detect progression Detect recurrence STAI Likert scale (subjective anxiety, 0-10) General Intervention trial: Directly before and after scan McGinty et al., 2014 136 Breast 0-IIIA Post-treatment Mammogram Detect recurrence Cancer Worry Scale (CWS) General Longitudinal: Immediately before and one week after mammogram (following negative results) McGinty et al., 2016 161 Breast 0-IIIA Post-treatment Mammogram Detect recurrence Two visual analog scale items about fear of recurrence CWS General Longitudinal: Seven time points; baseline, one month before, one week before, immediately prior to mammogram, and immediately after, one week after, and one month after receiving results Morreale et al., 2020 100 Lung Gastrointestinal IV Living with cancer CT or MRI Detect progression/assess treatment response Distress Thermometer HADS General Longitudinal: On imaging day and one week after receiving results Patel et al., 2016 539 Kidney I Living with cancer Axial imaging (CT or MRI), ultrasound Detect progression SF-12 QOL Questionnaire Mental Component Score General Longitudinal: Not specified with respect to scan Porter et al., 2003 55 (34 with breast cancer and 21 without cancer) Breast 0-III Post-treatment Mammogram Detect recurrence Daily stress (single item, 0-10) Psychological consequences questionnaire (PCQ) General (daily stress) Scan-specific (PCQ) Longitudinal: Daily measures for three days about one month before, day of, and day after mammogram Shelby et al., 2012 204 Breast I-III Post-treatment Mammogram Detect recurrence Stanford acute stress reaction questionnaire Scan-specific Longitudinal: Immediately before mammogram; adherence over next year Soriano et al., 2018 57 couples Breast 0-III Post-treatment Mammogram Detect recurrence Daily measure, six items adapted from Insight and Severity subscales of FCRI General Longitudinal: Daily measures over three weeks; two weeks before and one week after mammogram Soriano et al., 2019 57 couples Breast 0-III Post-treatment Mammogram Detect recurrence Daily measure, six items adapted from Insight and Severity subscales of FCRI PROMIS Anxiety Short Form General Longitudinal: Baseline anxiety; Daily measures over three weeks; two weeks before and one week after mammogram (results typically given same-day) Westerterp et al., 2008 82 Esophageal I-IV Living with cancer CT, PET, cervical ultrasonography, endoscopy, ultrasonography Staging/early treatment planning Likert item (anxiety; 1-5) Scan-specific Cross-sectional: two weeks after scan procedures Qualitative studies Allen, 2002 6 Breast Not specified; non-metastatic Post-treatment Mammogram Detect recurrence Semi-structured individual interview General Cross-sectional: Not specified with respect to scans Brandzel et al., 2017 41 Breast 0-III Post-treatment Mammogram and breast MRI Detect recurrence Semi-structured interview in focus groups Scan-specific Cross-sectional: Not specified with respect to scans Bui et al., 2021 16 Multiple; solid tumors III, IV Living with cancer CT Detect progression Semi-structured individual interview Scan-specific Cross-sectional: Within four months after scan Lai-Kwon et al., 2021 20 NSCLC IV Living with cancer CT; PET Detect progression Semi-structured individual interview Scan-specific Cross-sectional: Within three months after scans Mannion et al., 2022 20 Multiple Not specified; included those with Stage IV cancer (80%) Living with cancer Post-treatment Not specified Detect progression Detect recurrence Semi-structured individual interview Scan-specific Cross-sectional: Not specified with respect to scans Pascal and Endacott 2010 15 Multiple Not specified Post-treatment Variety including mammograms and other scans Detect recurrence In-depth individual interview General Longitudinal: Two interviews over six months, timing not specified with respect to scans Sterba et al., 2015 22 Colorectal I-III Post-treatment Not specified; regular follow up including CT and colonoscopy Detect recurrence Semi-structured interview in focus groups General Cross-sectional: Not specified with respect to scans Thompson, H.S. et al., 2006 10 Breast Not specified Post-treatment Variable as part of follow-up care; e.g., mammogram, x-ray, sonogram, CT, PET Detect recurrence Semi-structured individual interview General Cross-sectional: Not specified with respect to scans Wernli et al., 2017 41 Breast 0-III Post-treatment Mammogram and breast MRI Detect recurrence Semi-structured interview in focus groups Scan-specific Cross-sectional: Not specified with respect to scans Mixed methods studies Baun et al., 2020 38 Breast IV Living with cancer CT or combined PET/CT Detect progression/monitor treatment response Quantitative assessment focused on patients' use of electronic records for test results (not anxiety) followed by semi-structured individual interviews General Sequential: Not specified with respect to scans Bellhouse et al., 2020 29 NSCLC I-III Living with cancer Radiotherapy planning CT and two MRI scans approximately two weeks apart Other: Radiotherapy planning STAI-state Claustrophobia Questionnaire (CLQ) MRI anxiety questionnaire (MRI-AQ) CT anxiety questionnaire (CT-AQ) Semi-structured individual interview after last scan General (STAI-S, CLQ) Scan-specific (MRI-AQ, CT-AQ, interview guide) Longitudinal, concurrent: Baseline and after each MRI scan Bui et al., 2022 222 Multiple; solid tumors III, IV Living with cancer CT Detect progression Investigative Single item (experience scanxiety; yes/no) Modified Distress Thermometer Open-ended text response Other measures examined as correlates: six-item STAI, HADS, FOP-Q-SF Scan-specific (single item, modified Distress Thermometer, open text) General (FOP, STAI, HADS) Concurrent: Within four months after scan/scan results Koo et al., 2017 12 Bladder 0; non-muscle-invasive Living with cancer Post-treatment Cystoscopy Detect progression Detect recurrence Psychological consequences of screening questionnaire (PCQ) Customer satisfaction survey (CSS) with items on worry and discomfort from procedure Semi-structured interview in focus groups Scan-specific Sequential: After cystoscopy procedure Thompson, C.A. et al., 2010 70 Hodgkin's lymphoma Aggressive non-Hodgkin's lymphoma Not specified Post-treatment CT Detect recurrence STAI-trait Semi-structured individual interviews General (STAI) Scan-specific (interview guide) Concurrent: Not specified with respect to scan (mean time since last scan = 14.8 months, SD 25.3) cancers-15-01381-t004_Table 4 Table 4 Quantitative measures of scanxiety used in included articles, according to scan-specific vs. general assessments. Scan-Specific Measures (Worded with Respect to Scans) General Measures (Worded without Reference to Scans) Measure n, Articles Measure n, Articles Impact of Events Scale (IES; revised or short form) 2 State-Trait Anxiety Inventory (STAI) 6 Modified Distress Thermometer 2 Fear of Cancer Recurrence Inventory (FCRI) 3 Psychological Consequences of Screening Questionnaire (PCQ) 2 Hospital Anxiety and Depression Scale (HADS) 3 MRI Anxiety Questionnaire 1 Cancer Worry Scale (CWS) 2 CT Anxiety Questionnaire 1 Claustrophobia Questionnaire 1 Medical Imaging Radiation Worry item from the HINTS survey 1 Breast Cancer Psychosocial Assessment 1 Stanford Acute Stress Reaction Questionnaire 1 Health Fears items 1 Customer Satisfaction Survey (worry and discomfort items) 1 SF-12 Quality of Life Mental Component Score 1 Study-specific item (anxiety during procedure, 10-point Likert) 1 Visual analog scale (VAS) items on fear of recurrence 1 Study-specific item (distress about components of follow-up, 4-point Likert) 1 Fear of Progression Questionnaire 1 Study-specific item (anxiety about diagnosis, not at all to completely) 1 PROMIS Global Anxiety 1 Study-specific item (anxiety during procedure, 5-point Likert) 1 Study-specific item, daily stress (0-10 Likert) 1 Study-specific item (anxiety about scan modalities, 5-point Likert) 1 Study-specific item, subjective anxiety (0-10 Likert) 1 Study-specific item (whether experienced scanxiety; yes/no) 1 Distress Thermometer (general) 1 Note: Measures were used to assess psychological symptoms around the time of scans or with consideration of scans in the study design. Scan-specific assessments were specifically worded in reference to a scan; general measures did not include a specific focus. cancers-15-01381-t005_Table 5 Table 5 Recommended areas for future scanxiety research. Research Topics: Expanding Research to Areas Needing Additional Attention Suggested Areas Example Research Questions Investigate care partners' scanxiety levels and supportive care needs What are the scanxiety characteristics, levels, and support needs among care partners/family members? How do care partners' scanxiety compare to and influence that of the cancer survivor? Determine whether people with cancer experience similar anxiety and uncertainty while awaiting different types of tests How does anxiety fluctuate in the time period around cancer-monitoring blood tests and results delivery? What are the components or dimensions of anxiety around these procedures? What are the correlates and effects of anxiety around other tests? Explore how newer modes of scan results delivery (e.g., via electronic results release; via video or other remote clinical interactions) affect scanxiety Which patients experience decreased anxiety when receiving faster, automated test results (e.g., in patient portals)--and which patients experience elevated anxiety? What support strategies may patients need in order to engage with and benefit from this format of results delivery? Expand scanxiety research to under-investigated populations, time periods, and scan types What are the scanxiety experiences and coping strategies of those with hematological malignancies? How does anxiety fluctuate between the scan procedure and the scan results? What factors may exacerbate or buffer against increases? How do patients cope with anxiety in the context of investigative scans prompted by new or worsening symptoms? What are the longitudinal patterns of scanxiety that occur over time with repeated scans? Expand work on the effects of scanxiety and moderators of these effects For whom and how does scanxiety affect one's likelihood of adhering to follow-up care? How does scanxiety impact physical symptoms, communication in appointments, and other outcomes? Determine intervention targets and test whether interventions are effective Are brief interventions (e.g., just-in-time micro-interventions) acceptable, feasible, and efficacious for reducing anxiety at the time of scan procedures? Which coping strategies are most effective for managing uncertainty about what results may show? When is the optimal time to introduce a behavioral intervention with respect to scans? Research Methods: Strengthening how Scanxiety Studies Are Conducted Suggested Approaches Example Research Questions/Directions Harmonize measures and examine psychometrics How does scanxiety relate to close constructs such as fear of recurrence or progression, anticipatory anxiety, and state anxiety? Is it sufficient to use existing state anxiety measures to index scanxiety? Or are there advantages to developing specific measures that reflect multiple elements or specific components of scanxiety? Examine psychometrics of scanxiety measures Improve description of follow-up care procedures Strengthen descriptions of what typical follow-up procedures entail (e.g., exams, tests) In studies of general anxiety, include questions about whether an upcoming scan or scan discussion is occurring Detail time periods to include information on procedures and scan results delivery phases Describe the length of time between assessment time points with respect to pre-scan, post-scan, and results delivery time points. Report whether, when, and how results were delivered, and what they showed. How do waiting periods and results delivery methods influence scanxiety? How can clinicians or clinics structure the scan experience to help mitigate anxiety? Explore innovative measurement strategies Are daily diary and/or ecological momentary assessment approaches acceptable and feasible around the time of scans for older adults, those with advanced disease, and those on active treatment? Do these approaches reveal fluctuations and individual differences not evident from one-time assessments? Intervention Approaches: Developing and Testing Ways to Manage Scanxiety Promising Approaches Example Research Directions/Intervention Targets Screening to identify those experiencing or at higher risk for scanxiety Use scan-specific measures to identify those with scanxiety Prioritize at-risk individuals for interventions Tailoring strategies to stressful time periods Design interventions that address procedure-related and results-related components of scanxiety Optimize strategies for each time period (e.g., pre-scan; awaiting results) Behavioral / self-management strategies Promote self-efficacy for coping with progression/recurrence Bolster overall stress management skills (e.g., relaxation, pleasant activities) Facilitate just-in-time strategies for distinct periods (e.g., during or directly before scans) Clinic or system strategies Reduce scan-to-results waiting time Provide education/structure that promotes knowing what to expect for the procedure Engage in shared decision-making about scans and results delivery Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000103

The bovine endometrium has an important defensive role in the postpartum period that acts when an inflammatory process associated with tissue damage or infection by bacteria is produced. Endometrial cells release cytokines and chemokines that recruit inflammatory cells, which release danger-associated molecular patterns (DAMPs), such as adenosine triphosphate (ATP), and initiate and regulate the inflammatory response. However, the role of ATP in bovine endometrial cells is unclear. The aim of this study was to determine the effect of ATP on interleukin-8 (IL-8) release, intracellular calcium mobilization, ERK1/2 phosphorylation, and the role of P2Y receptors, in bovine endometrial cells. Bovine endometrial (BEND) cells were incubated with ATP and the IL-8 release was determined by the ELISA assay. ATP of 50 and 100 mM significantly increased IL-8 released in BEND cells (50 mM: 23.16 +- 3.82 pg/mL, p = 0.0018; 100 mM: 30.14 +- 7.43 pg/mL, p = 0.0004). ATP (50 mM) also induced rapid intracellular calcium mobilization in Fura-2AM-loaded BEND cells, as well as ERK1/2 phosphorylation (ratio 1.1 +- 0.04, p = 0.0049). Suramin (50 mM), a pan-antagonist of P2Y receptors, partially reduced the intracellular calcium mobilization, ERK1/2 phosphorylation (ratio 0.83 +- 0.08, p = 0.045), and IL-8 release (9.67 +- 0.02 pg/mL, p = 0.014) induced by ATP. Finally, BEND cells expressed higher mRNA levels of P2Y1 and P2Y2 purinergic subtype receptors, and lower levels of P2Y11 and P2Y12 receptors, as determined by RT-qPCR. In conclusion, these results showed that ATP activates pro-inflammatory responses in BEND cells, which are partially mediated via P2Y receptors, and BEND cells express the mRNA of subtypes of P2Y receptors, which could have a key role in bovine endometrial inflammation. ATP calcium purinergic receptors endometrial cell Fondo Nacional de Desarrollo Cientifico y Tecnologico (FONDECYT)1200905 This research was funded by Fondo Nacional de Desarrollo Cientifico y Tecnologico (FONDECYT) grant number 1200905. The APC was funded by FONDECYT 1200905. pmc1. Introduction Bovine uterine infections are a common health problem in the postpartum period and lead to economic losses due to treatment costs, lost milk production, and infertility . The endometrium, as well as being the site of embryo implantation and responsible for the regulation of the estrous cycle through the release of prostaglandins, has a defensive role against infection through classical innate immune mechanisms . After parturition, all mammals can develop an inflammatory process associated with tissue damage or infection by bacteria . Diverse cytokines and chemokines are produced by endometrial cells in response to tissue damage or infection, and an influx of inflammatory cells such as polymorphonuclear (PMN) are recruited into the endometrium, all of which favor an inflammatory environment . Interleukin-8 (IL-8), a strongly attractant chemokine for neutrophils, is released by bovine endometrial cells and produced at high levels in the endometrium in the postpartum period, and in cows with subclinical or clinical endometritis . PMNs also contribute to inflammation with the release of pro-inflammatory mediators such as adenosine triphosphate (ATP) , which acts as a danger-associated molecular pattern (DAMP), and initiates and regulates the inflammatory response along with other damage signals . ATP binds to purinergic receptors of type P2. There are 2 subtypes of P2 receptors: P2X and P2Y receptors. P2Y receptors are G protein-coupled receptors, and eight receptor subtypes have been identified in humans and rodents: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14 . In bovines, P2Y receptors have been identified in neutrophils, retinal endothelial cells, chromaffin cells, chondrocytes, and synoviocites . However, its presence in bovine endometrial cells is unknown. Some studies have reported the presence of P2Y and P2X subtype receptors in the human endometrium and the rat uterine epithelium . After binding to its receptors, ATP activates different signaling pathways. ATP induced intracellular calcium mobilization in human endometrial cancer cells . In human endometrial stromal cells, ATP activates the pathway PLC/PKC/ERK . ERK1/2 MAPK is a signaling pathway involved in the activation of transcription factors, such as the nuclear factor-kappaB (NF-kB), and the IL-8 gene has NF-kB binding sites in its promoter region that regulate its expression . However, little evidence exists regarding the role of ATP in endometrial innate immunity. Another nucleotide, UDP-glucose, stimulates IL-8 production in human endometrial epithelial cells, and neutrophil chemotaxis in an IL-8-dependent manner . We hypothesized that ATP activates inflammatory responses such as intracellular calcium release, ERK1/2 phosphorylation, and IL-8 production in bovine endometrial cells, and these responses could be mediated via P2Y subtype receptors. 2. Materials and Methods 2.1. Cell Culture Bovine endometrial (BEND) cells (ATCC(r) CRL2398TM) were obtained from ATCC (Manassas, VA, USA). BEND cells, a cell line derived from the uterine endometrium of a cow on d14 of the estrous cycle , were cultured according to the protocol recommended by the manufacturer and as described by Valenzuela et al. . Briefly, a 1:1 mixture of Ham's F12 and Eagle's Minimal Essential medium with Earle's BSS (D-valine modification) containing 1.5 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate (Sigma-Aldrich Co, St Louis, MO, USA) was used. The culture medium was supplemented with 0.034 g/L D-valine (Sigma-Aldrich Co, St Louis, MO, USA), 10% heat-inactivated fetal bovine serum (BioWest, Kansas City, MO, USA), 10% heat-inactivated horse serum (Gibco-Brl, Grand Island, NY, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Hyclone, Logan, UT, USA). Before each experiment, the culture medium was changed into a serum-reduced culture medium (2% serum) in the absence of antibiotics, and the cells were maintained for two hours. 2.2. IL-8 ELISA Assay BEND cells (n = 3 independent experiments) were cultured with vehicle (PBS) or different concentrations of ATP (0.1, 1, 10, 50, or 100 mM) (Cayman Chemical, Ann Arbor, MO, USA) for 24 h at 37 degC. LPS (500 ng/mL) (Escherichia coli O111:B4, Sigma-Aldrich, Saint Louis, MO, USA) was used as a positive control for the IL-8 assay. For specific experiments, BEND cells (n = 3 independent experiments) were cultured with 50 mM suramin for 15 min and then 50 mM ATP was added, followed by incubation for 24 h at 37 degC. IL-8 in the supernatants was assessed in duplicate using the Bovine IL-8 (CXCL8) ELISA BASIC Kit (MABTECH AB, Nacka Strand, Sweden), according to the instructions provided by the manufacturer. 2.3. Intracellular Calcium Mobilization BEND cells (1 x 107 cells/mL) (n = 3 independent experiments) were trypsinized (0.25% trypsin/EDTA), washed in Hank's balanced salt solution (HBSS) (136 mM NaCl, 5 mM KCl, 0.9 mM CaCl2, 0.4 mM KH2PO4, 0.3 mM NaH2PO4, 6 mM glucose, pH 7.4), and loaded with Fura-2-AM, according to the method described by Valenzuela et al. . Briefly, BEND cells were incubated with 2.5 mM Fura-2-AM (Invitrogen, Carlsbad, CA, USA) for 30 min at 37 degC in the dark, and then two washes of HBSS were performed. Finally, the cells were suspended in HBSS containing 1 mM probenecid (Invitrogen, Carlsbad, CA, USA). Each experiment was performed in a cuvette maintained at 37 degC with constant stirring in an LS55 spectrofluorometer (PerkinElmer Life Science, Waltham, MA, USA). After 60 s of basal measurement, every 0.2 s at 509 nm emission and 340/380 nm dual-wavelength excitation, vehicle (HBSS) or ATP (0.5, 1 or 50 mM) was added, and the signal registered for 240 s. In another set of experiments, the cells were incubated with vehicle (DMSO 0.1%) or suramin (10, 50, or 100 mM) for 15 min at 37 degC, and then the fluorescence intensity was detected for 60 s of basal measurement. ATP (50 mM ) was then added, and the signal registered for 140 s. 2.4. Immunoblot BEND cells cultured in a 60 mm plate (n = 3 independent experiments) were treated with vehicle (0.1 % DMSO) or 50 mM suramin for 15 min, and then 50 mM ATP (or vehicle PBS) was added, followed by incubation for 5 min at 37 degC. The cells were lysed according to Manosalva et al. . Briefly, cells were incubated with a lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 5 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM DTT) on ice, and centrifuged at 18,000x g for 20 min at 4 degC. Protein concentration was determined using the Bradford method (Sigma-Aldrich, Saint Louis, MO, USA). Fifty micrograms of protein were used on 10% SDS-PAGE gels. The proteins were transferred to PVDF membranes, and the immunoblot was performed according to the protocol recommended by the manufacturer of the antibody phospho-ERK1/2 (Cell Signaling, Beverly, MA, USA). The antibody against phospho-ERK1/2 was incubated overnight at 4 degC. Then, the membranes were washed and incubated with an HRP-conjugated secondary antibody (LI-COR, Lincoln, NE, USA). The bands of phospho-ERK1/2 were visualized using an Odyssey Fc infrared/chemiluminescent detection system (LI-COR Biosciences). The primary antibody was stripped, and the membranes were incubated with total anti-ERK1/2 antibody (Cell Signaling, Beverly, MA, USA) for 2 h at room temperature. A HRP-conjugated secondary antibody was used and the signal was detected as described above for phospho-ERK1/2 antibody. The band intensity was measured using image Studio Lite v5.2 software (LI-COR Biosciences). The results are shown in a representative image of three experiments, and the band intensity of phspho-ERK1/2 and ERK1/2 is shown in a bar graph as the ratio phspho-ERK1/2/ERK1/2. 2.5. Real Time-qPCR BEND cells (n = 3) grown in 100 mm-plates were used for total RNA isolation, using the EZNATM Total RNA Isolation Kit (Omega Bio-Tek, Norcross, GA, USA). To ensure removal of genomic DNA, all samples of total RNA were treated with DNase. For cDNA synthesis, 250 ng of total RNA were subjected to reverse transcription with Affinity Script RT (Agilent, Santa Clara, CA, USA), and then real-time PCR was performed in duplicate using the Brilliant II SYBRGreen qPCR K it (Agilent) with primers specific for bovine P2Y receptors subtype and the housekeeping ribonucleoprotein S9 (RSP9) (Table 1) . The following conditions were used: 95 degC for 3 min, 40 cycles of 10 s at 95 degC, and 60 s at 60 degC. Then, three additional steps (dissociation curve) were performed: 95 degC for 15 s, 60 degC for 60 s, and 95 degC for 15 s. The post-PCR melting curves confirmed the specificity of the single-target amplification. The efficiency of the reaction was determined by the StepOne software and ranged between 95 and 110% for the different primers. The abundance of each gene was calculated relative to RSP9 mRNA using the 2-DDCt method . The formula used is: relative abundance = 2 (-DCt), where DCt is calculated as the difference between the Ct of each P2YR (P2Y1R, P2Y2R, P2Y4R, P2Y6R, P2Y11R, P2Y12R, or P2Y13R) and RSP9 . The results are shown in a bar graph, as the relative expression of mRNA of each P2Y receptor. 2.6. Viability Assay BEND cells (n = 3 independent experiments) were cultured with vehicle (0.1% DMSO) or suramin (1, 10, 100, or 300 mM) for 30 min or 24 h at 37 degC, and cell viability was assessed, in duplicate, using the propidium iodide method as described by Valenzuela et al. . Briefly, 5 mM propidium iodide (Molecular Probes, Invitrogen, Carlsbad, CA, USA) were added and incubation was implemented for 15 min at 37 degC. A positive control for cell death was performed in cells incubated with 0.2% triton X-100 at 37 degC for 40 min. The propidium iodide signal was detected with a fluorescence multiplate reader (Varioskan(r) Flash, Thermo Fisher Scientific, Waltham, MA, USA) at 530 nm excitation/620 nm emission. The results are shown as % of cell viability. 2.7. Statistical Analysis The experiments (three independent experiments) were analyzed with a one-way analysis of variance and Dunnett's multiple comparison test (compared with the control or basal), performed with a significance level of 5%. An unpaired t-test was performed in selected groups of IL-8 assay and immunoblot; comparisons between groups are shown in each graph with square brackets and significance level. All analyses were performed with the GraphPad Prism 8.0 program. The results are shown as bar graphs of the mean +- SEM. 3. Results and Discussion 3.1. ATP Increased IL-8 Levels Bovine endometrial cells were treated with different concentrations of ATP for 24 h, and IL-8 release was determined in the supernatant. ATP (50 and 100 mM) significantly increased the production of IL-8 compared with the control (vehicle) (50 mM: 23.16 +- 3.82 pg/mL, p = 0.0018; 100 mM: 30.14 +- 7.43 pg/mL, p = 0.0004); similarly, the positive control LPS induced IL-8 release (p < 0.01), as has been described in several studies . The treatment of BEND cells with ATP plus LPS showed an additive effect on IL-8 release , for each concentration of ATP assessed. The contribution of different cells producing ATP into the uterine environment could have important consequences, including reducing bovine reproductive performance. ATP is released from inflammatory cells in response to bacterial peptides and inflammatory mediators . Likewise, ATP is released from endometrial epithelial cells, uterine blood vessels, and nerves innervating the uterus . ATP regulates classical endometrial functions such as implantation of the fertilized oocyte, endometrial fluid functionality, cell proliferation and differentiation in post-partum, apoptosis, and sperm functionality . However, few studies have evidenced the role of ATP in the endometrial inflammatory response. We observed a significant increase of IL-8 production in BEND cells stimulated with ATP, and in BEND cells treated with ATP plus LPS. High levels of IL-8 in the uterus promote an exacerbated inflammatory response, because more immune cells could be attracted. Inflammatory cells, such as neutrophils and macrophages, produce IL-8 under activation by stimuli associated with infection by bacteria or tissue damage ; therefore, the control of the uterine immune response is crucial for the resolution of inflammation. To assess a possible role of purinergic receptors in the increase of IL-8 induced by ATP, BEND cells were incubated with the P2Y pan-antagonist suramin for 15 min. Then, ATP was added and incubation carried out for 24 h. The IL-8 production was partially yet significantly reduced by suramin (p < 0.05), suggesting a partial role of P2Y receptors in this response. The partial suramin-induced reduction of IL-8 can be explained because ATP does not only activate P2Y receptors. ATP also activates P2X purinergic receptors and can be hydrolyzed into adenosine and activate different signaling pathways, which may contribute to gene expression . The participation of P2Y receptors in endometrial function has been previously suggested in other species. In human endometrial cells, the inhibition of the P2 receptor attenuated the ATP-induced activation of MAPK . Additionally, suramin blocked the stimulatory effect of ATP on prostaglandin production in a guinea pig's uterus . Thus, our results with suramin support the participation of P2Y receptors in IL-8 production in bovine endometrial cells. 3.2. ATP Induces Intracellular Calcium Mobilization and ERK1/2 Phosphorylation Since P2Y receptors are G-protein coupled receptors that activate second messengers, such as inositol triphosphate (IP3), and then lead to an fast increase of intracellular calcium released from the endoplasmic reticulum , we assessed intracellular calcium mobilization induced by different ATP concentrations. BEND cells rapidly increased intracellular calcium mobilization after ATP addition, ranging between 0.5-50 mM . To assess the participation of P2Y receptors, BEND cells were incubated with suramin for 15 min. Then, ATP was added and intracellular calcium was measured. Suramin reduced the intracellular calcium mobilization induced by ATP , suggesting the participation of P2Y receptors in the effect stimulated by ATP. Calcium has diverse functions in the uterus, such as the production of developing follicle hormones, estrogen synthesis, and contraction and relaxation . A study in bovines showed that the treatment of endometrial explant with the calcium ionophore A23187 increased the prostaglandin F2a synthesis induced by estradiol, thus demonstrating the role of calcium in hormone synthesis . At a cellular level, calcium regulates different cellular processes, such as cell proliferation and apoptosis, gene expression, and inflammasome activation . In bovine endometrial cells, it was shown that infection with E. coli increased the cytoplasmatic calcium, and the treatment with intracellular calcium chelators reduced the expression of IL-1b and IL-18, proposing the importance of calcium in pro-inflammatory gene expression . In addition, prostaglandin F2a induced IL-8 expression in endometrial adenocarcinoma cells via the PKC/calcium/NFAT signaling pathway . The reduction of intracellular calcium mobilization in BEND cells treated with suramin and ATP suggest the participation of P2Y subtype receptors in this response. Several previous studies done with suramin showed a reduction in ATP-induced calcium response in different cell types, such as human endocervical cell line, bovine trophoblast cell line, and canine macrophage cell line, and suggested the participation of P2 receptors in the rise of calcium . Calcium is a second messenger that activates intracellular signaling pathways, such as MAPK in human endometrial cells . Therefore, we assessed whether ATP could induce ERK1/2 phosphorylation in BEND cells. We observed that 50 mM ATP incubated for 5 min significantly increased ERK1/2 phosphorylation (p < 0.01) compared with the control, and this effect was partially but significantly reduced by the antagonist suramin (p < 0.05) . The band sizes detected in all experiments correspond to the size expected for ERK1/2 (44 and 42 kDa). Antibody against total ERK1/2 (independent of the phosphorylation status) was used as loading control, therefore, differences in the amount of protein loaded in each line of the gel do not interfere in the result, because the bands of each line were normalized to total ERK. In human endometrial stromal cells, it was shown for first time that ATP activates ERK1/2, and the ERK1/2 signaling pathway was involved in mediating ATP actions in the human reproductive system . Additionally, it was demonstrated that ATP activated ERK1/2 through the P2Y2 purinoceptor/PLC/PKC signaling pathway . In human endometriotic stromal cells, ATP (100 mM) increased ERK1/2 phosphorylation, which was involved in endometriosis pain . MAPK ERK1/2 has a key role in the expression of proinflammatory cytokines. The inhibition of the MAPK pathway in bovine endometrial epithelia and stromal cells reduced LPS-induced IL-8, IL-6, and IL-1b expression . The effect of suramin on cellular viability of BEND cells also was evaluated. BEND cells were incubated with vehicle (0.1% DMSO) or different concentrations of suramin (1, 10, 100, or 300 mM) for 30 min or 24 h. We observed that suramin did not reduce cell viability when compared with the vehicle . Therefore, the attenuation of IL-8 production, intracellular calcium mobilization, and ERK1/2 phosphorylation produced by suramin were effects mediated through the P2Y receptors. 3.3. Endometrial Cells Express P2Y mRNA Receptors Since extracellular ATP mediates its functions through binding purinergic type P2 receptors, we assessed the presence of mRNA of subtype P2Y receptors in BEND cells through RT-qPCR and analysis with the 2-DDCt method. BEND cells expressed higher levels of P2Y1 and P2Y2 purinergic subtype receptors, lower levels of P2Y11 and P2Y12 receptors, and P2Y4, P2Y6, and P2Y13 mRNA receptors were undetectable . P2Y receptors are predominantly activated by ATP or ADP, specifically P2Y-1, -2, -4, and -11 by ATP . A P2Y1 receptor can be activated by ATP, which is considered a partial agonist, but ADP present highest affinity by the receptor . Although the presence of P2Y receptors has been studied in human endometrial cells, in bovines, the expression has only been studied in other cell types (as mentioned in the Introduction). Human endometrial stromal cells express the P2Y2 receptor . Additionally, P2Y2, P2Y14, and P2X7 are expressed in the human endometrium with P2Y2 in human endometrial cancer cell lines , and P2Y1, P2Y2, P2Y4, and P2X7 in the rat uterine epithelium . Thus, our research shows, for the first time, the presence of P2Y receptor subtypes in bovine endometrial cells. P2Y subtype receptors couple to different G-protein. P2Y1 and P2Y2 are receptors linked to Gaq protein, and their activation stimulates the pathway of PLC/DAG/IP3/calcium . The increase of intracellular calcium induced by ATP in BEND cells correlates with the types of receptors expressed at a higher level. Specifically, P2Y2 receptor is expressed at the highest level compared with P2Y1. The P2Y11 receptor is considered an unconventional member of the P2Y family receptors, because it couples with both Gaq and Gas proteins, and its activation increases IP3 and cAMP, respectively ; however the expression of the P2Y11 receptor was lower in BEND cells. The P2Y12 receptor is coupled to Gai protein, and it is activated by ADP. P2 subtypes receptors induce classical signaling pathways and gene expression; P2Y1 and P2Y2 activate ERK1/2 in different cell types , and increase the expression of pro-inflammatory genes such as IL-6 and IL-8 . On the contrary, it has been suggested that the P2Y11 receptor has two types of responses, because it activates the pro-inflammatory NF-kB/ERK1/2 pathway and also activates anti-inflammatory signaling. Activation of P2Y11 receptor inhibited LPS-induced TNF-a production in M2 macrophages, which is proposed to occur through a regulated control of IL-1 receptors . 4. Conclusions This study showed that ATP increased IL-8 production, activated intracellular calcium mobilization, and induced ERK1/2 phosphorylation in BEND cells--effects that were partially reduced by a P2Y receptor antagonist. P2Y1 and P2Y2 receptors are expressed at high levels as compared to other subtypes of receptors in BEND cells. These results suggest a key role of P2Y receptors in endometrial inflammatory activation, which could be useful as a therapeutic strategy to regulate uterine inflammation through the modulation of P2Y receptors. Author Contributions Methodology, N.G., S.T. and P.A.; Data analysis, P.A., R.A.B. and M.A.H.; Conceptualization, M.A.H. and R.A.B.; Formal analysis, P.A. and M.A.H.; Writing--Original Draft Preparation, M.A.H.; Review and Editing, R.A.B. and M.A.H. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 ATP increases interleukin-8 (IL-8) production in BEND cells. (A) BEND cells were treated with vehicle (PBS) or different concentrations of ATP (0.1, 1, 10, 50, and 100 mM) for 24 h, and IL-8 production was assessed in the culture medium by ELISA assay. LPS (500 ng/mL) was used as a positive control. (B) BEND cells were incubated with vehicle (PBS) or ATP (10, 50, and 100 mM) without or with LPS (500 ng/mL) for 24 h, and IL-8 production was assessed in the culture medium by ELISA assay. (C) BEND cells were incubated with vehicle (DMSO 0.1%) or suramin (50 mM) for 15 min, and then ATP (50 mM) was added for 24 h, and IL-8 production was determined by ELISA assay. n = 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure 2 ATP increases intracellular calcium mobilization and ERK1/2 phosphorylation. (A) Fura 2AM-loaded BEND cells were treated with vehicle (PBS) and basal fluorescence was registered for 60 s; then, different concentrations of ATP (0.1, 1, or 50 mM) were added and the fluorescence was determined for 240 s. RFU = relative fluorescence units. (B) Fura 2AM-loaded BEND cells were incubated with vehicle (0.1% DMSO) or suramin (10, 50, or 100 mM) for 15 min. Then, the basal fluorescence was registered for 60 s and ATP (50 mM) was added, and the fluorescence registered for 140 s. V = vehicle. (C) BEND cells were incubated with vehicle (0.1% DMSO) or suramin (50 mM) for 15 min, and then vehicle (PBS) or ATP (50 mM) was added, and incubation was carried out for 5 min. Total proteins were analyzed by immunoblot with antibodies against phospho-ERK1/2 and total ERK1/2. c = control (0.1% DMSO). S = Suramin. Bands' intensities are shown as the ratio phospho-ERK1/2/ERK1/2 in the bar graph. (D) BEND cells were incubated with vehicle (0.1% DMSO) or suramin (1, 10, 100, or 300 mM) for 30 min or 24 h, and the cellular viability was measured by propidium iodide assay. * p < 0.05, ** p < 0.01 compared with the control. Images are representative of three independents experiments. Figure 3 Levels of expression of mRNA P2Y subtype receptors in BEND cells. Total RNA was isolated from BEND cells and RT-qPCR for P2Y subtype receptors was performed. Level of expression of each subtype receptor was determined by the 2-DDCt method. n = 3 independent experiments. animals-13-00841-t001_Table 1 Table 1 Primers used in the RT-qPCR assay. Name Primer 5'-3' Size Accession # P2Y1R F: TTCCACATGAAGCCGTGGAG R: GGCAGAGTCAGCACGTACAA 83 NM_174410.3 P2Y2R F: CAGTCCCCACACCCTTAGTCA R: CCGGGTATTTATGTGGGAGTCA 112 NM_001166525.1 P2Y4R F: CCCGTGACCTATGCAGTTGT R: GCGAAAGAGGAAGAGCCAGA 75 NM_001256557.1 P2Y6R F: GCTACTAAGGCGTGCGTTTC R: GGTTGCAGTCTACTTATCTCCCC 120 NM_001192295.1 P2Y11R F: AGTGGCCTCCAAGATGACTTTC R: GCTCCCGGCTGCAGAAG 103 NM_001204449.1 P2Y12R F: TACAGTCAGGCCACAAGAACA R: TGTCTTAGTTTAGCTGCTCACA 91 NM_001001174.2 P2Y13R F: TGCCAGTAGCTCCAACACAA R: AGGGTTGAGCAAGTTCCAGG 85 NM_001098152.1 RSP9 F: GCTGACGCTGGATGAGAAAGACCC R: ATCCAGCACCCCGATACGGACG 85 NM_001101152.2 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000104

The porcine epidemic diarrhea virus, PEDV, which causes diarrhea, vomiting and death in piglets, causes huge economic losses. Therefore, understanding how to induce mucosal immune responses in piglets is essential in the mechanism and application against PEDV infection with mucosal immunity. A method of treatment in our research was used to make an oral vaccine that packaged the inactive PEDV with microencapsulation, which consisted of sodium alginate and chitosan, and adapted the condition of the gut in mice. The in vitro release experiment of microcapsules showed that inactive PEDV was not only easily released in saline and acid solutions but also had an excellent storage tolerance, and was suitable for use as an oral vaccine. Interestingly, both experimental groups with different doses of inactive virus enhanced the secretion of specific antibodies in the serum and intestinal mucus, which caused the effective neutralization against PEDV in the Vero cell by both IgG and IgA, respectively. Moreover, the microencapsulation could stimulate the differentiation of CD11b+ and CD11c+ dendritic cells, which means that the microencapsulation was also identified as an oral adjuvant to help phagocytosis of dendritic cells in mice. Flow cytometry revealed that the B220+ and CD23+ of the B cells could significantly increase antibody production with the stimulation from the antigens' PEDV groups, and the microencapsulation could also increase the cell viability of B cells, stimulating the secretion of antibodies such as IgG and IgA in mice. In addition, the microencapsulation promoted the expression of anti-inflammatory cytokines, such as IL-10 and TGF-b. Moreover, proinflammatory cytokines, such as IL-1, TNF-a, and IL-17, were inhibited by alginate and chitosan in the microencapsulation groups compared with the inactivated PEDV group. Taken together, our results demonstrate that the microparticle could play the role of mucosal adjuvant, and release inactivated PEDV in the gut, which can effectively stimulate mucosal and systemic immune responses in mice. microcapsules PEDV mucosal immunity alginate chitosan National Natural Science Foundation of China31902169 Heilongjiang Province Science Fund for Excellent Young ScholarsYQ2020C008 Young Talents" Project of Northeast Agricultural University18QC39 This work was supported by the National Natural Science Foundation of China (Grant number:31902169), the Heilongjiang Province Science Fund for Excellent Young Scholars (Grant number: YQ2020C008), Young Talents" Project of Northeast Agricultural University (Grant number: 18QC39. pmc1. Introduction Porcine epidemic diarrhea virus, PEDV, is a highly contagious disease that infects the small intestinal epithelial cells of piglets during the first seven days of birth, and the mortality rate can range from 70% to 100% in three-day-old piglets . At the first week of birth, the major immune protection of piglets is dependent on maternal antibodies and innate immunity. The maternal antibody is induced by the inactivated and attenuated vaccines of PEDV through the intramuscular route or subcutaneous injection in the pregnant sow, but the maternal antibody is digested by gastric acid and pepsin before entering the intestine, which cannot effectively protect against PEDV infection. However, mucosal immunity can be built no more than three days after birth, and provides more effective protection than systemic immunity in piglets. Some studies have demonstrated that the first line of defense by IgA in the intestine would be more effective than that by IgG for neutralizing the infection of intestinal diseases . However, the problem of how to transport the inactivated PEDV virus into the intestine and activate mucosal immunity has not yet been solved. Moreover, the mechanism of mucosal immunity to PEDV in the gut of fetal pigs remains unclear. The process of specialized M-cells taking up and presenting PEDV to the dendritic cells and lymphoid follicles also needs to be better understood to learn how antigens arrive at the inductive sites for T-cell- and B-cell-dependent activation of IgG and IgA production. One interesting strategy is packaging vaccines to look like microcapsules, which have different properties for oral immunity, such as size, shape, and surface molecule organization . Chitosan and alginate are mixed to coagulate the ionic gel, which is a potential candidate vehicle for their main constituents due to their intrinsic immunomodulatory properties. Alginate is one of the most widely used carriers for the controlled release of different types of active agents. Moreover, sodium alginate, which is derived from marine brown algae and bacteria, has received attention as an antimicrobial in the food industry because of its unique physicochemical properties and biological activity. Chitosan (CS), also called poly glucosamine, is widely used to enhance the mucosal immune response due to its biocompatibility, biodegradability, and mucoadhesive properties . The production method of alginate-chitosan microcapsules by using ionic gelation is an interesting approach to compound oral delivery systems for inactivated PEDV vaccines. The classic technique involves first mixing the antigen of inactivated PEDV and alginate solution and then pouring it slowly into a solution of calcium chloride and chitosan. Finally, the alginate microcapsules are collected by decanting the supernatant and are washed with water. Hence, the preparation method of PEDV alginate microcapsules is very simple. The manufacture of microencapsulations showed that they used simple machinery, had a low cost, and a good safety profile, making them an attractive approach to oral immunization. In a previous study of the mucosal immune system, oral vaccination with recombinant Lactobacillus was more promising for stimulating specific immunization against pathogens in the first contact with mucosal surfaces . To improve the effectiveness of the oral vaccination delivery method, which protects against PEDV infection, avoiding digestive degradation in the acidic environment of the stomach is crucial (oral vaccination with alginate microsphere systems). There is also a novel microencapsulation method for rotavirus with anionic polymers and amines, including sodium alginate and spermine hydrochloride, which increases the antibody titers against rotavirus infection . Similarly, microencapsulations of PEDV can arrive at the gut of piglets and release the virus in an alkaline environment. Moreover, they can be taken up by mucosa-associated lymphoid tissues to activate mucosal immune responses against encapsulated PEDV, and be processed by Peyer's patches and M-cells in the small intestine . In this study, inactivated PEDV packaged in microencapsulations was used to explore an efficient oral vaccine delivery system to release the virus into the gut, which would be better for protecting against PEDV-induced specific mucosal immunity in mice. In addition, mucosal and systemic immune responses were monitored for specific antibodies, such as IgA and IgG, in the feces and serum of mice, and then used to analyze the neutralizing activity against PEDV. Most importantly, the micro-encapsulations of PEDV were used with the antigen of complete enterovirus particles to present into the mucosal immune cells in the gut. The functional immune cells in the specific immune response, such as dendritic cells and B cells, were studied to focus on the mechanism of complete virus particles in the recognition, presentation, and antigen processing with oral mucosal immunity against enterovirus infection in vivo. 2. Materials and Methods 2.1. Cell and Virus PEDV strain LJB/03 was previously isolated from diarrheic piglets in China and purified in our laboratory . Vero cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37 degC with 5% CO2. A PEDV strain (LJB/03) was isolated and stored in our laboratory. Confluent Vero cells grown in a 5 mL tissue culture flask were washed with PBS three times and inoculated with 1 mL of a mixture solution containing trypsin at a concentration of 10 mg/mL, PEDV inoculation culture (3 x 104 PFU), and DMEM without any serum. After incubating at 37 degC for 1 h, 4 mL of DMEM with trypsin at a concentration of 8 mg/mL was added. Before 70% cytopathic effects, the cells were maintained at 37 degC under 5% CO2 and monitored daily. Finally, the diseased cells were subjected to three rounds of freezing and thawing, and then the supernatants were collected after centrifugation for 10 min at 12,000 rpm. The virus titers were measured with the method of TCID50, and the PEDV titer was approximately 6.3 x 107 PFU. PEDV was inactivated with 0.1 M binary ethylenimine (BEI) to a final volume of 5%, which was incubated at 37 degC for 24 h. In addition, sodium thiosulfate was used to neutralize the excess BEI at 37 degC for 2 h. Inactivated PEDV virus was stored at -80 degC until use. 2.2. The Production of Microcapsules The inactivated PEDV (6 x 106 PFU and 6 x 107 PFU) and sodium alginate solution at 1.5% (wt/vol) were obtained by dissolving and passing the solution. The CaCl2-chitosan solution was obtained by adding CaCl2 and sodium alginate to final concentrations of 4% and 1% (wt/vol), respectively. After completely mixing the PEDV and the sodium alginate solution, the mixture was dropped at a constant speed (approximately 60-90 drops/min) through a medical 9-gauge syringe into a solution of calcium chloride and chitosan acetic acid. A suspension of sodium alginate-chitosan microcapsules was obtained after thorough stirring at a constant rate of approximately 300 r/min at room temperature for 30 min. The particle size was approximately 7.54 +- 2.87 mm, which was measured under a light microscope. Finally, the samples were separated, filtrated, and washed repeatedly with distilled water, precooled at -80 degC for 1 h, and freeze-dried. 2.3. Analysis of Encapsulation Efficiency and Protein Content The 100 mg freeze-dried microcapsules were weighed into a 50 mL centrifuge tube with 10 mL trisodium citrate solution (0.06 mol/L) and incubated at room temperature for 10 min. The microcapsules were ultrasonicated and centrifuged at 10,000 rpm for 10 min. Finally, approximately 1 mL of the supernatant (filtrated processing if there was another matter) was collected to determine the total protein content by the Bradford Protein Assay Kit (Coomassie Brilliant Blue) . The encapsulation efficiency (B) and the amount of carrier protein (Z) of the microcapsule were calculated according to the formula :B = W/J x 100%(1) Z = W/M(2) (W: protein content in the microcapsules; J: total amount of protein; M: total amount of microcapsules.) 2.4. In Vitro Release Experiment of Microcapsules in Different Saline Solutions, pH Values, and Room Temperature Storage Tolerances Three 50 mL centrifuge tubes containing 100 mg of PEDV microcapsules were added to 10 mL of pH 7.4 PBS solution, 0.75% physiological saline solution, and pH 2.3 hydrochloric acid solution, and then placed in the Oven-Controlled Crystal Oscillator at 37 degC and a speed of 100 r/m. The release of protein content in the solution was measured at 1 h, 3 h, 9 h, 1 d, 3 d, 6 d, 12 d, and 18 d. The protein content was analyzed by the Coomassie Brilliant Blue method , and then the release rate (RT) curve was drawn. The PEDV microcapsules of freeze-dried particles were stored without any media in the dark at room temperature for 5 months, and the storage performance was evaluated by measuring the changes in the PEDV protein release rate every month. The concrete method of PEDV protein release rate refers to analysis of encapsulation efficiency and protein content. The release rate (RT) was calculated according to the formula: RT = R/W. (R: release amount of PEDV protein; W: the protein content in the microcapsule) . 2.5. Immunization of Microcapsules in SPF Mice A total of 50 SPF mice, 5 weeks old, with approximately 18 g of body weight, were randomly divided into five groups. Each group contained 10 mice. The immunization regimen is shown in Table 1. The first group, the negative control group, was immunized with PBS buffer through oral administration. The second group was immunized with microcapsules without virus through oral administration. The third group was immunized with inactivated PEDV without encapsulation through oral administration. The fourth group was immunized with the encapsulated PEDV vaccine through oral administration, which encapsulated approximately 6 x 106 PFU of PEDV. The fifth group was immunized with approximately 6 x 107 PFU of encapsulated PEDV through oral administration, which encapsulated 100 mL of PEDV. All the groups were used to immunize mice via an intragastric route on Days 1 and 3 twice. On Days 0 (preimmunization), 3, 6, and 9, 200 mg serum samples and feces samples were collected from both the tail vein and the anus. The fecal samples were subsequently homogenized for 30 min in 400 mL of sterile PBS (pH 7.4) containing 0.01 mol/L EDTA-Na2 and then incubated for 12 h at 4 degC. The extracted supernatants of all fecal samples were collected by centrifugation at 15,000x g for 10 min and stored at -80 degC . The feces were supplemented with protease inhibitors for the subsequent ELISA analysis. The serum was stored at -80 degC for the subsequent ELISA and neutralization analyses. Intestinal lavage samples (mucus) were obtained from the intestine of euthanized mice. The mucus was suspended in 400 mL of sterile PBS (pH 7.4) containing 0.01 mol/L EDTA-Na2 for 2 h at 4 degC. Then, all mucus samples were collected by centrifugation at 15,000x g for 10 min, and the supernatant was stored at -80 degC with protease inhibitors for neutralization analysis. 2.6. Enzyme-Linked Immunosorbent Assay (ELISA) The ELISA plates were coated for 18 h at 4 degC with PEDV (6.3 x 107 PFU), which was previously cultured from Vero cells in the previous section. After the plates were blocked with 5% skim milk in PBS for 1.5 h at 37 degC, they were washed 3 times with PBST (PBS + 0.1% Tween 20). Sera and fecal samples (supernate of fecal) were added into wells in triplicate and used to test specific antibodies such as IgG and IgA. The sera and fecal samples were incubated for 1 h at 37 degC and washed as before. Goat anti-mouse IgG and IgA antibodies-HRP (Invitrogen) were added to each well (1:5000) and incubated for 1 h at 37 degC. After the final round of washing with PBST three times, the TMB substrate was used for color development, and the absorbance was measured at 490 nm. 2.7. PEDV Neutralization Assay The serum of mice fed with PEDV microcapsules and inactivated PEDV was collected to determine the neutralization ability of antibodies. The serum and intestinal lavage of mucus were obtained from the previously described immunization, and were filtered with a 0. 45 mm filter membrane. The concrete method was as follows: 50 mL samples (serum and intestinal lavage) were taken to a constant dilution from 1:2 to 1:512 and then added to a 96-well plate for eight replications with Vero cells. PEDV at a titer of 5 x 105 PFU was mixed within the DMEM culture medium, and 10% heat-inactivated bovine serum albumin was added to the cell plate, which was coated with the diluted serum and fecal sample solution and then incubated with the antibody and virus at 37 degC for 1 h. Then, 100 mL of Vero cells were added to the antibody-virus mixture and incubated in a 5% CO2 incubator at 37 degC for 3 days. Finally, the covered medium was discarded, and the wells were washed three times with sterile PBS (pH = 7.4) and dyed with 1% crystal violet solution. The quantity variance of plaque was analyzed to demonstrate their antibody level. 2.8. T-Cell Proliferation Five mice were dissected from each group on the seventh day after the second immunization. Single-lymphocyte suspensions were prepared from the spleen after the second immunization, as previously described . Lymphocytes were isolated from the spleens of five mice dissected from each group and incubated in triplicate in 96-well plates at 5 x 105 cells/well with Roswell Park Memorial Institute 1640 medium (RPMI-1640) plus 20% fetal calf serum (FCS) at 37 degC in a 5% CO2 incubator. Then, the cells were stimulated for 48 h with 0.5 and 1 mg/mL inactive PEDV in the experimental group (specific antigen stimulation) and control group (without antigen stimulation). Based on the manufacturer's instructions (Promega), the Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay was used to evaluate T-cell proliferation. The solution of A thiazolyl blue (MTT) was pipetted (10 mL) into each well to develop the color. The plates were incubated for 4 h before reading the OD490 values of the plate using the Magellan plate reader, and then the average was taken of the PEDV stimulation data to compare with that of the negative control wells. The proliferation rate was analyzed with the stimulation index: SI = (OD490 experiment group - OD490 culture media)/(OD490 control group - OD490 culture media) 2.9. Flow Cytometry and Cell Sorting The suspensions of single lymphocytes were derived from the spleens of immunizing mice that were dissected from each group on the seventh day of the second immunization . The isolated spleen T-cells (5 x 105 cells/mL) were cultured in RPMI 1640 culture media for flow cytometry staining. The incubated single-lymphocyte suspensions were stained with anti-CD11c (PE-conjugated) and anti-CD11b (FITC-conjugated) antibodies and anti-CD23 (APC-conjugated) and anti-B220 (PE-conjugated) antibodies in RPMI 1640 culture media without serum. All antibodies were purchased from Miltenyi Biotec. Single lymphocyte cells were stained with CD11c (PE-conjugated) and CD11b (FITC-conjugated) antibodies and sorted with FACStar to prepare CD11c+ and CD11b+ cells. The same method was used to prepare CD23+ B220+ cells and B220+ cells. All the samples were tested with FACSCanto (BD Biosciences) and analyzed by CellQuest software . In all cases, the purity of FACStar-sorted cells was >98%. The cells were sorted by FACSAria (BD Biosciences) into CD11c+, CD11b+, CD23+B220+, and B220+ populations in complete medium (RPMI, 1% penicillin-streptomycin, 1% glutamine, 50 mM b-mercaptoethanol). 2.10. Cytokine Assays Real-time qRT-PCR was used to determine the levels of cytokine genes and intestinal tight junction gene products in splenic lymphocyte and intestinal samples from immunized mice using a CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Total RNA was extracted from intestinal tissues and spleen with total RNA extraction kits (TaKaRa, Dalian, China) according to the manufacturer's instructions . Total RNA was converted to cDNA using the PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) . The cDNA products were used for real-time PCR with a SYBR Premix Ex TaqTM II reagent kit (TaKaRa, Dalian, China), and the specific primers used are listed in the Table S1 . The Livak method (2-CT method) was used to calculate the fold change compared to b-actin gene controls . 2.11. Statistical Analysis All the samples and experiments were analyzed in triplicate, and all the groups were examined as independent measurements to support adequate statistics in the experiments. The average duration of recovery and colonization over time were compared between groups using repeated measures analysis of variance with Bonferroni's correction using SPSS software (IBM, New York, USA). Data between the different groups were analyzed with one-way repeated measures analysis of variance (ANOVA) and the least significant difference (LSD) test with GraphPad software. Statistically significant effects (p < 0.05) were further analyzed . 2.12. Ethical Statement All animals were housed in negative-pressure isolators with HEPA filters in a BSL2. The protocols for animal experiments were approved by the Institutional Committee of Northeast Agricultural University (2018NEAU-131, 12 September 2018) and complied with the guidelines of the Northeast Agricultural University Administrative Committee of Laboratory Animals. 3. Results 3.1. The Excellent Storage Tolerances of Microcapsules in Different Saline Solutions, pH Values, and Room Temperature As a biomedical material, the ability of microcapsules to be released in saline and acid solutions is crucial in clinical applications. The release rates in different saline solutions, pH values, and storage times are shown in Figure 1. The trend of the in vitro release ability of microcapsules in the PBS group and normal saline group was similar, and the release rate was more than 50% on the 3rd day; on the 18th day, the release rate reached 95% and 91%, respectively. However, that of the hydrochloric acid group was only 37.5% on the 3rd day and just 63.2% on the 18th day. These results indicated that the microcapsules were easily released in saline solution, but most importantly, they were acid-stable. The release ability after different months of storage showed that the release rate remained higher than 83% in the first three months, and they still maintained the lowest release rate of 41% in the fifteenth month. These results indicated that the microcapsules have excellent storage tolerance in the dark at room temperature for 5 months. 3.2. The Microcapsules Stimulate the Specific Humoral and Mucosal Immunity The specific antibodies in intestinal lavage and serum were detected by ELISA at the end of the immunization. The ELISA results showed that with increasing days, the specific antibody level in both the inactivated PEDV and microcapsule groups continuously increased, as shown in Figure 2. For specific IgA, the PEDV (low) and PEDV (high) microcapsule groups had significantly higher levels than the inactivated group on the third day (p < 0.05), and the PEDV (high) microcapsules group had remarkably higher levels than the PEDV (low) microcapsules group (p < 0.05). The PEDV (low) microcapsules group had significantly higher levels than the inactivated PEDV (p < 0.05) on the sixth day, while the PEDV (high) microcapsules group had significantly higher levels than the inactivated group (p < 0.01). At the end of the detection on the ninth day, both PEDV microcapsules groups had significantly higher levels than the inactivated group (p < 0.01). Most importantly, the empty microcapsule group did not have detectable IgA antibodies during the whole immunization period. Figure 2B shows that the specific IgG levels in both PEDV microcapsules showed the same trend as the intestinal lavage results and were significantly higher than those in the inactivated PEDV group on the third day (p < 0.05). The specific IgG levels in the PEDV microencapsulated groups were significantly higher than those in the inactivated PEDV group on the sixth and ninth days (p < 0.01). These results suggest that the PEDV microcapsules have the ability to activate specific mucosal and systemic immune responses in a dose-dependent manner in mucosal immunity. 3.3. The Specific IgG and IgA Neutralized PEDV The neutralization of IgG and IgA is shown in Figure 3. The results indicated that the neutralization of IgG in the PEDV (high) microcapsule group was more effective than that in the PEDV (low) microcapsule group after the 1:8 dilution point against PEDV infection in the in vitro experiment. The 50% neutralization with IgG of the high PEDV group was probably 1:256, while the PEDV (low) microcapsule group was 1:128. The 50% neutralization of the PEDV (high) microcapsule group was higher than the PEDV (low) microcapsule group in the neutralization. The neutralization of IgA in both the PEDV (high) and PEDV (low) microcapsule groups was higher than that in the inactivated PEDV group, inhibiting PEDV infection in the in vitro experiment. The 50% neutralization of IgA for the inactivated PEDV group, PEDV (low) microcapsule group, and PEDV (high) microcapsule group gradually increased against PEDV infection in Vero cells at dilutions of 1:16, 1:32, and 1:64, respectively. The neutralization ability of IgG and IgA in the PEDV (high) microcapsule group was higher than that in the PEDV (low) microcapsule group. The neutralization of PEDV microcapsules was also dose-dependent, which indicated that the PEDV (high) microcapsule group had better systemic and mucosal immunity than the PEDV (low) microcapsule group. 3.4. PEDV Microcapsules Stimulated Immune Memory To further analyze whether microcapsules influence cell-mediated immunity, T-cell proliferation was analyzed to study immune memory with PEDV from five immunized mice for 29 days after the third immunization to obtain a single-cell suspension of lymphocytes for the in vitro experiment (Table 2). The suspension was stimulated with PBS (as a control), microcapsules (without PEDV), inactivated PEDV, PEDV (low) microcapsules, and PEDV (high) microcapsules. Different doses of 0.5 mg/mL and 1.0 mg/mL PEDV were used to simultaneously stimulate lymphocyte proliferation. At a dose of 0.5 mg/mL PEDV, the number of PEDV (low) microcapsules was significantly higher than that in the PBS group; moreover, the number of PEDV (high) microcapsules was significantly higher than that in the PBS group. There was no significant difference between the microcapsule groups, inactivated group, and PBS group with regard to the specific antibodies. With the stimulating dose of 1 mg/mL PEDV, both PEDV microcapsule groups were significantly higher than the PBS group. The microcapsules and inactivated group were not significantly different from the PBS group. The lymphocyte proliferation results indicated that both 0.5 mg/mL and 1 mg/mL PEDV could stimulate T-cell proliferation with immune memory for the immune response of PEDV microcapsules. Single-lymphocyte suspensions were isolated from animals after the last boost, plated as triplicates in a 96-well plate, and stimulated in vitro for 72 h with PEDV and con A as a positive control. T rate was analyzed with stimulation index: SI = (OD490 experiment group - OD490 culture media)/(OD490 control group - OD490 culture media). The differences between means were considered significant at * p < 0.05 and very significant at ** p < 0.01. 3.5. Microcapsules Enhanced the B Cell Differentiation CD11b and CD11c are cell surface markers of dendritic cells which were detected by flow cytometric analysis from different groups of immunizations. The results showed that PBS, microcapsules, and inactivated PEDV stimulated approximately 2.2% of CD11b+ cells, and PEDV (low) microcapsules and PEDV (high) microcapsules stimulated CD11b+ cells. Conversely, the trend in CD11c+ cells gradually increased from the PBS group (4.3%) to PEDV (high) microcapsules (34.6%). The results of CD23 and B220 cell markers indicated that all B220+ cells were stimulated for differentiation in addition to the inactivated PEDV group, and the PBS and microcapsule groups reached 57.4% and 54.4%, respectively. Research on B220+ cells indicated that inactivated PEDV could better inhibit antibody production with oral immunization compared with other groups. Most importantly, the percentage of B220+ cells in both the PEDV (low) microcapsule and the PEDV (high) microcapsule groups was higher than that in the inactivated PEDV group. Nevertheless, B220+ and CD23+ cells were significantly increased by stimulation with PEDV; moreover, the percentages of these three groups were slightly different from those of the inactivated PEDV group (46.6%), the PEDV (low) microcapsules (45%) and the PEDV (high) microcapsules (47.7%). 3.6. Microcapsules Inhibit the Inflammation To validate the results of inflammation after immunization, qRT-PCR was performed to analyze cytokine expression at the mRNA level . Compared to the samples from the different groups, our qRT-PCR results revealed significant differences in the expression of genes, such as IFN-g, IL-4, IL-1, TNF-a, IL-17, IL-10, TGF-b, occluding, and ZO-1. The expression analysis was clustered by hierarchical clustering using the complete linkage algorithm and Pearson correlation metric in R for the heat map with GraphPad software. The heat map results indicated that the IFN-g expression in the microcapsule group, PEDV (low) microcapsule group, and PEDV (high) microcapsule group was significantly higher than that in the inactivated group, and IFN-g expression increased with increasing doses of PEDV. IL-4 expression showed a similar trend in the microcapsule group, PEDV (low) microcapsule group, and PEDV (high) microcapsule group, which stimulated the Th2 immune response. IL-4 expression in the inactivated group was different from IFN-g expression, which indicated that the microcapsules could stimulate Th1 immune response. However, the two PEDV microcapsule groups exhibited different Th1 immune responses compared with the inactivated group. The microcapsules could effectively inhibit the inflammation stimulated by the inactivated virus, which could decrease the expression of IL-1, TNF-a, and IL-17 in the microcapsule groups. The anti-inflammatory cytokines IL-10 and TGF-b were significantly increased in the microcapsules groups compared with the inactivated group. The relative tight junction genes also showed the same expression trend in various groups. 4. Discussion The PEDV outbreak of 2013-2014 led to annual losses among worldwide swine producers. The loss of productivity from enteric diseases of PEDV in neonatal piglets costs swine producers millions of dollars . Previous development of live and attenuated vaccines for another diarrheal virus of pigs, transmissible gastroenteritis virus (TGEV), provided insights into the mechanisms of mucosal immunity with IgA and piglet protection . A previous study showed that the inactive PEDV was effective in stimulating the specific antibody of IgA in an immunized sow with attenuated vaccines protecting the piglets for lactogenic immunity . Lactogenic immunity from pregnant sows is induced via the gut-mammary gland secretory IgA (sIgA) axis, which is also a promising and effective way to protect suckling piglets from PEDV infection . Therefore, a successful PEDV vaccine must induce sufficient maternal or self-reproductive IgA antibodies . However, the PEDV did not use the same route as the TGEV immune plan. The main reason is that the antibody titers of PEDV did not reach the titers of TGEV to boost a successful immune response. More research has focused on recombinant vaccines with different delivery vectors to protect antigens not digested by gastric acid . However, the exposure dose of antigen was too low to stimulate specific antibodies, such as IgA and IgG. The antigen expressed by the recombinant vaccine was very exclusive. We explored a new form of possibility to deliver the virus into the gut, stimulating the specific mucosal immune response, which could directly neutralize the enterovirus at the site of infection. Alginate microcapsules using ionic gelation represent a new and interesting approach to oral delivery systems for inactivating PEDV carriers. The microcapsules could efficiently capsulate the inactive PEDV antigen, and the whole virus particle was contained in the microcapsule. The microcapsules had good release profiles of the virus particles in saline solutions, such as PBS and normal saline, and the release rate was higher than 50% within three days. Studies have shown that the specific antibodies of IgA reach a peak in the second after immunization . The most important feature was the microcapsules protecting the integrity of virus particles in hydrochloric acid, which prevents the virus particles from being released and subsequently digested in gastric acid . Phage encapsulation and subsequent release kinetics revealed that the microcapsules possess pH-responsive characteristics with phage release triggered in an intestinal pH range suitable for therapeutic purposes . A number of previous studies have used alginate as the main encapsulating agent either on its own or in combination with chitosan . The chitosan-alginate microspheres effectively protected the virus in simulated gastric conditions, showing the remaining viral titers. The chitosan-alginate microspheres acted as an acid barrier in the simulated gastric conditions, helping the virus arrive at the M cell in the intestinal mucosal immune system. With storage at room temperature, the PEDV microcapsules displayed a remarkably low loss of antigen in the first three months, and they had good storage tolerance, thus maintaining the quality of the PEDV antigen. Tan reported that tocotrienols encapsulated in chitosan-alginate microcapsules have effective storage tolerance in the yogurt matrix . PEDV initially attacks neonatal intestinal epithelial cells in piglets and the intestinal tract system, inducing diarrhea clinical signs, but systemic lymphoid organs cannot provide the effective antibody IgG to neutralize this virus in the neonatal pig. They mainly depend on the innate immunity of maternal antibodies. Thus, we chose microcapsules carrying inactive PEDV to stimulate the mucosal immune system of neonatal piglets. The specific antibodies against IgA and IgG showed that the microcapsules stimulated the mucosal and systemic immune systems producing specific antibodies, such as IgA and IgG . In the first mouse immunization experiment, the microcapsule groups had higher levels of antibodies than the inactivated PEDV group. Most importantly, the immunization of microcapsules in specific antibody production was dose-dependent, and the levels of specific IgA and IgG in the high-dose microcapsule group were significantly higher than those in the low-dose microcapsule group. There was a close connection between the specific neutralization of antibody titers and the dose dependence of microcapsules arriving at the mucosal surface. The results of 50% neutralization with specific IgG and IgA were also found during microcapsule immunization, and PEDV (high) microcapsule groups had better neutralizing activity than PEDV (low) microcapsule groups. The specific antibody of IgA had a greater efficiency of neutralization than the specific antibody of IgG in the same group. The main reason was that chitosan-alginate microcapsules stimulate humoral immunity at the mucosal area, which is mainly mediated by IgA antibodies as the predominant immunoglobulin, and serum-derived IgG also contributes to immune defense . Specific antibody experiments have indicated that microcapsules stimulate specific mucosal and systemic immunity in mice. However, the mice are not the infecting hosts of PEDV, which means there is just the possibility of an immune response in piglets. There are many differences in immunization and infection between mice and neonatal pigs. Virus particle carriers have shown higher potential as oral delivery systems of proteins and peptides, which are taken up by M-cells of Peyer's patches in the gut . As seen in the qRT-PCR results, the microcapsules of PEDV, which have been reported to immunize with microcapsules, were associated with higher levels of IFN-g production related to the T helper 1 (Th1)-type immune response . In contrast, immunization with microcapsules without PEDV promoted IL-4 secretion related to the Th2-type immune response. IFN-g production was higher than IL-4 production after PEDV microcapsule immunization. The cellular immune response (Th1) and humoral immune response (Th2) were disrupted, and the Th1 type immune response was predominant . In particular, the high-dose PEDV microcapsules enhance IFN-g expression, which means that the PEDV microcapsule immunization mediates the T helper 1 (Th1)-type immune response, and the chitosan-alginate plays an immunoregulatory role in extroversion to the cellular immune response. The mouse oral immunization of recombinant Lactobacillus casei expressing the Dendritic Cell-Targeting Peptide Fusing COE Protein of PEDV also mediated the Th1 immune response in piglets . The cytokine response was also analyzed to compare the inflammation, cellular and humoral immunity in oral immunization with PEDV microcapsules. IL-1 and TNF-a were also used to analyze the safety and inflammation with immunization, which did not stimulate the inflammatory response when using the oral PEDV microcapsules. Many more studies have demonstrated the chitosan-alginate induction of the Th1-type immune response for oral vaccination in mice, and studies on the effects of chitosan have indicated that chitosan-fed farm animals showed higher weight gains but a lower incidence of disease than unfed animals . DC-specific delivery has been considered a promising strategy for facilitating the efficient recognition, processing, and presentation of antigens by DCs, leading to enhanced antigen-specific immunity. The dendritic cell-targeted chitosan nanoparticles for nasal DNA immunization suggest that targeted pDNA delivery through a noninvasive intranasal route can be a strategy for designing low-dose vaccines . Without adjuvant immunization, the increase in CD11c+ during vaccination promotes germinal center induction and robust humoral responses . When injected, these alginate 'vaccination nodes' containing activated DCs attracted both host dendritic cells and a large number of T-cells to the injection sites in mice, while some of the inoculated DCs trafficked to the draining lymph nodes . Compared with the inactivated PEDV group, chitosan-alginate groups, such as high-dose PEDV microcapsules and low-dose PEDV microcapsules, significantly stimulated the CD11c+ increase after the last immunization. Taking the advantage of CD11c+ in vivo DC targeting into consideration, chitosan-alginate could presumably induce in vivo DC maturation more robustly than inactivated PEDV. The chitosan-alginate could first effectively induce DC maturation of CD11c+ through the interaction with immunoregulation of chitosan-alginate in the gut, which enhanced the specific immune response, inducing subsequent humoral immunity with mucosal immunity and the systemic immune response. 5. Conclusions The oral microencapsulation was packaged with different titers of inactivated PEDV, which consisted of alginate and chitosan arriving and presenting to the gut in mice. It induced specific humoral and mucosal immunity. Specific antibodies from immunized mice, such as IgA and IgG, neutralized PEDV in vitro. Oral immunization also stimulated immunologic memory in mice. Alginate and chitosan not only act as capsule wall materials but also enhance the viability of immune cells such as DCs and B cells with the function of adjuvants. To the best of our knowledge, this study of microencapsulation is the first to package enterovirus in the immunization of mice even though the host of PEDV is pigs. Therefore, the host animals, pigs, should be studied with inactivated and microencapsulated PEDV. All the data derived from this study can be an important reference point for further research in this area. Acknowledgments Student Innovation Practical Training of NEAU. Supplementary Materials The following supporting information can be downloaded at: Table S1. Primers used for q-RT PCR for inflammatory and functional analyses. Click here for additional data file. Author Contributions Data curation, Z.Q.; Formal analysis, Z.Q., G.L., J.X. and L.L.; Methodology, W.W., W.C. and X.J.; Resources, X.H.; Writing--original draft, Z.N. and X.J.; Writing--review and editing, X.J. and D.L. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Animals were housed in negative-pressure isolators with HEPA filters in a BSL2. Protocols for animal experiments were approved by the Institutional Committee of Northeast Agricultural University (2016NEAU-219, 13 September 2016) and complied with the guidelines of Northeast Agricultural University laboratory animal welfare and ethics of Northeast Agricultural University Administrative Committee of Laboratory Animals. Informed Consent Statement Not applicable. Data Availability Statement Once this manuscript is accepted, the data supporting the results of this study will be made publicly available in any publicly accessible repository. Conflicts of Interest The authors declare no financial or commercial conflict of interest. Figure 1 The stability of microcapsules shown by the release rate in (A) PBS, normal saline, hydrochloric acid, and release ability in different months (B). The differences between means were considered significant at * p < 0.05 and very significant at ** p < 0.01. Figure 2 Enzyme-linked immunosorbent assay measures of antibodies IgA and IgG in feces (A) and serum (B) after the final immunity. The differences between means were considered significant at * p < 0.05 and very significant at ** p < 0.01. Figure 3 The results of antibody neutralization activity of IgG (A) and IgA (B). Both neutralization percentages of IgA and IgG in the intestinal mucosa and serum. Figure 4 The flow cytometric analysis results for the percentage of CD11b+ and CD11c+ cell for immunization (A). (B) indicates the percentage of B220+ and CD23+ cell with the flowcytometric analysis. Figure 5 The expression analysis of genes by qRT-PCR analysis. For the expression analysis of genes, blue and red indicate decreased and increased expression, respectively. For qRT-PCR analysis of the expression of randomly selected novel genes from the immune and tight junction, data are presented as mean +- S.D. (n = 3). animals-13-00889-t001_Table 1 Table 1 Immunization regime of mice. Groups (n) Type of Vaccine 1st Vaccination 2nd Vaccination Route Dose Route Dose PBS (10) Negative control of PBS buffer O/A 0.2 mL O/A 0.2 mL Microcapsules (10) PBS encapsulated in alginate and chitosan O/A 0.2 mL O/A 0.2 mL Inactivated PEDV(10) Inactivated PEDV vaccine O/A 0.2 mL O/A 0.2 mL PEDV (low) microcapsule (10) PEDV vaccine encapsulated in alginate and chitosan with low titer virus (6 x 106 PFU) O/A 0.2 mL O/A 0.2 mL PEDV (high) microcapsule (10) PEDV vaccine encapsulated in alginate and chitosan with high titer virus (6 x 107 PFU) O/A 0.2 mL O/A 0.2 mL n = number of mice; O/A = oral administration. animals-13-00889-t002_Table 2 Table 2 Lymphocyte proliferation index. Groups Stimulation Index Value 0.5 mg/mL PEDV 1 mg/mL PEDV PBS(Control) 1.054 +- 0. 13 1.025 +- 0.11 Microcapsules 1.272 +- 0. 149 1.231 +- 0.156 Inactivated PEDV 1.349 +- 0.151 1.358 +- 0.166 PEDV (low) microcapsule 1.721 +- 0.171 * 2.529 +- 0.183 ** PEDV (high) microcapsule 1.987 +- 0.177 ** 3.161 +- 0.191 ** Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000105

(1) Background: Histological diagnosis and tumor grading are major prognostic and predictive factors in soft tissue sarcomas (STS), as they dictate the treatment strategies with a direct impact on patient survival. This study aims to investigate the grading accuracy, sensitivity, and specificity of Tru-Cut(r) biopsy (TCB) in primary localized myxoid liposarcomas (MLs) of the extremities and its impact on patient prognosis. (2) Methods: Patients with ML undergoing TCB and a subsequent tumor resection between 2007 and 2021 were evaluated. Concordance between the preoperative assessment and definitive histology was calculated with a weighted Cohen's kappa coefficient. Sensitivity, specificity, and diagnostic accuracy were calculated. (3) Results: Of 144 biopsies, the histological grade concordance rate was 63% (Kappa 0.2819). Neoadjuvant chemotherapy and/or radiotherapy impacted concordance with a downgrading effect in high-grade tumors. Among forty patients not treated in neoadjuvant settings, the sensitivity of TCB was 57%, the specificity was 100%, and the overall predictive values of positive and negative TCB were 100% and 50%, respectively. Misdiagnosis did not impact overall survival. (4) Conclusions: TCB may underestimate ML grading due to tumor heterogeneity. Neoadjuvant ChT and/or radiotherapy are associated with pathological downgrading; however, discordance in diagnosis does not modify patient prognosis because systemic treatment decision-making also includes other variables. soft tissue neoplasms myxoid liposarcoma pathology biopsy diagnosis histological grade diagnostic accuracy This research received no external funding. pmc1. Introduction Myxoid liposarcoma (ML) is the second most frequent subtype of liposarcoma, a malignant mesenchymal tumor originating in adipose tissue, presenting in 15-20% of cases and accounting for about 5% of all soft tissue sarcomas in adults . Epidemiologically, MLs are most frequent in the fourth and fifth decades of life, without differences between the two sexes, and they are also the most widespread subtype of liposarcoma in children and adolescents . The deep tissues of the extremities are the sites where ML localizes with the highest incidence, particularly in the thigh. In rare cases, they may localize subcutaneously and retroperitoneally . Patients affected by ML generally have a good prognosis, with an estimated 5-year overall survival rate of 78-96% in localized tumors . Local recurrences occur in 12-25% of cases, while distant metastases afflict 30-60% of patients, presenting even years after diagnosis. The regions mainly involved in metastasis are the bones, lymph nodes, lungs, and abdomen . Treatment of ML consists of a combination of surgery and radiotherapy (RT) associated with chemotherapy (ChT), according to clinical presentation. ML is highly sensitive to RT and partly sensitive to ChT . Histologically, ML is characterized by uniform, oval to round cells, with a variable number of small lipoblasts, set in a myxoid stroma, with a capillary-sized vascular network, organized in a distinctive plexiform pattern previously called chicken wire or crow's feet. The presence of FUS-DDIT3, or less commonly, EWSR1-DDIT3, is pathognomonic to this entity . Different threshold values of hypercellular areas (also known as round cell differentiation when neoplastic cells assume round cell morphological features) ranging from 5-25% have been set by different studies . The WHO's classification of soft tissue and bone tumors recommends that any amount of hypercellularity should be reported in the pathological report, and if it exceeds 5%, the tumor should be considered high-grade . The presence of >5% hypercellularity is associated with a significantly poorer prognosis, identifying patients at high risk, which is a determining factor in treatment planning . Pathological evaluation with molecular confirmation of specific fusion transcripts is necessary to define the histologic subtype and grade; it should be obtained before definitive treatment in the case of a suspected ML. The routine procedure for pathological diagnosis involves multiple preoperative core needle biopsies, generally using 14-16 gauge needles. . Tru-Cut(r) biopsy (TCB) is one of the most commonly used core needle procedures. It is a minimally invasive technique that can be easily performed under ultrasound guidance and with local anesthesia. The advantages of TCB include cost effectiveness, avoidance of diagnostic delays, low complication rates, and minimal invasiveness . Enough viable tissue representative of the lesion and available for histopathological and immunohistochemical evaluation is required. However, a core biopsy may not always accurately reflect the histological features of the tumor. It provides a relatively small sample of tumor tissue, and, taking into account intrinsic tumor heterogeneity, obtaining a representative sample can be difficult . This could result in a diagnostic inaccuracy in the form of underestimating the grade in patients with ML (downgrading error) and could result in a different treatment decision-making process, including the surgical approach and neoadjuvant ChT/RT . To our knowledge, no studies have reported the correlation between the discordance associated with tumor grade in ML and the impact on treatment planning. Therefore, this study aims to report our institutional experience and investigate the diagnostic accuracy, sensitivity, and specificity of the histologic TCB procedure and the potential impact of misdiagnosis on patient survival. 2. Methods 2.1. Patients and Methods The study was approved by the Institutional Review Board of the Rizzoli Orthopaedic Institute of Bologna (protocol code: CE AVEC 58/2022/Oss/IOR; date of approval: 14 February 2022). We retrospectively reviewed the histological and clinical records of 150 patients affected by myxoid liposarcoma treated between 2007 and 2021 and selected 144 cases of primary, localized MLs of the extremities with molecular confirmation of the diagnosis. Exclusion criteria included patients with metastasis at the onset or with a non-histologically proven diagnosis. Hypercellularity of greater than 5% was used as the threshold for labeling the ML high grade . The results of the grade determined in the core biopsy were compared to the complete resection final pathology reports. Follow-up data, including the status of patients at the last visit, were updated. Clinical data included patient demographics (age, gender), tumor properties (site, size, depth, and histology), diagnostic and therapeutic regimens (type of surgery, surgical margins, neoadjuvant and adjuvant therapy), and clinical outcome (status, local recurrence, and distant metastasis after treatment. Tumor size was assessed with a preoperative MRI. The histological diagnosis was confirmed by TCB. Outcome variables of determining malignancy, determining exact diagnosis, and treatment for core biopsy were measured against the final clinical diagnosis performed by analysis of the completely resected specimen in combination with the final clinical impression. 2.2. Biopsy Procedure Biopsy procedures were performed after a careful evaluation of MRI studies by orthopedic surgeons and radiologists with the aim of choosing the best approach. According to a standardized protocol, a percutaneous core needle biopsy was performed on the tumor by the orthopedic and radiologist oncology team. The core biopsies were performed using a 14-gauge Tru-Cut(r) soft tissue biopsy needle (Cardinal Health, Dublin, OH, USA) through the insertion site under ultrasound guidance, taking multiple samples (3-5 passes) throughout the tumor circumferentially, being careful to obtain adequate tissue for evaluation but not breach the far wall of the tumor. Power/color Doppler evaluation was performed during pre- and intra-procedural ultrasounds to avoid necrotic areas within the biopsy sample. 2.3. Therapeutic Procedures Patients were treated using a multimodality approach, including surgery, RT, and ChT. The choice of every surgical procedure was performed in an effort to obtain the broadest oncological margins. Surgical margins were defined according to the Musculoskeletal Tumor Society, based on Ennekin et al. classification : negative margins (microscopically negative) are indicated as R0-wide/radical; in case of the involvement of margins, a distinction is drawn between complete macroscopic resection with microscopic involvement (R1-marginal) and incomplete macroscopic resection (R2-intralesional). RT was administered according to the most appropriate technique available: in the preoperative setting, with a total dose of 50 Gy in 1.8-2 Gy fractions, and in the postoperative setting, with doses up to 66 Gy, based on the presentation, age, and resection margins. ChT treatment consisted of the combination of doxorubicin and ifosfamide or epirubicin and ifosfamide or trabectedin in the neoadjuvant and adjuvant setting. The surgery took place 4 weeks after the termination of the last cycle of ChT or the last fraction of RT . Follow-up procedures consisted of clinical examination and MRI with contrast enhancement of the primary tumor site and a chest CT (every 3 months for the first 2 years, every 4 months during the third year, every 6 months for the fourth and fifth years, and annually from years 6 through 10). Abdomen CT scan with contrast enhancement was performed every 6 months for the first 2 years, every 8 months during the third year, and annually during the rest of the follow-up until the tenth year. 2.4. Statistical Analysis The description of quantitative variables was performed using median and range. The qualitative variables were presented by means of the description of proportions. A comparison between the presurgical biopsy and postoperative histological analysis was performed. The correlation between groups was calculated using Cohen's Kappa coefficient. Complete agreement is considered as a Kappa score of 1. Kappa values close to or less than 0 show poor correlation. Sensitivity, specificity, positive predictive value, negative predictive value, and the overall accuracy of the clinical tests were calculated with the two-by-two table method. The final histology of the resected (surgical) specimen was used as the reference standard, and two-by-two contingency tables were reconstructed for TCB for the final histological diagnosis. A forward stepwise logistic regression analysis was used to determine the risk of error in predicting the various grades of ML. The concordance with the final diagnosis, as confirmed by postsurgical biopsy, was used as the dependent variable. The level of significance for each clinical test was set at 0.05. Statistical analysis was performed using STATA Software (version 17). 3. Results 3.1. Clinicopathological Features Our data included 144 localized myxoid liposarcomas (ML). Table 1 summarizes the main clinicopathological features of the patients and of the tumors analyzed. In our study, we evaluated 91 men and 53 women. The mean age at diagnosis was 48 years (16-78). Regarding tumor localization, 100 (69%) ML were found in the thigh, 33 (21%) in the lower limb, 10 (7%) in the buttock, and 1 (1%) in the arm. Most of the tumors (136 or 95%) were deep-sited. The size, determined by preoperative MRI, was larger than 10 cm in 83 (58%) patients, between 5 and 10 cm in 55 (38%) patients, and smaller than 5 cm in 6 (4%) patients. At the preoperative biopsy, 87 (60%) tumors were classified as high grade (>5% round cells), while 57 (40%) were low grade. Most patients were treated with surgical excision (99% of cases), while only two patients were treated with amputation due to the size and location (lower limb and thigh) of the tumor. A final histopathological diagnosis on the resected tissue specimens confirmed that 59 tumors (41%) were high grade and 85 (59%) were low grade. In overall cases, 104 (72%) resection margins were R0, 35 (25%) were R1, and 5 (3%) were R2. In cases of amputation (2/144; 2%), surgical margins were radical. Neoadjuvant RT was administered in 92 ML--59 high-grade and 33 low-grade (59 cases > 10 cm, 29 cases 5-10 cm, and 4 cases < 5 cm)--while postoperative RT was performed in 31 patients--20 high-grade and 11 low grade (18 cases > 10 cm, 13 cases 5-10 cm, and none < 5 cm). Forty-six (32%) ML patients were treated with neoadjuvant ChT, while 20 (14%) patients had postoperative ChT, and 2 (1%) patients had both pre- and postoperative treatment. Thirty-six and twelve patients were treated with the combination of ChT and RT in a neoadjuvant or adjuvant setting, respectively. Follow-up data were available for all patients. The median follow-up was 69 months (range 2-158 months). At the end of the study, 4% had died of cancer (6/144), 2% had died of unknown causes (3/144), 85% were alive without disease (122/144), and 9% were alive with disease (13/144). 3.2. Determination of Histologic Accuracy: Cytohistologic Correlation of Grade Diagnosis Following TCB, the 144 cases were grouped into two categories: Group A was high grade (n = 87), and Group B was low grade (n = 57). The histologic post-resection diagnosis of these cases was 59 high grades and 85 low grades. Specifically, there was a downgrading in 40 cases of Group A and an upgrading in 12 cases belonging to Group B. Details are reported in Table 2. Overall, the concordance rate between the two biopsies was 64% (Kappa 0.30). 3.3. Risk Factors Associated with Downgrading Logistical data analysis was performed to examine potential factors contributing to downgrading in Group A (Table 3). Neoadjuvant therapy was associated with pathological downgrading. Specifically, patients treated with neoadjuvant RT had a higher probability of downgrading compared to patients treated with neoadjuvant ChT (OR 4.66; 95% CI 0.09-0.21 p < 0.052). The trend was confirmed for patients treated with a combination of chemotherapy and RT (OR 5.30 p = 0.026) in comparison to only ChT. No confounding effect associated with measuring association was found after adjusting for the other variables in multiple regression analysis. 3.4. Impact of Misdiagnosis on Prognosis Of the 144 cases analyzed, the overall survival curves and estimation are reported in Figure 1 and Table 4. Considering 87 patients resulted in a high-grade at-core needle biopsy (Group 1), 45 "real" low-grade tumors (Group 2), and 12 undergoing "upgrade" (Group 3), misdiagnosis had no significant impact on prognosis . 3.5. Concordance Rate in the Group of Patients Not Treated We focused our study on the sub-cohort of patients treated without neoadjuvant therapies to explore grade rate concordance between the two biopsies without any confounding effect. A total of 40 ML patients were analyzed. Following TCB, upon histologic examination, the 40 cases were grouped into high-grade (n = 16) and low-grade (n = 24) groups. The histologic post-resection diagnoses of these cases confirmed 28 high grades and 12 low grades. Twelve cases did not show concordance with the final excisional biopsy and were, therefore, underestimated in the previous analysis (Table 6). The overall sensitivity of TCB was 57%. The overall specificity was 100%. The overall predictive value of a positive TCB was 100%, and the overall predictive value of a negative TCB was 50% (Table 6). No potential factors for grading errors were found. 4. Discussion Tumor grading assessment is critical in defining the best therapeutic approach in soft tissue sarcomas. The most common grading system, the FNCLCC (Federation Nationale des Centres de Lutte Contre le Cancer) classification, defined by a combination of tumor differentiation, mitotic count, and necrosis, remains controversial in grading myxoid liposarcomas and other specific histology. The presence of hypercellularity or round cell differentiation is linked to a worse prognosis in myxoid liposarcomas. However, different threshold values, ranging between 5% and 25%, have been set by independent studies . According to the WHO's classification of soft tissue and bone tumors, in the pathological report, any amount of hypercellularity should be reported; if it exceeds 5%, the tumor should be considered high-grade . TCB under ultrasound guidance with multiple tissue samples (14-16 gauge needles) is widely accepted as the gold standard for tumor sampling and diagnosis ; however, biopsy specimens do not always represent the entire tumor heterogeneity . Especially in myxoid liposarcomas, the presence of transitional areas between typical low-grade histology with modestly increased cellularity and high-grade round cell morphology showing cellular overlap, elevated nuclear grade, mitotic activity, and obscuring of the underlying vascular pattern can be confusing in evaluating the percentage of hypercellularity . Core needle biopsy accuracy, in combination with ultrasound guidance, could be useful to obtain the most representative samples of pathological tissue avoiding "blind" sampling techniques or incisional biopsy . The combination of these factors entails potential misdiagnosis of myxoid liposarcomas with a hypothetical impact on the patient's clinical history. In this study, we assessed a concordance rate of 64% between the biopsy and the final pathological report. In the low-grade TCB group, the discrepancy was 47%, with a substantial risk of underestimating the malignant potential of the tumor. Hoeber reported superior accuracy in biopsying soft tissue sarcomas (69-99%); however, in his paper, there is no mention of different results by histology subset. We did not find a correlation between ML misgrading and main clinicopathological characteristics (size, location, depth). Therefore, diagnostic inconsistencies could be related to tumor heterogeneity and/or the presence of neoadjuvant treatments. We determined that neoadjuvant therapy is associated with downgrading in the high-grade group of patients. ML is highly sensitive to RT treatment . In fact, we demonstrated that patients treated with neoadjuvant RT had a higher probability of downgrading compared to patients treated with neoadjuvant ChT. Previous studies have looked into the accuracy of biopsy techniques in terms of determining malignancy, grade, and subtype , but none have looked at the effect of neoadjuvant treatment. A reliable assessment of the percentage of hypercellularity would likely require adequate and extended sampling of the tumor, as performed in an open biopsy , or the definition of new morphologic criteria related to the grade of malignancy. The reported sensitivity for TCB compared to the final specimen biopsies is in the range of 82% and 92%, with a negative predictive value between 76% and 91% . In the present study, the specificity of TCBs was 100%, a result consistent with those previously reported . The sensitivity was 57%, indicating that there are as many true positives as there are false negatives (a = c) and that the test is not useful in detecting disease. The diagnostic accuracy of MLS biopsy is multifactorial, and the sensitivity of TCB in detecting high-grade lesions may depend either on technical or clinical features. Technical sensitivity correlates to the experience and ability of clinicians and to the tools available during the biopsy. The radiologist and the surgeon must be experts in sarcoma diagnosis to rigorously determine which portion of a tumor could better resemble its histology. Possibly, soft tissue tumors are an even easier sample to collect than other sarcomas, and TCB allows for collecting deeper parts of the mass in comparison to other techniques. We thus believe that the competence of the medical staff is determinant in increasing the sensitivity of high-grade lesion analysis. Since this study was performed in a highly specialized center for bone and soft tissue pathologies and considering that the medical staff was an expert in this field, we do not think that technical skills impacted the poor sensibility of the test. Clinical sensitivity, instead, is related to the material examined and to its quality. Underestimation of grade on TCB could be due to the lower quality of tissue sampled by TCB. The tissue obtained may not include the tumor's growing edge, or there may be insufficient tumor present to complete a formal count. Myxoid histology is associated with decreased accuracy because of the presence of transitional areas between low- and high-grade histology. High-grade subtypes are even harder to diagnose since the use of different threshold values corresponding to hypercellular areas could influence the sensitivity of the test. Some limitations must be acknowledged, including the study's retrospective design; therefore, some data could be fragmentary and difficult to trace. Moreover, the small number of patients participating in the study and the large time of follow-up considered, which could be linked to the development of different therapeutic approaches, are other limitations. Despite the above-mentioned limitations, based on our results and on previous reports, we believe that accurate diagnosis with TCB is not as simple as it may seem in this subset of malignancies. Yet, future application of more advanced tools, such as the combination of imaging analysis (radiomics) and pathological (pathomics) features, the use of new clinicopathological scoring, or revision of the histology grading system will be decisive in improving prognosis in myxoid liposarcoma . 5. Conclusions Regardless of the clinicopathological features, in a clinical setting with a multimodal approach, discrepancies in liposarcoma grading using TCB may occur in up to 36% of the cases with a determinant downgrading effect of preoperative chemotherapy and/or RT. Nevertheless, in cases of misdiagnosis of high-grade and low-grade tumors, overall survival is not affected because systemic treatment decision-making also includes other variables. In the absence of preoperative treatment, the sensitivity of TCB was 57%, and specificity was 100%. Acknowledgments We thank our patients and their families. Author Contributions Conceptualization, G.B.; methodology, R.L.; validation, G.B. and D.M.D.; formal analysis, R.L.; statistical analysis: R.L.; investigation, G.B., E.B., R.L., F.O., P.S. and A.R.; resources, A.R., P.S. and E.B.; data curation, R.L.; visualization, F.O. and G.B.; writing--original draft preparation, R.L., E.B. and G.B.; writing--review and editing, R.L., F.O., G.B., A.R. and P.S.; supervision, G.B. and D.M.D. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of Rizzoli Orthopedic Institute of Bologna (protocol code: CE AVEC 58/2022/Oss/IOR; date of approval: 14 February 2022). Informed Consent Statement Due to the retrospective design of the study, informed consent was not requested. Data Availability Statement The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Kaplan-Meier Overall Survival Curve. Figure 2 Kaplan-Meier curve stratified by histologic grade group. (A) Group 1 vs. Group 3 vs. Group 3, Log-rank test Pr > chi2 = 0.6061. (B) Group 1 + Group 2 vs. Group 3, Log-rank test pr = 0.3237. Group 1 = High-grade group (high-grade TCB); Group 2 = Low-grade group (low-grade TCB and final biopsy); Group 3 = Upgrade group (low-grade TCB and high-grade final biopsy). Log-rank test Pr > chi2 = 0.6061. cancers-15-01391-t001_Table 1 Table 1 Main Clinicopathological Features and Therapeutic Approaches. Factor Number of Patients % Patients 144 100 Gender Male 91 63 Female 53 37 Location Thigh 100 69 Lower limb 33 21 Buttock 10 7 Arm 1 1 Depth Deep 136 95 Superficial 8 5 Tumor Size >10 cm 83 58 5-10 cm 55 38 <5 cm 6 4 Preoperative Grade High 87 60 Low 57 40 Postoperative Grade High 59 41 Low 85 59 Surgery Excision 142 99 Amputation 2 1 Margin Wide/Radical (R0) 104 72 Marginal (R1) 35 25 Intralesional (R2) 5 3 Radiotherapy Preoperative 92 64 Postoperative 31 21 None 21 15 Chemotherapy Preoperative 46 32 Postoperative 20 14 Pre- and postop. 2 1 None 76 53 cancers-15-01391-t002_Table 2 Table 2 Grade Diagnosis of The Core Needle Biopsy Specimen and Surgical Specimen. Surgical Specimen Core Needle Biopsy Tot High grade Low grade Group A (High Grade) 87 47 (54%) 40 (46%) Group B (Low Grade) 57 12 (21%) 45 (79%) cancers-15-01391-t003_Table 3 Table 3 Factors Correlated to Histological Downgrading Limited to Group A. Univariate Analysis Factor N Discordant (N) Discordant (%) OR p-Value 95% CI Age <50 47 23 57 0.84 0.687 0.35-1.9 >50 40 17 43 Location Thigh (ref) 55 24 60 Lower limb 25 13 33 1.39 0.488 0.54-3.61 Buttock Arm 6 1 2 1 5 2 0.64 0.630 0.10-3.8 Size >10 cm (ref) 51 26 65 5 < to < 10 cm 31 11 28 0.53 0.174 0.21-1.32 <5 cm 5 3 7 1.44 0.701 0.22-9.37 Depth Deep 82 39 98 3.6 0.25 0.38-33.8 Superficial 5 1 2 Neoadjuvant therapy ChT (ref) 12 3 7.5 RT 23 14 35 4.66 0.052 0.9-21 ChT + RT 36 23 57.5 5.30 0.026 1.3-24 None 16 0 Reference (ref); 95% confidence interval (95% CI); radiotherapy (RT); chemotherapy (ChT). cancers-15-01391-t004_Table 4 Table 4 Overall Survival: Kaplan-Meier Survival Estimation. Survival Function 95% Confidence Interval 2 years 99 94-99 5 years 96 90-98 10 years 93 81-97 cancers-15-01391-t005_Table 5 Table 5 OS Rates Stratified by Histologic Grade Group. Factor (N) 5-Years OS (%) 95% CI 10-Years OS (%) 95% CI Grade group 1 (87) 96 87-98 96 87-98 2 (45) 97 78-99 90 62-97 3 (12) 90 47-98 90 47-98 Grade group 1 + 2 (132) 96 89-98 93 80-97 3 (12) 90 47-99 90 47-99 Group 1 = High-grade group (high-grade TCB); Group 2 = Low-grade group (low-grade TCB and final biopsy); Group 3 = Upgrade group (low-grade TCB and high-grade final biopsy). OS: overall survival; 95% Interval of confidence (95% CI). cancers-15-01391-t006_Table 6 Table 6 Accuracy of Tru-Cut(r) Biopsy Techniques Determining Malignancy When Compared to The Final Diagnosis. Analysis of 40 MLS Not Treated with Neoadjuvant Therapy. True Diagnosis (Surgical Specimen Histology) High-Grade Low-Grade Tot TCB High-grade 16 (TP) 0 (FP) 16 Low-grade 12 (FN) 12 (TN) 24 Tot 28 12 40 Tru-Cut(r) biopsy (TCB), TP: true positive; FN: false negative; TN: true negative. Sensitivity: 57%, 95% CI (37-76). Specificity: 100%, 95% CI (74-100). Predictive values of positive results: 100%, 95% CI (79-100). Predictive values of negative results: 50%, 95% CI (29-71). Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000106

Idealism and relativism are components of ethical ideologies which have been explored in relation to animal welfare and attitudes, and potential cultural differences. The present study investigated how ethical ideologies influenced attitude toward animals among undergraduate students. With the help of stratified random sampling, 450 participants were selected from both private and public sector universities in Pakistan. Research instruments consisted of a demographic sheet, the Ethics Position Questionnaire (EPQ), the Animal Attitude Scale--10-Item Version (AAS-10), and Animal Issue Scale (AIS). The study hypotheses were explored by employing various statistical analyses like Pearson Product Moment Correlation, independent sample t-test, ANOVA, and linear regression. Results revealed that there was a significant positive relationship between ethical ideologies (idealism and relativism) and attitude toward animals in students. Results further showed that students who consumed meat less frequently scored higher on relativism as compared to those who consumed meat more frequently (however, the effect size was small). It was also found that senior students held more idealistic ideologies as compared to freshman students. Finally, idealism positively predicted concern for animal welfare among students. The current study shed light on how ethical ideologies can shape and influence animal welfare. It further highlighted the potential cultural differences for the study variables by allowing for comparison with other published studies. By understanding these dynamics better, researchers will be better equipped to help students become informed citizens that may also influence future decision-making processes. ethical ideologies attitude toward animals animal welfare cultural differences undergraduate students There are no funding sources to disclose. pmc1. Introduction The nature of human-animal interactions is diverse. Animals have been an essential part of human lives for centuries, from being hunting partners to companion animals at home and livestock raised for human consumption at farms. Given this bond and inter-dependence, significance of attitude toward animals and other related influential factors is increasingly being recognized within the field of human-animal relationships as well as animal welfare . More recently, researchers have increasingly highlighted people's positive attitude toward animals due to their numerous physical and psychological benefits for individuals as well as society. These benefits included enhanced physical health, increased happiness, reduced loneliness and anxiety, and enhanced social interactions . Similarly, animals are an integral part of people's lives in Pakistan. They serve various roles as pets, livestock, transportation and food animals, even being part of religious practices. However, despite playing such crucial roles in society, very little is understood about how people view and treat animals in general and how these views affect animal welfare. One variable that is pertinent to understanding attitude toward animals is understanding the effect of ethical ideologies. Forsyth (1980) highlights two aspects of ethical ideologies to explain differences in moral reasoning within his Ethics Position Questionnaire: idealism and relativism . Individuals who hold idealistic ideologies firmly believe that positive outcomes always follow behaviors based on principles and ethics . On the contrary, individuals with relativistic ideologies believe that decisions of moral nature should be taken while keeping in view certain rules and values prevalent in their respective society . People holding idealistic view believe that their decisions always lead to positive outcomes and those who hold a relativistic approach believe that moral decisions are situational and based on local principles . Individuals are further distinguished into four groups based on their level of idealistic and relativistic ideologies: (1) Individuals that hold high idealistic and relativistic ideologies are called situationists; (2) individuals with higher idealistic ideologies but lower relativistic ideologies are referred to as absolutists; (3) subjectivists possess lower idealistic and higher relativistic tendencies; (4) lastly, individuals who have both low idealistic and relativistic ideologies are known as exceptionists . Individuals who were situationists believe that it is acceptable to not follow moral rules if it leads to better outcomes. Absolutists believe that rules of morality should be followed even if there are rewards which can be attained by not following them. Subjectivists are of the view that there will be unpreventable negative consequences for ethical decisions given that every person follows their own different moral principles. For exceptionists, whether an action is morally accurate or not depends on the outcomes it produces . Individuals' attitude towards animals is affected by the type of group they identify with. For example, absolutists view experiments involving animals as highly immoral, as compared to individuals belonging to other ethical ideology groups . This idealistic or relativistic view toward ethical actions affects people's views and attitude toward animals and animal welfare . One study showed that individuals higher on idealism usually expressed more concern for animal use than those higher on relativism . Given the strong moral principles reported by people holding idealistic tendencies, it will have a significant impact on welfare concerns for others as well as animals . Further, it was found that younger people had more awareness regarding welfare of animals as compared to older people . Considering this, analyzing and understanding students' perception and ethical tendencies will have similar implications. Moreover, it is important for students to understand ethical issues and to implement a change that supports animal welfare. Young adults are at a crucial stage of transitioning into adulthood and have a vested interest in the future of the society. They are further exposed to a multitude of information through different channels that lead to formation of views and opinions about critical topics. To bring about such a change requires considering students' existing understanding, including their views on moral reasoning and ethical concerns. This understanding, and consequently implementation for change, is crucial for promoting educational standards, including 'decision-making competence' in the future . Hence, the present study aimed at understanding the ethical ideologies of students while raising awareness amongst said students about animal welfare concerns, which may consequently influence future decision making when it comes to ethical issues. These moralistic tendencies were further influenced by other factors such as age, gender, religion, pet ownership, and geographic location , all of which were included and investigated in the present study. Women were found to be more concerned regarding animal welfare as compared to men . Moreover, young adults exhibited more positive attitude toward animals as compared to middle-aged and older adults . Another important consideration for comparison was the culture and society. Within developed countries, individuals were highly aware and concerned about the wellbeing of animals, and they formed their views regarding animals based on this awareness instead of analyzing animals in term of their respective advantages and disadvantages . Studies in developed countries such as USA and the Netherlands showed significant impact of idealism, and not relativism, on attitude toward animals indicating that relativism was not a critical factor in this respect. However, a study conducted in China showed how relativism had a negative relationship with individuals' attitude toward animals . As China is economically prospering so their focus of attention was on attaining the latest technological advancements due to which their awareness regarding animal welfare was highly limited. People in such countries formed their views regarding animals based on the advantages they can derive from them instead of considering their wellbeing. Given that Pakistan is a developing country with significant cultural differences from developed countries, the way human demographics interacted with ethical ideologies in forming attitudes towards animals will also differ. The current study aimed at highlighting any cultural influence on ethical ideologies by comparing the outcomes with other published studies. 1.1. Objectives To investigate the relationship between ethical ideologies and attitude toward animals. To investigate the effect of interaction of demographic variables (such as frequency of meat consumption and semester) in relation to ethical ideologies and attitude toward animals. To investigate and highlight any cultural differences among the study variables. 1.2. Hypotheses There will be a positive relationship among idealism, relativism, and attitude toward animals in students. Students who score higher on idealism will report positive attitude toward animals. Students who consume less meat will score higher on relativism. Senior students will score higher on idealism as compared to freshman students. Idealism will positively predict attitude toward animals in students. 2. Methodology 2.1. Research Design The present study implemented a cross-sectional correlational research design. The study was cross-sectional in nature because the data were collected from research participants at one point in time in which the relationship between study variables was explored without any manipulation . The study gained approval from the National Bioethics Committee (Ministry of National Health Services, Regulations and Coordination Islamabad, Government of Pakistan). 2.2. Instruments The instruments used in the present study include a demographic sheet, Ethics Position Questionnaire (EPQ), the Animal Attitude Scale--10-Item Version (AAS-10), and Animal Issue Scale (AIS). 2.2.1. Demographic Sheet The demographics included were age, educational institution, degree program, semester, place of residence, religion/source of inspiration, degree of religiousness, pet ownership, type of pet, level of attachment with pet and frequency of meat consumption. Degree of religiousness was measured on a Likert-type question, ranging from "Not at all Religious" to "Highly Religious". The level of attachment was measured using a 10-point rating scale, ranging from 0 (Not at all Attached) to 10 (Highly Attached). Level of comfort was measured similarly, ranging from 0 (Low Comfort Level) to 10 (High Comfort Level). Finally, frequency of meat consumption was measured on a 5-point Likert scale, from "Never" to "Every Day". 2.2.2. Ethics Position Questionnaire (EPQ) This instrument was used to determine the ethical ideology position held by individuals . It is a 20 item self-report questionnaire on which respondents were asked to rate items on a 5-point Likert scale, ranging from 1 (completely disagree) to 5 (completely agree). It has two subscales: idealism and relativism. The idealism dimension asked respondents to give their responses on item such as "Risks to another should never be tolerated, irrespective of how small the risks might be" and "One should not perform an action which might in any way threaten the dignity and welfare of another individual". The relativism dimension required respondents to express whether they agreed or disagreed with statements like "Moral standards should be seen as individualistic; what one person considers to be moral may be judged to be immoral by another person" and "What is ethical varies from one situation and society to another". In the current study, Cronbach's alpha of the scale is 0.90; for idealism subscale it is 0.88 and for relativism subscale it is 0.87. 2.2.3. Animal Attitude Scale--10-Item Version (AAS-10) This questionnaire was used for measuring attitude toward animals. It is a self-report 20 item, 5-point Likert scale, ranging from 1 (strongly disagree) to 5 (strongly agree). Respondents were required to express their degree of agreement on items like "It is morally wrong to hunt an animal just for sport" and "It is unethical to breed purebred dogs for pets when millions of dogs are killed in animal shelter each year". Items 2, 3, 4, 7 and 8 were reverse scored. The higher the score on the scale the greater the concern for animal welfare. In the current study, the Cronbach's alpha was 0.86. 2.2.4. Animal Issue Scale (AIS) This questionnaire was used for measuring attitude toward animals in respondents. It had a total of 43 items and respondents rated items on a 5-point Likert scale ranging from 1 (extremely acceptable) to 5 (extremely unacceptable). It further had eight sections which included use of animals, disrupting animal integrity, killing animals, compromising animal welfare, experimenting on animals, changing animals' genotypes, animals and the environment (harming animals to protect the environment), and societal attitudes toward animals (harming animals for social purposes). Greater score on the scale indicated a higher concern for wellbeing of animals . Respondents rated items such as "Inflicting pain, injury or disease on animals", "Killing animals because they are not native to the area where they live" and "Destroying the habitat of endangered animal species". A reliability of 0.94 was reported in the current study for this questionnaire. 2.3. Sample and Demographic Characteristics With the help of random sampling, a total sample of 450 participants (men = 76, women = 374) within an age range of 18-36 years was collected from both public and private universities of Pakistan. The inclusion criteria for the present study pertained to any individual who was enrolled in an undergraduate program in a university and who was at least 18 years of age. For distribution of sample along demographic variables, see Table 1. Table 1 illustrates the distribution of sample (N = 450) along various demographics. A majority of the participants were women (83%). The majority of students were doing their Bachelor of Science in psychology while the least number of students were enrolled in a Bachelor of Arts in psychology degree and Bachelor of Science in journalism degree. The highest frequency of student belonged to the 1st to 3rd semester. Most of the students resided in an urban section. The greatest number of students were moderately religious and did not own a pet. Finally, majority of the students consumed meat two to three times a week. 2.4. Procedure For university sample selection, a list of accredited public and private universities in Pakistan for each province was obtained through the Higher Education Commission (HEC) website, and with the help of stratified random selection, three universities were selected from each province to be included in the sample. The purpose for this selection was to increase the representativeness of the sample. Afterwards, the responsible authorities were contacted for permission and their assistance requested for conducting the research while providing all the necessary information through the proper channels/protocols. In cases where institutions declined to participate in the research or there was a lack of response from a particular university within the specified amount of time, another university was selected from the remaining list with the help of stratified random selection until there was a balanced sample from each province. Institutes assenting to participate in the study were requested to provide a list of their active departmental classes across all semesters and using a stratified random sampling technique, classes were selected for inclusion in the study. Based on the institutional protocol, we requested either the focal person or the instructor to distribute the survey questionnaires either in printed form or through an online link generated through Qualtrics software among participants. Any queries on the part of the focal person/instructor were cleared beforehand to optimize clear communication with the participants. The survey consisted of the demographic sheet, the Ethics Position Questionnaire (EPQ), the Animal Attitude Scale--10-Item Version (AAS-10), and Animal Issue Scale (AIS). The average time required for filling the survey was 15-20 min. Data were analyzed using appropriate statistical methods. With the help of latest version of SPSS Statistical software, the collected sample data were tested for normal distribution and where required, were translated into normal distribution. Levene test was used to determine homogeneity of the variances. Pearson Product Moment Correlation was computed for investigating the relationship among research study variables. Independent sample t-test and Analysis of variance (ANOVA) were performed to determine respondents' ethical ideologies and demographics that may affect their attitudes toward animals. Simple and multiple linear regression were employed for relating participants' responses in EPQ to their responses on AIS and AAS-10 to identify which variables determined attitude toward animals by employing the above-described model and utilizing an alpha value of 0.05 for variables to enter the model. The finding of the research study was shared with participants through their respective email address. 3. Results The present study aimed at exploring attitude toward animals among university students and the possible influence of ethical ideologies on such attitudes. Cronbach alpha reliabilities for research instruments were computed (see Table 2). Table 2 shows psychometric properties for the scales used in the study. All the scales and their respective subscales had satisfactory reliability ranging from 0.94 to 0.86. 3.1. Correlation among Ethical Ideologies (Idealism and Relativism), Attitude toward Animals, and Concern for Animal Welfare For investigating relationship among research variables Pearson Product Moment Correlation was computed (see Table 3). Table 3 shows a significantly stronger positive correlation between ethical position scale and the subscale idealism (r (448) = 0.85, p = 0.00). A significant positive correlation between ethical position scale and its respective subscale relativism (r (448) = 0.86, p = 0.00) was also found. Furthermore, results showed a significantly moderate positive relationship between ethical position (ethical ideologies) and attitude toward animals (r (448) = 0.38, p = 0.00). A significantly moderate positive relationship also existed for ethical position (ethical ideology) and concern for animal welfare (r (448) = 0.35, p = 0.00). Furthermore, idealism had a significantly moderate positive relationship with attitude toward animals (r (448) = 0.39, p = 0.00). A small significantly positive relationship was evident between relativism and attitude toward animals (r (448) = 0.25, p = 0.00). Moreover, a moderate significantly positive relationship was found between idealism and concern for animal welfare (r (448) = 0.43, p = 0.00). A small significantly positive relationship was found between relativism and concern for animal welfare (r (448) = 0.18, p = 0.00). Lastly, a stronger significantly positive relationship was found between attitude toward animals and concern for animal welfare (r (448) = 0.74, p = 0.00). 3.2. Difference among Ethical Ideologies, Attitude toward Animals, and Concern for Animal Welfare for Frequency of Meat Consumption in University Students Independent sample t-test was used to study differences for ethical ideologies (idealism and relativism), animal attitude and concern for animal welfare between low level meat consumption and high level meat consumption groups (see Table 4). Table 4 shows significant differences along frequency of meat consumption for ethical position and idealism. Results indicated that students consuming less amount of meat had a greater score on ethical position ideologies as compared to students who consumed more meat. However, the value of Cohen's d was 0.22 (<0.50) which indicated small effect size. Furthermore, findings revealed that students consuming less meat held greater relative ideologies as compared to students who consumed more meat. The value of Cohen's d was 0.24 (<0.50) which indicated small effect size. Nonsignificant findings were found for the remaining variables. 3.3. Difference along Semester/Stage of Program for Idealism in University Students One-way ANOVA and post hoc analysis (Tukey Kramer for significant results only) were carried out to study the role of stage of the program/semester for idealism in university students (See Table 5). Table 5 reveals that senior students held more idealistic ideologies as compared to freshman students. Furthermore, the value of e2 was 0.01 (<0.20) which showed small effect size. Post-hoc comparison indicated significant mean group differences for freshman group with senior group. 3.4. Predictive Role of Ethical Position on Attitude toward Animals Simple linear regression was computed for investigating predictive role of ethical position on attitude toward animals in university students (see Table 6). Table 6 show the impact of ethical position on attitude toward animals in university students. The R2 value of 0.38 revealed that the predictor, ethical position, explained 38% variance in outcome variable which was attitude toward animals with F (1, 448) = 75.3, p > 0.001. Findings indicated that ethical position positively predicted attitude toward animals (b = 0.38, p > 0.001). 3.5. Predictive Role of Ethical Position on Concern for Animal Welfare Simple linear regression was computed for investigating predictive role of ethical position on concern for animal welfare in university students (see Table 7). Table 7 shows impact of ethical position on concern for animal welfare in university students. The R2 value of 0.13 revealed that ethical position as a predictor explained 13% variance in concern for animal welfare which was the outcome variable with F (1, 448) = 64.7, p > 0.001. The results showed that ethical position positively predicted concern for animal welfare (b = 0.35, p > 0.001). 3.6. Predictive Role of Idealism and Relativism on Attitude toward Animals Multiple linear regression was computed for investigating predictive role of idealism and relativism on attitude toward animals in university students (see Table 8). Table 8 shows effect of idealism and relativism on attitude toward animals in university students. The R2 value of 0.16 showed that idealism and relativism explained 16% variance in the outcome variable which was attitude toward animals with F (2, 447) = 43.8, p < 0.001. The findings revealed that idealism positively predicted attitude toward animals (b = 0.36, p < 0.001) while nonsignificant results were found for relativism regarding attitude toward animals (b = 0.09, p > 0.05). 3.7. Predictive Role of Idealism and Relativism on Concern for Animal Welfare Multiple linear regression was computed for investigating predictive role of idealism and relativism on concern for animal welfare in university students (see Table 9). Table 9 shows impact of idealism and relativism on concern for animal welfare in university students. The R2 value of 0.19 showed that idealism and relativism explained 19% variance in the outcome variable that was concern for animal welfare with F (2, 447) = 52.3, p < 0.001. Results revealed that idealism positively predicted concern for animal welfare (b = 0.45, p < 0.001), whereas relativism did not significantly predict concern for animal welfare (b = -0.03, p > 0.05). 4. Discussion The aim of the present study was to investigate how components of ethical ideologies (idealism and relativism) influenced attitude toward animals in the student population. Results supported the first hypothesis of the study that a significant positive relationship existed between idealism and attitude toward animals (see Table 3). This finding was in line with results of previous studies which showed that individuals holding idealistic ethical tendency had positive attitude towards animals, and they were more concerned with animal wellbeing . People having an idealistic ideology were selflessly worried regarding the welfare of others without any cost-benefit analysis , so it was expected that such people showed excessive concern towards the wellbeing of animals as well. In addition, these individuals had a firm belief that harm could always be prevented, hence, they considered animal issues (like killing animals, keeping animals as pets, experimenting on animals, and such) very seriously while being concerned about animal welfare . Hypothesis two of the study predicted there will be a significant positive relationship between relativism and attitude toward animals. The results confirmed this (see Table 3). This result was inconsistent with findings of previous studies highlighting a nonsignificant relationship between the two variables in the US . Furthermore, this result was also inconsistent with findings from a study conducted in China which found a significant negative relationship between relativism and attitude toward animals . This result may be accounted for by cultural differences. As Pakistan is an Islamic state and the religion promotes fair as well as humane treatment of all living things especially animals , people having relativistic ideologies hold a positive attitude towards welfare of animal due to their faith, even if they view animals in relation to the benefits they provide with situational factors in mind for moral decision making. Another explanation could be that Asian countries follow collectivist ideologies and universal principles are not that important for people residing in such countries . Individuals living in Western countries follow individualist ideologies which focus on universal principles when it comes to interpreting various situations . Hence, the relationship between relativism and attitude toward animals in students from Asian and Western countries could possibly differ. The study outcomes further supported Hypothesis 3 which highlighted a significant difference along frequency of meat consumption for relativism among students (see Table 4). However, it should be noted that the effect size was small and as such, should be taken into consideration. One possible explanation is related to the price of meat. Certain meat products were perceived as luxury item in countries such as China and people consumed such items in a reduced amount as they cost more. Pakistan being a developing country contains many people, especially students, belonging to lower-middle class and middle-middle class who cannot afford to eat luxury items on a regular basis . An alternative explanation could be that students high on relativism were vegetarians who consumed no meat because of reasons like health or moral concerns while on the other hand very few students avoided meat because of being concerned with animal welfare . The study results indicated a significant difference along stage of program (semester) for idealism among students (see Table 5). In support of these findings, a previous study indicated that students more progressed in their studies and at a later stage of education showed more concern towards harming animals . This could be because students at more advanced stages of their education had more access to information regarding animal wellbeing as compared to students who were freshly admitted into a university . Therefore, senior students developed greater idealistic tendencies regarding animal welfare as compared to students who were freshman, sophomore, or junior levels due to being more informed. Lastly, idealism positively predicted favorable attitude toward animals in students (see Table 8). This result was consistent with findings of previous studies which showed that idealism had a positive association with attitude toward animals . Individuals who believed that their ethical behaviors, based on universal principles, led to more desirable outcomes held favorable attitude towards animals and showed greater concern for their welfare . 4.1. Limitation and Suggestion For the present study, all information was obtained from a self-reported questionnaire, resulting in the possibility of response bias. Including multiple assessments over certain times and deeper exploration through employing qualitative methods will provide a richer and thorough understanding of ethical ideologies and related factors. Since the survey was administered during the pandemic while universities were still adjusting to the transition, this may have impacted the responses. A consideration of the possible influences of changes within university dynamics due to the pandemic needs to be made. Furthermore, COVID-19 interfered with the data collection process by limiting access to the target sample. Due to this limitation, some of the research data was collected through online questionnaires while the remaining was collected in-person. There might have been an uncertainty in responses obtained from participants based on the mode of completing the research questionnaire. Another limitation of the study was that confounders were not accounted for during the analysis. The study outcomes showed a significant positive relationship of relativism with attitude toward animals which contradicted previous research findings. This indicated that the way relativism interacted with attitude toward animals and concern for animal welfare differed across developing countries, especially Muslim countries, and may require further investigation for cultural and religious differences. In addition, the unequal distribution of participants in demographic groups could have affected the research outcomes. Participants ought to be balanced across various human demographics to acquire a comprehensive understanding regarding the interaction of the study variables. 4.2. Implications The present study highlighted the significance of ethical ideologies for influencing students' attitude toward animals and concern for animal welfare. This is an important finding for future researchers who can further explore role of relativism in forming positive attitude towards animals in collectivist culture. The findings of the current study can be used to spread awareness regarding animal welfare and ways of improving concern among people regarding animal wellbeing through enhancing their ideological beliefs like idealism and relativism. In addition, relativism had a significant positive relationship with the frequency of meat consumption. Beliefs and culture of a country have a great impact on the consumption practices of its citizens so further research could be beneficial for exploring this domain, with findings of the current study being a starting point in this direction. More studies are required for further understanding the relationship between these and other variables within a collectivist, faith-based country. 5. Conclusions To the best knowledge of the authors, the current study is the first one to explore ethical ideologies alongside attitude toward animals among students in Pakistan. The study showed that there was a positive relationship between ethical ideologies (idealism and relativism) and positive attitude toward animals. Individuals who believed that their moral behaviors always led to desirable outcomes as well as held universal moral principle were more concerned about animal welfare and held more positive attitudes towards animals. Further, individuals who believed that moral decision should be based on situational factors were also concerned for animal welfare. It was also evident from findings of study that students in advanced stages of their program held greater idealistic ideologies as compared to students in their initial semesters. Lastly, idealism was found to predict positive attitude and concern for animal wellbeing among students. Author Contributions Conceptualization, A.K. (Aliya Khalid) and P.M.; methodology, A.K. (Asiya Khalid); formal analysis on software, A.K. (Asiya Khalid); writing--original draft, A.K. (Asiya Khalid); validation, A.K. (Aliya Khalid); investigation, A.K. (Aliya Khalid) and A.K. (Asiya Khalid); resources, A.K. (Aliya Khalid) and A.K. (Asiya Khalid); data curation, A.K. (Aliya Khalid) and A.K. (Asiya Khalid); writing--review and editing, A.K. (Aliya Khalid) and P.M.; supervision, P.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study was conducted according to the guidelines of and approved by the National Bioethics Committee (Ministry of National Health Services, Regulations and Coordination Islamabad, Government of Pakistan) under Ref: No.4-87/NBC-527/20/714. Informed Consent Statement Consent was taken from the research participants before conducting the study. Data Availability Statement The data presented in this study are available from the corresponding author upon request. Conflicts of Interest The authors declare no conflict of interest. animals-13-00927-t001_Table 1 Table 1 Frequencies and Percentages along Demographic Variables (N = 450). Demographics f (%) Gender Men 76 (17) Women 374 (83) Degree program Advance diploma in clinical psychology 11 (2) Bachelor of arts (psychology) 1 (0.2) Bachelor of business administration 5 (1) Bachelor of science (psychology) 382 (85) Bachelor of science in computer 4 (1) Bachelor of science (Journalism) 1 (0.2) Master of science (psychology) 46 (10) Semester 1st-3rd 198 (44) 4th-6th 109 (24) 7th-8th 143 (32) Place of residence Urban 374 (83) Rural 76 (17) Degree of religiousness Prefer not to say 46 (10) Not religious at all 4 (0.9) Slightly religious 42 (9) Somewhat religious 39 (9) Moderately religious 258 (57) Highly religious 61 (14) Own pet Yes 125 (28) No 325 (72) Amount of consumption of meat Never 14 (3) Once a week or less 185 (41) 2-3 days a week 195 (43) 4-6 days a week 36 (8) Everyday 20 (4) animals-13-00927-t002_Table 2 Table 2 Psychometric properties for scales and their subscales (N = 450). Scale M SD Range Cronbach's a Ethical Position Scale 80.5 10.8 30-100 0.90 Idealism Subscale 43.0 6.23 11-50 0.88 Relativism Subscale 37.5 6.35 12-50 0.87 Animal Attitude Scale 35.8 5.82 16-50 0.86 Animal Issue Scale 156.5 23.0 72-211 0.94 animals-13-00927-t003_Table 3 Table 3 Correlation among ethical position, idealism, relativism, attitude toward animals, and concern for animal welfare (N = 450). Variables 1 2 3 4 5 1 Ethical Position - 2 Idealism 0.85 ** - 3 Relativism 0.86 ** 0.46 ** - 4 Attitude toward Animals 0.38 ** 0.39 ** 0.25 ** - 5 Concern for Animal Welfare 0.35 ** 0.43 ** 0.18 ** 0.74 ** - ** p < -0.01. animals-13-00927-t004_Table 4 Table 4 Level of meat consumption along ethical position, relativism, idealism, attitude toward animals, and concern for animal welfare (N = 450). Low Level Meat Consumption High Level Meat Consumption Variables M SD M SD t (448) p Cohen's d Ethical Position 81.8 10.2 79.4 11.2 2.28 0.02 0.22 Idealism 43.4 5.67 42.7 6.64 1.33 0.19 Relativism 38.3 6.14 36.8 6.45 2.57 0.01 0.24 Attitude toward Animals 36.2 6.96 35.6 5.71 1.11 0.27 Concern for Animal Welfare 155.9 23.1 157.1 22.9 -0.55 0.58 animals-13-00927-t005_Table 5 Table 5 Mean, standard deviation and one-way ANOVA in idealism across semesters (N = 450). Freshman Sophomores and Juniors Seniors Variables M SD M SD M SD F (2, 447) e2 Post-Hoc Idealism 42.3 6.40 42.7 6.55 44.0 5.55 3.11 * 0.01 1 < 3 * p < 0.05. animals-13-00927-t006_Table 6 Table 6 Regression coefficient of ethical position on animal attitude (N = 450). Variable B b SE Constant 19.3 *** 1.92 Ethical position 0.20 *** 0.38 0.02 R2 0.38 *** p < 0.001. animals-13-00927-t007_Table 7 Table 7 Regression coefficient of ethical position on concern for animal welfare (N = 450). Variable B b SE Constant 95.6 *** 7.64 Ethical position 0.76 *** 0.35 0.09 R2 0.13 *** p < 0.001. animals-13-00927-t008_Table 8 Table 8 Regression coefficient of idealism and relativism on attitude toward animals (N = 450). Variables B SE t p 95% CI Constant 18.5 1.91 9.67 0.00 [14.7, 22.3] Idealism 0.33 0.05 7.33 0.00 [0.24, 0.42] Relativism 0.08 0.04 1.77 0.08 [-0.01, 0.17] Note. CI = Confidence Interval. animals-13-00927-t009_Table 9 Table 9 Regression coefficient of idealism and relativism on concern for animal welfare (N = 450). Variables B SE t p 95% CI Constant 89.7 7.44 12.1 0.00 [75.1, 104.3] Idealism 1.66 0.18 9.36 0.00 [1.31, 2.01] Relativism -0.12 0.17 -0.69 0.49 [-0.46, 0.22] Note. CI = Confidence Interval. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000107

The discovery of the Robertsonian translocation (rob) involving cattle chromosomes 1 and 29 and the demonstration of its deleterious effects on fertility focused the interest of many scientific groups on using chromosome banding techniques to reveal chromosome abnormalities and verify their effects on fertility in domestic animals. At the same time, comparative banding studies among various species of domestic or wild animals were found useful for delineating chromosome evolution among species. The advent of molecular cytogenetics, particularly the use of fluorescence in situ hybridization (FISH), has allowed a deeper investigation of the chromosomes of domestic animals through: (a) the physical mapping of specific DNA sequences on chromosome regions; (b) the use of specific chromosome markers for the identification of the chromosomes or chromosome regions involved in chromosome abnormalities, especially when poor banding patterns are produced; (c) better anchoring of radiation hybrid and genetic maps to specific chromosome regions; (d) better comparisons of related and unrelated species by comparative FISH mapping and/or Zoo-FISH techniques; (e) the study of meiotic segregation, especially by sperm-FISH, in some chromosome abnormalities; (f) better demonstration of conserved or lost DNA sequences in chromosome abnormalities; (g) the use of informatic and genomic reconstructions, in addition to CGH arrays, to predict conserved or lost chromosome regions in related species; and (h) the study of some chromosome abnormalities and genomic stability using PCR applications. This review summarizes the most important applications of molecular cytogenetics in domestic bovids, with an emphasis on FISH mapping applications. animal cytogenetics cattle river buffalo sheep goat FISH mapping PCR PON1_486 GENOBUThe study was supported by the project PON1_486 GENOBU. pmc1. Introduction The application of cytogenetics to domestic animals emerged about 60 years ago with the study of normal stained chromosome preparations from some cases of domestic animals with reproductive defects . However, the discovery of the Robertsonian translocation (rob) involving cattle chromosomes 1 and 29 and the demonstration of its deleterious effects on fertility was what piqued the interest of many scientific groups and focused their attention on studying the chromosomes of domestic animals. This approach was particularly useful for selecting bulls to be used for artificial insemination, as it could avoid the transmission of chromosome abnormalities (i.e., rob1;29) from bull carriers to their progeny. Evolutionary studies also benefitted from advancements beyond normal chromosome staining. Among the various studies, the most important was the study of the Bovidae family by Wurster and Benirske , who looked at the diploid number and shape of chromosomes. They concluded that while the diploid number varies from 38 to 60 among all bovid species, the number of chromosome arms (Fundamental Number = NF) varies only between 58 and 62, with three exceptions; therefore, they hypothesized a high degree of autosome arm conservation among all bovid species. This hypothesis was later confirmed with the application of chromosome banding techniques , which ushered in a new era of chromosome studies in various domestic animal species, allowing (a) the establishment of standard karyotypes of the most important domestic species as a point of reference for various applications; (b) better characterization and identification of the chromosomes involved in chromosome abnormalities of domestic animals , particularly domestic bovids , pigs , horses , and dogs ; (c) the study of the chromosome homologies between related and unrelated species ; and (d) the study of chromosome fragility in animals exposed in vivo or in vitro to particular mutagens . The molecular cytogenetics, particularly the introduction of fluorescence in situ hybridization (FISH), offered a deeper investigation of the chromosomes of domestic animals through: (a) the physical mapping of specific DNA sequences on chromosome regions; (b) the use of specific chromosome markers for the identification of chromosomes or chromosome regions involved in chromosome abnormalities, especially when poor banding patterns are produced; (c) better anchoring of radiation hybrid (RH) and genetic maps to specific chromosome regions; (d) better comparisons of related and unrelated species by comparative FISH mapping and/or Zoo-FISH techniques; (e) the study of meiotic segregation, especially by sperm-FISH, in some chromosome abnormalities or aneuploidies in both oocytes and embryos; (f) better demonstration of conserved or lost DNA sequences in chromosome abnormalities by CGH (comparative genomic hybridization) or SNP (single-nucleotide polymorphism) arrays; (g) the use of informatic and genomic reconstructions, in addition to CGH arrays, for the prediction of conserved or lost chromosome regions in related species; and (h) the study of chromosome abnormalities and genomic stability using PCR (polymerase chain reaction). This review summarizes the most important applications of molecular cytogenetics in domestic bovids, with particular emphasis on FISH mapping applications. 2. The Fluorescence In Situ Hybridization (FISH) Technique The FISH mapping technique is based on two main principles: the target and the probe. The target can be a whole chromosome (or chromosome arms) or a specific chromosome region. The probe is prepared according to the size of the target and is typically: (a) cDNA (generally applied when the target gene is a multi-copy); (b) cosmids with DNA insert sizes of 20-40 kb; (c) bacterial artificial chromosomes (BACs) with DNA insert sizes of 100-300 kb; (d) yeast artificial chromosome (YAC) clones (these are actually not used because they have a low cloning efficiency and show a high level of chimerism); (e) chromosome painting probes (obtained by cell sorter or chromosome microdissection techniques) that can visualize parts of or entire chromosomes; and (f) CGH arrays to check for genomic gains or losses. The probes are labeled directly with fluorochromes or indirectly with molecules that bind to the probe via fluorochrome-conjugated antibodies. The probe is specific for the target, based on complementary DNA base pairing, which allows the fluorescence-labeled probes to hybridize and form specific fluorescent signals on specific chromosome regions. The advent of the fluorescence in situ hybridization (FISH) technique, initially applied to human chromosomes , noticeably expanded cytogenetics research and investigations applied to domestic animals due to the possibility of revealing specific chromosome regions, entire chromosomes, or chromosome arms according to the choice of probe. One of the great advantages of the FISH technique is that it can be applied to interphase cell nuclei, meiotic preparations (sperm and oocytes), embryos, and elongated chromatin fibers, in addition to metaphase chromosomes, thereby allowing more complete cytogenetic investigations of animal cells. The following sections describe the main uses of FISH in domestic bovids. 2.1. FISH and Chromosome Abnormalities The first study to apply FISH for the precise identification of the chromosomes involved in a chromosome abnormality was published by Gallagher et al. , who discovered an X-autosome translocation (X;23) using both Q-banding and a BoLA Class I cDNA probe. The probe shows hybridization signals to the normal chromosome 23 and to the translocated autosomal material present on the X chromosome, allowing a more precise localization of MHC (major histocompatibility complex) in cattle than was achieved earlier by genetic mapping. Several subsequent studies also applied FISH to obtain better confirmation of the chromosome(s) involved in abnormalities (especially when banding was poor) and identification of the break points, especially in reciprocal translocations. Table 1 shows the main studies that applied FISH mapping, either alone or in combination with other classical cytogenetic techniques (e.g., C-banding, G-banding, R-banding, and Ag-NORs), to study the chromosome abnormalities of domestic bovids in somatic cells at the metaphase or interphase nuclei of germinal cells, such as sperm and oocytes, or embryos at different cell stages. A more complete classification of all chromosome abnormalities studied by classical cytogenetic techniques alone or (in some cases) with other molecular cytogenetic techniques is provided by Iannuzzi et al. . Two examples of the importance of the use of FISH for the correct identification of the chromosomes involved in chromosome abnormalities of cattle were a case of autosome trisomy and two types of Robertsonian translocations. A case of autosome trisomy 28 in an abnormal calf, revealed by both R-banding and FISH mapping with a specific molecular marker , was identified, and the same abnormality was reported earlier as trisomy 22 using only the banding technique . Two robs earlier reported as rob (4;8) and rob (25;27) in cattle were later corrected as rob (6;8) and rob (26;29), respectively, using C-, G-, and R-banding and FISH mapping with specific molecular markers and the use of HSA painting probes . Table 1 shows that FISH mapping applications were used for the diagnosis of chromosome abnormalities in both metaphase (the majority) and interphase cells, the latter applied to lymphocyte nuclei , sperm , oocytes, and embryos. Concerning the studies on meiotic preparations, those performed on the synaptonemal complexes (SCs), especially in spermatocytes, were particularly important for establishing the regularity of the pairing processes during the pachytene substage of meiotic prophase in animals carrying chromosome abnormalities (reviewed in ). Recent analyses of meiotic preparations have been performed using immune fluorescence approaches and have provided more detailed information on SCs . Other studies have addressed the fragile sites in the chromosomes of domestic animals (reviewed by ), and limited studies have used CGH and SNP arrays to establish possible genomic losses occurring during chromosome rearrangements (Table 1). FISH mapping was also very important for the definitive establishment of the agreement between various chromosome nomenclatures due to some discrepancies found during the Reading conference and the subsequent ISCNDA1989 (the inverted position between BTA4 and BTA6, as well as the correct position of BTA25, BTA27, and BTA29). This aspect was vital for the clinical cytogenetics of domestic bovids, as it allowed a correct identification of the chromosomes involved in chromosome abnormalities. During the Texas conference , specific molecular markers (only type I loci) were selected for each bovine syntenic group and each cattle chromosome based on previous standard chromosome nomenclatures. The next advance was the application of FISH mapping by two labs that used 31 selected BAC clones (from the Texas Conference) on RBG- and QBH-banded cattle preparations . The chromosome-banding homologies among bovids (cattle, sheep, goats, and river buffalo) were then used to establish a definitive standard chromosome nomenclature for the main domestic bovid species . Subsequent studies using FISH mapping and the same Texas markers on river buffalo, sheep, and goat R-banded chromosomes definitively confirmed the chromosome homologies among domestic bovids, as established at the ISCNDB2000 . 2.2. FISH in Physical Mapping The identification of the DNA structure paved the way for the development of in situ hybridization technology. In the early stages of its development, this technology allowed the localization of genes using radioactive probes . It was also used in studies of domestic animals , but the greatest diffusion of the physical mapping of genes awaited the development of fluorescent probes . At that moment, we entered the golden years of gene mapping, and domestic animals were not excluded. One of the first examples was the localization of bovine alpha and beta interferon genes , and this localization was rapidly replicated in buffalos, goats, and sheep . Subsequently, many other localizations were obtained using this technology . Considering the practical impossibility of compiling a complete list of all gene localizations obtained using this technology, some significant examples are listed in Table 2. Localization sometimes involved a single gene or a family of genes . Other reports, however, mapped many genomic markers . A point to remember is that FISH technology has significantly benefited from the availability of BAC genomic libraries--elements that represent the ideal source for the construction of the probes. Among these, the INRA library and the CHORI-240 have played relevant roles. The publication of genomes has since inevitably diminished interest in using this technology for mapping genetic factors, although genetic factor mapping continued for species whose genomes were sequenced later, such as the water buffalo . However, this technology has proved useful in several aspects, including: a) the identification of errors in genomic assembly ; b) the refinement of genome assembly ; and c) the mapping of sequences not included in genomic assemblages . Clearly, the interest today is very limited in locating a genetic factor in a species whose genomic sequence is available, but this does not mean that FISH technology is no longer indispensable for solving other problems related to the organization of genomes. The mapping of genomic elements by FISH has also been used successfully for the physical mapping of data obtained by other technologies. The first examples concerned the physical anchoring of a genetic map to a chromosome and the mapping of a synteny group to a specific chromosome . Subsequent examples of the combined use of FISH and genetic maps followed . 2.3. Comparative FISH Mapping Two main methods have been applied thus far to obtain a FISH mapping comparison between related and unrelated species: Zoo-FISH, which uses chromosome painting probes, and FISH mapping, which uses specific molecular markers of both type I and type II. Zoo-FISH is a molecular technique that provides an easier comparison between related and unrelated species from a macro point of view. The term was first reported by , based on earlier studies that used genomic chromosome painting probes, obtained by cell sorter chromosomes, to compare related species . Zoo-FISH was first applied in domestic animals when human chromosome painting probes became commercially available. This approach demonstrated the conservation of several human chromosome segments in both domestic bovids (Table 3) and other domestic species (reviewed in ). The use of human-chromosome painting probes allowed the identification of a substantial number of human chromosome segments (around 50) in bovid chromosomes . Zoo-FISH has also been applied to correctly identify some chromosomes involved in the chromosome abnormalities shown in Table 1. The availability of specific painting probes obtained by both cell sorting and/or by the microdissection of specific chromosomes (or chromosome arms) from domestic animals extended these studies to investigations between related species (Table 3). For example, in cattle, Zoo-FISH was applied to study X-Y aneuploidy in sperm and in oocytes (Table 1). An interesting approach was demonstrated in two studies characterizing two cases of goat/sheep and donkey/zebra hybrids using multicolor FISH (M-FISH), starting from painting probes obtained from microdissected river buffalo chromosomes (or chromosome arms) and from flow-sorted donkey chromosomes, respectively. Chromosome painting probes allow the delineation of large, conserved chromosome regions between related and unrelated species, as reported above. The use of comparative FISH mapping using several chromosome markers to map a single type I or type II locus along the chromosomes allows a more accurate establishment of the gene order within chromosome regions, thereby confirming that chromosome rearrangements occurred to differentiate related or unrelated species in key evolutionary studies (Table 3). These detailed comparisons have confirmed a high degree of autosome (or chromosome arm) conservation among all bovid species. The main autosome difference found thus far in bovids was a chromosome translocation of a proximal chromosome region from Bovinae chromosome 9 to Caprinae chromosome 14, as demonstrated by both chromosome banding and, in particular, by a molecular marker (COL9A1) mapping proximal to Bovinae chromosome 9 and proximal to Caprinae chromosome 14 (reviewed in ). This translocation involved a genome region of about 13 MB and was followed by an inversion in Caprinae chromosome 14, as demonstrated earlier . This chromosome event was common to all remaining Bovidae subfamilies, leading to the conclusion that the Bovinae subfamily is an ancestor to the remaining Bovidae subfamilies (reviewed in ). In contrast to autosomes, sex chromosomes are differentiated by more complex chromosome rearrangements. Indeed, the Caprinae X chromosome (as for all remaining X chromosomes of the other Bovidae subfamilies) is differentiated from the ancestor Bovinae X (very probably a large acrocentric chromosome, such as that of the water buffalo) by at least three chromosome transpositions and one inversion (reviewed in ). Detailed FISH mapping data are also useful for better anchoring of both genetic and RH maps . The availability of detailed cytogenetic maps in bovid species allowed a better comparison of the bovid and human chromosomes, especially using type I loci. These comparisons facilitated the translation of genomic information from the human genome to the genomes of domestic animals, especially in those with no genome sequencing available. These comparisons also revealed a very high number of chromosome rearrangements that differentiate bovid species from humans. Indeed, the conservation of entire chromosomes or large regions of them between bovid and human chromosomes, as revealed by Zoo-FISH, was the result of complex chromosome rearrangements that differentiated human and bovid species according to their gene order. An example is presented in Figure 5 which illustrates the comparison of FISH mapping between HSA2q and BTA2. As seen, when utilizing the Zoo-FISH technique with the HSA2q painting probe, almost all BTA2 is painted , indicating a high degree of chromosome conservation between the chromosomes of the two species. By conducting the same comparison using comparative FISH mapping and examining the gene order along the chromosomes of the two species, we observe a distinct gene order between the two species, thus revealing complex chromosome rearrangements that differentiated the chromosomes of the two species during their evolution. 2.4. Fiber-FISH The various FISH mapping techniques developed for human cytogenetics (reviewed by ) include SKY-FISH (spectral karyotyping FISH), Q-FISH (quantitative FISH), M-FISH (multicolor FISH), heterochromatin-M-FISH, COBRA-FISH (combined binary ratio labeling FISH), cenM-FISH (centromere-specific M-FISH), and fiber-FISH. Among these techniques, only fiber-FISH and M-FISH have been applied to domestic bovids. The use of fiber-FISH yields high-resolution maps of chromosomal regions and related genes on a single DNA fiber. This approach establishes the physical location of DNA probes with a resolution of 1000 bp. It is particularly useful for detecting gene duplications, gaps, and variations in the nuclear genome. The DNA fibers are obtained from nucleated cells by releasing the DNA fibers from the nucleus, stretching them mechanically, and then fixing them on slides . Table 4 summarizes the studies that have used this technique in domestic bovids. 2.5. CGH Arrays The CGH array technology, an evolution of in situ comparative genomic hybridization (CGH), is a method of cytogenetic investigation that emerged in the 1990s to overcome the limitations of common banding cytogenetic analyses, especially those involving the presence of genomic imbalances, such as duplications or deletions . In situ CGH technology has many similarities to FISH: the support used is the same, i.e., denatured metaphases fixed on slides and the approaches to label the probes are identical. However, in this case, the probes are produced using complete genomic DNA deriving from two subjects: typically, one healthy and one relating to the subject being investigated. The two DNAs are labeled with two different fluorochromes and then hybridized simultaneously on the slide. In the hybridization phase, a competition is therefore created between the probes, and in the presence of a normal chromosomal segment, an intermediate color is obtained, while in the presence of chromosomal alterations, a fluorescence closer to one of the two colors used is obtained. Although this technology has been widely used and has provided important results, its major limitation lies in the resolution. CGH array technology follows the same principle, but the support is no longer represented by slides but by synthetic DNA fixed on slides. Initially, the chips for CGH array analyses contained DNA extracted from BAC to provide as uniform a representation of the genome as possible . Current CGH array analyses are performed using devices containing oligonucleotides chosen that uniformly cover the whole genome and achieve resolutions of 5-10 kb . More information about this technology and its use is provided by . In species of zootechnical interest, CGH array analyses became common following the appearance of the first commercial arrays, and these analyses are conducted essentially for two purposes: the identification of copy number variation (CNV) polymorphisms and the characterization of chromosome anomalies. CNVs are polymorphic variations present very frequently in the genomes of higher organisms . In humans, approximately 4.8-9.7% of the genome contains CNVs . The introduction of commercial arrays has allowed the use of this technology to obtain a great amount of information about the distribution of CNVs in species differences and how these variations are related to phenotypic traits. The transfer of this technology to the animal field and the availability of commercial arrays has led to the publication of several reports (Table 5). 3. Combined Informatic and Genomic Information The publication of animal genomes has made available a very large series of data that required the development of sophisticated analysis techniques and often required the use of computers with large processing capacities. The first bio-informatic analyses were used to assemble thousands of short genomic sequences, produced by modern high-throughput sequencing technologies, into genomes. Today, most of these programs are available free of charge through web pages that function as interfaces between the user and calculation tools . Currently, dozens of bio-informatics programs are available to analyze the data contained in genomic assemblies, and many of these are accessible through various web platforms. Making a complete list is very complicated, in part because this is a rapidly evolving discipline that introduces, almost daily, new analytical tools. 3.1. Visualization of Genomes The genomic sequences produced by the various assemblies can be visualized using one of the available websites available, including Genome Data viewer , UCSC Genome Browser , and Ensembl . Currently, these websites provide the ability to view and process data relating to several genome assemblies (Table 6). These genome viewers are constantly evolving and contain several tools within them that allow the user to obtain highly relevant genetic data and information. This includes, but is not limited to, the possibility of: (a) identifying the structure of genetic factors (in terms of exon-intron boundaries); (b) identifying SNP polymorphisms in a particular region of the genome; (c) identifying the position of BACs by mapping the BES (Bac Ends Sequences, particularly useful when the user wants to choose the BACs to use in FISH analysis); (d) observing the genomic regions expressed in particular types of tissues; (e) analyzing the relationships between different assemblies of the same species; (f) visualizing the relationships between similar regions in different species (comparative genomics); and (g) viewing the repeating regions. In this review, we do not specify a best genome viewer, as this will often depend on personal needs and experience. However, as each genome viewer has its own specific analysis tools, sometimes the best solution is to use all three to obtain more complete information. 3.2. Use of Genomic Assemblies The availability of genomic assemblages has, on the one hand, limited the interest in the physical mapping of genomic elements, but has, on the other hand, allowed the evolution of a very large number of genetic and genomic analyses. Probably one of the most common uses (even if not directly related to cytogenetics) is to design primers for use in PCR amplifications. This operation can be performed using different software, both available for free and for a fee. Among those available free of charge, the most frequently used is Primer3 . The availability of genomic assemblages also makes rapid evolutionary investigation possible (i.e., visualizing, in a simple and rapid way, the similarities that exist between the various genomic regions of different species). The publication of genomes has certainly had a great impact on cytogenetics (both negatively and positively). If the golden era of gene mapping has ended, the possibility of rapidly identifying BACs for use as probes in FISH experiments has certainly provided great benefits to cytogenetics, as it avoids long and tedious testing of BAC libraries. This aspect has allowed the rapid characterization of some chromosomal anomalies, such as a centromere repositioning event in cattle , a reciprocal translocation, also in cattle , and cryptic evolutionary rearrangements between cattle and sheep . Finally, the rapid localization of BACs on genomes has allowed the development of complex approaches for the identification of chromosomal abnormalities, which are also difficult to identify . Obviously, these are not all the possible uses of genomic assemblies, but they represent the best examples in relation to cytogenetics. Each genomic assembly contains substantial information that can be used for very specific purposes and avoids the need for probes that would be complex to synthesize. The continuous evolution of these data analysis tools creates difficulty in any attempt to compile their possible uses. 3.3. Tools for Genomic Data Analyses Simultaneously with the publication of the genomes, bio-informatics tools were developed for the analysis of the vast amount of data generated--data that are characterized by both their great variety and their large quantity. One of the main repositories of tools for analyzing genomic data is Galaxy . This repository provides access to bio-informatic analysis tools, which are constantly updated. SNP variations represent the major source of variation in genomes, and the genomes of the species covered in this review are no exception. Currently, identifying these sources of variation is quite simple (through modern high-throughput sequencing techniques at ever-lower cost), but this does not characterize the effect that these variations can cause. For this scenario, the variant effect predictor (VEP available on the Ensembl website) software is helpful . Without a doubt, BACs represent one of the most useful tools for molecular cytogenetics, and, as previously mentioned, their identification in genomes is currently greatly facilitated. However, the current situation would not be possible without the existence of two important institutions that have dedicated part of their activities to the construction, maintenance, and distribution of BAC libraries: the BACPAC Resources Center (BPRC, (accessed on 2 March 2023)) and INRA (accessed on 2 March 2023)). Through these two institutes, BACs belonging to different libraries can be obtained. 3.4. Whole-Genome Sequencing In recent years, the decreasing costs of sequencing have made it possible to analyze many subjects. The purposes of these sequencings are different; in many cases, the aim is the identification of signatures of selection , but other purposes are represented, such as: (a) the identification of genetic variants in specific genes ; (b) the verification of data obtained regarding the identification of SNPs with chip arrays ; (c) the identification of the run of homozygosity in breeds intended for different productions ; (d) prediction and QTL mapping ; and (e) the identification of copy number variants and transcriptome characterization . Similar analyses were performed on sheep and goats . Additionally, in this case, the water buffalo seems to be slightly behind, as there are very few papers available on it . 4. PCR-Based Methods and Molecular Cytogenetics The polymerase chain reaction (PCR) is a method largely used to make millions of copies of a specific DNA sample in a fast and economical way for the detection, quantification, and typing of infectious diseases and genetic changes. Current PCR-based methods are distinguished as: (a) first-generation PCR, (b) second-generation quantitative PCR (qPCR), and (c) third-generation droplet-based digital PCR (dPCR). PCR detects endpoint, qualitative, or semi-quantitative assays by gel electrophoresis, separating DNA fragments according to size. The qPCR measures DNA/RNA in real time using PCR methods, fluorescent dyes, and fluorometry for relative quantification and quantitative assays with standard curves. The dPCR splits a PCR sample labeled with fluorescent dye into millions of microsamples to digitize the pool of DNA molecules with a single or no copy in each droplet. It quantifies the DNA/RNA copy number faster than qPCR based on standard curves . In recent years, PCR-based methods have replaced the classic cytogenetic techniques for detecting chromosome abnormalities and aneuploidy due to greater precision, lower cost, and faster data than are possible with cytogenetic methods, because of the small quantities of DNA (30 ng) required from any stored or fresh biological samples. PCR-based approaches are most commonly used in bovid studies to examine sex chromosomes in early-sex-determination assays to detect aberrations (Table 7). Telomere assessment is another critical goal of cytogenetics research due to the central roles of telomeres in chromosome stability, aging, cancer development, apoptosis, and senescence. The telomeres consist of thousands of noncoding repetitive sequences of DNA composed of six nucleotide motifs (TTAGGG)n localized at the ends of chromosomes and are responsible for maintaining DNA integrity during each cell division. They are associated with several proteins, with the most abundant being the shelterin complex, which is made up of six different polypeptides. Telomeres also contain other genomic structures, such as T-loops, D-loops, G-quadruplexes (G4), R-loops, and long noncoding RNA (TERRA) . In farm animals, telomere length (TL) did not receive much interest initially due to the difficulty in determining the natural limits of their lifespans. However, a recent study related TL to health, genome stability, and aging in cattle aged between 2 and 13 years and transformed TL into a sensitive biomarker for longevity and wellness (critical traits of selective breeding), responding to the "One Health" approach (improving animal welfare) . TL is not often used as a unique marker of aging in humans because of its poor predictive accuracy due to increased telomere shortening in elderly humans as a consequence of age-related diseases (e.g., cancer, atherosclerosis, autoimmune disorders, obesity, chronic obstructive pulmonary disease, diabetes, hematological disorders, and neurodegenerative diseases) . By contrast, TL proved to be a relevant biomarker of the general state of farm animals due to their lack of age-related pathologies . Approaches for measuring TL include: (a) telomere restriction fragment (TRF) length ; (b) length analysis by Southern blotting; (c) fluorescent in situ hybridization (FISH) by flow cytometry (flow-FISH) or in metaphase cells (Q-FISH) ; and (d) PCR-based methods. Most of these methods have several limitations. For example, TRF and flow-FISH are labor-intensive and expensive; Southern blot analysis requires large amounts of genomic DNA, and Q-FISH works only on chromosomes (metaphase stage). Of the available methods, the PCR-based ones are the fastest, most recent, and least costly and require only small quantities of DNA (30 ng) from stored or fresh biological samples . The qPCR method amplifies telomere repeats relative to a single-copy gene (reference gene) according to a method described by Cawthon et al. and follows the MIQE guidelines . One limitation of qPCR is the inconsistent repeatability and reproducibility of different TL measurement methods, producing a high variation in results . Several studies on humans and animals indicated that the DNA extraction method might affect TL measurements using q-PCR, as DNA yields were higher using the non-silica membrane kit (salting-out method), and DNA integrity on electrophoresis gels varied . A recent study showed comparable results for DNA quality and purity (tested using a NanoDrop instrument and electrophoresis gels) in cattle blood and milk samples using two different extraction kits (a salting-out kit for blood and a silica membrane kit for milk samples) due to the difficulty of extracting DNA from milk matrices. The DNA quality results were similar in both matrices, demonstrating a synchronous trend between them for the first time . 5. Current Developments and Knowledge Gaps Molecular cytogenetics is approaching its first 30 years of history and during this period, it performed important functions that evolved over time. It therefore seems normal that in the coming years, we will witness further developments; however, some approaches will always be current and irreplaceable. The FISH technology represents, and will represent, the main methodology for the verification of chromosomal anomalies eventually identified with other approaches, just as the CGH array technology that will be increasingly used for the identification of genomic variants linked to a particular phenotype. Molecular cytogenetics could be very useful for the study of those species which have not yet benefited from the genomic revolution, or which are still in its early stages: in this sense, the water buffalo (Bubalus bubalis) is the main example. Despite possessing a great economic importance, its genome has been decrypted and made available only recently, and the application of other technologies is very late. A further gap that can be filled is the development of a technological approach that can allow the identification of all chromosomal types identifiable by cytogenetic analyses. A similar approach has already been published , but only the transfer of SKY-FISH technologies from humans to bovids will bridge this gap. Finally, the certain decrease in costs will mean that even the species considered in this review will be able to benefit from long-read genomic sequencing, such as PacBio and Oxford Nanopore . 6. Conclusions The study of the chromosomes of domestic bovids is about to enter its seventh decade, and, as expected, it has undergone a notable evolution along the way. This evolutionary process for this discipline is mainly a result of the appearance of technologies that have significantly increased the potential of applied cytogenetics. Banding techniques, FISH, CGH arrays, and PCR have radically changed animal cytogenetics, making them irreplaceable tools for understanding the genetics of bred animals. Therefore, considering the history of cytogenetics, a quite easy prediction is that even the next evolutions will be dictated by technological advances. Predicting the next technological leap is difficult, but if we were to make a prediction, it would be that long-read genomic sequencing technologies will have important impacts on cytogenetics. Cytogenetics will likely retain its functionality, particularly in the confirmation of genomic results and the characterization of cytogenetic anomalies, as well as in evolutionary studies. This is because the most significant genetic mutations have accumulated at the chromosome level during the evolution of species. Finally, the implication and progresses from animal cytogenetics can be summarized as follows:In the pre-genomic era, FISH technology represented the almost exclusive technology available for the localization of genes in genomes. Prior to the availability of low-cost genomic sequencing, molecular cytogenetics was the only approach for identifying similarities between karyotypes of different species. The technologies of molecular cytogenetics represent the best approach for the characterization of chromosomal abnormalities. Despite scientific progress in similar disciplines, molecular cytogenetics will always find its place and represent an inescapable investigation methodology. Author Contributions Conceptualization, P.P, L.I. and A.I.; writing--original draft preparation, P.P, L.I. and A.I.; writing--review and editing, P.P. and L.I. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement An Institutional Review Board statement was not required. Data Availability Statement Data sharing is not applicable to this article as no new data were created or analyzed in this study. Conflicts of Interest The authors declare no conflict of interest. Abbreviations aCGH array Comparative Genomic Hybridization BAC Bacterial Artificial Chromosome BBU Bubalus bubalis Chromosome, 2 n = 50 BES Bac Ends Sequences BIN Bos indicus Chromosomes, 2 n = 60 BTA Bos taurus Chromosome, 2 n = 60 CA Chromosome Abnormalities (chromosome breaks) CBA C-banding by Acrine Orange Staining CHI Capra hircus Chromosomes, 2 n = 60 Fiber-FISH Extended Chromatin Fiber-FISH FISH Fluorescence In Situ Hybridization GBG G-banding by Early BrdU-Incorporation and Giemsa Staining HSA Human sapiens Chromosome, 2 n = 46 IVP In Vitro Production MHC Major Histocompatibility Complex MI Mitotic Index MN Micronuclei OAR Ovis aries Chromosomes, 2 n = 54 PCR Polymerase Chain Reaction PNA Peptide Nucleic Acids QBH Q-banding by Early BrdU-Incorporation and Hoescht Staining RBA R-banding by Late BrdU-Incorporation and Acridine Orange Staining RBG R-banding by Late BrdU-Incorporation and Giemsa Staining RH Radiation Hybrids SCA Synaptonemal Complex Analysis SCE Sister Chromatid Exchange SKY-FISH Spectral Karyotyping SNP Single Nucleotide Polymorphism YAC Yeast Artificial Chromosome Figure 1 FISH mapping with a BAC clone mapping proximal to BTA29 (large arrow) and proximal to q-arms (BTA1) of rob (1;29) (small arrows). Indeed, a small chromosome region of 5,4 Mb translocated from proximal BTA29 to the proximal region of BTA1 (with an inversion), originating rob (1;29) . Different colors indicate different BACs. Figure 2 FISH mapping in an interphase nucleus of a female river buffalo affected by X-trisomy. Note the three hybridization signals due to the X chromosome PGK marker. Figure 3 Sperm-FISH in a river buffalo bull carrying a rob (1p;18) using BAC probes for BBU 1p (red), BBU 1q (green), and BBU 18q (yellow) chromosomes. Normal sperm nucleus with 1/1/1 fluorescent phenotype and separate signals on left. Unbalanced sperm nucleus with 1/0/1 fluorescent phenotype on right. Figure 4 FISH mapping of type II loci in river buffalo R-banded chromosomes. FITC signals (arrows) of the markers and RBH banding were separately acquired by two different microscope filter combinations. Then signals were precisely superimposed to R-banded chromosomes (Drawn from Iannuzzi et al., Cytogenet Cell Genet. 102, 65-75, 2003, DOI: 1 0.1159/000075727, S. Karger AG, Basel ). Figure 5 Comparative FISH mapping between HSA2q and BTA2. Note the different gene order between the two chromosomes due to complex chromosome rearrangements occurred during the chromosome evolution of the two species (Drawn from Di Meo et al., Animal Genetics 37, 299-300, 2006, Wiley Online Library ). Figure 6 Details of the fiber-FISH performed on a lymphocyte nucleus of cattle affected by arthrogryposis using a BAC clone containing the survival of motor neuron gene (SMN). The presence of two groups of linear hybridization signals (arrows) supports the hypothesis that SMN was at least duplicated . Figure 7 Identification of the PAR region present on BTAX and BTAY. The PAR region (yellow box) is identified by comparing DNA obtained from a male subject and that obtained from a female subject using a SurePrint G3 Bovine CGH Microarray 180 k (Agilent Technologies, Santa Clara, CA, USA). Parma P. Personal communication. animals-13-00944-t001_Table 1 Table 1 FISH mapping approaches applied for the detection of chromosome abnormalities in domestic bovids. The type of chromosome abnormality, the techniques used (including FISH), the main results, and authors are reported. Species Chromosome Abnormality Techniques Used Main Results References Cattle t(X-BTA23) in two normal cows QBH, FISH Better position of MHC-locus Minute fragment Bovine SAT-DNA Visualization of fragment rob(4;10) Bovine bivariate flow painting probes on R-banded karyotype Discovery of a new rob iso(Yp) GTG, FISH with repeat sequences Visualization of iso(Yp) Trisomy 20 QBH, FISH Malformed calf with cranial defects rob(2;28) Q-, R-banding, telomeric probe Monocentric translocation rob(1;29), rob(6;8), rob(26;29) GBG, RBG, CBA, FISH, HAS painting probe correct identification of two of the three robs earlier published Mixoploidy Dual-color FISH with BTA6/BTA7 painting probes 72% of IVP blastocysts were mixoploid, versus 25% in vivo Mixoploidy/polyploidy Dual-color FISH with BTA6 and 7 painting probes on in vitro embryo cells Numerical chromosome aberrations were detected as early as day 2 post insemination (pi) rcp(1;5)(q21;qter)(q11;q33) CBA, GBG, RBG, FISH with HSA3 and HSA12 painting probes Bull and dam carriers, the latter with poor fertility invY(Yq11-q12.2) CBG, RBA, FISH 12 young males of which one (carrier) had female traits Trisomy 28 CBG, RBA, FISH New chrom. identification of a previous studied case of abnormal calf t(Xp+;23q-) FISH with painting probe, SCA Oligospermic bull rcp(Y;9)(q12.3:q21.1). CBA, RBG, FISH Azoospermic bull Polyploidy Painting probe BTA6 and BTA7 by microdissection on in vitro embryos Polyploidy was significantly higher in trophectoderm (TE) cells than in embryonic disc (ED) cells rob(1;29) FISH with SAT-I, III, IV Different pattern of satellite DNA families in several chromosomes, model of rob(1; 29) origin Mosaicism 2 n = 60/2 n = 60 t (2q-;5p+) FISH with painting probes BTA2 and BTA5 Translocation mosaicism in a bull XXY-Trisomy X-Y painting probes Testicular hypoplasia fragm/hypoploidy/hypoploidy-mixoploidy; hyperploidy/hyperploidy-mixoploidy Karyotyping, FISH with X-Y painting probes in nuclear transfer embryos Anomalies occurred in NT embryos varied according to the donor cell culture and paralleled the frequency of anomalies in donor cells rob(1;29) CBG, GTG, FISH with a rob(1;29) painting probe Presence of rob(1;29) in Gaur (Bos gaurus) rob(1;29) CBA, RBA, FISH Origin of rob(1;29) by complex chromosome rearrangements rob(1;29) Sperm-FISH Low percentage of abnormal sperm in two carriers rcp(9;11)(q27;q11) RBG and FISH De novo origin of the rcp Mosaicism XX/XY cells FISH with a male-specific BC1.2 DNA sequence in interphase cell nuclei Diagnosis of freemartin rcp(11;21)(q28-q12) CBA, RBA, Ag-NORs, FISH Normal bull but with absence of libido; reduced fertility (very low presence of spermatozoa in germinal elements) rob(1;29) microdissection, DOP-PCR, cloning and sequencing, sperm-FISH Detection of sperm-carrying rob(1;29) rcp(2;4)(q45;q34) G-banding, SCA, and chromosome painting Detection of a new rcp in bull Aneuploidy Dual-color FISH with Xcen/Y painting probes in sperm Study the aneuploidy in different breeds rcp(4;7) RBG, FISH (painting probe), aCGH Normal male and no genomic loss in the rcp Aneuploidy Dual-color FISH with Xcen and BTA5 painting probes Study of aneuploidy in oocytes of two breeds Aneuploidy FISH with BTAX, BTAY, and BTA6 painting probes on sperm of several young bulls Aneuploidy frequencies in young fertile bull spermatozoa were relatively low rcp(Y;21)(p11;q11) G-banding, FISH Normal young bull but lower testosterone level at 12 months rcp(11;25)(q11, q14~21) CBA, RBA, FISH, NOR der11 with two C-bands for a break at the centromere of BTA25; cow with reduced fertility Aberrant oocytes Dual-color FISH of X-cent/BTA5 painting probes Similar rate of aneuploidy in different cattle breeds rob(1;29) FISH, aCGH New results of the origin of this rob by transposition, inversion; no gene-coding regions were disrupted during the rearrangements Xp-del (inactive X) CBA, RBA, FISH del found in both dam and calf (normal cow) X-Y aneuploidy Dual-color FISH with Xcen-BTAY painting probes Testing X-Y ratio and aneuploidy Aneuploidy Dual-color FISH with Xcen and five autosome painting probes Similar rates of chromosomal aberrant secondary oocytes in two indigenous cattle breeds Mixoploidy FISH with BTAX and BTA6 painting probes First zygotic cleavage (FZC) is a marker of embryo quality by demonstrating a significantly lower incidence of aberrations in early embryos Aneuploidy/polyploidy CA, SCE, MN, MI, FISH Effect of the tebuconazole-based fungicide: monosomies and trisomies on BTA5 and 7 rcp(5;6)(q13;q34) RBG, FISH, aCGH Normal young bull with balanced rcp rcp(13;26)(q24;q11) CBG, GTG, painting probes BTA13 and 26, telomeric probe De novo rcp in both dam and calf der(11)t(11;25)(q11;q14-21) CBA, RBA, FISH Abnormal female calf Chromosome damages SCE, MN, FISH with BTA1, 5, 7 painting probes No significant chromosome fragility with use of thiacloprid Abnormal BTA17 in a young bull CBA, R-banding, FISH, PNA-telomeric probe, aCGH, SNP array Centromere repositioning X-monosomy Karyotyping, FISH, SNP genotype data Sterile for abnormal internal sex adducts rob(3;16) Sperm-FISH Low rate of unbalanced gametes produced by adjacent segregation (5.87%) and interchromosomal effect (ICE) on BTA17 and BTA20 Trisomy 20 Q-banding, FISH Malformed fetus, cranial defects Trisomy 29 FISH/genomic analysis Malformed female calf showing dwarfism with severe facial anomalies rob(1;29); rcp(12;23) FISH, use of BAC clones mapping prox- and dist- regions of all cattle autosomes and X Identification of chromosome abnormalities in all autosomes and BTAX tan(18;27) CBA, RBA, FISH Male calf with congenital hypospadias and a ventricular septal defect River buffalo X-monosomy CBA, RBA, FISH Normal body conformation and external genitalia, ovaries not detectable, sterile rob(1p;23) CBA, RBA, Ag-NORS, FISH Complex chromosome abnormality with fission on BBU1 and centric fusion of BBU1p with BBU23 in both dam and female calf; reduced fertility in the dam rob(1p;18) CBA, RBA, FISH Famous bull eliminated from reproduction for the presence of the same chrom. abnormality in part of progeny Chromosome abnormalities Zoo-FISH Sequential approach with 13 chromosome river buffalo painting probes to detect river buffalo chromosome abnormalities rob(1p;18) Sperm-FISH in motile and total fraction sperm Limited effects on the aneuploidy in gametes on the motile fraction sperm River/Swamp buffalo Aneuploidy M-FISH Study of aneuploidy in river and swamp buffalo oocytes Sheep Chromosome abnormality Production of all sheep chromosome painting probes from cell sorter technique Easy identification of chromosome abnormalities rob(8;11) G-bands, painting probes 8 and 11, SAT-I and SAT-II SAT-I proximal on both arms with SAT-II covering the centromere Diploid-polyploid mosaicism Zoo-FISH with bovine painting probes X/Y and 1;29 on nuclei of in vivo and in vitro embryos In vitro embryos showed significant higher number of abnormal embryos than in vivo ones del(10q22) Use of ovine BAC clone in addition to genetic analyses Micro-chromosomal deletion responsible for EDNRB gene lack rcp(4q;12q)(q13;q25) CBA, RBA, FISH with both specific markers and PNA-telomeric probe Characterization of a new rcp in a young sheep rcp(18;23)(q14;q26). CBA, RBA, FISH with bovine painting probe Reduced fertility Chromosome abnormalities in bovids Partial river buffalo chromosome painting probes from microdissection Detection of chromosome abnormalities in bovids animals-13-00944-t002_Table 2 Table 2 Gene mapping obtained with FISH in domestic bovids. Type I and type II markers are expressed with polymorphic (SSRs, microsatellite, STSs) sequences, respectively. Gene/Genes/Marker Species Reference Lysozyme gene cluster BBU Uridine monophosphate synthase BTA Uridine monophosphate synthase BBU BTA1 to 7 BTA Microsatellites BTA Microsatellites BTA Beta-defensin genes BTA; OAR Alpha-S2 casein BTA; BBU Fas/APO-1 BTA Interferon gamma OAR Interleukin-2 receptor gamma BTA Beta-lactoglobulin pseudogene BTA, OAR, CHI Bone morphogenetic protein 1 BTA TSPY BTA, OAR, CHI VIL OAR, CHI, BBU Type I markers BTA Prion protein gene BTA, OAR, CHI, BBU IL2RA, VIM, THBD, PLC-II, CSNK2A1, TOP1 BTA NF1, CRYB1, CHRNB1, TP53, P4HB, GH1 OAR, BBU PAX8 BTA, OAR, CHI Type I markers BTA PREF1 BTA PRKCI BTA MHC BTA Type I markers OAR, CHI CACNA2D1 BTA SLC26a2 BTA SMN BTA, OAR, CHI, BBU Type I markers BBU Type I and II markers OAR PRPH BTA CYP11b/CYHR1 BTA SRY, ANT3, CSF2RA BTA Autosomal loci (11) BTA, OAR, CHI, BBU Autosomal loci (88) OAR Autosomal loci (68) BBU BMPR1B, BMP15, GDF9 BTA, OAR, CHI, BBU animals-13-00944-t003_Table 3 Table 3 Comparative FISH mapping in domestic bovids with related and unrelated species. Author/s Results Mapping omega and trophoblast interferon genes in cattle and river buffalo Mapping of lactoperoxidase, retinoblastoma, and alpha-lactalbumin genes in cattle, sheep, and goats Mapping omega and trophoblast interferon genes in sheep and goats Mapping LGB and IGHML in cattle, sheep, and goats Mapping CASAS2 gene to the cattle, sheep, and goat chromosome 4 Mapping MHC-complex in cattle and river buffalo Mapping inhibin-alpha (INHA) to OAR2 and BTA2 Mapping inhibin subunit beta b to OAR2 and BTA2 Mapping beta-lactoglobulin pseudogene in sheep, goats, and cattle Mapping ZNF164, ZNF146, GGTA1, SOX2, PRLR, and EEF2 in bovids Mapping of the alpha-S2 casein gene on river buffalo and cattle Mapping of beta-defensin genes to river buffalo and sheep chromosomes suggest a chromosome discrepancy in cattle standard karyotypes Mapping STAT5A gene maps to BTA19, CHI19, and ORA11 Mapping in Y chromosomes of cattle and zebu by microdissected painting probes Mapping of villin (VIL) gene in river buffalo, sheep, and goats Mapping prion protein gene (PRNP) on cattle, river buffalo, sheep, and goats Mapping BCAT2 gene to cattle, sheep, and goats Comparative mapping in X chromosomes of bovids Comparative mapping between BTA-X and CHI-X Survey of chromosome rearrangements between ruminants and humans Comparative mapping between cattle and pig chromosomes using pig painting probes Extensive conservation of human chromosome regions in euchromatic regions of river buffalo chromosomes Mapping of six expressed gene loci (NF1, CRYB1, CHRNB1, TP53, P4HB, and GH1) to river buffalo and sheep chromosomes Comparison of human and sheep chromosomes using human chromosome painting probes Mapping four HSA2 type I loci in river buffalo chromosomes 2q and 12 Mapping BCAT1 in cattle, sheep, and goats Comparative mapping in bovid X chromosomes reveals homologies and divergences between the subfamilies Bovinae and Caprinae Mapping 16 type I loci in river buffalo and sheep Mapping 13 type I loci from HSA4q, HSA6p, HSA7q, and HSA12q on in river buffalo Mapping forty autosomal type I loci in river buffalo and sheep chromosomes and assignment from sixteen human chromosomes Mapping eight genes from HSA11 to bovine chromosomes 15 and 29 International chromosome nomenclature in domestic bovids based on Q-, G-, and R-banding and FISH with 31 specific Texas marker chromosomes Mapping 28 loci in river buffalo and sheep chromosomes Sheep/human comparative map in a chromosome region involved in scrapie incubation time shows multiple breakpoints between human chromosomes 14 and 15 and sheep chromosomes 7 and 18 Physical map of the survival of motor neuron gene (SMN) in domestic bovids Assignment of the 31 type I Texas bovine markers in sheep and goat chromosomes by comparative FISH mapping and R-banding Mapping 195 genes in cattle and updated comparative map with humans, mice, rats, and pigs Mapping of F9, HPRT, and XIST in BTAX and HSAX clarifies breakpoints between the two species 15 gene loci were mapped in the telomeric region of BTA18q and HSA19q Comparative G- and Q-banding of saola and cattle chromosomes as well as FISH mapping of 32 type I Texas markers Mapping of fragile histidine triad (FHIT) gene in bovids Chromosome evolution and improved cytogenetic maps of the Y chromosome in cattle, zebu, river buffalo, sheep, and goats Physical map of mucin 1, transmembrane (MUC1) among cattle, river buffalo, sheep, and goat chromosomes and comparison with HSA1 Mapping of LEP and SLC26A2 in bovidae chrom. 4 (BTA4/OAR4/CHI4) and HSA7 Mapping 11 genes to BTA2, BBU2q, OAR2q, and CHI2, and comparison with HSA2q Mapping among humans, cattle, and mice suggests a role for repeat sequences in mammalian genome evolution Mapping sheep and goat BAC clones identifies the transcriptional orientation of T cell receptor gamma genes on chromosome 4 in bovids Mapping of twelve loci in river buffalo and sheep chromosomes: comparison with HSA8p and HSA4q Mapping 25 new loci in BTA27 and comparison with both human and mouse chromosomes An advanced sheep cytogenetic map and assignment of 88 new autosomal loci Cross-species FISH with cattle whole-chromosome paints and satellite DNA I probes was used to identify the chromosomes involved in the translocations of some tribe Bovinae species Extended river buffalo cytogenetic map, assignment of 68 autosomal loci and comparison with human chromosomes FISH with 28S and telomeric probes in 17 bovid species. NORs are an important and frequently overlooked source of additional phylogenetic information within the Bovidae Mapping DMRT1 genes to BTA8 and HSA9 Comparative DM domain genes between cattle and pigs Assignments of new loci to BBU7 and OAR6 and comparison with HSA4 Mapping 22 ovine BAC clones in sheep, cattle, and human X chromosome Mapping and genomic annotation of bovine oncosuppressor gene in domestic bovids Cytogenetic map in sheep as anchor of genomic maps also using different genomic resources from other species Molecular cytogenetics in goats and comparative mapping with human maps Mapping of 6 loci containing genes involved in the dioxin metabolism of domestic bovids Extended cytogenetic maps of sheep chromosome 1 and their cattle and river buffalo homologues: comparison with the OAR1 RH-map and HSA2, 3, 21, and 1q Mapping between BTA5 and some Antilopinae species using Sat-I and SAT-II sequence and BTA-painting probes Comparison of centromeric repeats between cattle and other Bovidae species Advanced comparative map in X chromosome of Bovidae Physical map of BMPR1B, BMP15, and GDF9 fecundity genes on cattle, river buffalo, sheep, and goat chromosomes Physical mapping of 20 unmapped fragments in Btau 4.0 Genome Assembly in cattle, sheep, and river buffalo Physical map of LCA5L gene in cattle, sheep, and goats New cryptic difference between cattle and goat karyotypes Small evolutionary rearrangement between BTA21 and homologous OAR18 Assignment of 23 endogenous retrovirus to both sheep and homologous chromosomes regions of river buffalo animals-13-00944-t004_Table 4 Table 4 Studies using the fiber-FISH on domestic bovids. Species Author/s Results Cattle Genomic organization of the bovine aromatase Molecular characterization of STAT5A- and STAT5B-encoding genes Demonstration of survival of motor neuron gene (SMN) duplication in a calf affected by arthrogryposis Demonstration of multiple TSPY copies on the Y chromosome Sheep DNA fiber barcodes indicated a chromosomal deletion animals-13-00944-t005_Table 5 Table 5 Identification of CNV. Specie Reference Note Cattle 3 Holstein bulls Cattle 90 animals: 11 Bos taurus breeds, 3 Bos indicus breeds, and 3 composite breeds for beef, dairy, or dual purpose Cattle 20 animals: 14 Holsteins, 3 Simmental 2 Red Danish and 1 Hereford Cattle 47 Holstein bulls Cattle 24 animals from Chianese breeds Cattle 3 Angus, 6 Brahman, and 1 composite animal Sheep 36 animals Sheep 12 animals Goat 10 animals animals-13-00944-t006_Table 6 Table 6 Independent genomic assemblies that can be analyzed through the main genomic visualization sites. Specie 1 Genome Assembly 2 Origin GDW 3 UCSC 4 ENS 5 BTA ARS-UCD1.3 USDA ARS yes no no ARS-UCD1.2 USDA ARS no yes no Btau_5.0.1 Cattle Gen. Seq. Int. Consortium yes no no Btau_4.6.1 Cattle Gen. Seq. Int. Consortium no yes no Btau_4.0 Cattle Gen. Seq. Int. Consortium no no yes UMD_3.1.1 University of Maryland yes yes no UMD_3.1 University of Maryland no no yes Baylor 4.0 Baylor College of Medicine no yes no OAR ARS-UI_Ramb_v2.0 University of Idaho yes no no Oar_rambouillet_v1.0 Baylor College of Medicine yes no yes Oar_v4.0 Int. Sheep Gen. Consortium yes yes no CAU_O.aries_1.0 China Agricultural University yes no no CHI ARS1.2 USDA ARS yes no no ARS1 USDA ARS no no yes CHIR_1.0 Int. Goat Gen. Consortium yes no no BBU NDDB_SH_1 Nat. Dairy Dev. Board, India yes no no UOA_WB_1 University of Adelaide yes no no BIN Bos_indicus_1.0 Genoa Biotecnologia SA yes no no 1 BTA = cattle; OAR = sheep; CHI = goat; BBU = water buffalo and BIN = Zebu. 2 Only genomic assemblages at the chromosomal level were considered and not those limited to scaffolds. 3 Genome data viewer. 4 USCS genome browser. 5 Ensembl genome browser. animals-13-00944-t007_Table 7 Table 7 PCR-based approaches on bovids for the detection of chromosomal aberrations. Species Objective Sample PCR-Based Method Reference Cattle Sex-determination Embryos PCR Cattle Freemartinism diagnosis Blood PCR Cattle Sex-determination Embryos PCR Cattle Sex-determination Spermatozoa PCR Cattle Chimerism diagnosis Blood qPCR Cattle XX/XY chimerism diagnosis Blood PCR Cattle SRY-positive hermaphrodite diagnosis Blood PCR Cattle XY (SRY-positive) diagnosis Blood PCR Cattle Freemartinism diagnosis Blood qPCR Cattle Freemartinism diagnosis Blood dPCR Cattle Sex-determination Spermatozoa dPCR Cattle Mosaic karyotype (60,XX/60,XX,+mar) diagnosis Skin tissue PCR Cattle Mosaicism (60,XX/90,XXY) diagnosis Blood, skin, buccal epithelial cells, and hair follicles dPCR Cattle XX/XY chimerism diagnosis Blood and hair follicles dPCR Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000108

The study analyzes red deer responses to disturbances during the day and different exposures to tourists, to establish the more appropriate times to carry out activities inside the Paneveggio deer enclosure. The alarm reactions of red deer were observed after presenting different types of visual stimuli inside and outside the fence, in order to answer some questions: Which stimuli produce the strongest reactions from the animals? Do animals differently react to stimuli presented outside and inside the fence? On which days and times are the animals more sensitive to disturbances? Are there different reactions between the males and females? The results suggest that the red deer adversely react to the disturbance at different degrees of intensity in relation to day, sex, tourist and where the stimuli are presented. It was observed that during the days with the highest tourist presence, the animals were particularly alarmed; discomfort accumulation produced the highest number of alarm reactions on Monday. For these reasons, it would be opportune to manage the pasture on Tuesday, Wednesday and Thursday, scheduled at specific times of day, preferably far from the estimated presence of tourists. red deer alarm behavior disturbance vigilant attitudes pasture restoring This research received no external funding. pmc1. Introduction The intensification of recreational activities in wild areas is resulting in an increase in disturbance to wildlife . Human disturbance deriving from such activities is supposedly perceived by animals as a predation risk . Therefore, animals tend to adapt to such disturbances by adopting a repertoire of behaviors aimed at avoiding predators and therefore reinforcing survival chances . Due to deforestation and hunting activities, the alpine red deer (Cervus elaphus) population dramatically dropped at the beginning of the year 1800 and the species was considered extinct in the southern part of the Alps . The species population has been experiencing a stable recovery since the 1950s as a result of spontaneous recolonization and reintroductions, with a peak of circa 3000 individuals estimated in 2008 . For educational/didactic purposes, a captive population of red deer is kept at Paneveggio Pale di San Martino, which is part of the San Martino Natural Park (IT). Red deer are Intermediate Opportunistic Mixed feeders with a forage selectivity related to food availability and quality . The kept red deer receive a feed supply by the keepers; this causes the inappropriate exploitation of trophic resources inside the enclosure, which may lead to a loss of biodiversity because the deer select the more palatable plants, causing an inhomogeneous disturbance to the grass species. In addition, the pasture inside the enclosure is not mowed periodically, favoring the spread of tall grasses such as Bellardiochloa variegata, Brachypodium rupestre and Deschampsia cespitosa . Worsening in forage composition and increased abundance of high fibrous tall grasses could result in a decline in animals' body condition, as a consequence of the increase in rumen keratinization degree, which limits the absorption of volatile fatty acids . Indeed, the keratinized outer layers of the epithelium act as a protective layer for the mucosa from exogenous mechanical stimuli, while the volatile fatty acids obtained from cellulose digestion are metabolized by the deeper layers. Therefore, when the degree of keratinization increases, the absorptive ability decreases . The study was requested by the Paneveggio Pale di San Martino National Park in order to plan the restoration activities needed to repristinate a suitable pasture for the animals. However, human presence inside the enclosure might produce stress in the animals and negatively affect their welfare. If an animal is not in a healthy state, prolonged stress may result in the triggering of adaptive processes, which can cause significant harm to its body state . For this reason, any activity that may affect animal welfare should be timed based on the animals' response to disturbances. For this purpose, the alarm reactions of the red deer captive population inside the deer enclosure in Paneveggio Pale di San Martino Natural Park were observed after presenting different types of visual stimuli inside and outside the enclosure. The animals' behaviors, such as vigilance, alert and alarm reactions, were observed since they may be linked with underlying "states", such as fear and stress . For this reason, we attempted to answer the following questions:Which stimuli produce the strongest reactions from the animals? Do animals differently react to stimuli presented outside and inside the enclosure? What are the days and times in which the animals are more sensitive to disturbances? Do different groups react to disturbances in different ways? This information will be useful when planning the times and methods of each necessary intervention within the enclosure, from restoring the quality and composition of the pasture to maintenance interventions on the fence, but also for organizing entry for scientific or educational purposes. Maintaining a low level of disturbance is also the most ethical choice when caring for captive animals in order to ensure "freedom from fear and distress", one of the five fundamental freedoms formulated by Webster . 2. Materials and Methods 2.1. Study Area The study was conducted in Paneveggio Pale di San Martino Natural Park (IT), a protected area situated in the eastern part of the Alps. In particular, the captive red deer population inside the Paneveggio enclosure (46deg18'24'' N, 11deg44'24'' E) was observed. The enclosure is managed by the "Technical and Management Office of the State Forests Provincial Agency", in Trento province, and has a 6-hectare extension ; a quarter of it is composed of woodlands, while the remaining is pasture for the animals. A well-hidden lake is used by the animals to drink. The enclosure can accommodate up to a maximum of twenty animals. Tourists are allowed to walk around the whole perimeter of the enclosure, with some easily walkable parts and some less accessible ones. Tourists are not allowed to enter the enclosure and their presence is variable depending on the day of the week, time of day and month. An observation platform is present, allowing the observation of the animals from a respectful distance . 2.2. Observation Procedure During the study, the red deer population was composed of 18 individuals, 3 adult males, 10 adult females and 5 fawns. All deer present in the enclosure during the study were born in the enclosure and were fed only by their dams during the lactation period. The animals tended to separate into a nursery group (females and fawns) and a male group. The oldest of the males was the dominant one. The animals are subjected to intense exposure to tourists throughout the year, with higher incidences in the summer months. The study was performed during the touristic season of 2015, for 7 days during the month of July. Red deer alarm reactions were observed after presenting different stimuli in order to visually determine how the animals' reactiveness changed throughout the day and the days of the week. Three experienced observers, who had a good knowledge of the group of red deer, were present during the observation sessions, one presenting the stimulus and one hidden and recording the alarm reactions, while the third observer took photos and videos of the animal group in order to look for particular group patterns afterwards. The observers played the same roles for the whole study period. Animals' responses were observed with the naked eye by the observer presenting the stimulus, and with binoculars by the recording observer. Following Hodgetts's study, stimuli that produced the greatest responses were selected, according to the possibility of reproducibility. Responses to three visual stimuli, i.e., (1) a person standing still (S) outside and inside the enclosure, (2) a person moving towards the animals (M) outside and inside the enclosure, (3) an umbrella opened (U) outside and inside the enclosure, were examined (Table 1). Alarm was detectable mostly by observing changing head and neck positions and ears' movements; all these features were punctual and fast, but easily perceptible . The observed alarm reactions were (i) head held high or parallel to the body, (ii) prominent neck, (iii) straight ears, (iv) ear twitching (rapid movement of both ears). All the described alarm reactions were noticeable in three different vigilant attitudes, i.e., (a) vigilant lying, (b) vigilant standing and (c) vigilant moving; the three vigilant attitudes are described in Table 2. The three vigilant attitudes may give an insight into the intensity of the alarm. The animals are more relaxed when lying down, while they are more sensitive to disturbances when standing and moving . Reactions to disturbances were recorded in 30 min observation sessions; they were distributed from 9 am to 6 pm depending on the presence of tourists. At least one observation session was performed before, during and after the exposure to tourists per day. Different observation sessions were performed for the nursery and the male groups. When possible, an observation session for every time slot (before, during and after) was performed for both groups, depending on the arrival of tourists and the possibility of observing the animals, since the enclosure offers good hiding spots. During every observation session, all the stimuli were presented to the animals in random order. A detailed list of observation sessions is reported in Table S1 in the Supplementary Materials. Responses were observed after presenting each stimulus; the next stimulus was presented only after the group stopped showing alarm reactions. Alarm reactions were counted and their intensities were recorded and divided into 3 levels of intensity depending on the transition from one vigilant attitude to another: Level 1, the animal showed an alarm signal but did not change its vigilant attitude; Level 2, the animal moved from a vigilant attitude to a more reactive one (from lying to standing, from standing to moving); Level 3, the animal ran away. The stimuli were presented both outside and inside the enclosure to test the difference in the number of alarm reactions and alarm levels. The experiment only started when the animals showed no alarm signals and were in a relaxed state. When entering the enclosure, the observer walked first beside the fence. This was important because the sound of the gate could alarm the animals. In this way, the animals were able to return to a relaxed state before starting testing with the stimuli inside the enclosure. Only when the animals were relaxed did the observer start moving toward them and presenting the stimuli. One observer always remained outside the enclosure. The time of day was recorded, as well as the time with respect to exposure to tourists (before, during, after). The visibility conditions were recorded as good or bad depending on the presence/absence of rain or fog. 2.3. Statistical Analysis Data were analyzed in R (Version R4.1.2) . A preliminary model, using Generalized Linear Mixed Models (GLMM, lme4 package ), was used to test the influence of the variables of observation session ID and group size as random effects. Marginal and conditional coefficients of determination were calculated (using r.squaredGLMM, MuMIn package), and no difference was found between the two. For this reason, the variables were excluded as they did not explain the variance in the data. A simpler generalized linear model (GLM, lme4 package) was therefore run without random factors; the comparison of the two models based on the AIC value confirmed the preference for the simpler model. The variable of visibility conditions was initially included in the model but was then excluded during the variable selection procedure. Based on the descriptive statistics, the variable groups that induced a lower average number of alarm reactions (Wednesday, before, afternoon, males, outside, standing) were included in the intercept as a baseline. 2.4. Effects of Disturbance on the Number of Alarm Reactions Since the dependent variable (sum of alarm reactions i-iv) comprised count data, data were analyzed with Generalized Linear Models (GLM) using a Poisson link function to test for the effects of the various explanatory variables on the total number of alarm reactions shown by animals. A full model (with variables and their interactions) and a reduced model were produced (using the drop1 function) and selected based on their AIC values. Significance was set at p < 0.05. A post hoc pairwise comparisons test was then run using the glht function (Multcomp package, R, version R4.1.2). To understand which variable significantly affected the number of alarm reactions, seven variables were tested as fixed effects: type of stimulus (see Table 1), whether the stimulus was presented inside or outside (inside/outside), day of the week (day), time of the day (morning/afternoon), group type (nursery group or males), exposure to tourists (before, during or after tourists' presence) and the interaction between the type of stimulus and inside/outside. 2.5. Effects of Disturbance on the Intensity of Alarm Reactions To understand which variables significantly affected the number of responses by the animals pertaining to the three levels of intensity, canonical redundancy analysis (RDA) on the "stimulus x number of alarm reactions x level of intensity" matrix was performed, using the above-mentioned fixed effects as constraining variables (rda function of vegan R package, version 2.6-2). RDA is an extension of regression analysis that allows us to analyze the relations between multivariate response data (response intensity of animals in our study) and an explanatory variable data set. A preliminary backward selection of the explanatory variables and their interactions was performed starting from the full model using the ordistep function (vegan package) and then checking the reduced model to detect possible collinearities among the explanatory variables using the vif.cca function. No collinear variables were found. The significance of the reduced model was tested using the anova.cca function (vegan package), as well as axes' and terms' significance using, respectively, the by="axis" and by="term" arguments and running 999 permutations per test. The adjusted R-squared value of the model was obtained by using the function RsquareAdj. 3. Results 3.1. Behavioral Overview During the observation sessions, other unexpected alarm signals were observed; they were not included in the statistical analysis, but were very useful to better understand the animal behavior, so we decided to include them in this subsection. Such signals were performed by one animal in response to a higher disturbance and were probably aimed at alerting the whole group to the danger. The signals that we could observe were as follows: Urination before flight ; The stamping of the hoof on the ground ; Defecation during flight . Such signals were normally performed by the dominant male or by a female sentinel, a female without fawns in the nursery group, which remains always in an alert state, and was ready to warn the group of an approaching danger . Males and females had completely different reactions to disturbances. With good visibility conditions, the closest approach distance inside the enclosure to the males' group was in fact ca. 7 m, while that to the nursery group was ca. 60 m; distances were recorded with a measuring tape after the observation sessions. The two groups also had different flight strategies. The males tended to frequently stop during the flight to check the disturbance source , while the nursery group, after initiating a flight, ran away immediately without stopping until a safe spot was reached. Moreover, other differences within the groups were noticeable. In the male group, the last one fleeing was always the dominant one, which was also the least sensitive to disturbances. In the nursery group, the females without fawns were less sensitive to disturbances. With poor visibility conditions, animals were less sensitive to disturbances. During a rainy day, in fact, the distance between the nursery group and the observer was ca. 30 m. Moreover, animals were less disturbed by known stimuli, even when close, while new stimuli alarmed the animals even when far from the enclosure. However, they tended to become habituated to particular stimulus after a few expositions. It was observed that deer perceived the enclosure as a defensive limit that tourists could not cross, but did not discern whether a person, if close to the enclosure, was inside or outside. This was also observed when entering the enclosure: if the observer walked beside the fence, the animals quickly went back to a relaxed state after their first reaction to the gate sound. When the observer started walking toward the animals, they started displaying increasing alarm reactions. A brief video of the described observation is available in the Supplementary Materials (Video S1). 3.2. Descriptive Statistics A total of 27 observation sessions were analyzed. The mean number of alarm reactions per observation session was 9.07. During the weekend and on Monday, the animals were more sensitive to the stimuli; in fact, the mean number of alarm reactions was the highest on Monday (mean = 15.6; interquartile range (IQR) = 14.0) and Sunday (mean = 9.7, IQR = 5.0). Instead, the deer were less sensitive on Wednesday (mean = 6.2; IQR = 4). Exposure to tourists affected the animals' reactions to disturbances. They were particularly reactive to disturbances during the exposure (mean = 11.5; IQR = 7.3), compared to before (mean = 6.9; IQR = 4). The nursery group was more easily alarmed (mean = 12.4; IQR = 8) compared to the males (mean = 6.7; IQR = 4). The type of stimulus produced visible differences. All the stimuli performed inside the enclosure induced higher alarm responses, and stimulus M produced the highest number of reactions (mean = 11.7, IQR = 9), almost double compared to stimulus S (mean = 6.8, IQR = 5.5). We did not observe a remarkable difference between morning and afternoon, but the stimuli presented in the morning alarmed the animals more (mean = 9.2, IQR = 8) compared to the stimuli presented in the afternoon (mean = 8.9, IQR = 6.7). The stimuli presented inside the enclosure (mean = 13.5, IQR = 10.5) produced more than the double the alarm reactions compared to the stimuli presented outside the enclosure (mean = 5.9, IQR = 4). The results of the descriptive statistics of the effects of key variables on the number of alarm reactions are summarized in Table 3. 3.3. Effects of Disturbance on the Number of Alarm Reactions In order to test for the effect of several variables on the number of alarm reactions shown by the animals, GLM were used. The reduced model was selected on the basis of the AIC value. Stimulus U had a higher impact on the number of alarm reactions compared to S (p < 0.001) and to M (n.s.), while stimulus S had a lower effect than the other two types of stimuli (p < 0.001). The difference between morning and afternoon was not significant. The number of alarm reactions recorded on Monday was significantly higher than on Friday (p = 0.005), Wednesday (p < 0.001), Tuesday (p < 0.001) and Thursday (p = 0.002) and it was slightly significant on Saturday (p = 0.05) and Sunday (p = 0.07). On Wednesday, a lower number of alarm reactions was recorded, compared to Friday (p = 0.02), Saturday (p < 0.001), Sunday (p < 0.001), Thursday (n.s.) and Tuesday (n.s.). On Sunday, a significantly higher number of alarm reactions was recorded compared to Tuesday (p = 0.02). The tourists' presence affected the deer's reactivity; in fact, during the tourists' presence, the animals showed a higher number of alarm reactions compared to before (p < 0.001) and to after (n.s.). After the tourists' passage, the animals remained more reactive than before (n.s.). The two groups reacted to stimuli in different ways; in fact, the nursery group showed a significantly higher number of alarm reactions compared to the males (p < 0.001). The animals showed a higher number of alarm reactions when the stimuli were presented inside the enclosure (p < 0.001). Stimulus S presented outside significantly produced the lowest impact on the animals' alarm reactions. The deer in fact presented a lower number of alarm reactions to it compared to all the stimuli presented inside S (p < 0.001), M (p < 0.001) and U (p < 0.001), and the other stimuli presented outside the enclosure, M (p = 0.009) and U (p = 0.004). Stimulus M presented inside had the highest impact on the number of alarm behaviors, significantly higher than stimulus S presented outside (p < 0.001), stimulus S presented inside the enclosure (p < 0.001), stimulus M presented outside (p < 0.001) and stimulus U presented outside (p < 0.001). GLM results are shown in Table 4, while the respective pairwise comparisons are shown in Table S2 in the Supplementary Materials. 3.4. Effects of Disturbance on the Intensity of Alarm Reactions The RDA model explained globally 46% (adj. R-squared, p = 0.001) of the variance in the number of alarm reactions per level of intensity. The RDA graph depicted a main gradient along axis 1, related to the number of alarm reactions and the intensity of the response, with the maximum number and intensity linked to the positive values. The variables that most positively responded to the number and intensity of reactions were as follows: during the tourists' presence; the position inside the enclosure (inside) and its interactions with the type of stimulus (stimulus M) and with the days of the week (Friday and, secondly, Sunday); and the reaction displayed by the nursery group, along with its interaction with time (morning). The least intense reaction (intensity level 1) was mostly displayed after the exposure to tourists and on Friday, Saturday and Sunday. 4. Discussion The results of this study suggest that the red deer adversely reacted to the disturbance by tourists to different degrees depending on the day of the week. Animals showed greater sensitivity to disturbances when many tourists were present; the higher number of alarm reactions and the higher alarm intensity observed indicated higher discomfort. We observed that the days with more tourist arrivals (Friday, Saturday and Sunday) led to higher alarm rates of animals, presenting a higher number of alarm reactions, as highlighted by the GLM results. During these days, the stimuli presented inside the enclosure induced a more intense response than during the other days, suggesting that the animals were disturbed to the point of changing their current state (changing attitude or fleeing) than on every other day. This is visible in the RDA graphs, where "Friday:inside", "Sunday:inside" and "Saturday:inside" are positively related to the two highest intensity levels (2 and 3). However, as the GLM indicates, the highest number of alarm reactions was seen on Monday; this suggests that stressors' effects build up during the weekend, leaving the deer particularly sensitive to disturbances, despite lower exposure to tourists. This is consistent with Sibbald et al. , who found that the disturbance effects were still visible after 24 h after exposure to hillwalkers. The disturbance effects' accumulation may also explain the differences between the alarm reactions displayed before, during and after exposure to tourists. The animals were less sensitive to stimuli before tourists' arrival and more sensitive during the tourists' presence, with higher intensity during this particular condition. As shown in the GLM, the variable "during tourists' presence" was significantly higher than previously seen and positively related to the alarm reaction intensity levels 2 and 3 in the RDA graph. Tourists induced a change in attitude in the deer, with possible negative effects on their welfare, derived from a change in time spent grazing, resting or nursing. The animals still appeared sensitive to the disturbance after the tourists had left, but with a lower intensity level (intensity level 1). The time of the day did not particularly affect the animals' alarm reactions, but we observed a higher response on Monday morning. Stankowich's study demonstrated that female deer and young animals appear to flee to greater distances than males . Similarly, females with more vulnerable offspring exhibit greater flight responses than males and conspecific females . In line with this, the nursery group of our study, which included mothers, dry females and fawns, was significantly more sensitive to disturbances compared to the group composed of males. Females in the nursery group were visibly less approachable than males, and showed a higher alarm level by quickly fleeing after being exposed to the stimulus, as shown in the RDA graph, with the variable "nursery group" positively related to intensity level 2 and 3. The distance of the closest approach to the male group was in fact ca. 7 m, while that to the nursery group was ca. 60 m with good visibility conditions. The results suggest that the deer responded differently to different visual stimuli. They were less reactive to stationary movements; a person standing (S) alarmed the animals less and with a lower intensity, while the movements toward the animals (M) produced a significantly higher number of alarm reactions at a higher intensity level, with the variable "stimulus M" positively related to alarm intensity 2 and 3. It would be more energy-efficient for the animal to remain alert to the sight of a predator but only react strongly (i.e., run away) when the predator approaches, being an imminent threat to the animal . It was observed that deer perceived the enclosure fence as a form of protection; in fact, the stimuli performed outside the enclosure alarmed the animals less than the ones performed inside, with a lower number of alarm reactions and a lower intensity level. The stimulus M performed inside the enclosure particularly alarmed the deer, as shown in the GLM results and in the RDA graph, with the interaction "M:Inside" being the most related to alarm intensity 3. This is consistent with Whittington and Chamove's study , where deer took longer to react to disturbances when offered shelter and after a habituation period. The same conditions were found in the population object of this study. The animals were observed displaying excretion alarm signals, such as urination before flight and defecating during it. In other studies, ungulates were observed displaying the same alarm behaviors--for example, in goitered gazelle (Gazella subgutturosa) , pronghorn (Antidorcas americana) and fallow deer (Dama dama) . Generally, urination and/or defecation occurs as a response to stress in animals. Thus, excretion frequency can indeed be an index for fearfulness . In this situation, the behaviors displayed may be aimed at alerting the group to an approaching danger, since, after the signal, all the other animals were observed fleeing. Experiments with cattle demonstrated that the urination of stressed individuals may increase the stress levels of other conspecifics, which in turn become more fearful . Prolonged stress may lead to negative outcomes for the animal's body and, in trying to overcome such stress, the body will be placed in a survival state, and if it is not in a condition to cope with such a stressor, the animal will go through an exhaustion phase . Prolonged and chronic stress is an established risk factor for the development of depression-like states , even when not visibly shown by the animal. We observed that the dominant male was particularly less sensitive to disturbances compared to the whole group of animals. However, as stated by Nemets et al. , dominants, even if resistant to transient stressors, are more subject to the actions of prolonged stress, with the subsequent development of a depression-like state. Inactive subordinates, susceptible to transient stressors, contrarily show greater resistance to prolonged disturbance, without negative effects on their psycho-emotional status . Furthermore, clinical and preclinical studies have shown that females have higher sensitivity to stress . It is crucial to avoid stress during crucial periods, such as gestation, birth and nursing; for this reason, it would be advisable to avoid carrying out any activity inside the enclosure in such periods (in the study case, from May to mid-July). Mating season (in the study case, September to October) may be dangerous for humans inside the enclosure, due to the high aggressivity displayed by males. In this particular location, it is not possible to work during the winter due to the snow cover. Therefore, for this study case, the best time slots to perform activities inside the enclosure are March and April (if snow is not present) and August. August, despite being the more suitable one, is of interest to a great number of tourists. Consequently, it is important to establish the days/times that may be particularly suitable, avoiding the accumulation of disturbances (high tourist presence, humans inside the enclosure, etc.), which may increase the negative effects on the animals' welfare. Based on the results obtained from data elaboration, we can conclude that the best day to perform activities inside the enclosure is Wednesday, being the day on which the red deer showed alarm reactions at a lower intensity level. In addition, the alarm reaction number observed on Wednesday was the lowest of all the other days, and significantly lower compared to the reactions observed on Friday, Saturday, Sunday and Monday. However, Tuesday and Thursday could also be suitable days to carry out activities inside the enclosure, considering that the number of alarm reactions was significantly lower than the one observed on Monday. Conversely, the days on which to avoid carrying out activities that may stress the animals, inside the enclosure, are Friday, Saturday, Sunday and Monday. For this study case, taking into consideration the functional area distribution inside the enclosure, it would be recommended to concentrate the required mowing actions of different areas on different days, allowing the entrance of a few operators each day, in order to ensure that the animals have the possibility of hiding in a safe place. Some measures may be helpful in controlling disturbances by tourists and preventing direct contact with the animals, taking into consideration some tourists' inappropriate behavior, such as touching the more confident individuals through the fence or feeding the animals inappropriate food (even if not allowed). Building a second fence at least 2-3 m away from the principal deer enclosure might help to provide more space between the humans and the animals. In the study case, additional observation platforms should be constructed and placed so that visitors can view the animals from a respectful distance. 5. Conclusions According to the study results, we can assert that the deer's reactivity was affected by the tourists' presence; a significantly higher number of alarm reactions were in fact observed during the tourists' presence. The nursery group showed a significantly higher number of alarm reactions compared to the males. The animals showed a higher number of alarm reactions when the stimuli were presented inside the enclosure with respect to those presented outside. Finally, the days on which the animals were less sensitive and showed a lower number and intensity of alarm reactions were Wednesday, Tuesday and Thursday. In order to buffer the negative effects on the deer's well-being, it would be opportune to plan any action inside the enclosure during the days on which the animals are less sensitive to disturbances. Although it may be necessary for different purposes to enter into the enclosure, this certainly creates a certain level of disturbance for the animals. Therefore, it is necessary to carry out any action inside the enclosure when the disturbance does not further increase the state of discomfort and fear of the animals, which must be as free as possible from such conditions. Despite the observation period being quite limited, the obtained results might serve as a baseline for more in depth and prolonged studies, as well as to enhance the knowledge about the species' behavior. In addition, the proposed observational method could be applied to other locations and on other captive species, in order to identify the main behavioral patterns of animals and to establish the best times to carry out different activities inside the enclosures. Acknowledgments The authors wish to thank Paolo Kovatsch and Andrea Felicetti of the "Technical and Management Office of the State Forests Provincial Agency" (TN, Italy) for their excellent technical support, in addition to Milena Compagnucci for her contribution to observation activity. Supplementary Materials The following supporting information can be downloaded at: Table S1: List of observation sessions, Table S2: Tukey post hoc pairwise inter-group comparisons, Video S1: Alarm reactions of red deer. Click here for additional data file. Author Contributions Conceptualization, A.M. and P.S.; methodology, S.M. and D.C.; formal analysis, S.M. and F.M.T.; investigation, S.M., D.C. and P.S.; data curation, S.M. and D.C.; validation, S.M. and C.P.; writing--original draft preparation, S.M., C.P. and P.S.; writing--review and editing, S.M., P.S. and C.P.; supervision, P.S. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was included in the activity agreement (ndeg74 28 November 2011) between the University of Camerino and Provincia autonoma di Trento. Informed Consent Statement Not applicable. Data Availability Statement Data supporting the reported results are kept in the archive of Sara Moscatelli. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Aerial photo of the enclosure. On the left, a woodland is present, and the central part is an open pasture, and, on the right, a small lake is present. The blue line represents the easily walkable perimeter of the enclosure, while the red line corresponds to the less accessible path; mark shows the observation platform. Figure 2 Observation platform. Figure 3 The stimulus of the umbrella opened inside the enclosure. Figure 4 Alarmed subordinate male. The neck is prominent and the head is held high; the ears are directed toward the source of disturbance. Figure 5 Three different alarm signals. (a) The urination before flight displayed by the dominant male. (b) The stomping of the hoof on the ground performed by a subordinate male. (c) The defecation during flight performed by a female without a fawn. Figure 6 A female sentinel remains in an alert state while the other females eat. Figure 7 Two males stopping and looking back to the source of disturbance while fleeing. Figure 8 Redundancy analysis graph of the matrix "stimulus x number of animals' responses per level of intensity", using the following variables: type of stimulus, inside/outside, day of the week, time of the day (morning/afternoon), group type (nursery group or males), exposure to tourists (before, during or after tourists' presence) and the interaction between the type of stimulus and inside/outside. animals-13-00903-t001_Table 1 Table 1 Visual stimuli presented to the deer. Stimulus Stimulus Description S A person standing still M A person moving toward the animals U An umbrella opened animals-13-00903-t002_Table 2 Table 2 Types of vigilant attitudes. Vigilant Attitude Criteria Vigilant lying Body stretched on the ground with head held high; occasional turning of the head and ear twitching. Vigilant standing Standing still with either head held parallel to the body or high; neck prominent; sometimes freezing the posture with occasional ear twitching. Vigilant moving Rapid pacing (either brisk walking or running) with head held high and neck very prominent and ears straight. animals-13-00903-t003_Table 3 Table 3 Descriptive statistics, mean, median, Q1 (first quartile), Q3 (third quartile), IQR (interquartile range) of number of alarm reactions shown by deer as a reaction to the key variables. Alarm reactions before, after and during exposure to tourists, on Friday, Saturday, Sunday, Monday, Tuesday, Wednesday and Thursday, in the morning and in the afternoon, by males and nursery group, to the different stimuli, and to the stimuli presented inside or outside. Median Mean Q1 Q3 IQR Before 6 6.929 4 8 4 After 8 8.242 4 10 6 During 9 11.5 8 15.25 7.25 Stimulus S 5 6.795 3.5 9 5.5 Stimulus M 9 11.66 7 16 9 Stimulus U 8 8.059 5 9 4 Morning 8 9.176 4 12 8 Afternoon 8 8.957 4 10.75 6.75 Males 5 6.754 4 8 4 Nursery 10.5 12.38 8 16 8 Friday 8 8.467 4 12.5 8.5 Saturday 8 9.231 6 9 3 Sunday 9 9.765 7 12 5 Monday 15 15.62 8 22 14 Tuesday 8 7.643 4 9.75 5.75 Wednesday 5 6.231 4 8 4 Thursday 4.5 6.333 3.75 8 4.25 Inside 12 13.05 8 18.5 10.5 Outside 5 5.907 4 8 4 Stimulus S = a person standing, stimulus U = an umbrella opened, stimulus M = a person moving toward the animals. animals-13-00903-t004_Table 4 Table 4 Fixed effects table for the generalized linear model (GLM) fitted to the number of alarm reactions displayed by deer for type of stimulus, stimulus presented inside/outside the enclosure, time of the day, day of the week, exposure to tourists, group type and interaction between type of stimulus and inside/outside. Significance level set at p < 0.05. Estimate Std. Error z Value Pr(>|z|) (Intercept) 0.69492 0.25227 2.755 0.005876 Morning 0.07765 0.1531 0.507 0.612049 Friday 0.35715 0.15704 2.274 0.02295 Monday 0.72524 0.13959 5.196 <0.001 Saturday 0.48973 0.14639 3.345 <0.001 Sunday 0.51879 0.13895 3.733 <0.001 Thursday 0.22988 0.1802 1.276 0.202058 Tuesday 0.21046 0.1657 1.27 0.204053 After 0.28083 0.17769 1.58 0.114011 During 0.35799 0.10013 3.575 <0.001 Nursery 0.38549 0.08932 4.316 <0.001 Inside 0.58977 0.12993 4.539 <0.001 Stimulus M 0.44603 0.13289 3.356 <0.001 Stimulus U 0.56067 0.15675 3.577 <0.001 Inside:Stimulus M 0.10292 0.16405 0.627 0.530428 Inside:Stimulus U -0.28722 0.21913 -1.311 0.189956 Stimulus U = an umbrella opened, stimulus M = a person moving toward the animals. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000109

The sequence of Arabic numerals from 1 to 19 was taught to six chimpanzees, three pairs of mother and child. Each chimpanzee participant sat facing a touchscreen on which the numerals appeared in random positions within an imaginary 5-by-8 matrix. They had to touch the numerals in ascending order. Baseline training involved touching the adjacent numerals from 1 to X or from the numeral X to 19. Systematic tests revealed the following results: (1) The range 1 to 9 was easier than 1 to 19. (2) Adjacent numerals were easier than nonadjacent ones. (3) The "masking" (memory task) caused deterioration of performance. All these factors depended on the number of numerals simultaneously presented on the screen. A chimpanzee named Pal mastered the skill of ordering two-digit numerals with 100% accuracy. Human participants were tested in the same experiment with the same procedure. Both species showed relative difficulty in handling two-digit numerals. Global-local information processing is known to be different between humans and other primates. The assessment of chimpanzee performance and comparison with humans were discussed in terms of the possible difference in the global-local dual information processing of two-digit numerals. chimpanzee Arabic numeral touchscreen masking task working memory transitive inference decimal number system word-letter processing global-local processing cognitive tradeoff theory Grant-in-Aid for JSPS (Japan Society for Promotion of Science) Research FellowJP14J00488 JP16H06283 JP24000001 JP20002001 JP16002001 The present study was financially supported by Grant-in-Aid for JSPS (Japan Society for Promotion of Science) Research Fellow JP14J00488 to A.M. It was supported by grants MEXT/JSPS JP16002001, JP20002001, JP24000001, and JP16H06283 to T.M. pmc1. Introduction There are many studies on numbers from a developmental and evolutionary perspective. This study aimed to teach the numerical sequence from 1 to 19 in the decimal system to six chimpanzees. They had already learned the numerical order in the range 1 to 9 but the present study expanded this from 1 to 19. The original goal for chimpanzees was to touch the Arabic numerals on the display 1 to 19 in this order and understand the decimal number system. To test for a possible species difference in processing the numerals from 1 to 19, chimpanzee performance was directly compared with that of humans using the same task, apparatus, procedure, and place. Before introducing the research background of the existing literature on numbers, we explain and define some technical terms. "Number" means the concept of numbers. "Numerosity" refers to a property of a stimulus that is defined by the number of discriminable elements it contains . "Numerals" are Arabic numerals, which are the media for representing numbers. For a further detailed explanation of the decimal number system, we also use another term: "digit". A digit is a single numeral from 0 to 9. Therefore, in the decimal system, a numeral may be represented by two or more digits, such as 12 and 345. The number 12, for example, is referred to as a two-digit (or double-digit) numeral in this article. In sum, digits and numerals are used to refer to numerosity and to represent numbers. However, it must be noted that digits and numerals may not always have a number concept. For example, suppose that there are three classrooms at a school, such as classes 1, 2, and 3, or A, B, and C. The numerals in this example are not based on any cardinal or ordinal scale of numbers; instead, they are used on a nominal scale of numbers to distinguish three different things. The present study does not directly focus on numbers as it aimed to teach a sequence of Arabic numerals from 1 to 19. Therefore, this study is related to the existing literature on sequence learning and the sequential order of items . Numbers have been studied in humans and nonhuman animals . The human number concept has been studied from developmental perspectives . Without conscious counting, human infants can discriminate, represent, and remember a small number of items . Verbal counting may have precursors during infancy based on subitizing, the direct perception of numbers. Beyond subitizing, young children start to count one by one and comprehend numbers as part of their linguistic capability. The concept of numbers has been studied from an evolutionary perspective as well. Studies on numbers have been carried out with a wide range of species, including invertebrates , fish , pigeons , a grey parrot , rats , and monkeys and apes . In addition to behavioral studies, there are neurophysiological studies on monkeys that support the existence of a specific substrate of the number concept in the brain . Chimpanzees and monkeys can provide a unique opportunity for applying various tests in the same situations as humans. Ferster (1964) first used the binary number system in which two chimpanzees learned from 1 (001) to 7 (111) by turning three lights on and off . By introducing Arabic numerals, it is possible to match the chimpanzee studies precisely to studies in humans. Matsuzawa (1985) and Boysen and Berntson (1989) started to use Arabic numerals to represent numbers. Chimpanzee Ai of the Primate Research Institute of Kyoto University (KUPRI) learned to use Arabic numerals arranged on a keyboard connected to a computer . She learned to use the numerals from 1 to 6 on the keyboard and combined the skill with naming colors and objects shown in a display window. This task is referred to as symbolic matching-to-sample (symbolic MTS). She succeeded to press the corresponding keys for "red" + "pencil" + "5" in sequence for describing five red pencils that were shown to her. Ai mastered the skills of both ordinals and cardinals, and coding and decoding . The ordinal sequence of numerals was expanded from 1 to 9 . Additionally, Ai learned the meaning of the numeral 0 . The other chimpanzees of KUPRI have also learned numbers in various tasks in this line of studies . The major findings of earlier studies on numbers in nonhuman primates (mainly chimpanzees and macaques) can be summarized with the following three points. First, they can master the skill of using Arabic numerals. Second, they may use the numerals for both cardinality and ordinality and in both productive uses and receptive uses. Third, there have been no studies teaching the use of decimals to systematically increase the repertoire of using numerals beyond 9. What kind of numerosity judgment is used by animals: subitizing, counting, or magnitude estimation? This is not clear, because the numerical repertoire of the animals is still small, with a few exceptions . One solution is to extend the numerical sequence as a larger repertoire, as a first step for further study following numerosity judgment. With respect to previous efforts, the present study is unique in being the first attempt to expand the numerical sequence from one-digit numerals to two-digit numerals up to 19. It should be stressed that this study is about the sequential order of the numerals, not numerosity judgment. The task is not about cardinality, such as when "counting" objects. Sequential learning of numerals may provide the basis for the psychophysical sense of numerosity and the corresponding use of symbolic numbers . Young human children recite numerical sequences, saying "one-two-three-four-five-six-seven-eight-nine-ten", and so on, without fully understanding the meaning. The sequence of numerals may play a fundamental role in the future understanding of the number concept. The present study aimed to show this kind of precise numerical sequencing in chimpanzees. Numbers can be described in various ways. For example, the number "ten" can be described in the binary system as 1010. In the octal system, it is 12. In the decimal system, it is 10. In the duodecimal and hexadecimal systems, it is A. The decimal number is a structuralized notation system: there is a "spiral staircase" or a "clockwise" structure. Each stair of the single-digit numerals goes up and round to the next stair of two-digit numerals from 10 to 19, to further stairs from 20 to 29, from 30 to 39, and so on. The question is whether the chimpanzee can master this kind of ordering system of decimal numerals. The present study aimed to teach three mother-child pairs of chimpanzees to use the numerical sequence 1 to 19 and to understand the notation system of the decimal. The participants had already learned the numerical sequence of 1 to 9 and one step forward to the sequence from 10 to 19 . Based on the previous research, the present study aimed to establish a way to examine and evaluate chimpanzees' learning of 1 to 19 in the decimal system. The performance of processing two-digit numerals was directly compared between humans and chimpanzees in the same tests with the same apparatus and procedure. This cross-species comparison was planned to clarify the underlying mechanism for processing two-digit numerals. The present study postulates that the key issue is global-local dual information processing in humans and nonhuman animals . The well-known phenomenon called the Navon effect was discovered by David Navon, who measured the speed at which people process global and local information. When objects are arranged in groups, they possess global features and local features. For example, a group of trees has local features (the individual trees) and the feature of a forest (the trees together). In this framework, a group of digits (two-digit numerals) has local features (the individual digits) and a global feature (the digits together make a numeral that has the real meaning). Humans are faster at identifying features at the global than at the local level (in other words, they show global precedence). In contrast, the existing literature on nonhuman primates (chimpanzees , baboons , and capuchin monkeys ) suggests they identify the local level faster than the global. Thus, this study is a pilot study to explore possible evolutionary origins of the difficulty in processing two-digit numerals. 2. Materials and Methods 2.1. Participants: Chimpanzees The participants were three mother-child chimpanzee pairs. The study period lasted three years and eight months, from April 2011 to November 2014. All three children were 10 years old at the beginning and reached 14 years of age by the end (Table 1). They were not fully independent from their mothers, so the mother-child pairs came together to the test booths. During the present study period, the pairs came to the booth as follows: 630 days in total for Ai-Ayumu, 477 days for Chloe-Cleo, and 498 days for Pan-Pal. In prior work, all six participants had learned the sequence from 1 to 9 . An adult female, Ai, had learned the meaning of 0 as well, so her range was 0 to 9. She also mastered skills in the ordinal and cardinal aspects of numbers . The other five chimpanzees had experience touching the numerals in ascending order, but no prior experience using the numerals for the cardinal task. As the first step toward 1 to 19, all six chimpanzees gained experience in generalizing the skill to the set of numerals from 10 to 19 by introducing the two adjacent numerals successively: 9-10, 10-11, 11-12, 12-13, and so on up to 18-19. The initial training procedure on two-digit numerals is described in detail in a separate article . Thus, they were ready to be trained and tested on the numerical sequence from 1 to 19 in the present study. The rearing condition and the experience of the six chimpanzees are described in Matsuzawa et al. . The present study is a part of a larger project studying chimpanzee cognition at KUPRI . The participants experienced different kinds of cognitive experiments in parallel during this study period . Please refer to the details in Appendix A.1. In general, KUPRI chimpanzees had a maximum of seven feeding opportunities in a day: four laboratory tests plus three daily meals. The KUPRI schedule included two series of sessions in the morning and another two in the afternoon for cognitive tests. One series of sessions either in the morning or in the afternoon was allocated to each pair for the present study. This arrangement simulated the daily cycle of natural feeding behavior in wild chimpanzees . The chimpanzees were free and spent the time between sessions in the enriched outdoor enclosure with trees, shrubs, and a stream . When the present study started, the chimpanzee group had 14 members , including 3 generations of chimpanzees of patrilineal lineage, similar to a wild chimpanzee community. The three children had grown up in this socially enriched environment. All chimpanzees understand their names . The experimenters called the name of a particular chimpanzee to invite them into the test booth. The chimpanzees were completely free to choose whether or not to participate in tests. However, as this was a daily routine, all chimpanzees were willing to come to the booth. This study followed the Guideline of Care and Use of Nonhuman Primates, KUPRI, and was approved by the Animal Welfare and Care Committee of KUPRI (see Ethics statement). 2.2. Participants: Humans There were 6 human participants, 24-29 years old, including both sexes (4 females and 2 males). All were right-handers. They were students and staff of KUPRI. We collected the chimpanzee data first and then tested the humans in 2015 (April to June). The humans were tested with the nonmemory task only (see details of the test procedure in Section 3.4). The task was to touch the numerals on the screen in ascending order. The range was either 1 to 9 or 1 to 19. The number of numerals was 3, 4, or 5. The numerals were adjacent or nonadjacent. There was no memory load, so the task was an easy one for adult humans. The human participants were naive to the task but they had plenty of opportunities in the institute and their daily life to use the touchscreen and Arabic numerals. We did not explicitly train the participants to become experts in quickly touching the numerals. Computer use, such as regularly playing computer games, may lead to decreased response latency in some visual/motor tasks; however, this was not the purpose of the present study, which aimed to evaluate the performance of naive humans in this setting of two-digit numerals without specific training (see Ethics statement). 2.3. Stimuli The stimuli were 19 Arabic numerals from 1 through 19. The font was MSP Gothic. They appeared on a CRT monitor touchscreen. The numerals were displayed as white stimuli on a black background. The height of the numerals was 3.5 cm. The resolution of the monitor was 1023 by 768 pixels. The numerals appeared randomly in one of the 40 imaginary positions in a matrix of five rows and eight columns. There was a so-called start key to initiate a trial: it was a white circle that appeared at the sixth bottom row of the CRT. 2.4. Number, Numerosity, Numeral, and Digit As we described in the Introduction, there are various related and confusing terms to describe the study of numbers. Please see the definition in the Introduction about "Number", "Numerosity", "Numeral", and "Digit". 2.5. Apparatus A "touchscreen" has an input device called a "touch detector or touch panel" and an output device called a "monitor or screen" . A touch by the participant was detected by a touchscreen (Mitsubishi Electric Engineering 15-inch LCD touchscreen monitor: TSD-FT157-MN and TSD-AT1515-MN, Tokyo, Japan). As shown in Figure 1, the touchscreen was encased in a translucent acrylic box and set just behind a translucent panel which prevented the chimpanzee from strongly banging the screen. The participant touched the screen through a window opened at the lower part of the box. For an adult chimpanzee sitting in front of the monitor, the center of the monitor was at eye level. The distance from the eyes to the monitor was about 30 cm. The history of this apparatus is described in Section S5.1 of Supplementary Materials S5. To test a mother and child at the same time, we used a "twin booth" consisting of two identical booths located side by side. Each booth was 1.8 m by 1.8 m by 2.0 m in height. Spontaneously, all three mothers came to the far side of the twin booth and the three children chose the near side to the entrance. Human participants were tested by using the same apparatus following the same procedure at the same place, but alone. The task was controlled by standard PC-type computers running under Windows XP(c) and Windows 7 (c) OS. The program controlling the experimental session was made with Visual Basic (c) 6.0. The entire experiment was preprogrammed with the previously determined stimulus sequence and position sequence files. The food reward was delivered by an automatic feeder (Bio Medica: BUF-310-P50, Osaka, Japan) connected to the computer. The feeder had a disc of 50 small compartments, and a brush rotation automatically delivered a piece of food to the place just below the touch panel. Thus, daily cognitive test sessions were fully automated in preplanned ways and with no interference by experimenters. This means that there was no social cueing. 2.6. General Procedure To come to the experimental booth, the chimpanzees walked through a corridor about 50 m long which connected the outdoor enclosure to the booth. Each trial went as follows. A white circle appeared on the monitor. When the participant touched it with their fingers, the circle disappeared, and several Arabic numerals immediately appeared in random positions on the monitor. There were 5 by 8 imaginary matrix positions on the monitor in which each numeral appeared. The correct response was to touch the 'smaller' number first, followed by the 'larger' number(s). CRF (Continuous Reinforcement Schedule) was applied: every correct response was rewarded. The correct choice was signaled by a chime and followed by automated food delivery. The food reward was a piece or a half-piece of raisin or a very small piece of apple (1.5 g per piece on average). A wrong response was signaled by a buzzer sound and followed by a 3 s blackout and a return to the start key. The inter-trial interval (ITI) was set at 1 s. After the ITI, the next trial started: a white circle appeared on the monitor. A session consisted of 50 trials without exception in all training and assessment tests. It was completed in 3 to 5 min. The inter-session interval was 1 to 3 min. Participants received four to six continuous sessions each day on average. After completing the 30 to 60 min series of sessions for the cognitive tests, the participants rejoined the other chimpanzees in the outdoor enclosure . Before starting each session, the experimenter ran a Q and A with the computer program to set up the task parameters. Parameters of a session were as follows: the number of trials in a session (fixed at 50 trials), ITI (fixed at 1 s), fixed ratio (FR) for reinforcement (fixed at FR1), time out (fixed at 3 s blackout), correction trial (no correction trial; an error trial did not repeat and the next new trial started), sequence files of stimulus presentation and the position of numerals appearing in the imaginary 5-by-8 matrix on the screen (All conditions of stimulus and position were randomized in a session). 3. Method: Baseline Training and Assessment Tests 3.1. Baseline Training of Touching Adjacent Numerals: VarNumMix (VNM) Task 3.1.1. VNM-Startfix Task During the present study, chimpanzees received daily training in numerical ordering using the touchscreen system. The numerals appeared on the screen and the task was to touch them in ascending order. The task was named the "VarNumMix (VNM)", in which adjacent numerals appeared in every trial, although the number of numerals varied in each trial. Each trial was unique and randomized within a session. For example, the "VNM 1 to 14" task means that a trial could be either 1, 1-2, 1-2-3, 1-2-3-4...or 1-2-3-4-5-6-7-8-9-10-11-12-13-14. It must be noted that all of the numerals were randomly scattered on the screen . VNM tasks of the present study started from "VNM 1 to 9" to "VNM 1 to 10", "VNM 1 to 11", "VNM 1 to 12", and so on, step by step. This sequence in the VNM task was characterized by the "Startfix" condition in which the numerals always started from the numeral 1. Therefore, this is specifically called the "VNM-Startfix" task hereafter. 3.1.2. VNM-Endfix Task In another type of VNM task, the end of the sequence was always fixed as the numeral 19. Thus, this is called the "VNM-Endfix" condition. For example, "VNM-Endfix 13 to 19" means that a trial could be either 19, 18-19, 17-18-19, 16-17-18-19...and so on up to 13-14-15-16-17-18-19. This training focused on teaching the end part of the long numerical sequence from 1 to 19. In the present study, we started with "VNM-Endfix 16-19". After reaching the criterion of 90% accuracy in a 50-trial session, the task became one step more difficult, meaning VNM-Endfix 15 to 19, and so on. 3.1.3. Baseline Training to Maintain Motivation The baseline training of VNM tasks was characterized by adjacent numerals. In both Startfix and Endfix conditions, chimpanzees learned to touch the adjacent numerals in ascending order. Nonadjacent numerals were not used in the training but were used for further assessment tests. This training method of VNM tasks aimed to keep accuracy and motivation high by reducing the difficulty of the task. It is very important to keep motivation high for chimpanzees to participate in cognitive tasks. For example, suppose that all 19 numerals appeared scattered across the screen at one time and the chimpanzees were asked to touch them in ascending order. The chance level of the correct order is 19P19 equal to one out of 1.21645 x 1017, which is approximately one out of some quadrillions, too much for some chimpanzees in the training phase, which could easily lead to loss of motivation to participate. Thus, the present study used the VNM training method to mix difficult trials with easy ones within a session. 3.1.4. Baseline Training to Assess Daily Fluctuations Baseline training occurred each day. The chimpanzees received baseline training on the VNM task whenever they came to the booths. This was done to improve their performance at ordering numerals while evaluating daily fluctuations in performance in each individual. In general, the criteria for moving to the next condition were kept constant, i.e., more than 90% correct in a session. However, in some chimpanzees, the criterion gradually shifted to 85% and then 80% accuracy in a session, depending on their performance. The whole experiment was designed to make the tasks gradually more demanding based on each individual's daily performance (see Section 4.1 and Section 4.2). 3.2. Assessment Tests: Range, Adjacency, Number of Numerals, and Memory In parallel to the baseline daily training, chimpanzees underwent systematic tests to assess their understanding of numerical sequence . The assessment tests aimed to evaluate the progress of learning the numerical sequence of 1 to 19. The tests were carried out under the condition of differential feedback (positive reinforcement training); a correct answer was rewarded with food and an error was not. Therefore, in terms of the reinforcement history, it was an assessment test and also training on the numerical sequence by differential feedback. In the assessment tests, we focused on four factors that could be influencing performance on numerical ordering. First, the range of numerals was either 1 to 9 or 1 to 19. Second, the adjacency was either adjacent numerals or nonadjacent ones. Third, the number of numerals tested was 3, 4, or 5. Fourth, the 'memory' refers to whether the task was a nonmemory (nonmasking) task that required no memory at all or a task that required memorizing of numerals. In the memory condition, after touching the 'smallest' numeral, the other numerals were immediately masked by a black-and-white checker pattern. Therefore, the chimpanzees had to remember which numeral appeared in which position of the monitor before starting the touch. The memory condition is called the "masking task" . All chimpanzees were tested about factors of adjacency and memory in the range 1 to 9 . They underwent some sessions for masking tasks in the range 1 to 9 during the present study to assess any aging effect on working memory. However, the two-digit numerals in the range 1 to 19 were only used in the present study. The four factors were systematically introduced in the assessment tests. For example, in the test condition "5, nonadjacent numerals, in the range 1 to 19, with nonmemory", five numerals, such as 5-9-12-13-19, were scattered in random positions on the screen. There were four factors: range, adjacency, the number of numerals, and memory, giving 2 x 2 x 3 x 2 = 24 conditions. Each condition was tested in one session of 50 trials. The order of testing the 24 conditions was not fully randomized but moved from easier to more difficult ones. The assessment test was conducted twice to confirm performance stability (September 2013 and March 2014, a 6-month interval). 3.3. Assessment of Adjacent Numerals Including the Numeral 10 In addition to the four factors, there is a unique problem with numerals in the decimal number system: the carryover at the numeral 10. We gave an assessment test to evaluate the difficulty of processing the numeral 10 in chimpanzees. The task was to touch four adjacent numerals in the range 1 to 19. Thus, there were 16 patterns in this task: 1-2-3-4, 2-3-4-5, 3-4-5-6, and so on up to 16-17-18-19. One of them was randomly presented in a session of 50 trials. We ran this test 10 times, for a total of 500 trials for each chimpanzee. Thus, each of the 16 patterns was tested 31 or 32 times. We collected the data for each pattern at the end of the study period. This is an assessment test and also a part of the intensive training of adjacent numerals including the numeral 10. 3.4. Comparison of Humans and Chimpanzees in Terms of Accuracy and Response Latency Chimpanzee performance was directly compared with human performance. All six chimpanzees received the assessment tests for four factors. However, two adult chimpanzees, Chloe and Pan, did not master the sequence from 1 to 19 completely. This means that the VNM-Startfix task and VNM-Endfix task did not cover the entire range of the numerals from 1 to 19. Therefore, these two chimpanzees were excluded from further tests for human-chimpanzee comparisons, leaving the four chimpanzees Ai, Ayumu, Cleo, and Pal, who mastered the skill of ordering the numerals in the range 1 to 19. They were compared with six humans (H1, H2, H3, H4, H5, and H6) who received the same test. Chimpanzee data were the same as a part of the assessment test. Human data were collected under the same procedures as chimpanzees. The chimpanzees and humans were compared in the condition of nonadjacent numerals. There were 12 conditions in total. The present study focuses on one condition among them, namely "four nonadjacent numerals in the range 1 to 19 with a nonmemory task". In this task, the four numerals were randomly chosen from 1 to 19. Therefore, there are many combinations of four numerals: 19C4 = 3876. The 3876 patterns can be classified into five groups. The first group is labeled as "Under10", which means that four numerals are all one-digit numerals such as 2-5-6-9. The second group is "Cod1", which means that a two-digit numeral is included as one of the four numerals such as 1-5-7-13. The third group is "Cod2", which means that two two-digit numerals are included as two of the four numerals such as 5-7-12-18. The fourth group is "Cod3", which means that three two-digit numerals are included such as 3-10-15-19. Finally, the fifth group is "Over10", which means that four numerals are all two-digit numerals such as 11-14-17-18. The major difference among the five groups is the total number of digits to be processed. The more two-digit numerals, the more digits to be processed. Note that the total number of digits that appeared on the screen increased from four, five, six, and seven to eight along with the five conditions: from all one-digit to all two-digit conditions. The human participants received the verbal instruction: "Please touch the numerals in ascending order". There was no instruction to make quick decisions. Humans and chimpanzees were tested at the same place using the same apparatus and following the same procedure. The differences were no verbal instruction for chimpanzees and no food reward for humans. 3.5. Summary of Methods To sum up the Methods, Table 2 describes all tasks described in Section 3. The list includes a basic description of the task and which chimpanzees received the task. It shows short names for the tasks, which are referenced throughout the text. The tasks are ordered in increasing complexity. The table is provided for looking up the different tasks while reading the manuscript. Concerning the comparison of the two species, three points should be noted. First, chimpanzees were trained to touch two-digit numerals in VNM tasks, whereas humans received no such training because they were already familiar with this response. Second, chimpanzees were assessed on four factors, compared to three factors for humans (memory load not tested). The two species were compared in the same condition of nonadjacent numerals. Third, only chimpanzees were assessed for the effect of memory load, intensively surveyed for the role of the numeral "10", and repeatedly assessed on tests on four factors. These tests are designed to identify the difficulty of processing two-digit numerals for chimpanzees. Details of the training method and metadata are summarized in Appendix A.2. 4. Results 4.1. Baseline Training of Touching Adjacent Numerals: VarNumMix (VNM) Task 4.1.1. Summary of the VNM Tasks: Accuracy of Each Chimpanzee in Each Stage All six chimpanzees were trained to touch numerals from 1 to 19 in ascending order. The baseline training was to touch the adjacent numerals on the screen from 1 to X. X is the maximum number in the sequential ascending order. As described in the Methods section (Section 3.1), the task was called the "VarNumMix task (VNM in short)", in which each trial was varied in terms of the number of numerals. The task of "VNM-Startfix 1 to 16" means that each trial always started from the numeral 1 (which means the start numeral was fixed) and could be either 1-2, 1-2-3, 1-2-3-4, and so on, or 1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16 within a session of 50 trials. There was also the "VNM-Endfix" task. Table 3 summarizes each chimpanzee's accuracy in each stage of training. 4.1.2. VNM-Startfix Task The baseline training of the VNM-Startfix task continued up to 1 to 16 for chimpanzee Ai. Figure 4 shows Ai's performance as the representative. Accuracy gradually dropped as a function of the number of numerals to be processed. Ai reached the level of sequentially touching numerals from 1 to 16, but not more. We stopped at this point to avoid too much pressure on the chimpanzee. The other chimpanzees followed the same pattern. Ayumu reached the stage of 1 to 18, and Pal reached 1 to 17. Data for each chimpanzee and the average performance of the six chimpanzees are shown in Table 3 and Appendix A.3. See the trials in the Supplementary Materials (Video S1: video clip of Ayumu for the VNM-Startfix 1 to 17 task). 4.1.3. VNM-Endfix Task We introduced the "Endfiix condition" in addition to the training with the "Startfix condition". This task tested adjacent numerals from X to 19 to teach the end part of a long sequence. The task always ended with the numeral 19. Figure 5 shows the performance of Ai as a representative. She started with the task of "VNM-Endfx 16 to 19" and ended with "VNM-Endfix 9 to 19". The longest numeral of the "VNM-Endfix 9 to 19" is 12 numerals (9-10-11-12-13-14-15-16-17-18-19). We stopped the baseline training of VNM-Endfix for Ai at this point. The other chimpanzees followed similarly. The data for each chimpanzee and the average performance of the six chimpanzees are shown in Table 3 and Appendix A.3. See the trials in the Supplementary Materials (Video S2: video clip of Pal for the VNM-Endfix 11 to 19 task). 4.1.4. Comparison of Startfix Task and Endfix Task In the Baseline training, the two tasks, VNM-Startfix and VNM-Endfix, helped the chimpanzees to learn sequential touching from 1 to X and also from X to 19. The two tasks were complementary to each other but not equal. The Startfix condition was easier than the Endfix condition for all chimpanzees because the Startfix condition had been trained for a long time since the beginning of training the chimpanzees on numeral orders. Another reason is in the starting numeral of each trial. In the Startfix condition, every trial started from the numeral 1, so the chimpanzees had to find the numeral 1 among the numerals scattered on the screen. There was no varied condition, but the numeral 1 was always found on the screen. In contrast, in the Endfix condition, every trial changed the 'smaller' number while keeping the last numeral 19 fixed. It was difficult for the chimpanzees to find which was the 'smaller' number on the display in this case because it always changes from one trial to the next. Figure 6 shows the comparison of Ai's performance in the two conditions. There is an overlapping zone for 9, 10, and 11 numerals. Performance on the Startfix condition (94% on average) was better than the Endfix condition (76% on average) in the corresponding overlapping zone. Although the Endfix condition was demanding, the chimpanzees mastered the task. This means that the chimpanzee can find the 'smaller' number even though there are many numerals simultaneously on display. Each individual's data are shown in Appendix A.3 (Table A1), and the average data are shown in Appendix A.3 . 4.1.5. Best Performance in Touching Numerals in the Range 1 to 19 The other chimpanzees' data showed a similar pattern to Ai. The longest sequence tested were as follows: Ayumu, 1 to 18 and 8 to 19; Pal, 1 to 17 and 9 to 19; Ai, 1 to 16 and 9 to 19; Cleo, 1 to 15 and 10 to 19; Chloe, 1 to 13 and 13 to 19; Pan, 1 to 12 and 14 to 19, respectively. Five of the six chimpanzees show overlap in the range 1 to 19: Ai, Ayumu, Chloe, Cleo, and Pal: only Pan failed to reach this level. They could touch the two-digit numerals in the range 1 to 19 if the sequence was divided into two parts. Ayumu touched 1 to 17 precisely in this order , which was the best performance obtained so far in processing two-digit numerals in one trial. 4.2. Range, Adjacency, Number of Numerals, and Memory 4.2.1. Four Factors As described in the Methods section (Section 3.2), four factors may influence numerical ordering performance: range, adjacency, number of numerals, and memory. Table 4 shows the results of the second assessment test (The results of the first test are in Appendix A.4 Table A2). The interval between the two tests was about six months. During this time, average performance was slightly improved by about 2% accuracy. The correlation between the two tests was very high: r = 0.941. To avoid presenting the same results twice, we describe the data from the second and the final test as follows. Table 4 shows all individual data in which all four factors had a significant effect. First, for all six participants, the range 1 to 19 was more difficult than 1 to 9. Second, the nonadjacent numerals were more difficult than adjacent ones. Third, the memory task was more difficult than the nonmemory task. Fourth, in all conditions, when the number of numerals increased to three, four, and five, the task became more difficult. There was almost no exception in the conditions of 2 x 2 x 2 x 3 = 24 cases (range x adjacency x memory x number of numerals) in all six individuals. There were some exceptional cases among 144 cells. For example, in three cases Ayumu performed better on the masking (memory) task than the nonmasking (nonmemory) task. For Ayumu, the memory task was as easy as the nonmemory task, due to a sort of ceiling effect (100, 98, and 96% accuracy in a session of 50 trials), alongside inevitable small daily fluctuations in performance. Although the tendency was the same among these six chimpanzees, there was a clear individual difference in the average performance level. Overall performance in the assessment test was best in Ayumu, with 85% accuracy on average over all 24 conditions. The second best was Pal (79%), followed by Ai (75%) and Cleo (75%), with the remaining two adults, Chloe (61%) and Pan (57%), further behind. There was a correlation between the performance of VNM tasks (Table 3) and the assessment test (Table 4): better learners in training showed better performance on the tests. This result showed that the children outperformed their mothers. 4.2.2. Range Figure 7 shows the effect of range: comparison of a nonmasking task in the range 1 to 9 and the range 1 to 19 (see data in Table 4). For example, in the five-numeral condition, the display showed five numerals, such as 1-3-5-8-9 or 4-7-12-13-16. The wide range 1 to 19 was more difficult than the narrow range 1 to 9. Performance decreased as a function of the number of numerals: three, four, or five. 4.2.3. Adjacency Figure 8 shows the comparison of adjacent and nonadjacent numerals in the range 1 to 19 (see data in Table 4). For example, in the five-numeral condition, the display showed five numerals, such as 7-8-9-10-11, in the adjacent condition and 8-10-13-16-19 in the nonadjacent condition. The nonadjacent condition was more difficult than the adjacent one. This tendency was also confirmed in the range 1 to 9 (see Table 4). As we predicted, performance decreased as a function of the number of numerals: three, four, or five. 4.2.4. Memory Figure 9 shows the effect of memory: comparison of nonmasking and masking tasks in the conditions of range 1 to 19 and nonadjacent numerals (see the bottom six rows of Table 4). For example, in the five-numeral condition, the display showed the five numerals 5-12-13-16-19. In the masking (memory) task, the participant had to remember the five numerals before touching the first one. Performance decreased as a function of the number of numerals. It also decreased in the nonmasking control task. For the chimpanzees, it was difficult to touch, for example, the numerals 5-8-12-14-19. Therefore, the decrement in performance was not solely due to memory but was partly due to the difficulty of touching five numerals which included two digits in the decimal number system. In short, the memory ability of chimpanzees can be tested with the range of 1 to 19, but a proper control condition is required to subtract the contribution of the nonmemory factor; see Figure 3 and the Supplementary Materials (Video S3: video clip of Pal for the nonmemory task of four nonadjacent numerals in the range 1 to 19, and Video S4: video clip of Pal for the memory task in the same condition). 4.3. Adjacent Numerals Showed Difficulty in Processing the Numeral 10 We gave an assessment test to evaluate the difficulty of processing the numeral 10 in chimpanzees. The task was to touch four adjacent numerals in ascending order. There were 16 patterns in this task: 1-2-3-4, 2-3-4-5, 3-4-5-6, ..., 16-17-18-19, randomly presented in a session (see Section 3.3). Figure 10 shows accuracy (% correct) with four adjacent numerals. It shows the accuracy of 16 patterns of four adjacent numerals in the range 1 to 19. We tested four chimpanzees, Ai, Ayumu, Cleo, and Pal, who mastered the numeral order in the range of 1 to 19 by showing good overlapping in the range of numerals in the VNM-Startfix and VNM-Endfix tasks. There were 16 patterns in this task, which can be classified into three groups. The first is "Group 1 to 9" (Group 1), in which the four adjacent numerals are within the range 1 to 9. There were six patterns from 1-2-3-4 to 6-7-8-9. The second is "Group 11 to 19" (Group 2), in which the four adjacent numerals are within the range 11 to 19. There were six patterns from 11-12-13-14 to 16-17-18-19. The third is in between: "Group including 10" (Group 3), in which the four adjacent numerals include the numeral 10. There were four patterns in this group: 7-8-9-10, 8-9-10-11, 9-10-11-12, and 10-11-12-13. Average accuracy was 91.7% (SD = 5.5%) in "Group 1 to 9" and 92.1% (SD = 5.6 %) in "Group 11 to 19". The chimpanzees processed the two-digit numerals (11, 12, 13....19) as easily as the one-digit numerals (1, 2, 3....9) if the numeral 10 was not one of the four numerals. They might, for example, put the left side of the two-digits aside and make the judgment by focusing on the right side. It must be noted that this assessment test was performed for adjacent numerals and not nonadjacent numerals. In contrast, the average accuracy dropped to 80.9% (SD = 13.3%) in Group 3. If the statistical analysis is applied to the data, it shows a significant difference between the groups: p = 0.022 in Group 1 and Group 3, and p = 0.020 in Group 2 and Group 3, in the t-test. The chimpanzees mastered the skill of processing the numerals 1 to 19. Even more, the processing of 1-9 and 11-19 was equal in terms of accuracy (not the response latency described later). However, if the numeral 10 was included in the patterns, it was a little difficult for them. Although the tendency was common to all four chimpanzees, there was a marked individual difference: compared to the other three chimpanzees, Cleo had extreme difficulty in processing the numeral 10. Because of this individual difference, it may not be adequate to apply statistical tests for the group level. However, in addition to Cleo, Figure 10 shows that the sequence of 8-9-10-11 was the most difficult one for the other three chimpanzees too. The accuracy of this sequence was about 80 % correct for the three chimpanzees. It must be also noted that the chance level of touching four numerals in the correct order is about 4%. Therefore, in conclusion, chimpanzee performance was very high even in difficult patterns. 4.4. Comparison of Humans and Chimpanzees: Accuracy The difficulty of processing two-digit numerals (the numeral 10 and more) was shown in humans too. Figure 11 shows the accuracy (% correct) of performance in chimpanzees (n = 4) and humans (n = 6), showing both the individual data and the average. All participants were tested on nonadjacent numerals in 1 to 19 in the four-numeral condition. For example, the display showed four numerals such as 2-5-7-8, 4-8-9-15, 6-8-12-15, 5-10-12-19, 13-16-17-19, and so on. There are many combinations of four numerals: 19C4 = 3876. Further details of human performance are given in Appendix A.5 and Supplementary Materials S5. Among the 3876 patterns, there are one-digit-only numerals, such as 2-5-7-8, which are labeled "Under10". Combinations such as 4-8-9-15 are called "Cod1," which means they contain one two-digit numeral. Combinations such as 6-8-12-15 are labeled "Cod2" (contains two two-digit numerals). Combinations such as 5-10-12-19 are labeled "Cod3" (three two-digit numerals). Combinations such as 13-16-17-19 are called "Over10": all stimuli are two-digit numerals (see Section 3.4). In the four-numeral conditions, there were 24 possible sequences of four numerals (4P3), with only one correct order. Therefore, the chance level of correctly touching four numerals was only about 4%. As shown in Figure 11, the performance of chimpanzees was very high; much higher than chance. Humans are good at touching four numerals in ascending order, with accuracy in the range of 94 to 100%. Humans showed no difficulty in touching four numerals in ascending order if they consisted of either single digits only (Under10) or double digits only (Over10). However, humans made some interesting mistakes in the intermediate conditions (Cod1, Cod2, Cod3) containing mixtures of one-digit and two-digit numerals, except for one individual (H3) who took a very long time to make decisions (see response latency results in the next section). In other words, even for human participants, there was some 'difficulty' in touching four numerals in ascending order in the range 1 to 19. This was most marked in Condition "Cod2" with numerals such as 6-8-12-15, in which the participant may have touched "12" first rather than "6". This kind of confusion can occur even in human adults when judging ascending order of a mixture of one-digit and two-digit numerals in the decimal system. However, humans correctly identified the numerical order if all four numerals are 10 or more (Condition "Over10"). The Over10 condition was as easy as the Under10 condition at least in terms of accuracy. This means a complete understanding of the carry-over of the numeral 10 in this visual discrimination task. For chimpanzees, it was clear that none had difficulty touching four numerals in ascending order if they consisted of single digits (Under10). All four participants showed 100% accuracy. They perfectly understood the numerical sequence from 1 to 9. However, they made mistakes in the other conditions (Cod1, Cod2, Cod3, and Over10) which contained the two-digit numerals. Processing the two-digit numerals was more difficult than the one-digit numerals. This result is congruent with the result of testing the factor of the range "1 to 9" vs. "1 to 19" (see Section 4.2). The chimpanzee Pal is an interesting exception. She showed 100% accuracy in Over10. This result indicates that she is as good as humans at understanding the decimal number system and she scored 100% correct in the two-digit numerals (in the range 10 to 19). Pal's performance was V-shaped, similar to humans. Chimpanzee Ai showed a similar tendency. In contrast, the accuracy of Cleo monotonically decreased as the number of two-digit numerals increased. The result corresponds to her poor performance in the assessment test of four adjacent numerals including the numeral 10 (see Section 4.3). Processing two-digit numerals are difficult for chimpanzees, but Pal shows the possibility of becoming close to human performance. However, a further detailed comparison of the two species is needed for the conclusion. 4.5. Comparison of Humans and Chimpanzees: Response Latency Figure 12 shows individual data and averages for response latency (msec) in chimpanzees (n = 4) and humans (n = 5). The response latency was compared in the condition of four nonadjacent numerals in the range 1 to 19. Response latency is defined as the duration from the display onset to the first touch. Only correct trials were included in this analysis and the data from error trials were excluded. All six human participants showed a monotonic increase in response latency as a function of the number of two-digit numerals, 0 through 4 . The major difference between the five groups is the total number of digits to be processed (see Section 3.4). The more two-digit numerals, the more digits to be processed. The total number of digits that appeared on the screen increased from four, five, six, seven, and to eight at maximum along with the five conditions. Participant H3 was the only person with 100% accuracy in all conditions. This was due to the unique style of taking a very long time before the execution, with no mistakes; thus, response latency was much longer than for the others. There was a tradeoff between accuracy and response latency. H3's latency in each condition was extremely long at 5 to 6 s (see Appendix A.5: 4696 msec in Under10, 5425 msec in Cod1, 5891 msec in Cod2, 5735 msec in Cod 3, and 6505 msec in Over10). Despite longer latencies, the tendency was the same as the other five human participants. The latency simply increased as a function of the number of two-digit numerals, but as the values were outside the scale of the other participants, H3's data were omitted from the analysis of response latency in Figure 12. For chimpanzees, the data showed two patterns. Chimpanzees Ai and Pal followed the same tendency as humans: a monotonic increase in response latency as a function of the number of two-digit numerals 0 through 4. The more two-digit numerals, the more processing time is needed. Pal's data were very similar to human data; her response latency increased from 492 to 1195 msec as a function of the two-digit numerals. Ai's latency increased from 727 to 1328 msec. Their performance was similar to humans both in accuracy and response latency. In contrast, the two chimpanzees Ayumu and Cleo deviated from the human tendencies. Ayumu's latencies were flat regardless of the conditions, ranging from 594 to 672 msec. In the case of Cleo, latency was in the range of 554 to 859 msec and showed no monotonic increase. They did not take long to make a decision. The comparison of response latency between naive adult humans and chimpanzees shows a clear difference between the two species. All four chimpanzees were quicker than all six humans in all five conditions. Latency was about 700 msec for chimpanzees and about 1000 msec for humans on average. These results suggest that chimpanzees prioritize a quick response over a correct response. Their latencies remained low and more or less unchanged, even with deteriorating performance, whereas humans slowed down with increasing difficulty. This may indicate that chimpanzees make faster decisions than humans when processing the two-digit numerals in the visual discrimination task. However, we must acknowledge that the chimpanzees' extensive, daily interactions with the device over several years may have had an impact on their motor skills, which in turn may have a positive effect on their response latencies. 4.6. Individual Differences in Chimpanzees The six chimpanzee participants were not homogeneous. The basic notable information on individual differences follows. Ayumu (male) was almost at puberty. He enjoyed staying with estrus females and was sensitive to noise, such as screams and barks from the outside. Pan was not much attracted by the food reward, and sometimes did not eat it, especially in the hot summer season. She preferred to stay in the air-conditioned experimental room without participating in the trials. Interestingly, she could perform the task for social praise or with verbal encouragement. Along with individual differences, two possible major influences on the results should be mentioned. One is the aging effect. The child chimpanzees always outperformed their mothers. As shown in Table 3, all three children were better than their mothers in baseline training. In the assessment test (see Table 4), average performances were: Ai 75% vs. Ayumu 85%, Chloe 61 % vs. Cleo 75 %, and Pan 57 % vs. Pal 79%, respectively. However, we did not run a statistical analysis because there were only three pairs. The other influencing factor is shared "personality" within mother-child pairs. If we refer to families A (Ai and Ayumu), P (Pan and Pal), and C (Chloe and Cleo), family willingness to participate and perform in cognitive tests was in the order: A > P > C; again, however, we only mention this trend without statistical analysis. 5. Discussion 5.1. Sequence Order and Transitivity from Adjacent Numerals to Nonadjacent Numerals The present study showed that chimpanzees can master the sequence order of numerals in the range 1 to 19 (see Section 4.1). Their understanding of the order was assessed by systematically manipulating four factors: range (1 to 9 vs. 1 to 19), adjacency (adjacent vs. nonadjacent numerals), number of numerals (3, 4, or 5 numerals), and memory (introducing the masking task). The study showed that training on adjacent numerals spontaneously transferred to nonadjacent numerals (see Section 4.2). The baseline training provided the order of adjacent numerals. Chimpanzee Ai accurately touched each of the 16 simultaneously presented numerals from 1 to 16 in the VNM-Startfix task and the 11 numerals from 9 to 19 in the VNM-Endfix task. We never asked her to touch 19 numerals from 1 to 19 within a trial to avoid too much pressure on her. Interestingly, chimpanzee Ai invented new tactics to avoid some difficulties in the test. She understood the task nature of the VarNumMix tasks and made an "easy mistake" to finish the difficult trial quickly. She skipped it to move on to the next trial, which was expected to be easier. This "skipping behavior" may be an example of metaknowledge: knowing the nature of the task. The chimpanzees showed clear evidence of transitivity (see Section 4.2). The baseline training on touching adjacent numerals spontaneously transferred to nonadjacent numerals. This phenomenon is called "transitivity" or "transitive inference" , known to be within the capabilities of many animal species without training. Transitivity applies not only to cognitive tasks but also to social life . For example, the social ranking order among nonhuman primates is called the dominance hierarchy . Group-living animals do not have to learn about every possible social pair in their group but can infer the dominance order from witnessing a limited number of encounters. The evolutionary adaptation sensitive to this kind of order may provide the basis for a transitive inference of numerals in the present study. 5.2. Morphological Structure of the Decimal System in Terms of Visual Perception The present study introduced the following 19 numerals: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 to the chimpanzees. The last nine two-digit numerals are morphologically similar from a visual perspective to the first nine one-digit numerals. If you disregard the left half of the complex numeral, the digit "1", the sequential order of the numerals can be understood based on previous knowledge. The morphological feature of the decimal system in visual perception might help the chimpanzees in addition to the transitive inference in general. The chimpanzees mastered the skill of the sequence of the Arabic numerals in the range 1 to 19, although the numeral 10 still caused difficulties for some chimpanzees such as Cleo, Chloe, and Pan (see Section 4.3). However, other chimpanzees mastered the skill very well, namely Ai, Ayumu, and Pal. They did not rely on rote memorization of the sequence, as they showed transfer to nonadjacent numerals (see Section 4.2). The transfer might be helped by the visual features of the decimal system. Further examination of the contribution of visual features (in other words, the morphology of the decimal system) was carried out by comparing humans and chimpanzees using the same procedure (see Section 4.4). 5.3. Difficulty in Processing the Numeral 10 The critical point at this stage was the numeral "10" and the introduction of the digit "0". With the five numerals 5, 9, 12, 14, and 18, for example, you should not touch "12" first. You have to understand the two-digit numeral. Two-digit numerals should be 'larger' (positioned later in the sequential order) than one-digit numerals. Even more, the numeral "10" is located between 9 and 11. The numeral 0 has a crucial role in this regard. The chimpanzee Ai had learned the meaning of "0" . She mastered the ordinary scale of 0 to 9. She could understand and use "0" properly in naming tasks and choose "0" for nothing. She was fluent in both productive use and receptive use of numerals . In short, Ai had acquired and established the number concept and utilized Arabic numerals to represent it. Additionally, because she had also learned the numeral 0, only Ai quickly learned the meaning of 10 . She easily expanded her knowledge of the number "0 to 9" to "10 to 19" in the decimal system. The other five chimpanzees never explicitly learned the meaning of "0". However, the present study showed that their performance was approaching Ai's thanks to the accumulated experience of the baseline training over the course of three years and eight months. At the second assessment test at the end of the present study, Ayumu outperformed Ai, and Pal was equal to Ai (see the summary in Table 3). This means that the chimpanzees did master the numerical sequence from 1 to 19 as Ai did, even without explicit training about the numeral 0. This appears reasonable when considering the acquisition of numerical sequences in human children. Without learning the meaning of 0 in advance, children can establish the numerical order including 10. They recite the sequence "one-two-three-four-five-six-seven-eight-nine-ten" without the concept of zero. 5.4. Global and Local: Dual Processing of Two-Digit Numerals How do we understand the cognition of numerals in chimpanzees? The question can be framed as how chimpanzees perceive two-digit numerals in comparison to humans. The comparative experiment examined the performance when processing one-digit and two-digit numerals. Both accuracy and response latency showed characteristics shared by the two species. Processing two-digit numerals was harder than one-digit numerals. Here, we postulate that this result can be understood with reference to global and local processing (see Section 1) . Global vs. local processing was originally devised by Gestalt psychologists. Navon (1979) published a paper titled: "Forest before trees: the precedence of global features in visual perception" . Human perception is analytic and also holistic. The global precedence hypothesis (Navon effect) emphasizes the perceptual primacy of wholes. However, the comparative study of nonhuman primates, namely chimpanzees , baboons , and capuchin monkeys , shows perceptual primacy not in wholes but in local components. In other words, monkeys and apes may perceive each tree first and seldom think about the forest . In our opinion, global-local processing might be an analog to the processing of visual words and letters in humans. Visual word recognition is a basic process involved in reading . In the English language, a word contains some of the 26 letters of the Roman alphabet. There is a parallel processing of the global feature--the word--and the local feature--the letter . We postulate that dual processing, such as word-letter, must exist in the visual processing of the two-digit numerals. Decimal numerals consist of 10 elementary digits of 0 to 9, and the combination makes an infinite number of integers, such as 13, 610, 2584, 10,946, and so on. Because of the dual process of global-local, it takes more energy and time to recognize two-digit than one-digit numerals. The present study demonstrated that chimpanzees mastered the order of numerals in decimals in the range 1 to 19. However, the comparison with humans suggested difficulty in processing two-digit numerals in terms of global-local dual processing. In the cognitive tradeoff of accuracy and latency, humans take time for dual processing of both global and local features. Some chimpanzees such as Ai and Pal could do this too, while others such as Ayumu and Cleo could not. In general, all chimpanzees prefer to make quick decisions that focus on the local features of two-digit numerals. Humans' proficiency in dual processing of global-local features results in them taking time to make the right decisions. There appears to be a cognitive tradeoff between the "local-quick" and "dual-slow" processing of global-local features . The behavioral repertoire of touching the numerals from 1 to 19 may open a new window to understanding species-specific ways of information processing. 5.5. Individual Differences and Aging Effect One major factor in the observed individual differences is age: child chimpanzees outperformed their mothers. This effect of aging was reported for other cognitive tasks in the same six chimpanzees, concerning auditory-visual position learning , sequential learning of one-digit numerals , and memory retention . Another major factor was shared by the mother-child pairs. Mothers and their children showed similarities in both behavior and cognitive performances. For example, the C family (Chloe and Cleo) was characterized by quick decisions. Chimpanzees, like humans, possess broad intellectual capacities that are affected by their personalities . These results highlight the importance of considering individual differences, including personality when evaluating responses in cognitive and behavioral tests. What kind of personality is involved may become a hot topic in future studies. 5.6. An Outlook for Future Studies The present study used a sequential ordering task with Arabic numerals. One original aim was to understand the number concept in chimpanzees. What kind of numerosity judgments exist in animals: subitizing, counting, or magnitude estimation? The answer remains unclear, because the numerical repertoire of animals is small, with a few exceptions . One solution was to extend the numerical sequence to produce a larger repertoire. This was accomplished in the present study by covering the range from 1 to 19. What follows should be the introduction of cardinal numbers to assess the numerosity judgment to many items up to 19. The introduction of zero (the digit "0") in the numerical sequence must be an important step, as is extending two-digit numerals into the range of 20 to 29, 30 to 39, and so on. Is there any transfer from the experience of 1 to 19? Working memory has been tested in the range 1 to 9, revealing the extraordinary memory of young chimpanzees . The memory test can be extended to two-digit numerals. This was partly done in the assessment test which had five numerals; however, it became clear that processing two-digit numerals is not so easy for chimpanzees, and it is not purely a matter of working memory. Further direct comparisons of humans and chimpanzees may be needed in this field. The results suggest that chimpanzees prioritize a quick response over a correct response. Their latencies remain short and more or less unchanged whereas humans slow down their response with increasing task difficulty. We concluded that there may be a cognitive tradeoff between "local-quick" vs. "dual-slow" processing of global-local features. If so, it would be interesting to study completely naive humans, in which participants receive no verbal instruction for the task and only minimal instruction for the touchscreen device; thus, they would have to figure things out for themselves with auditory feedback/reward alone, similar to the chimpanzees. Such a study could track learning curves over time and see if humans still prioritize accuracy over solving the task quickly. For comparison, we have long-term chimpanzee data from the very early stages of their training. The comparison of completely naive humans and chimpanzees could illuminate the evolutionary origins of human visual information processing. To close the article, we mention the relevance of the study paradigm used here in terms of animal welfare. We used a twin-booth system (see Appendix A.6) designed to keep the mother-infant pairs free and comfortable: the child was not fully separated from their mother. However, as the children grow up, the twin booths can be utilized for studies of cooperation and communication . The two booths can be separated and connected by electronic vertically sliding doors. The study of social intelligence in the twin booths together with the touchscreen system might be an important research method. The cognitive study of chimpanzees must be accompanied by the promotion of environmental enrichment. The key is the freedom to join the experiments . 6. Conclusions The present study taught the numerical sequence from 1 to 19 to six chimpanzees: three pairs of mother and child. They had previous experience of touching the numerals 1 to 9 in ascending order. Then, the decimal number system was introduced and the chimpanzees learned to touch the numerals 1 to 19. The sequential touching was taught in two different conditions. One was to touch from 1 to the numeral X (Startfix condition). The other was to touch from the numeral X to 19 (Endfix condition). Daily baseline training was based on the two conditions of adjacent numerals. A systematic test examined the following four factors: range (1 to 9 vs. 1 to 19), adjacency (adjacent vs. nonadjacent), number of numerals (three, four, or five numerals), and memory (nonmemory task vs. memory task). All four factors were important. The narrow range (1 to 9) was easier than the wide range (1 to 19). Adjacent numerals were easier than nonadjacent numerals for expressing the ascending order. As the number of numerals increased, performance decreased. Memory tasks caused deterioration of performance. The further examination focused on processing the numeral 10. Performance was relatively low when the numeral 10 was involved. Taken together, with some difficulties, chimpanzees can master the sequence order in the range 1 to 19 in the two-digit Arabic numerals. Direct comparison was carried out between humans and chimpanzees using the same apparatus and the same procedure. It was revealed that both humans and chimpanzees have relative difficulty processing two-digit numerals compared to one-digit numerals. The results were discussed in the framework of the global-local problem in information processing. Humans are good at processing multilevel information, including dual global and local levels. In contrast, chimpanzees tend to focus on local features and make quick decisions, much faster than humans. The difference might be due to the cognitive tradeoff of chimpanzee-like "local but quick" vs. human-like "dual but slow" information processing. Acknowledgments This study is a part of the Ph.D. thesis of A.M. A.M. is currently supervised by Shinya Yamamoto. A.M. received the Grant-in-Aid for JSPS Research Fellow JP14J00488. T.M. received the Grant-in-Aid of MEXT/JSPS: JP16002001, JP20002001, JP24000001, and JP16H06283. The study was also supported by the database of GAIN, JSPS-GCOE (A06, Biodiversity), JSPS-HOPE, JSPS core-to-core program of CCSN, JSPS-WISH, and JSPS-PWS (U04). T.M. thanks Caltech eLibrary, Chubu Gakuin University, Northwest University Program (G2022040013L), and CARTA of UCSD for help with preparing the manuscript. Our thanks are due to Sumiharu Nagumo for his help with computer programming, Masaki Tomonaga, Masayuki Tanaka, Ikuma Adachi, Misato Hayashi, Dora Biro, Nobuyuki Kawai, Tomoko Imura, Yuko Hattori, Sana Inoue, Christopher Martin, Mari Hirosawa, Makiko Uchikoshi, Tadashi Ogura, Takaaki Kaneko, Fumihiro Kano, Yumi Yamanashi, and Lira Yu for their support in conceiving and conducting the study. Special thanks are due to Tomoko Takashima who helped with the data collection during the entire period of the present study. Misato Hayashi, Mari Hirosawa, Mai Nakashima, Etsuko Ichino, and Akemi Hirakuri also helped us to perform the experiments in the South playroom of KUPRI. The video recording was performed by Miho Nakamura during the entire period. The authors also appreciate James Anderson for his English editing and his advice on the manuscript. Special thanks are due to Juri Suzuki, Takako Miyabe, Norihiko Maeda, Atsushi Yamanaka, Akihisa Kaneko, and the veterinary staff and caregivers of the KUPRI chimpanzees: they work daily seven days a week for many years and decades continuously since 1977 when the Ai project started. Finally, we thank the chimpanzees who participated in the present study. Supplementary Materials The following supporting information can be downloaded at: Supplementary Video S1: video footage of chimpanzee study (chimpanzee Ayumu touchscreen task of VNM-Startfix 1 to 17); Supplementary Video S2: video footage of chimpanzee study (chimpanzee Pal touchscreen task of VNM-Endfix 11 to 19); Supplementary Video S3: video footage of chimpanzee study (chimpanzee Pal touchscreen task of four nonadjacent numerals in the range 1 to 19 nonmemory task); Supplementary Video S4: video footage of chimpanzee study (chimpanzee Pal touchscreen task of four nonadjacent numerals in the range 1 to 19 with memory task); Supplementary Document S5: additional explanation and the detailed figures and tables of the article . Click here for additional data file. Author Contributions Conceptualization, A.M. and T.M.; methodology, A.M. and T.M.; software, A.M.; formal analysis, A.M. and T.M.; investigation, A.M. and T.M.; data curation, A.M. and T.M.; writing--original draft preparation, A.M. and T.M.; writing--review and editing, A.M. and T.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study for chimpanzees was approved by the ethics committee of the Primate Research Institute of Kyoto University (2011-136, 2012-044, 2013-031, 2014- 034). It was undertaken by the Japanese Act on the Welfare and Management of Animals and the Guideline of Care and Use of Nonhuman Primates, Kyoto University Primate Research Institute. All work undertaken for this study was purely non-invasive. Informed Consent Statement Informed consent was obtained from all human subjects involved in the study following the "Code of ethics and conduct" designated by the Japanese Psychological Association. Available online: (accessed on 27 November 2022). This study for human subjects was also approved by the ethics committee of the Primate Research Institute of Kyoto University (2014-034, 2015-063). Data Availability Statement All of the fundamental data sets that we analyzed are available on the following site in Figshare Dataset: Muramatsu, Akiho; Matsuzawa, Tetsuro (2023): Arabic numerals 1 to 19 in chimpanzees. figshare. Dataset. The video data is available: Further detailed data are available from the corresponding author upon reasonable request. Conflicts of Interest The authors declare no conflict of interest. Appendix A Appendix A.1. Details of the Parallel Studies of Chimpanzee Participants The overall study period was from April 2011 to November 2014. Details of parallel studies are presented here to show any possible interference or conflict among the different kinds of cognitive experiments in which the six chimpanzees participated during this period . The topics were as follows in mainly chronological order. The one-digit Arabic numerals were used to explore a SNARC-like effect as found in the spatial mapping of numbers in humans. A small number was also found to be processed in the left visual hemifield in chimpanzees . Chimpanzees were tested on (and mostly mastered) self-paced rhythmic tapping on a keyboard , symbolic matching-to-sample tasks involving colors and symbols, especially the effect of the exclusion principle , object manipulation in a face-to-face situation and action grammar , face-processing and the inversion effect , visual processing of objects , and perception of the freshness of food . They were also tested on collaboration, involving a shared serial ordering task between two chimpanzees who alternately touched the numerals from 1 to 8 which appeared on each half of the divided touchscreen . The emergence of social learning in two chimpanzees together was extended to synchronization of simultaneous finger-tapping , especially in a face-to-face setup . Observations made of mother-infant interactions in the laboratory were compared with mother-infant pairs in the wild: Bossou, Guinea, West Africa . In sum, the participant chimpanzees in the present study experienced various cognitive tasks; each individual has a history of training and testing. However, not all six chimpanzees experienced all of the tasks described above, and importantly, no previous studies tested the two-digit numeral processing originally reported in the present study and the preliminary training . There should be no interference/confounding bias from the other tasks. Appendix A.2. Details of the Training Procedure The training procedure can be summarized as follows. Regardless of training and tests, a session consisted of 50 trials, corresponding to the number of small reward compartments on the food dispenser. After the dispenser was loaded with food rewards, the experiment ran in a fully automated way. See the Supplementary videos S1, S2, S3, and S4. How long it took to train depended on each chimpanzee and their different temperaments and histories; individual data on the course of training on each task are presented in Section 4. In general, the criteria for success in training sessions required to move from one condition to the next was getting 90% correct in a session. The criterion was gradually lowered to 85% or more depending on the difficulty of the task. This was done to keep the chimpanzees motivated. During the assessment tests, there was no criterion; we simply measured the performance under particular conditions. Throughout the study, the training technique was the so-called PRT (positive reinforcement training) using the reinforcement schedule of CRF (continuous reinforcement; in other words, Fixed Ratio 1). The reward quality and quantity were kept constant in all conditions. We did not manipulate rewards such as by increasing the number of rewards when the task was getting difficult. Instead, we gradually shifted the difficulty of the task while setting the 90% correct criterion to maintain motivation. For example, the VNM-Startfix 1 to 9 task resulted in more than 90% accuracy in all participants. Starting from there, the tasks gradually increased in difficulty depending on the performance of each participant. Appendix A.3. Performance on VNM-Starfix Task and VNM-Endfix Task Figure A1 provides the average accuracy of six chimpanzees in the two tasks. Table A1 shows the data of each chimpanzee in each condition of VNM tasks. Figure A1 The average performance of six chimpanzees in VNM tasks. animals-13-00774-t0A1_Table A1 Table A1 Data of each six chimpanzees in each condition of VNM tasks. Name VNM-Startfix Task VNM-Endfix Task Condition 1 to 9 1 to 10 1 to 11 1 to 12 1 to 13 1 to 14 1 to 15 1 to 16 1 to 17 1 to 18 1 to 19 16 to 19 15 to 19 14 to 19 13 to 19 12 to 19 11 to 19 10 to 19 9 to 19 8 to 19 Ai Mean 94 93 93 88 80 77 71 66 na na na 80 88 84 82 81 79 74 74 na SD 0.0 6.4 3.4 5.9 4.6 4.9 5.3 5.6 na na na 9.6 3.2 6.2 4.8 7.9 5.9 6.4 3.4 na Ayumu Mean 92 96 92 84 81 80 80 79 77 63 na 97 97 95 86 88 84 80 78 65 SD 0.0 3.5 3.4 7.3 7.2 5.7 5.4 5.0 6.5 5.7 na 1.2 1.9 1.2 6.0 4.1 5.6 6.7 6.2 7.0 Chloe Mean 79 82 80 71 65 na na na na na na 65 80 80 72 na na na na na SD 6.9 4.6 4.8 6.0 5.5 na na na na na na 15.8 8.5 5.9 7.5 na na na na na Cleo Mean 80 81 81 76 71 67 61 na na na na 72 88 88 79 75 73 63 na na SD 6.6 6.4 8.3 5.0 7.1 7.3 7.0 na na na na 17.2 4.2 3.5 8.4 7.1 6.0 4.5 na na Pan Mean 82 84 80 68 na na na na na na na 73 72 61 na na na na na na SD 7.1 4.1 4.7 5.4 na na na na na na na 10.2 6.8 5.6 na na na na na na Pal Mean 83 88 88 83 78 78 74 69 63 na na 69 94 91 84 84 79 75 76 na SD 1.2 3.3 3.5 6.8 5.3 5.4 4.9 4.0 5.7 na na 12.2 2.0 6.2 5.4 5.9 6.5 7.0 5.5 na Average Mean 85 87 86 78 75 76 72 71 70 63 na 76 87 83 81 82 79 73 76 65 SD 3.6 4.7 4.7 6.1 5.9 5.8 5.6 4.8 6.1 5.7 na 11.0 4.4 4.8 6.4 6.3 6.0 6.1 5.0 7.0 Appendix A.4. Details of the Main Finding of the Assessment Test This appendix is about the four factors which influenced chimpanzee performance, namely range, adjacency, number of numerals, and memory. Table 4 in the main text shows the results, and the main text focused on each factor: range , adjacency , and memory , respectively. However, as their combination might be important to determine their relative contribution, the combined data are visualized in Figure A2. Figure A2 is the combined presentation of Figure 7, Figure 8 and Figure 9, and includes all conditions. It aims to give the visualization of Table 4 (the data of the 2nd and the final assessment test). Table A2 presents the results of the first assessment test which correspond to the second assessment test carried out six months later (see Table 4 in the main text). The correlation between the two assessment tests was very high: r = 0.941. This means the results of the four factors were robust. The direct comparison of the first assessment test and second assessment test is provided in the Supplementary Materials (S5.2). Figure A2 Data from all six chimpanzees were combined to show all four factors: range, adjacency, number of numerals, and memory. The X-axis shows the number of numerals. The Y-axis shows accuracy (% correct). Regardless of the experimental condition, the chimpanzee had to touch the numerals from 1 to 19 in ascending order. animals-13-00774-t0A2_Table A2 Table A2 Data for all six chimpanzees in the first assessment test corresponding to the second test (Table 2 in the main text). There was a six-month interval between the two tests. Four factors influenced the performance of numerical ordering. Each cell shows accuracy (% correct). These data are from the first test; corresponding data from the second test carried out six months later are available in Table 2 in the main text. The data of the two tests were very similar and the results were robust. The correlation was r = 0.941. Task Chimpanzees Range Adjacency Memory Number of Numerals Ai Am Chloe Cleo Pan Pal Average 1-9 Adjacent Nonmask 3 96 96 96 98 88 98 95.3 4 90 92 94 100 92 96 94.0 5 90 90 84 86 88 96 89.0 Mask 3 98 98 88 88 84 92 91.3 4 76 94 58 82 56 86 75.3 5 60 84 36 56 24 76 56.0 Non-adj Nonmask 3 100 100 96 94 94 98 97.0 4 98 94 90 88 98 96 94.0 5 84 96 82 84 92 92 88.3 Mask 3 94 98 90 92 86 88 91.3 4 90 92 68 76 48 80 75.7 5 58 80 26 58 16 66 50.7 1-19 Adjacent Nonmask 3 94 98 68 90 68 92 85.0 4 92 96 54 80 48 98 78.0 5 72 92 52 70 30 80 66.0 Mask 3 88 96 66 90 50 94 80.7 4 62 92 40 68 22 80 60.7 5 32 62 8 44 8 60 35.7 Non-adj Nonmask 3 82 86 50 64 56 82 70.0 4 62 74 30 50 52 60 54.7 5 50 44 18 24 20 42 33.0 Mask 3 86 64 66 58 60 74 68.0 4 30 40 30 34 26 54 35.7 5 16 8 14 22 8 24 15.3 Average 75 82 59 71 55 79 70.0 Appendix A.5. Details of Human Data: Response Latency This appendix describes the details of the human data. Participant H3 was out of range in comparison to the other five participants in terms of response latency. His latency data were therefore excluded from a further analysis involving this measure. Figure A3 Raw data of six human participants: response latency. H3 showed uniquely long latencies. Appendix A.6. Details of the Experimental Setup Figure A4 The experimental setup. This is a twin booth. Ayumu chimpanzee is in the left booth, with his back on the camera. Ai chimpanzee is in the right booth, facing the touchscreen. The automated experiment is supervised by a staff member, Tomoko Takashima, who arranges the food rewards on the disc of the food dispenser. The experimenters engage in Q&A with the computer to set up the session parameters. During the session, the experimenters sit quietly and watch the chimpanzees directly or look at the monitor. The video images on the monitors were taken from the rear side of the booths. This photo is a panorama view, so the image is slightly distorted. Figure 1 The touchscreen apparatus. It was encased in a translucent acrylic box and set just behind a translucent panel which prevented the chimpanzee from strongly banging the screen. The participant touched the screen through a window opened at the lower part of the box. Here, chimpanzee Ayumu is touching the numerals presented on the display in ascending order using his left index finger. The figure was cut out from the video clip (see Supplementary Materials Video S1). The following video clip is available to the public: accessed on 6 December 2022. Figure 2 Chimpanzees' outdoor enclosure at KUPRI. A group of 14 chimpanzees lived in an enriched environment. A participant chimpanzee came to the test booth based on their own free will. Figure 3 Assessment tests: range, adjacency, number of numerals, and memory. Chimpanzee Pal is touching the numerals in ascending order. (a) Four nonadjacent numerals in the range 1 to 19 in the nonmemory task. (b) Four nonadjacent numerals in the range 1 to 19 in the "masking" memory task. Individuals have different ways of touching numerals; Pal uses her left middle finger while keeping her palm up. Figure 4 Data of chimpanzee Ai in the baseline training of VNM-Startfix task. The task started from VNM 1 to 9, in which the numerals appeared as either 1, 1-2, 1-2-3, 1-2-3-4, 1-2-3-4-5, 1-2-3-4-5-6, 1-2-3-4-5-6-7, 1-2-3-4-5-6-7-8, or 1-2-3-4-5-6-7-8-9. In the case of VNM 1 to 10, one more numeral sequence, 1-2-3-4-5-6-7-8-9-10, was added. The chimpanzee had to touch the numerals from 1 to X in ascending order. The X-axis showed the number of numerals presented at the maximum level in the task. The Y-axis showed the accuracy (% correct). The boxplot shows the quartile range of performance and the line shows the average accuracy. Figure 5 Data of chimpanzee Ai in the baseline training of the VNM-Endfix task. The chimpanzee had to touch the numerals from X to 19 in ascending order. Y-axis showed the accuracy (% correct). The boxplot shows the quartile range of performance and the line shows the average accuracy. Figure 6 Data of chimpanzee Ai in the baseline training of two kinds of VNM tasks. In the VNM-Startfix task (red symbols), the numerals always started with 1. In the VNM-Endfix task (blue symbols), the numerals always ended in 19. In both cases, the chimpanzee had to touch the numerals in ascending order. Y-axis shows accuracy (% correct). Each dot shows performance in each session. Performance on the two tasks was plotted in terms of the maximum number of numerals (meaning the longest sequence) to be processed. Figure 7 Data from all six chimpanzees were combined in the assessment test of range. The range was either "1 to 9" or "1 to 19". Adjacency was kept as nonadjacent numerals. The X-axis (3, 4, and 5) shows the number of numerals presented on the screen. The Y-axis shows accuracy (% correct). Figure 8 Data from all six chimpanzees were combined in the assessment test of adjacency. Either adjacent numerals (solid lines) or nonadjacent numerals (dotted lines) were presented. The range was from 1 to 19. The X-axis shows the number of numerals presented on the screen. The Y-axis shows accuracy (% correct). Figure 9 Data from all six chimpanzees were combined to show the factor of memory, i.e., whether the task was an ordinary nonmasking task (solid symbols) or the masking task (open symbols) that required memorizing the numerals. The X-axis shows the number of numerals, and the Y-axis shows accuracy (% correct). The condition is the range 1 to 19 and the nonadjacent numerals. Figure 10 Individual data for four chimpanzees, showing the difficulty of ordering sequences containing the numeral 10. Each of the 16 patterns was tested 31 or 32 times in a total of 500 trials (see Section 3.3). Despite their intensive training in the sequence from 1 to 19, chimpanzees performed relatively poorly on patterns that included the numeral 10. Cleo's data show that she found sequences containing the numeral 10 particularly difficult. The X-axis shows the sequence of four adjacent numerals, and the Y-axis represents accuracy (% correct). Figure 11 Comparison of chimpanzees (Left) and humans (Right) in the task of touching four nonadjacent numerals in the range 1 to 19. Individual accuracy data (% correct) are plotted in the bar graph, with average performance in the solid lines. The X-axis shows the conditions of the four presented numerals (see main text). Human performance shows a V-shape: performance on all one-digit and all-two-digit numerals was perfect, but performance on the in-between conditions deteriorated. Chimpanzee performance deteriorated as a function of the number of two-digit numerals, but Pal's performance was V-shaped, similar to humans. Pal showed 100% in the Over10 condition, just like humans. In contrast, Cleo's performance monotonically decreased and showed 0% accuracy in the Over10 condition. There are two missing data: data for Ayumu in the Over10 condition and H2 in the Under10 condition are unfortunately missing because of the purely randomized procedure. These two extreme conditions of Under10 and Over10 had very low occurrence in a test session of 50 trials. A total of 3876 possible patterns of 4 numerals were purely randomized in this test. Thus, there happened to be no trials that matched these conditions in the particular participants. However, the general trend is preserved. Figure 12 Comparison of chimpanzees (Left, n = 4) and humans (Right, n = 5) in the task of touching four nonadjacent numerals in the range 1 to 19. Individual response latencies (in msec, Y-axis) are plotted in the bar graph, and average performance is shown in the solid lines. The X-axis shows the conditions of the four presented numerals, as described in Section 3.4 and Figure 11. Chimpanzees responded faster than humans. In humans, the latency for all one-digit numerals was about 1000 msec, but for all two-digit numerals, it increased to about 1500 msec. In chimpanzees Ayumu and Cleo, the latency remained constant at about 700 msec throughout the conditions, much shorter than in naive adult humans. Ai and Pal showed a similar tendency to humans. There were three missing data because of the purely randomized procedure: there happened to be no trials or no correct trials that matched these conditions in the particular participants (Ayumu, Cleo, and H2). Participant H3's value was well outside the range of the other participants (see Appendix A.5), so H3's data were omitted from this graph and further analysis of response latency. The exact latencies of all six humans including H3 are shown in the Supplementary Materials (S5.3). animals-13-00774-t001_Table 1 Table 1 List of participant chimpanzees in KUPRI. Age is at the beginning of the present study. GAIN stands for Great Ape Information Network, which is a database of all chimpanzees living in Japan (see: accessed on 6 December 2022). GAIN is equivalent to ChimpCare in the USA (see: accessed on 6 December 2022). "Numerals experience" means the experience before starting the learning of two-digit numerals. Name Sex GAIN ID Mother/ID Birth Age Numerals Experience Ai female 0434 na 1976? 34 0-9 Chloe female 0441 Charlotte/na 12 December 1980 30 1-9 Pan female 0440 Puchi/0436 7 December 1983 27 1-9 Ayumu male 0608 Ai/0434 24 April 2000 10 1-9 Cleo female 0609 Chloe/0441 19 June 2000 10 1-9 Pal female 0611 Pan/0440 9 August 2000 10 1-9 animals-13-00774-t002_Table 2 Table 2 Summary of training tasks and assessment tests. All six chimpanzees received training and tests in the same chronological order. However, the progress was different among chimpanzees due to their availability, their willingness to participate, and their learning speed. The first row shows the number of days on which chimpanzees came to the test booths. Other cells represent the number of sessions required for each stage. "na" means not applicable, i.e., not done for that particular chimpanzee. All sessions consisted of 50 trials. Chimpanzees Section Task Name, Abbreviation, and Definition Ai Ayumu Chloe Cleo Pan Pal Total days of coming to the test booth in the study period 630 630 477 477 498 498 3.1. Baseline training of touching adjacent numerals: VarNumMix (VNM) task in the range of 1 to 19 VNM task which characterized by the "Startfix" condition The sequence always started from the numeral 1 VNM-Startfix 1 to 9 1 1 14 46 16 3 VNM-Startfix 1 to 10 3 3 19 23 18 8 VNM-Startfix 1 to 11 4 4 37 35 165 3 VNM-Startfix 1 to 12 4 13 217 43 254 20 VNM-Startfix 1 to 13 97 17 221 41 na 55 VNM-Startfix 1 to 14 117 108 na 178 na 77 VNM-Startfix 1 to 15 300 46 na 116 na 108 VNM-Startfix 1 to 16 151 109 na na na 18 VNM-Startfix 1 to 17 na 151 na na na 190 VNM-Startfix 1 to 18 na 136 na na na na VNM-Startfix 1 to 19 na na na na na na total (number of sessions) 677 588 508 482 453 482 VNM task which characterized by the "Endfix" condition The end of the sequence was always fixed as 19 VNM-Endfix 16 to 19 22 3 56 12 56 39 VNM-Endfix 15 to 19 9 4 25 7 157 3 VNM-Endfix 14 to 19 15 3 90 9 39 4 VNM-Endfix 13 to 19 26 9 72 24 na 12 VNM-Endfix 12 to 19 22 9 na 80 na 16 VNM-Endfix 11 to 19 52 16 na 98 na 74 VNM-Endfix 10 to 19 92 28 na 8 na 50 VNM-Endfix 9 to 19 17 125 na na na 148 VNM-Endfix 8 to 19 na 8 na na na na VNM-Endfix 7 to 19 na na na na na na total (number of sessions) 255 205 243 238 252 346 3.2. First Assessment tests (4 factors): range, adjacency, number of numerals, and memory 24 24 24 24 24 24 3.2. Second Assessment tests (4 factors): range, adjacency, number of numerals, and memory 24 24 24 24 24 24 3.3. Assessment of adjacent numerals including the numeral 10 10 10 na 10 na 10 The task was 4 adjacent numerals in the range of 1 to 19 with nonmemory task 3.4 Comparison of humans and chimpanzees 12 12 12 12 12 12 The task was 3,4, or 5 nonadjacent numerals in the range of either 1 to 9 or 1 to 19 with nonmemory task Notes: Assessment tests (4 factors): The 1st test was done in September 2013 and 2nd test in March 2014; na: not applicable. animals-13-00774-t003_Table 3 Table 3 Summary of accuracy (% correct) in baseline training tasks (VNM tasks) in each chimpanzee. Each cell represents a condition and a chimpanzee. The tables show average performances. "na" means not applicable, i.e., not done for that particular chimpanzee. Task Name Chimpanzees Condition Ai Ayumu Chloe Cleo Pan Pal Average VNM task which characterized by the "Startfix" condition The sequence always started from the numeral 1 VNM-Startfix 1 to 9 94 92 79 80 82 83 85 VNM-Startfix 1 to 10 93 96 82 81 84 88 87 VNM-Startfix 1 to 11 93 92 80 81 80 88 86 VNM-Startfix 1 to 12 88 84 71 76 68 83 78 VNM-Startfix 1 to 13 80 77 65 71 na 78 74 VNM-Startfix 1 to 14 77 80 na 67 na 78 76 VNM-Startfix 1 to 15 71 80 na 61 na 74 72 VNM-Startfix 1 to 16 66 79 na na na 69 71 VNM-Startfix 1 to 17 na 77 na na na 63 70 VNM-Startfix 1 to 18 na 63 na na na na 63 VNM-Startfix 1 to 19 na na na na na na Average accuracy in total 83 82 75 74 79 78 78 VNM task which characterized by the "Endfix" condition The end of the sequence was always fixed as 19 VNM-Endfix 16 to 19 80 97 65 72 73 69 76 VNM-Endfix 15 to 19 88 97 80 88 72 94 87 VNM-Endfix 14 to 19 84 95 80 88 61 91 83 VNM-Endfix 13 to 19 82 86 72 79 na 84 81 VNM-Endfix 12 to 19 81 88 na 75 na 84 82 VNM-Endfix 11 to 19 79 84 na 73 na 79 79 VNM-Endfix 10 to 19 74 80 na 63 na 75 73 VNM-Endfix 9 to 19 74 78 na na na 76 76 VNM-Endfix 8 to 19 na 65 na na na na 65 VNM-Endfix 7 to 19 na na na na na na Average accuracy in total 80 86 74 77 69 82 78 animals-13-00774-t004_Table 4 Table 4 Data for all six chimpanzees in the second assessment test. Four factors influenced numerical ordering performance. First, the range of numerals was either 1 to 9 or 1 to 19. Second, the adjacency was either adjacent or nonadjacent. Third, 'memory' means whether the task was an ordinary task (nonmasking) or the masking task, which required the memorizing of numerals. Fourth, the number of numerals was either three, four, or five. In all conditions, the chimpanzee had to touch the numerals from 1 to 9 or from 1 to 19 in ascending order. Each cell shows accuracy (% correct). These data are from the second test; corresponding data from the first test performed six months earlier are available in Appendix A.4. Task Chimpanzee Participants Range Adjacency Memory Number of Numerals Ai Ayumu Chloe Cleo Pan Pal Average 1-9 Adjacent Nonmask 3 98 98 94 98 90 98 96.0 4 96 90 86 92 88 98 91.7 5 88 90 78 90 82 94 87.0 Mask 3 90 100 76 92 78 98 89.0 4 74 88 60 88 42 90 73.7 5 62 90 22 68 24 62 54.7 Non-adj Nonmask 3 94 96 92 98 92 94 94.3 4 96 96 92 92 86 94 92.7 5 92 94 92 78 88 90 89.0 Mask 3 94 100 82 90 80 84 88.3 4 86 100 60 82 42 80 75.0 5 54 86 36 58 8 66 51.3 1-19 Adjacent Nonmask 3 96 98 76 98 64 98 88.3 4 86 100 54 82 64 98 80.7 5 76 92 50 78 42 88 71.0 Mask 3 88 98 46 94 80 92 83.0 4 68 86 30 74 42 78 63.0 5 34 50 24 44 8 48 34.7 Non-adj Nonmask 3 90 96 64 78 78 92 83.0 4 72 86 62 54 58 70 67.0 5 52 84 36 36 36 50 49.0 Mask 3 72 76 76 66 62 80 72.0 4 34 48 48 44 28 38 40.0 5 14 8 18 16 16 26 16.3 Average 75 85 61 75 57 79 72.1 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000110

Poor quality straw can be made more digestible and palatable through delignification using white rot fungi as a biological treatment in SSF. The decomposition of organic matter by white rot fungi is improved when a carbon source is added. Reducing the fermentation cycle can also help retain more nutrients in straw feed. To increase rumen digestibility and nutrient utilization, corn straw and rice straw were subjected to SSF with white rot fungi (Phanerochaete chrysosporium) for 21 days. The type of carbon source (glucose, sucrose, molasses, or soluble starch) was optimized, and the nutrient composition and in vitro fermentation parameters of the fermented straw were assessed. In the fermented corn straw and rice straw supplemented with different carbon sources, the results showed a decrease in lignin content, dry matter, cellulose, and hemicellulose loss, and an increase in crude protein content after 21 days. Total volatile fatty acid and ammonium nitrogen concentrations increased significantly (p < 0.01) during in vitro fermentation. Overall, the most enhanced nutritional values for corn straw and rice straw were observed after 14 days of SSF in the groups using molasses or glucose as a carbon source. solid-state fermentation Phanerochaete chrysosporium in vitro straw fermentation Research Fund of Engineering Research Center of the North-East Cold Region Beef Cattle Science & Technology Innovation, Ministry of Education and the "111" Project (D20034), ChinaD20034 This study was funded by the Research Fund of Engineering Research Center of the North-East Cold Region Beef Cattle Science & Technology Innovation, Ministry of Education and the "111" Project (D20034), China. pmc1. Introduction Crop waste, including straw, hulls, and husks, is often stacked or burned causing environmental pollution. China is a sizable agricultural nation with a focus on grain production, and the enormous amount of straw generated leads to severe environmental pollution. Statistics indicate that 7.35 x 1011 kg of straw was fed to agriculture animals in China in 2021, demonstrating the enormous development potential of straw feed . Crop straws are the primary source of roughage for ruminants; however, their fiber digestibility in the rumen typically does not exceed 50% . Approximately 40-50% of the fiber in legumes and 60-70% of the fiber in grass can be digested by ruminants; however, some fibres in the straw, such as lignin that cannot be digested, regardless of how long they remain in the rumen. By functioning as a physical barrier and cross-linking with polysaccharides via ferulic acid bridges, lignin is hypothesized to prevent the microbial degradation of fibrous polysaccharides . Various pretreatment techniques have been employed to overcome lignin barriers, including physical (grinding and irradiation), chemical (acids, bases, oxidation, and organic solvents), physicochemical (extrusion, hydrothermal, and steam blasting), and biological (microbial and enzymatic) techniques . However, these technologies have several drawbacks, such as high cost, high energy need, economic unattainability, and environmental unfriendliness . Biological pretreatment of ruminant roughage with fungi offers a low-energy and secure substitute to avoid these drawbacks . White rot fungi offer the greatest potential among naturally existing fungi for selective lignin removal. Since Kirk and Moore (1972) showed that white rot fungi may selectively degrade lignin and improve rumen degradation of swamp and birch trees, numerous researchers have worked to increase the digestibility and nutritional value of lignocellulosic resources using white rot fungi. Poor quality straw can be made more digestible and palatable through a biological delignification process using white rot fungi in solid-state fermentation (SSF) . The degradation of neutral detergent fibers (NDF) in ruminal straw was found to increase by 13% in vitro when fibrolytic enzymes from several white rot fungi were added to wheat straw . To increase in vitro gas production from wheat straw, Nayan et al. (2018) treated various wheat straws with the white rot fungi Ceriporiopsis subvermispora and Lentinula edodes . Shrivastava et al. (2011) improved wheat straw digestibility and nutrient content through SSF for 15 days with Ganoderma sp. rckk . White rot fungi may be used to increase the digestibility and nutrient utilization of roughage in the rumen. The degradation mechanisms have not yet been fully elucidated because the structures are complicated and numerous enzymes, free radicals, and tiny molecules are involved in the degradation of lignin by white rot fungi . Although there are many different types of white rot fungi, most grow slowly, are marginally resistant to weeds, degrade lignin, and produce large amounts of hemicellulose and cellulose. These characteristics result in a lengthy process for white rot fungi to ferment straw and necessitate sterilization conditions, both of which severely restrict the promotion and use of white rot fungi for straw fermentation . Additionally, while white rot fungi specifically break down lignin during the nutritional growth phase, they can also extensively degrade hemicellulose and cellulose . As a result, the incubation period has a significant impact on the selectivity of white rot fungi. Liang et al. (2010) demonstrated that the addition of 0.007% rhamnolipid disaccharide to Phanerochaete chrysosporium-fermented straw increased lignin peroxidase activity by 86% and lignin degradation by 54% . In summary, in order to investigate whether the addition of different carbon sources and reduced fermentation cycles could improve the ability of white rot fungus to degrade organic matter and reduce nutrient losses. A common strain of white rot fungus, P. chrysosporium , and corn straw and rice straw were chosen in this experiment for a 21-day solid-state fermentation with different carbon sources (glucose, sucrose, molasses, or soluble starch) in order to increase the quality of straw fermentation by white rot fungi. The aim of this study was to determine the optimal fermentation conditions for corn and rice straw with white rot fungi to improve nutrient availability and digestibility for use in ruminant feeds. 2. Materials and Methods 2.1. Treatment of Corn Straw and Rice Straw Corn straw and rice straw were produced in the town of Chaoyangchuan, Yanji, China. The straw was dried naturally, cut into 3-5 cm pieces, crushed in an ultrafine grinder, and passed through a 60-mesh sieve (0.25 mm aperture) for further use. The straw was then treated with P. chrysosporium (CICC 14076, China Centre of Industrial Culture Collection, Beijing, China) with a concentration of 2 x 108 cfu/mL. As this experiment was carried out using dried maize straw, the substrate itself had a low water content, so a fermentation broth of 70% of the mass of the fermented substrate was added. For the control group, 25 g of corn straw or rice straw was added to 15 mL of the fermentation solution (volume of 10% white rot fungal mixture and 0.1% urea in distilled water). Solutions including different carbon source types (A: glucose; B: molasses; C: sucrose; and D: soluble starch) (Laboratory conservation) were mixed with 25 g of corn straw or rice straw to make up the eight treatment groups. Each solution contained 3% of the respective carbon source, 0.1% urea, and 10% white rot fungus in 15 mL of distilled water. 2.2. In Vitro Fermentation Experimental Design A one-way experimental design was used to investigate the effects of different carbon sources and white rot fungi on the rumen fermentability of SSF straw over 21 days. After mixing the corn straw and rice straw with their respective fermentation solutions, they were placed in a plastic bag and kept at room temperature (28 degC) for 21 days for fermentation. Samples were collected every 7 days. Corn straw and rice straw were divided into control group and four treatment groups (A: glucose; B: molasses; C: sucrose; D: soluble starch) and samples were collected at 0, 7, 14, and 21 days. Every group with three parallel groups each were subjected to in vitro fermentation using 1 g of straw as substrate, and the reaction was terminated after 72 h. Determination of fermentation indicators pH, NH3-N, IVDMD and VFA. For the in vitro digestion test, 1 g of fermentation substrate was accurately weighed, poured into a fiber filter bag, sealed, and packed into a pre-warmed (39 degC) in vitro digestibility tube. An automatic dispenser was used to add 70 mL of mixed artificial rumen culture solution to the digestibility tube, which was then mixed and immediately incubated in a thermostatic water bath incubator (39 degC) for 72 h. The fermentation mixture was then centrifuged at 12,000x g for 10 min at 4 degC. The resulting supernatant was used to determine pH and volatile fatty acid (VFA) and ammonia nitrogen (NH3-N) concentrations. The pH was determined using a pH meter, and NH3-N was determined using the phenol-sodium hypochlorite colorimetric method . The organic acids were determined using a TC-C18 column (250 mm x 4.6 mm x 5 mm; Agilent Technologies, Santa Clara, CA, USA) at 50 degC with methanol as mobile phase A and 0.01 mol/L aqueous KH2PO4 (pH adjusted to 2.70 with phosphoric acid) as mobile phase B at a flow rate of 0.7 mL/min. A total of 5 mL of straw filtrate was centrifuged and mixed with 1 mL of 25% metaphosphoric acid; 1 mL of this mixture was centrifuged at 4 degC for 15 min at 12,000x g and then the supernatant was filtered through a 0.22 mm aqueous membrane. The organic acid concentration was immediately determined using a 25 mL automatic injection needle (Agilent Technologies, Santa Clara, CA, USA) with a volume of 5 mL, calculated using an external standard. In addition, the 72 h fermentation substrate was dried at 105 degC for 8 h to assess the dry matter digestibility . In vitro digestibility of dry matter (NH3-N) was calculated using the following equation: IVDMD (%) = (sample dry matter weight - residue dry matter weight)/sample dry matter weight x 100. 2.3. Chemical Analysis The dry matter (DM) content of the samples was determined via oven drying at 105 degC for 16 h (method G-003/1). The N concentration of milled straw was determined using the Kjeldahl method, and crude protein (CP) was calculated as N x 6.25. Ash content was measured after treatment of the samples in a muffle furnace at 600 degC for 6 h . Ash-free neutral detergent fiber (ANF) , ash-free acid detergent fiber (ADF), and lignin (Van Soest 1973) were determined as previously described. ADL is the residue of ADF minus ADF digested by 72% sulfuric acid and then subjected to ashing. Hemicellulose content was calculated as the difference between NDF and ADF, and cellulose was calculated as the difference between ADF and acid detergent lignin (ADL). 2.4. Statistical Analysis The trial data were collated in Microsoft Excel (Microsoft, Redmond, WA, USA) and analyzed by one-way analysis of variance (ANOVA) using SPSS 21.0 (IBM, Armonk, NY, USA). Differences were considered statistically significant if p <= 0.05. 3. Results and Discussion DM, ADF, and NDF concentrations in straw typically decrease, while CP content rises, when straw is fermented by white rot fungi . According to this study's findings, addition of carbon sources for corn and rice straw fermentation decreased dry matter, ADF, and NDF losses, while increasing protein content when compared to the respective control group. When straw was supplemented with any of the four different carbon sources, the CP content of the straw increased over time and was significantly higher than it was at 0 days (p < 0.01), but the DM content of all groups declined as fermentation time increased (p < 0.01) (Table 1). These results were similar to those reported by Zhao et al. (2020) . According to previous reports , one cause of DM loss is the secretion of enzymes by white rot fungi during asexual growth, which break down macromolecules in corn stover to release carbon and nitrogen. The increase in protein content is likely due to the conversion of the carbohydrates degraded by the white rot fungi into CO2 and H2O. Hence, the increase in CP content and decrease of NDF and ADF content in the substrate with increasing fermentation time. The NDF and ADF content in the substrates significantly decreased in this study (p < 0.01; Table 1 and Table 2), which is consistent with a previous report by Wang et al. (2021) ; supplementation with a carbon source reduced this loss. The order of ability of the different carbon source groups to increase the CP content of corn stover was as follows: molasses > glucose > sucrose > soluble starch > control. The ash was composed of mineral bars and inorganic matter. While there was no significant change in the ash content between the control and glucose groups after 14 days of fermentation (p > 0.05), the ash content of the soluble starch, molasses, and sucrose groups increased significantly with increasing fermentation time (p < 0.01), and was significantly higher in the molasses group than in the other groups at 14 days (p < 0.01; Table 1). The order of ability of the different carbon source groups to increase the CP content of the rice straw was as follows: glucose > molasses > control > sucrose > soluble starch. The ash contents of rice straw in the added carbon source groups were significantly higher than that of the control group after both 14 and 21 days of fermentation (p < 0.01; Table 2). Changes in the chemical composition of straw caused by fungi have been reported . The results of this study showed an increasing loss of Cellulose, Hemicellulose, and Lignin in all corn straw and rice straw treatment groups during the 21 days of fermentation (Table 3 and Table 4), and the difference was significant (p < 0.01), but the loss in control group was greater than that in the four treatment groups (A: glucose; B: molasses; C: sucrose; D: soluble starch). White rot fungi can produce a wide range of lignocelluloses. The complete degradation of cellulose, hemicellulose, and lignin during fermentation results in a significant loss of carbohydrates . In this study, the loss rates of cellulose, hemicellulose, and lignin were calculated to assess which carbon source could reduce the loss of hemicellulose and cellulose from corn stover and enhance the degradation of lignin by the white rot fungus . At the 14th day of fermentation, the loss rate of cellulose and hemicellulose of corn straw were significantly higher in control group than the other treatment groups . The lignin degradation rate in the molasses group was higher than that in the sucrose, glucose, soluble starch, and control groups . The fiber loss in rice straw was greatest in the soluble starch group followed by the control, sucrose, molasses, and glucose groups. Hemicellulose loss was greatest in the soluble starch group followed by the control, molasses, sucrose, and glucose groups. Lignin degradation was highest in the sucrose group than in the glucose, molasses, and control groups, and lowest in the soluble starch group . The molasses and glucose groups showed the lowest loss of hemicellulose and cellulose and the highest degradation of lignin in corn straw and rice straw fermented by white rot fungi. It has been shown that carbon sources can control the secretion of polysaccharide-hydrolytic enzymes by wood-rotting fungi and that ligninase production by white rot fungi can be effectively stimulated by glucose supplementation, with continuous low concentrations of glucose supplementation achieving better enzyme production . In this study, the lignin degradation rates of the carbon supplementation groups were higher than those of the control group, and the loss of cellulose and hemicellulose was reduced. Therefore, it is reasonable to speculate that the addition of a carbon source to straw fermentation with white rot fungus could enhance the production of plasmalogenase and thus enhance the selective degradation of lignin. IVDMD is an important index of nutrient value of roughage, and it is mainly affected by cellulose content and lignification degree. In general, the lower the degree of lignification, the higher the IVDMD value and the better the quality of roughage . The results of this experiment showed that the IVDMD of the added carbon source groups were higher than that of the control group at the same fermentation time (p < 0.01; Table 5 and Table 6). Additionally, the IVDMD of all groups increased with time and was significantly higher on day 21 than on day 0 (p < 0.01; Table 5 and Table 6). This was consistent with the observed changes in lignin content. At the 14th day of fermentation, the IVDMD value of corn stover in the molasses-added group was twice that of control group (p < 0.01; Table 5). These results were consistent with those reported by Datsomor et al. (2022) . In the rumen, VFAs are produced through the fermentation of dietary carbohydrates; they provide 70-80% of the energy for ruminants, reflect the nutritional value of the feed and the state of rumen fermentation, and are mainly influenced by feed NDF content . In the present study, VFA concentrations peaked at 14 days of fermentation in all corn straw and rice straw treatment groups (p < 0.01; Table 7 and Table 8), and were significantly higher in the groups with added carbon sources than in the control group at the same fermentation time. The glucose group had a significantly higher VFA concentration than all other groups at 14 days of fermentation (p < 0.01; Table 7 and Table 8). Acetic acid is a major source of energy for ruminants and propionic acid must be present to make full use of acetate in fat synthesis. Therefore, both these acids affect the digestion and metabolism of ruminant diets. In all treatment groups, acetic acid content of corn was highest at 14 days of fermentation and then decreased (p < 0.01; Table 7 and Table 8), while propionic and butyric acid content gradually increased over the 21 days of fermentation (p < 0.01; Table 7 and Table 8). The molasses group had higher proportions of acetic, propionic, butyric, and valeric acids than all other treatment groups after 14 days of fermentation (p < 0.01; Table 7 and Table 8). pH and NH3-N are important indicators of the rumen environment. They are used to regulate the homeostasis of rumen environment, and NH3-N can be absorbed and utilized by rumen epithelial cells. In this study, all groups with carbon source additives had a higher pH at day 14 of fermentation than the control group, and the values observed were within the normal range for optimal rumen metabolism (5.5-7.5). In all treatment groups, NH3-N concentrations peaked at 14 days of fermentation (p < 0.01; Table 7 and Table 8) and then gradually decreased corn, with higher NH3-N concentrations observed in the groups with carbon sources than in the control group. This study showed that rumen microbial activity is positively correlated with the CP content of the rumen fermentation substrate. An increase in microbial activity has been shown to increase the NH3-N concentration and promote the synthesis of rumen microbial proteins , which is consistent with the results of the present study. There was a decrease in NH3-N concentrations at 14-21 days in this experiment, which is unlike the previous report by Thao et al. (2014). Therefore, it is reasonable to speculate that the addition of a carbon source to straw fermentation by the white rot fungus could improve the rumen digestibility and nutritional value of the straw. This experiment mainly studied the influence of this treatment method on the composition and in vitro digestibility of straw, but did not explore the changes of metabolites and differences of straw degradation products after white rot fungus added carbon sources, and In-depth exploration of the fermentation mechanisms of white rot fungus. 4. Conclusions Addition of carbon sources to SSF with P. chrysosporium reduced the loss of DM, ADF, NDF, cellulose, and hemicellulose and increased the CP content of corn straw and rice straw. Maize and rice straw in the molasses and glucose groups exhibited significantly improved digestibility and forage quality in vitro, with the highest forage value at the 14th day of fermentation. This study determined the optimal fermentation time and carbon source type with white rot fungi in corn and rice straw to improve the utilization and digestibility of nutrients for ruminant feed. Acknowledgments This Supported by Chungbuk National University 2022. Author Contributions Conceptualization: X.L. Data curation: Y.W., J.Y., L.T. and S.N. Formal analysis: X.L., Q.L. Methodology: Q.L. Software: J.Z. and Y.W. Investigation: B.S., J.Z. and L.T. Writing--original draft: Y.W., J.Y. Writing--review & editing: S.C. and X.L. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of Yanbian University using the approval code YBU20160303. Informed Consent Statement Not applicable. Data Availability Statement The datasets generated and/or analyzed during the conduct of the study are included in this published article. Upon reasonable request, the datasets of this study are available from the corresponding author. Conflicts of Interest No potential conflict of interest relevant to this article was reported. Figure 1 Loss rate of cellulose, hemicellulose, and lignin from corn stover during 21 days of solid-state fermentation with Phanerochaete chrysosporium. (A) Cellulose loss rate. (B) Hemicellulose loss rate. (C) Lignin loss rate. Figure 2 Loss rate of cellulose, hemicellulose, and lignin from rice straw during 21 days of solid-state fermentation with Phanerochaete chrysosporium. (A) Cellulose loss rate. (B) Hemicellulose loss rate. (C) Lignin loss rate. animals-13-00888-t001_Table 1 Table 1 Chemical composition of corn straw fermented by white rot fungi with the addition of different carbon sources (g/kg DM) after 0, 7, 14, and 21 days. Item Treatment Sampling Day SEM p 0 7 14 21 DM Control 80.56 a 74.18 b 63.43 c 59.88 c 2.534 <0.01 Glucose 84.95 a 73.16 b 62.32 c 59.76 c 3.210 <0.01 Molasses 86.39 a 74.76 b 64.31 c 63.58 c 2.909 <0.01 Sucrose 85.49 a 72.36 b 62.2 c 60.94 c 3.010 <0.01 Soluble starch 85.09 a 71.59 b 62.58 c 62.58 c 2.804 <0.01 SEM 0.725 0.621 0.985 0.534 _ _ p NS NS NS NS _ _ CP Control 2.41 d 2.88 c 3.17 Cb 3.31 Ba 0.10 <0.01 Glucose 2.31 d 3.05 c 3.54 Ab 3.90 Aa 0.18 <0.01 Molasses 2.18 d 3.00 c 3.48 Ab 3.90 Aa 0.19 <0.01 Sucrose 2.23 c 3.08 b 3.21 BCab 3.41 Ba 0.140 <0.01 Soluble starch 2.40 c 3.03 b 3.33 Ba 3.41 Ba 0.122 <0.01 SEM 0.04 0.03 0.04 0.07 _ _ p NS NS <0.01 <0.01 _ _ Ash Control 5.78 A 5.54 5.94 AB 5.49 0.088 NS Glucose 4.69 BC 5.41 5.54 AB 5.63 0.134 NS Molasses 4.62 BCb 5.52 a 5.78 ABa 5.84 a 0.153 <0.01 Sucrose 5.13 Bb 5.51 ab 6.12 Aa 5.51 ab 0.128 <0.05 Soluble starch 4.40 Cc 5.63 a 5.07 Bb 6.01 a 0.187 <0.01 SEM 0.137 0.062 0.117 0.073 _ _ p <0.01 NS <0.05 NS _ _ ADF Control 56.30 a 52.28 a 43.67 Bb 33.76 Bc 2.632 <0.01 Glucose 58.03 a 54.83 a 46.61 ABb 41.6 Ab 2.029 <0.01 Molasses 56.62 a 53.65 a 49.50 Ab 38.60 Ac 2.073 <0.01 Sucrose 58.13 a 53.65 b 50.95 Ab 38.48 Ac 2.210 <0.01 Soluble starch 56.23 a 50.82 b 50.19 Ab 33.48 Bc 2.596 <0.01 SEM 0.347 0.471 0.817 0.879 _ _ p NS NS <0.01 <0.01 _ _ NDF Control 85.87 a 75.99 Ba 64.52 Cb 53.51 Cd 3.263 <0.01 Glucose 87.52 a 82.94 Ab 78.58 Ac 62.29 ABd 2.878 <0.01 Molasses 85.71 a 80.73 ABb 77.63 Ac 65.58 Ac 2.241 <0.01 Sucrose 87.16 a 77.02 Bb 73.63 Bc 60.51 Bd 2.885 <0.01 Soluble starch 87.37 a 77.77 Bb 74.54 ABc 58.98 Bc 3.086 <0.01 SEM 0.281 0.741 1.364 1.099 _ _ p NS <0.01 <0.01 <0.01 _ _ Mean values with different superscripts (A-D) in the same row differ within sampling days (p < 0.01). Means with different superscripts (a-d) in the same column differ within the treatments (p < 0.05). SEM, standard error of means. NS, not significant. animals-13-00888-t002_Table 2 Table 2 Chemical composition of rice straw fermented by white rot fungi with the addition of different carbon sources (g/kg DM) after 0, 7, 14, and 21 days. Item Treatment Sampling Day SEM p 0 7 14 21 DM Control 78.27 a 72.71 Bb 65.12 c 63.29 ABc 1.839 <0.01 Glucose 79.04 a 73.53 Bb 61.54 c 61.00 Bc 2.357 <0.01 Molasses 80.75 a 71.82 Bb 61.6 c 61.63 ABc 2.802 <0.01 Sucrose 78.67 a 75.62 Ab 63.45 c 64.48 ABc 2.032 <0.01 Soluble starch 80.28 a 74.36 ABb 63.31 c 65.17 Ac 2.567 <0.01 SEM 0.314 0.380 0.455 0.501 _ _ p NS <0.01 NS <0.01 _ _ CP Control 2.90 c 2.99 Bbc 3.15 BCb 3.41 Ca 0.06 <0.01 Glucose 2.93 c 3.09 ABbc 3.24 Bb 3.68 Aa 0.09 <0.01 Molasses 2.95 c 3.29 Ab 3.50 Aa 3.56 Ba 0.07 <0.01 Sucrose 2.95 bc 2.86 Bc 3.02 Db 3.32 Ca 0.05 <0.01 Soluble starch 2.97 2.95 B 3.03 CD 3.16 D 0.04 NS SEM 0.02 0.05 0.05 0.05 _ _ p NS <0.05 <0.01 <0.01 _ _ Ash Control 9.38 Ac 10.86 ABa 10.99 Ca 10.33 Cb 0.197 <0.01 Glucose 9.52 Ac 10.63 Bc 12.18 Aa 11.86 ABa 0.332 <0.01 Molasses 7.47 Cc 11.71 Ab 11.81 ABab 12.47 Aa 0.600 <0.01 Sucrose 8.43 Bc 10.51 Bb 11.21 BCab 11.92 ABa 0.402 <0.01 Soluble starch 7.61 BCc 10.76 Bb 10.84 Cab 11.53 Ba 0.461 <0.01 SEM 0.238 0.132 0.146 0.196 _ _ p <0.01 <0.01 <0.01 <0.01 _ _ ADF Control 66.80 ABa 58.89 BCb 56.92 Ac 41.24 Cd 2.799 <0.01 Glucose 65.98 ABa 63.89 Ab 55.97 Ac 49.29 Ad 2.005 <0.01 Molasses 66.93 ABa 63.66 Ab 58.24 Ac 50.83 Ad 1.865 <0.01 Sucrose 67.99 Aa 57.64 Cb 52.74 Bc 44.86 Bd 2.538 <0.01 Soluble starch 64.28 Ba 60.25 Bb 57.43 Ab 42.30 Bc 2.521 <0.01 SEM 0.399 0.697 0.538 1.042 _ _ p <0.05 <0.01 <0.01 <0.01 _ _ NDF Control 84.61 ABa 74.37 Cb 71.58 Bc 53.07 d 3.445 <0.01 Glucose 81.95 Ba 79.17 Aab 76.76 Ab 63.32 c 2.180 <0.01 Molasses 86.49 Aa 78.79 ABb 75.75 Ac 60.59 d 2.849 <0.01 Sucrose 83.93 ABa 76.44 ABCb 71.9 Bbc 67.98 c 1.831 <0.01 Soluble starch 83.35 ABa 75.90 BCb 74.07 ABb 57.36 c 2.875 <0.01 SEM 0.484 0.532 0.604 1.396 _ _ p <0.05 <0.01 <0.01 <0.01 _ _ Note: same as Table 1. animals-13-00888-t003_Table 3 Table 3 Cellulose, hemicellulose, and lignin content of corn straw. Item Treatment Sampling Day SEM p 0 7 14 21 Cellulose Control 29.40 a 28.67 Aa 18.20 Bb 10.74 Cb 2.367 <0.01 Glucose 30.34 a 32.07 ABa 25.46 Aab 22.25 Ab 1.284 <0.01 Molasses 29.21 a 27.55 ABCa 25.56 Aa 16.44 Bb 1.530 <0.01 Sucrose 29.78 a 26.37 BCa 23.71 Ac 16.27 Bd 1.512 <0.01 Soluble starch 28.11 a 23.45 Ca 25.13 Aa 8.99 Cb 2.268 <0.01 SEM 0.342 0.830 0.835 1.316 _ _ p NS <0.01 <0.01 <0.01 _ _ Hemicellulose Control 29.23 a 23.72 ABa 20.85 Cb 19.75 Bb 1.196 <0.01 Glucose 29.48 a 28.12 Aab 31.97 Aa 20.69 ABb 1.405 <0.01 Molasses 29.08 27.08 AB 28.13 AB 26.98 A 0.415 NS Sucrose 29.04 a 23.37 Bb 22.69 BCb 22.03 ABb 0.926 <0.01 Soluble starch 31.14 a 26.94 ABb 24.35 Bb 25.5 ABb 0.833 <0.01 SEM 0.334 0.616 1.149 0.878 _ _ p NS <0.05 <0.01 <0.01 _ _ Lignin Control 21.12 Ba 19.54 Bb 18.07 Bc 17.54 ABc 0.424 <0.01 Glucose 18.35 Ca 17.34 Ba 15.61 Cb 13.72 Cc 0.538 <0.01 Molasses 22.78 Aa 20.58 Aa 18.16 Bb 16.32 Bb 0.758 <0.01 Sucrose 23.22 Aa 21.77 Ab 21.13 Ab 16.71 Bc 0.740 <0.01 Soluble starch 23.72 Aa 21.75 Ab 20.00 ABc 18.48 Ad 0.601 <0.01 SEM 0.528 0.508 0.518 0.443 _ _ p <0.01 <0.01 <0.01 <0.01 _ _ Note: same as Table 1. animals-13-00888-t004_Table 4 Table 4 Cellulose, hemicellulose, and lignin contents of rice straw. Item Treatment Sampling Day SEM p 0 7 14 21 Cellulose Control 41.85 ABa 34.85 Bb 33.31 Ab 19.34 Bc 2.467 <0.01 Glucose 37.33 Ca 35.86 ABa 25.82 Cb 24.50 Ab 1.750 <0.01 Molasses 41.82 ABa 34.57 Bb 29.32 Bc 21.29 ABd 2.279 <0.01 Sucrose 39.32 BCa 27.92 Cb 24.412 Cc 19.45 Bd 2.215 <0.01 Soluble starch 43.25 Aa 37.35 Ab 34.57 Ab 19.13 Bc 2.702 <0.01 SEM 0.610 0.879 1.090 0.600 _ _ p <0.01 <0.01 <0.01 <0.01 _ _ Hemicellulose Control 17.81 ABa 15.48 a 14.66 Bab 11.84 Bb 0.690 <0.01 Glucose 15.96 Bb 15.28 b 20.79 Aa 14.02 Bb 0.831 <0.01 Molasses 19.56 ABa 15.13 ab 17.51 ABab 19.76 Bb 1.203 <0.01 Sucrose 15.93 Bb 18.81 ab 19.16 ABab 23.12 Aa 0.913 <0.01 Soluble starch 19.07 ABa 15.65 ab 16.65 ABab 15.06 Bb 0.553 <0.01 SEM 0.470 0.493 0.668 1.280 _ _ p <0.01 NS <0.01 <0.01 _ _ Lignin Control 15.57 Da 13.18 Db 12.61 Bb 11.56 Cc 0.450 <0.01 Glucose 19.15 Ba 17.41 Ba 17.97 Aa 12.93 BCb 0.746 <0.01 Molasses 17.64 Ca 17.38 Bb 17.10 Ac 17.07 Ac 0.072 <0.01 Sucrose 20.25 Aa 19.2 Ab 17.12 Ac 13.50 Bd 0.778 <0.01 Soluble starch 13.42 Ea 12.14 Eb 12.01 Bb 11.64 Cc 0.204 <0.01 SEM 0.659 0.727 0.689 0.546 _ _ p <0.01 <0.01 <0.01 <0.01 _ _ Note: Same as Table 1. animals-13-00888-t005_Table 5 Table 5 In vitro fermentation characteristics of corn stover. Item Treatment Sampling Day SEM p 0 7 14 21 IVDMD, % Control 13.75 Cb 13.53 Eb 14.77 Ca 15.20 Da 0.2249 <0.01 Glucose 13.99 Bc 26.37 Bb 32.55 Aa 30.11 ABab 2.1762 <0.01 Molasses 13.95 BCc 30.31 Ab 33.32 Aa 33.46 Aa 2.4387 <0.01 Sucrose 14.72 Ad 20.52 Dc 24.29 Bb 24.97 Ca 1.2797 <0.01 Soluble starch 14.29 ABc 22.39 Cb 25.65 Bb 28.03 BCa 1.5540 <0.01 SEM 0.097 1.516 1.516 1.691 _ _ p <0.01 <0.01 <0.01 <0.01 _ _ Total VFA concentration, mmol/L Control 18.39 Cd 19.48 Cc 21.79 Ea 20.61 Eb 0.3904 <0.01 Glucose 19.35 Ad 25.72 Ac 35.56 Aa 31.40 Ab 1.8548 <0.01 Molasses 19.61 Ac 26.13 Ab 30.27 Ba 20.11 Dc 1.3358 <0.01 Sucrose 18.63 BCd 23.42 Bb 24.35 Da 21.84 Cc 0.6576 <0.01 Soluble starch 19.33 ABd 22.23 Bc 27.50 Ca 25.88 Bb 0.9640 <0.01 SEM 0.143 0.679 1.280 1.134 _ _ p <0.01 <0.01 <0.01 <0.01 _ _ pH Control 6.67 b 6.41 b 6.75 Ba 6.8 Ba 0.0505 <0.01 Glucose 6.69 ab 6.39 c 6.65 Bb 6.9 Ba 0.0584 <0.01 Molasses 6.75 a 6.38 b 6.74 Ba 6.83 Bb 0.0536 <0.01 Sucrose 6.68 b 6.49 c 7.11 Aa 6.66 Cbc 0.0700 <0.01 Soluble starch 6.72 b 6.34 c 7.06 Aa 7.30 Abc 0.1121 <0.01 SEM 0.024 0.023 0.050 0.059 _ _ p NS NS <0.01 <0.01 _ _ NH3-N, mmol/L Control 9.64 BCc 13.82 Ea 13.59 Ca 12.5 Bb 0.5023 <0.01 Glucose 11.18 ABd 16.44 Cb 17.31 Ca 12.28 Bc 0.7920 <0.01 Molasses 12.10 Ac 18.41 Ab 20.27 Aa 7.75 Cd 1.5085 <0.01 Sucrose 8.76 Cc 17.52 Ba 13.4 Bb 14.29 Ab 0.9692 <0.01 Soluble starch 9.82 ABd 15.49 Da 14.26 Db 12.71 Bc 0.6386 <0.01 SEM 0.360 0.429 0.714 0.588 _ _ p <0.01 <0.01 <0.01 <0.01 _ _ Note: same as Table 1. animals-13-00888-t006_Table 6 Table 6 In vitro fermentation parameters of rice straw. Item Treatment Sampling Day SEM p 0 7 14 21 IVDMD, % Control 15.62 ABd 26.34 Ea 21.73 c 23.41 Cb 1.1843 <0.01 Glucose 15.02 Cc 35.55 Ba 35.86 a 28.49 Bb 2.5478 <0.01 Molasses 15.27 ABCd 40.06 Aa 29.68 c 30.60 Ab 4.1407 <0.01 Sucrose 15.83 Ac 28.34 Db 32.72 a 28.5 Bb 1.9077 <0.01 Soluble starch 15.23 BCd 30.07 Cb 32.69 a 24.39 Cc 2.0191 <0.01 SEM 0.089 1.348 2.070 2.820 _ _ p <0.01 <0.01 NS <0.01 _ _ Total VFA concentration, mmol/L Control 15.46 Cc 16.44 Db 19.58 Da 16.54 Cb 0.4680 <0.01 Glucose 16.33 Bd 25.18 Bb 30.68 Aa 18.5 Bc 1.7079 <0.01 Molasses 17.277 Ac 26.38 Ab 30.33 ABa 16.50 Cd 1.7821 <0.01 Sucrose 16.50 Bd 22.27 Cb 26.11 BCa 12.4 Dc 1.5883 <0.01 Soluble starch 15.34 Cd 26.1533 Cc 25.16 Ca 19.64 Ab 1.3829 <0.01 SEM 0.193 0.996 1.133 0.661 _ _ p <0.01 <0.01 <0.01 <0.01 _ _ pH Control 6.68 b 6.89 Aa 7.04 Ba 7.01 Aa 0.0445 <0.01 Glucose 6.83 b 6.8 Ab 7.17 Aa 6.85 Bb 0.0482 <0.01 Molasses 6.77 a 6.53 Cb 6.64 Dab 6.53 Db 0.0321 <0.01 Sucrose 6.68 ab 6.53 Bb 6.84 Ca 6.82 BCa 0.0400 <0.01 Soluble starch 6.71 ab 6.64 Bb 6.71 Dab 6.75 Ca 0.0130 <0.01 SEM 0.023 0.040 0.054 0.042 _ _ p NS <0.01 <0.01 <0.01 _ _ NH3-N, mmol/L Control 9.11 Ca 9.49D a 7.55 Eb 5.55 Ec 0.4691 <0.01 Glucose 9.62 Bb 12.47 Ca 12.42 Ba 9.52 Ab 0.4344 <0.01 Molasses 10.40 Ab 14.57 Aa 14.34 Aa 7.46 Cc 0.8902 <0.01 Sucrose 8.71 Dc 12.37 Ca 10.82 Cb 6.47 Dd 0.6710 <0.01 Soluble starch 9.60 Bb 13.61 Ba 9.23 Dc 8.23 Bd 0.6183 <0.01 SEM 0.154 0.457 0.635 0.369 _ _ p <0.01 <0.01 <0.01 <0.01 _ _ Note: same as Table 1. animals-13-00888-t007_Table 7 Table 7 Proportion of volatile acids in corn stover. Item Treatment Sampling Day SEM p 0 7 14 21 Acetic acid, % Control 70.00 Ac 73.03 Cb 75.09 Ca 74.90 Ca 0.628 <0.01 Glucose 69.00 Bc 75.33 Ab 77.29 Ba 76.84 Aa 1.008 <0.01 Molasses 68.90 Bc 75.10 ABb 78.40 Aa 78.29 Aa 1.169 <0.01 Sucrose 70.50 Ac 73.84 BCb 75.00 Ca 75.24 Ca 0.580 <0.01 Soluble starch 68.80 Bc 74.14 Bb 75.93 Ca 75.72 Ca 0.887 <0.01 SEM 0.189 0.243 0.329 0.313 _ _ p <0.01 <0.01 <0.01 <0.01 _ _ Propionic acid, % Control 18.49 BCc 18.72 Bb 19.18 Bb 20.17 a 0.210 <0.01 Glucose 19.33 Ab 19.76 Ab 20.81 Ab 23.25 a 0.536 <0.05 Molasses 18.24 Cd 19.06 ABb 19.89 ABb 20.78 a 0.29516 <0.01 Sucrose 18.95 ABb 18.79 Bb 19.69 ABab 21.52 a 0.426 NS Soluble starch 19.27 Ab 19.27 ABb 19.30 Bb 21.92 a 0.40391 <0.05 SEM 0.120 0.116 0.182 0.443 _ _ p <0.01 <0.01 <0.01 NS _ _ Butyric acid, % Control 7.25 ABb 7.33 Bb 8.04 Ca 8.16 Da 0.128 <0.01 Glucose 6.58 Bc 7.76 Bc 8.81 Ba 8.92 Ba 0.293 <0.01 Molasses 6.51 Bc 8.69 Ab 9.4.0 Aa 9.51 Aa 0.364 <0.01 Sucrose 7.37 Aa 6.55 Cb 7.43 Da 7.52 Da 0.132 <0.01 Soluble starch 6.60 Bb 7.80 Bb 8.21 Ca 8.30 Ca 0.217 <0.01 SEM 0.116 0.191 0.185 0.186 _ _ p <0.01 <0.01 <0.01 <0.01 _ _ Valeric acid, % Control 0.84 A 0.85 BC 0.84 0.85 B 0.0045 NS Glucose 0.78 BC 0.90 A 1.07 0.96 A 0.0427 NS Molasses 0.75 Cb 0.85 Cb 1.52 a 0.93 Ab 0.0958 <0.01 Sucrose 0.80 B 0.86 BC 1.53 0.87 B 0.1724 NS Soluble starch 0.85 A 0.86 B 0.86 0.86 B 0.0042 NS SEM 0.011 0.005 0.146 0.012 _ _ p <0.01 <0.01 NS <0.01 _ _ Note: same as Table 1. animals-13-00888-t008_Table 8 Table 8 Proportion of volatile acids in rice straw. Item Treatment Sampling Day SEM p 0 7 14 21 Acetic acid, % Control 65.16 c 77.93 b 79.99 Ca 79.80 Ca 1.856 <0.01 Glucose 65.85 c 80.23 b 82.19 Ba 81.74 Ba 2.044 <0.01 Molasses 64.04 c 80.00 b 83.30 Aa 83.19 Aa 2.403 <0.01 Sucrose 64.70 b 78.74 b 79.90 Ca 80.14 Ca 1.959 <0.01 Soluble starch 64.85 c 79.04 b 80.83 Ca 80.62 BCa 2.019 <0.01 SEM 0.188 0.220 0.316 0.301 _ _ p NS <0.01 <0.01 <0.01 _ _ Propionic acid, % Control 19.70 Bc 21.68 Bb 22.22 Ba 22.37 Ca 0.33 <0.01 Glucose 22.47 Ab 22.66 Ab 23.91 Aa 24.04 Aa 0.26 <0.05 Molasses 21.92 Ab 22.02 ABb 22.92 ABa 22.97 BCa 0.16 <0.01 Sucrose 20.29 Bc 21.69 Bb 22.80 ABa 23.15 Ba 0.35 <0.01 Soluble starch 21.72 Ac 22.16 ABb 22.40 Bb 22.85 BCa 0.13 <0.01 SEM 0.286 0.112 0.189 0.156 _ _ p <0.01 <0.01 <0.05 <0.01 _ _ Butyric acid, % Control 9.06 ABb 9.41 Dc 10.45 Ca 10.17 Ea 0.176 <0.01 Glucose 8.39 Bd 10.07 Bc 12.61 Aa 12.17 Bb 0.512 <0.01 Molasses 8.32 Bc 11.42 Ab 11.52 Bb 12.56 Aa 0.481 <0.01 Sucrose 9.19 Ab 9.29 Db 9.39 Db 10.62 Da 0.191 <0.01 Soluble starch 8.41 Bd 10.83 Ca 11.26 Ba 11.45 Ca 0.375 <0.01 SEM 0.121 0.225 0.293 0.244 _ _ p <0.05 <0.01 <0.01 <0.01 _ _ Valeric acid, % Control 1.01 c 1.10 bc 1.19 Bb 1.21 Ba 0.0296 <0.05 Glucose 0.94 b 0.99 ab 1.28 Aa 1.28 Aa 0.0553 <0.01 Molasses 0.91 b 1.09 a 1.25 Ba 1.27 Aa 0.0482 <0.01 Sucrose 0.96 b 0.95 b 1.19 Ba 1.21 Ba 0.0471 <0.05 Soluble starch 1.01 1.11 1.20 C 1.20 B 0.0322 NS SEM 0.030 0.034 0.010 0.010 _ _ p NS NS <0.01 <0.01 _ _ Note: same as Table 1. 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PMC10000111

The present study aimed to investigate whether the exogenous addition of amylase enhances the nutritional value of pea seeds for broiler chickens. In total, 84 1-day-old male broiler chickens (Ross 308) were used for the experimental study. During the first phase of the experiment (1-16 d), all birds in each treatment were fed with a corn-soybean meal reference diet. After this time, the first treatment (control) was still fed the reference diet. In the second and third treatment, 50% of the reference diet was replaced with 50% pea seeds. In addition, the third treatment was supplemented with exogenous amylase. Animal excreta were collected on 21 d and 22 d of the experiment. The birds were sacrificed at the end of the experiment (23 d), and samples of ileum content were collected. The experimental results showed that the exogenous addition of amylase significantly improved (p < 0.05) the apparent ileal digestibility (AID) of the crude protein (CP), starch, and dry matter (DM) of pea. In addition, an improvement in the AID of essential amino acids in pea seeds (except Phe) was observed. The trend in the AMEN values was also noted (p = 0.076). It can be concluded that supplementation with exogenous amylase improves the nutritional value of pea seeds in broiler chicken nutrition. pea amylase nutritional value digestibility poultry Ministry of Agriculture and Rural Development149/2011 This work was supported by the Program of the Ministry of Agriculture and Rural Development for 2016-2020, Resolution No. 149/2011 of 9 August 2011. pmc1. Introduction Pea (Pisum sativum L.) can be successfully grown and harvested in almost all the climatic zones across the world. Currently, the main protein source that is used in animal nutrition is soybean meal (SBM). However, alternative protein sources obtained particularly from non-GMO plants have been the subject of interest for many authors conducting studies in poultry nutrition . Pea seeds are characterized by a relatively high content of crude protein and starch, and they can serve as a potential energy source when included in feed. It has been reported that starch is the most important energy source for poultry , and starch widely determines the apparent metabolizable energy (AMEN) value. Starch is usually digested by the endogenous enzymes, but the efficiency of digestion depends on many factors, such as growth phase , composition of diet, and the chemical structure of starch . A small portion of starch present in pea seeds is defined as resistant starch (RS), which, depending on the subtype, is harder to digest or not digested at all . Recently, several methods have been explored to enhance the nutritional value of pea seed meals. Technological processes are very effective because they induce changes in the molecular structure of starch. Previous studies have shown that the extrusion process significantly reduced the content of RS in pea seeds , improving the digestibility of starch, CP, and, crude fat in broiler chickens . However, these techniques are expensive and led to increases in the cost of production. Compared with this process, enzyme supplementation may be more economically advantageous. Amylase can be highly beneficial for pea seeds because of the presence of a relatively high starch concentration. However, reports regarding the use of amylase alone in diets based on pea seeds are limited. Earlier studies demonstrated the positive impact of amylase when used in soybean-corn diets. Some authors showed that the addition of amylase improves growth performance , the digestibility of starch and CP , and AMEN value in broiler chickens. In contrast, Kaczmarek et al. (2014) showed that amylase supplementation in a corn-soybean meal diet did not cause any impact on starch and CP digestibility, or on AMEN values. Based on the above-mentioned results and due to the lack of information about the effects of amylase supplementation when added to a diet based on pea, the present study hypothesized that exogenous amylase could exhibit a positive influence on the nutritional value of pea seeds when included in the feed of broiler chicken. Moreover, the amount of excreted sialic acid will allow for the estimation of endogenous losses after amylase supplementation. The aim of the present study was to determine the influence of amylase on nutrient digestibility, energy value, and the excretion of sialic acid in pea-seed-based diets for broiler chickens. 2. Materials and Methods All animal procedures were conducted in accordance with the guidelines proposed by the Polish Council of Animal Care . Pea seeds The samples of pea seeds (Pisum sativum L.) cv. Mentor were obtained from the Polish Plant Breeding Station located in Tulce, and harvested in 2019 in Poland. The seeds were initially ground by using a hammer mill (RG11 model; Zuptor, Gostyn, Poland) with a screen size of 2.0 mm. The chemical composition and the antinutritional factors regarding the concentration of pea are shown in Table 1. 2.1. Bird Management This experiment was conducted in a completely randomized design with a control group and two experimental groups, which differed by the addition of exogenous a-amylase. The study was conducted on 84 1-day-old male broiler chickens (Ross 308) with an initial individual weight of 42 +- 2 g, which were obtained from a Polish hatchery (Dan Hutch, Wolsztyn, Poland). Each group was composed of 14 replicates, with 2 birds in each replicate. All the environmental conditions were controlled and kept constant and were in agreement with the recommendations of AVIAGEN . During the first 7 days of the experiment, the temperature was maintained at about 33 degC and then gradually reduced to 23 degC. The chickens were exposed to light for 24 h during the first 7 days, and then the time of light exposure was gradually reduced to 16 h. Birds were kept in metabolic cages in one common room, and collection trays were installed in every cage. Access to the feed and water was ad libitum during the entire experiment. 2.2. Diets All diets were prepared according to AVIAGEN recommendations , such that they meet the nutritional needs of the chickens. The chemical composition and the nutritional value of the reference diet (control) are presented in Table 2. Birds in treatment 1 (control) were fed with a reference diet from 1 d to 16 d of the experiment. Birds in treatment 2 and treatment 3 were fed with the same reference diet from 1 d to 16 d of the experiment, and after this period, 50% of the reference diet was replaced with pea seed. The third treatment was supplemented with exogenous amylase, according to the dosage recommended by the manufacturer of the enzyme (0.14 g/kg; 80 KNU/kg as fed). The enzyme was obtained from a commercial source (Rononzyme HiStarch; DSM Nutrition, Heerlen, The Netherlands). Titanium dioxide (3 g/kg as fed) was added to each diet in each treatment, which serves as a nonabsorbable marker to determine the digestibility coefficients and AMEN value. 2.3. Samples Collection For excreta collection, collection trays were installed under each cage, and excreta were collected thrice a day at 21 d and 22 d of experiment. One sample was collected from each replicate group from each treatment. Hence, the total number of samples collected was 14. At the end of the experiment (23 d), all the birds were slaughtered by cervical dislocation. The ileum was removed and the ileal digesta (15 cm, adjacent to the ileocecal junction) was acquired as samples for further analysis. To ensure that a sufficient amount of material was available for chemical analysis, digesta samples of two birds were combined to form a single sample, and thus the total number of samples obtained was 14. Immediately after sample collection, each sample was frozen and then lyophilized. 2.4. Chemical Analysis All samples were ground and passed through a 0.5 mm sieve. Chemical analysis of pea seeds (ran in duplicate) was carried out to determine the amounts of DM, CP, ether extract, crude ash, neutral detergent fiber (NDF), aNDF (NDF with heat-stable amylase and expressed inclusive of residual ash), and acid detergent fiber (ADF; expressed inclusive of residual ash), which was performed according to AOAC standard methods 934.01, 976.05, 920.39, 984.27, 973.18, and, 942.05, respectively . These methods were used to determine the content of DM and CP in digesta and excreta. The assay kit (Megazyme International, Dublin, Ireland) was prepared using thermostable a-amylase and amyloglucosidase according to the standard guidelines of AOAC using method 996.11. This method was used to determine the starch content in pea seeds, diet formulations, and digesta. RS assay kit (Megazyme International, Wicklow, Ireland) was prepared according to the method 2002.02 , with a slight modification in the time of incubation, as presented by Weurding et al. (2001) , and was used for the determination of the content of RS in pea seeds. Gross energy was calculated in pea seeds, feed, and excreta by using an adiabatic bomb calorimeter (KL 12 Mn; Precyzja-Bit PPHU, Bydgoszcz, Poland) standardized with benzoic acid. The tannins were evaluated according to the method described by Kuhla and Ebmeier (1981) . Phytates were determined according to the method presented by Haug and Lantzsch (1983) . RFOs were determined using a high-resolution gas chromatography technique according to the method suggested by Zalewski et al. (2001) . Phosphorus was determined using the colorimetric method with ammonium molybdate, and calcium by the spectrophotometric method using a split AES Agilent instrument. Non-starch polysaccharides (NSP) in pea seeds were determined by using gas-liquid chromatography-neutral sugars, according to method presented by Englyst and Cummings (1988) , with modifications presented by Slominski and Campbell (1990) . Colorimetry method was used for uronic acids, according to method presented by Scott (1979) . Postcolumn derivatization using ninhydrin reagent (according to method 994.12) was performed on an AAA-400 Automatic Amino Acid Analyzer (INGOS s.r.o., Prague, Czech Republic) to determine the amino acid content in pea seeds, experimental diets, and digesta samples. The concentrations of titanium dioxide in diet formulations, digesta, and excreta samples were determined by using the method described by Short et al. (1996) , and each sample was prepared according to the procedure presented by Myers et al. (2004) . The excreted free and total sialic acid content was evaluated according to the method presented by Jourdian et al. (1971) after extracting crude mucins from excreta . The determination of WEV was carried out as follows: 1 g of sample was mixed with 5 mL distilled water for 1 h at 40 degC and was centrifuged at 10,000 g for 10 min at 4 degC. The supernatant was withdrawn and viscosity was determined in a Brookfield Digital DV-II+ cone/plate viscometer (Brookfield Engineering Laboratories Inc., Stoughton, MA, USA) maintained at 40 degC at a shear rate of 12 x s-1. WEV units are mPas*s = cP = 1 x 100 dyne s cm2. 2.5. Calculations and Statistical Analysis Apparent ileal digestibility (AID) values in reference diet (treatment 1) for DM, CP, starch, and amino acids were calculated from the ratio of titanium dioxide concentration in the diet to its concentration in digesta (indigestible marker method) by using the following formula (with starch (S) as an example):AID=1-TiO2 diet TiO2 digestaxSdigestaSdietx100% where S represents the content of starch and TiO2 is the dietary marker. The AID values of DM, CP, starch, and amino acids in pea seeds (treatment 2 and 3) were determined (difference method) using the following formula:AIDStarch=AIDStarch dietxCStarch diet-AIDStarch referencexCStarch referencex0.50CStarch diet-CStarch referencex0.50 where AIDStarch--digestibility of starch; AIDStarch diet--digestibility coefficient of starch in diet; CStarch diet--concentration of starch in diet; 0.50--amount of investigated pea seeds in diet; AIDStarch reference--digestibility coefficient of starch in reference diet; and CStarch reference--concentration of starch in reference diet. The difference method approach was used to calculate the AMEN and apparent ileal digestibility coefficient of various dietary components contained in pea. The AMEN value was corrected to zero nitrogen balance using 34.4 kJ/g of N retained and was calculated by using the following equation (treatment 1):AMEN=GEdiet-GEexcretaxTiO2 dietTiO2 excreta-0.0344xNdiet-NexcretaxTiO2 dietTiO2 excreta where GE represents the gross energy (kcal/kg), N represents nitrogen, and TiO2 is the dietary marker. The AMEN value for treatment 2 and 3 was estimated using the formula:AMEN Pea seeds=(AMEN Pea diet-AMEN reference dietx0.5/0.5) where 0.50 is the proportion of the investigated pea seeds in the pea diet and 0.50 is the proportion of the reference diet in the pea diet. The obtained data were explored to discard any possible outliers. The experimental results were analyzed by using statistical software SAS, (2012). Pooled standard error of mean and group mean values were calculated. Significant differences between means values of the factors investigated in this study were estimated by performing a t-test at a significance level of p <= 0.05. The following formula was used:t=Kh 1-Kh 2s12n1+s22n2 where t-Student's t-test value Kh 1-Kh 2 is the difference in the means between the two groups being compared; s12,s22 are variance estimates from each independent group; and n1,n2 are the respective sample sizes for each independent group. In this study, the standard error of the mean (SEM) was established as a measure of error. 3. Results The overall mortality was found to be low (<2%) and was not attributed to any specific dietary treatment (data not shown). All research procedures were conducted according to EU directives, and the protocol for this study was approved by the Local Ethics and Animal Experimentation Committee at Poznan University of Life Science. 3.1. Nutritional Value and Secretion of Sialic Acid The effect of enzyme addition on the AID values in pea seeds of DM, CP, and starch is presented in Table 3. The addition of exogenous amylase significantly increased the AID value of DM (from 0.691 to 0.730; p < 0.05). A similar result was observed for the AID value of CP, from 0.800 to 0.864. Additionally, the AID value of starch was significantly higher from 0.852 to 0.886 (p < 0.05). No significant differences in the AMEN values were observed between supplemented and non-supplemented diets. However, a tendency was observed (p = 0.076). No significant differences in the amount of excreted total and free sialic acids were observed as a result of amylase addition (Table 3). 3.2. Amino Acid Digestibility The effect of amylase supplementation on the AID of amino acids is presented in Table 3. The addition of amylase significantly increased the AID of almost all essential amino acids (except Phe; p < 0.05). A maximum improvement in AID (8% increase) was observed for Thr and Ile. Additionally, a high (7.00%) increase was noted for Lys and Val. In the case of the non-essential amino acids of pea seeds, a significant difference was observed only for Pro, an almost 12.00 % improvement (p < 0.05). 4. Discussion Pea seeds are considered to be an important source of protein. The CP concentration was found to be in the reported range of about 208.0-276.0 g/kg as fed depending on the cultivar or type of pea (white or colored flower) . The concentration of starch was found to be higher than the value presented by another study . The content of resistant starch was low (136.6 g/kg as fed) when compared with that observed in a previous study . The pea seeds are a good source of essential amino acids, such as Leu, Lys, and Arg . The main RFO found in the pea seeds was stachyose, which was also found by Hejdysz et al. (2015) . The total concentration of NSP was much lower than in data presented by others authors . Indeed, the influence of the cultivar should be distinguished among the factors influencing the concentration of nutrients, as well as antinutritional factors. Amylase is an enzyme that specifically degrades starch, and supplementation with amylase can have a positive impact on the growth performance of broiler chickens , which can be attributed to higher AMEN value. No significant differences were noted between non-supplemented and supplemented pea seeds for AMEN value in the present study. However, there was a tendency (p = 0.076), which is likely related to enhanced starch digestibility. The lack of significant difference in AMEN, and at the same time an important difference in the AID of starch, was interesting. However, we can speculate that RS in the group without the enzyme was fermented in the caeca and, due to this fact, the energy value level was close between groups. It has been earlier confirmed that fiber and CP also contribute to energy value measurements, although the apparent digestibility of energy alone does not provide a complete response regarding the efficacy of enzyme use . In this study, the addition of amylase significantly increased the AID of starch by 8.00% (p < 0.05), similar to the results of Gracia et al. (2003) and Amerah et al. (2017) . The crucial factor that determines the level of digestion is the ratio of amylose to amylopectin, and increasing the ratio of these two compounds reduced starch digestibility. Gunawardena et al. (2010) reported that the ratio of amylose to amylopectin in pea seeds was 31:69, due to which pea native starch is classified as type C. C-type starches do not undergo hydrolysis easily unlike A-type starch, which is found in corn. Since type-A starch is more susceptible to degradation, greater digestibility of this compound is observed in corn-based diets. In addition, a previous study by Amerah et al. (2017) reported that exogenous amylase increased starch digestibility by about 1.12%, while an improvement of 8.00% was noted in this study. However, the level of starch digestibility was found to be much higher in the above-mentioned study. Therefore, it may be concluded that the overall starch digestibility will always be higher with type-A starch. However, it should be noted that the addition of exogenous amylase could also provide good results with type-C starch. Additionally, since exogenous amylase was able to degrade RS effectively, a higher digestibility was observed. On the other hand, Stefanello et al. (2015) found the positive effect of amylase supplementation on the AMEN value in conventional diets. However, Kaczmarek et al. (2014) and Amerah et al. (2017) did not observe any increase in the AMEN value when amylase was supplemented in conventional diets. It can be assumed that many factors could affect the AMEN value, such as the source and the type of starch present in the feed, the different components used in the diet, and the techniques used for the processing of raw materials. However, the crucial factors that determine the AMEN value could be the concentration of ANF and RS. The addition of exogenous amylase significantly increased (p < 0.05) the AID of DM and CP by about 5.64% and 3.99% in pea seeds, respectively. An improvement in the AID of DM is the result of an overall improvement in the AID of starch and CP. Gracia et al. (2003) and Amerah et al. (2017) also reported an improvement in the digestibility of CP in studies where conventional diets based on corn and SBM were used. Starch granules contain almost 3 g of protein per 1 kg of starch . As a result of the hydrolysis of starch glycosidic bonds, the portion of protein molecules that were involved in the formation of complexes with starch are now be available for further digestion processes. Another factor that may contribute to improvements in the AID of CP is the fact that the addition of amylase could, by increasing the digestibility of starch and RS, have an effect on the viscosity of the digesta. This could have resulted in changes in the passage time of digesta through the gastrointestinal tract, thus prolonging the time for the action of endogenous proteases. An increase in the concentration of exogenous amylase in diets used for chickens resulted in the lower production and activity of endogenous amylase . In addition, Jiang et al. (2008) showed that amylase supplementation reduces the mRNA expression of amylase. The results of the present study indicate an increasing in the AID of almost all essential amino acids (except Phe). We can assume that a reduction in the production of endogenous amylase can be attributed to the enhanced AID of amino acids. The probable mechanism can be due to the reduced utilization of amino acids for the production of amylase by the pancreas. However, this finding should be confirmed in future studies. As mentioned earlier, starch binds to specific domains of the amylase enzyme. The cleavage of glycosidic bonds in the presence of amylase increases the availability of the substrate to enzymatic hydrolysis by proteases, which further leads to an increase in the digestibility of amino acids. Liu et al. (2020) reported that increasing the concentration of RS decreases the digestibility of CP. Amylase supplementation could effectively decrease the levels of RS in the feed. Schramm et al. (2021) reported that amylase addition increased the digestibility of RS. Therefore, we can conclude that a reduction in the levels of RS resulted in an increase in the digestibility of CP and amino acids. A previous study indicated that sialic acid excretion was lower after the extrusion process, which can be associated with lower levels of RS . Additionally, sialic acid can be considered to be an indicator of endogenous losses . However, no effect of amylase supplementation on sialic acid secretion was observed in the present study, which indicates that ANF concentration, rather than RS concentration, is a strong determiner of this parameter; however, this requires confirmation. In conclusion, the addition of exogenous amylase enhances the apparent ileal digestibility of DM, starch, and CP in pea seeds. In addition, the significantly higher apparent ileal digestibility of almost all essential amino acids was observed. The tendency to obtain a higher AMEN value was also observed. Therefore, exogenous amylase can improve the nutritional value of pea and affects the nutrients and amino acid utilization in broiler chicken nutrition. Acknowledgments The experiment was supported by the project "Integrated Program of the Poznan University of Life Sciences for Innovative Wielkopolska", implemented under OP KED, co-financed by the European Union under the European Social Fund. Author Contributions Conceptualization, K.P., M.H. and S.N.; methodology, K.P., M.H. and S.A.K.; software, M.H.; validation, M.H. and S.A.K.; formal analysis, K.P., M.H., S.N., S.A.K. and A.J.C.; investigation, K.P., M.H., S.N. and S.A.K.; resources, M.H.; data curation, K.P. and M.H.; writing--original draft preparation, K.P; writing--review and editing, M.H., S.N., S.A.K. and A.J.C.; visualization, K.P. and M.H.; supervision, M.H.; project administration, M.H. and S.A.K.; funding acquisition, M.H. All authors have read and agreed to the published version of the manuscript. Informed Consent Statement Not applicable. Data Availability Statement Data is available upon request from the corresponding authors. Conflicts of Interest The authors declare that there are no conflicts of interests. animals-13-00816-t001_Table 1 Table 1 Chemical composition, amino acids profile, NSP content, and antinutritional factors 1. Item Content (g/kg as fed) Dry matter 872.00 Crude protein 242.80 Crude fat 9.90 Ash 29.70 Ca 2.00 P 4.80 Starch 487.00 ADF 80.30 NDF 199.10 Resistant starch 136.60 WEV (cP) 1.34 Essential amino acids (g/kg as fed) Lys 16.34 Thr 9.25 Ile 10.39 Val 11.78 Leu 17.89 Phe 12.09 His 6.90 Arg 22.53 Gly 11.19 Non-essential amino acids (g/kg as fed) Tyr 7.41 Ala 12.36 Asp 33.17 Glu 42.81 Ser 13.06 Pro 5.58 Non-starch polysaccharides (g/kg as fed) Total 120.40 Arabinose 25.90 Xylose 10.80 Mannose 0.80 Galactose 5.10 Glucose 53.60 Uronic acids 23.80 Antinutritional factors (g/kg as feed) RFO 4.50 Raffinose (% of RFO) 14.47 Stachyose (% of RFO) 44.86 Verbascose (% of RFO) 40.67 Phytic P 2.50 Tannins 0.08 1 Each value represents mean of 2 replicates. WEV = water extract viscosity. RFO = raffinose family oligosaccharides. animals-13-00816-t002_Table 2 Table 2 The composition and nutritional value of reference diet. Components (g/kg as Fed) Corn 600.00 Soybean meal 292.60 Soybean oil 41.60 Fish meal 29.40 Monocalcium phosphate 10.30 Limestone (<2 mm) 5.10 Premix-broiler 1 10.00 Salt 2.90 Sodium bicarbonate 0.10 D, L-Methionine 2.70 L-Lysine-HCl 1.70 L-Threonine 0.60 TiO2 3.00 Determined nutritional value (g/kg as fed) Metabolizable energy (kcal/kg) 3011.85 Dry matter 882 (0.70) 2 Starch 423 (0.89) Crude protein 212 (0.90) Amino acids Lys 12.80 (0.72) Thr 8.73 (0.64) Val 9.00 (0.71) Ile 8.94 (0.69) Leu 17.70 (0.73) Phe 10.80 (0.73) His 6.80 (0.71) Arg 15.20 (0.80) Gly 1.05 (0.67) Tyr 6.82 (0.70) Ala 11.00 (0.71) Asp 22.30 (0.70) Glu 34.40 (0.80) Ser 10.50 (0.66) Pro 13.20 (0.69) Calculated nutritional value (g/kg as fed) Crude fat 72.90 NDF 99.00 ADF 37.30 Ash 28.00 Ca 9.20 P-avail. 4.30 Na 1.60 Cl 3.00 1 Provided per kg diet: IU: retinol 11,250, cholecalciferol 2500; mg/kg: tocopherol 80, menadione 2.50, cobalamin 0.02, folic acid 1.17, choline 379, D-pantothenic acid 12.5, riboflavin 7.0, niacin 41.67, thiamin 2.17, D-biotin 0.18, pyridoxine 4.0, ethoxyquin 0.09, Mn 73, Zn 55, Fe 45, Cu 20, I 0.62, Se 0.3, salinomycin 60. 2 Apparent ileal digestibility of dry matter and ingredient in reference diet. animals-13-00816-t003_Table 3 Table 3 Apparent ileal digestibility (AID) of dry matter, crude protein, starch, amino acids, and AMEN (kcal/kg) levels, and the excretion of total and free sialic acid (micromol/g TiO2) from broiler chickens fed pea seeds without (-) and with (+) enzyme supplementation 1. Item - + SEM p AID Dry matter 0.691 0.730 0.013 0.048 Crude protein 0.800 0.864 0.020 0.031 Starch 0.852 0.886 0.011 0.039 Lys 0.868 0.930 0.013 0.001 Thr 0.811 0.874 0.019 0.025 Ile 0.827 0.890 0.015 0.010 Val 0.832 0.890 0.015 0.012 Leu 0.835 0.881 0.017 0.012 Phe 0.839 0.881 0.017 0.107 His 0.844 0.895 0.012 0.004 Arg 0.912 0.942 0.008 0.046 Gly 0.820 0.871 0.015 0.024 Tyr 0.705 0.742 0.015 0.010 Ala 0.840 0.886 0.018 0.079 Asp 0.864 0.889 0.014 0.215 Glu 0.918 0.940 0.014 0.280 Ser 0.789 0.829 0.020 0.174 Pro 0.712 0.795 0.024 0.024 Energy value AMEN 2476.83 2579.54 38.215 0.076 Excretion of sialic acid Free 434.7 431.4 16.9 0.895 Total 496.2 497.5 18.0 0.959 SEM = Pooled standard error of mean; AMEN = nitrogen corrected metabolizable energy. 1 Each value represents mean of 14 replicates. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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