Proceedings of the 11th International Congress of the World Equine Veterinary Association

www.ivis.org Proceedings of the 11th International Congress of the World Equine Veterinary Association 24 27 September 2009 Guarujá, SP, Brazil Next Meeting : Nov. 2-6, 2011 - Hyderabad, India Reprinted in IVIS with the Permission of the Meeting Organizers

Contemporary aspects of Embryo Transfer; a Brazilian perspective. MP Reprodução Eqüina Ltda Bauru-São Paulo- Brasil MV Marcelo de Araújo Pessôa MV André de Paula Cannizza MV Gabriel Felício Introduction: MP Reprodução Equina is a partnership, localized in Bauru, middle-west of São Paulo State, which works mainly with equine reproduction. History: The commercial embryo transfer program, was started in Brazil, by João Fleury, in 1985, using the surgical ET Technique. In 1996, the non-surgical technique became more popular and, now, we do not have any news about someone using surgical technique in Brazil. In 2008/09 breeding season approximately 10.000 of embryo transfers were made, becoming Brazil one of the leaders in number of ET in the world. We started, in 1996, our commercial program using non-surgical transfer. That year, we got our first ET pregnancy after 13 flushing and 7 transfers. It was a little frustrating. In 1997, we got better results, and then our program started getting more clients, making more flushing e getting more pregnancies. Last breeding season we finish with 523 pregnancies at 40 days, after 1152 flushing. From 1997 to 2008, we change the main breed worked, going from PH to QH. We started making a good number of pregnancies, using clients recipients, and flushing a lot of mares out of our breeding center. At this time bigger we were more problems we found. The Routine: In Brazil, depending the breed involved the procedure to manage the donor mares to be flushed is different. The most vets involved with Mangalarga, Mangalarga Marchador and Campolina breeds manage the donor mares in the client s Ranch. The most of those Ranches own their recipients and all the procedure is realized there. In the QH, PH, Jumping breeds the most of donor mares, are shipped to breeding center to be managed. We are working on both type of management. We are 3 vets, involved on the service. We own our breeding center, localized in the family Ranch owned, and we take care of 16 more different Ranches. Our routine includes: palpations, inseminations, collection of semen to ship or fresh use, flushing mares and transferring embryos. In all of those places, we work in an every other day schedule. In small ranches, where we have a more malleable schedule, the visit is not so frequent. On those Ranches we flush some mares, but the most of then will finish the year pregnant. We manage some popular QH stallions and around 200 doses of semen are shipped every month from August to February. We also received about 40 % from the semen we to use to inseminate the mares. We have to work every day, even Sundays and holidays from august to February. It happens, with the most of vets involved with equine reproduction in Brazil. In our practice, we still were not able to change that, to provide us at least one day off every week. The Breeding Center routine: We worked at our Breeding Center with about 120 client mares and 15 owned by the Ranch. About 35 of them finished the breeding season carrying your own baby, some after were flushed to produce ET pregnancies. The recipient mares owned by us are kept in our breeding center. We can keep around 160 recipient mares at the same time. They are kept in dry lots, with 15 mares housed in each

one. The recipient herd has been feed with hay, grain and barley. 328 ET pregnancies were done in this herd in 2008, including embryos collected in the breeding center or collected outside and transported to be transferred at the Center. In the morning we evaluate the mares, collect the stallions to inseminate or ship the semen, flush the mares, and do the transfers. Every veterinary involved, has their schedule, previously, determined. Everything has to be done on this day. We palpate in the non-breeding day only mares to be bred with shipped or frozen semen. Also the recipients which will be transferred in non-breeding day, have to be checked on breeding day. In the other day, we work in a big Ranch that kept around a 100 recipient mares at the same time, and about 170 breeding mares. On this place were made 150 ET pregnancies and 110 mares are carrying your own babies. This Ranch owns his stallions, donor mares, recipient herd, and also provides services for his clients. In the afternoon, we have to visit the small ranches, palpating mares, collecting stallions, doing some clinic and flushing mares. Almost all these embryos collected, are transported to be transferred at our breeding center. The Brazilian model of producing embryos: - Pre-Breeding Season Management (Artificial Light Treatment ): Around April 15 th, 150 recipient mares are available to begin our program. They are divided in 15 mares by lot and kept in a dry lot. Were chosen following the criteria: returning mares which have foaled an ET foal or new mares bought at least 90 days earlier and adapted to our management. Had been fed in the same way and they will not change their lot until get pregnant. The light program started on May 20 th, every year since from 2005. We tried, in 2004, start the lights in winter solstice (around June, 10th) as is recommended by literature, but the recipients started cycling around 60 days later. It was a little late to our program, because in middle of July we already have some mares to flush. The light program has worked pretty well for us. We strongly think that the adaptation time is responsible for the good results we have early in the breeding season. We chose the mares which we think were the best ones, they are cycling early, they are adapted, and this is the only time in the breeding season, we have a lot of ovulated recipients to choose. In 2008-09 breeding season 120 recipient mares were put on lights on May, 20th. In the first palpation between July, 10th and 21st almost 80% of them were cycling, 80% got pregnant in the 1st ET and more than 90% get pregnant at all. (see table 1) Table 1. No. of mares Rate Total mares 120 Cycling middle 94 78,3% of July Not cycling 26 21,66% middle of July Do not cycle at 5 4,16% all Pregnant 1 st. ET 96/119 80,6 % Pregnant 2 nd.et 21/22 95,45% EED 1 st. ET 8/96 7,39% - Reproductive management of donor mares: The donor mares nutritional management and housing is Ranch responsibility. We

orientate it, looking for the best, but daily board is charged by the Ranch. We also put the most of donor mares housed in the Ranch on the lights. The donor mares are evaluated every other day. All of management is recorded. When ready to be bred, the ovulation is induced using the most of the times Deslorelin (1mg, IM). If fresh semen is to be used, they will be inseminated on the same day, if cooled semen is the choice it will be done on the same day or the next day depending of the stallion s schedule. The ovulation is induced earlier, if the cooled semen will take more than 12 hours to be used after the collection. The goal, to this cooled semen, is the mare ovulate no more than 24 hr post AI. The necessary uterine treatments can be done in the morning or in the afternoon, depending of the problem of the donor mare. Between 7 and 9 days, after the ovulation is detected, we flush those mares to look for an embryo. On this way we work, we try pay a lot of attention when are checking the follicles. If we think the mare can ovulate soon, we plan to flush her 7 days after the ovulation is detected by palpation. Sometimes the donor mare had detected its ovulation late and a big embryo can be flushed (until 6 mm of diameter). -Reproductive management of recipient herd: The recipient mares feeding management and housing is the Breeding Center responsibility. The recipients are identified by numbers forged in the croup. They are evaluated every other day. We record the results from the palpations, and mainly the ovulation. As we do not palpate every day, we avoid use mares recorded with 8 days pos-ovulation, at least a fresh ovulation was palpated. If the schedule is so tight, we plan to transfer the embryos on these mares 7 days after they have the ovulation determined. As a lot of transported embryos are transferred, and it is done normally on the nonbreeding day, the recipients are checked one day earlier in the breeding day. We do not make any uterine treatment in recipient mares. We just use prostaglandin (as a luteolytic agent ), oxytocin (as a ecbolic agent ) and deslorelin ( to induce ovulation ). Most of the mares that not are pregnant after the second ET are eliminated from the program. In a few occasions we can give them one more chance. It includes mares that foaled by ET before, mares recently bought who had a hard time (a lot of stress) to adapt to our program, or when we do not get buy mares to replace the pregnant ones. But we try all the time be so selective. More chance we give worse will be our results. We are absolutely conscientious that the stress of the recipient mares is the most important deleterious problem in any ET program. It can be caused by nutrition, environmental temperature, rain, diseases, or other factors do not determined yet. In our program, as we can not keep at the same time more than 150 mares, we have to replace the pregnant mares around 90 days after we initiate the breeding season (middle of October). It causes a big problem to us. At this time we have to buy mares in bad body condition, they never been in a management like ours, eating hay, grain and barley, and the most are not cycling. And a lot of them will experiment Strangles Disease. Quarantine can not be done, because at this time of the year about 65% of the recipients that started the breeding season are already pregnant, and a lot of donors are available to be worked. Commonly in November, the pregnant rate goes abruptly down. After that some increase happens, but never more as the breeding season were initiated. ( Graph 1 ). Graph 1

Comparison between months 100% 80% 60% 40% Pregnancy rate EED rate 20% 0% aug/08 sep/08 oct/08 nov/08 dec/08 jan/09 feb/09 mar/09 - ET procedures from collection to transfer: The technique is familiar to everybody. In our practice, mare s flushing, embryo manipulation and transferring it to the recipient is almost 100% of the times done for the same vet. We do not use to sedate the donor mares. A filter in line which is opened in the top is our preference. We had problem before recovering embryos ruptured during the flushing and decided to do not use negative pressure in the filter. The cuff of the collection catheter is inflated with 35 cc air approximately, that becomes easier if the repositioning of the catheter in uterus need be done. To flush the mare we use Lactated Ringer solution warmed between 32-38 C. One liter each flush is used, no more than 3 flush. As much as possible, of the solution, needs to be recovered. After finish the flush, if the fluid recovered is cloudy we wash it with another liter of LR, until it looks clear. 30 cc of fluid is kept in the filter; it is rotate in a non-clock wise and deposited in a petri dish. More 30 cc of LR is deposited in the filter and the procedure is repeated. We also rotate the petri dish in a non-clock wise for many times trying to move the embryos for the center of the dish. Smaller is the embryo easier to move it to center. If embryo can not be localized at the center of the petri dish we search in all the dish. If an embryo is in the petri dish most of the times it is not necessary. After the embryo is visualized it will be manipulated. A holding solution named Holding Plus (Vitrocell) is our preference. It s 0,4% BSA solution. We wash the embryo on this solution for seven times. If the fluid recovered was not clear we prefer wash a lot of more times, at least 20. Then the embryo is kept in a cryogenic tube with 2,0 cc of holding. The embryo can be maintained on this tube, at room temperature, until 4 hours. If it will take longer we prefer refrigerate it until 15-16 degree Celsius. The embryo transfer is realized in a recipient mare previously examined. We look for a good uterine and cervix tonus, and a homogeneous image of the uterus. We prefer mares that are d4 to d7 post-ov. All of our recipients receive a dose of acepromazine ( 20 mg i.m. ) just before the transfer. The embryos until 1400 microns of diameter are transferred using a stainless steel reusable insemination gun from IMV, embryos from 1400-3500 microns of diameter are transferred using AI pipette. Bigger than that we have adapted swab devices. The transfer procedure must be realized fast and without any damage to the cervix and uterus of the recipient mare. We think easier and fast you can do is better. We localize the cervix, do not introduce the finger in the external os, just the insemination gun. If necessary, the cervix

can be manipulated from the vagina and the embryo is left into the uterine body. No attempt to reach the uterine horn is done. The mares are checked 4 to 6 days later and if they are pregnant they are taken out from the lot and go to a pregnant lot. Our intention is decrease the movement of those mares. They are check again once time a week until 40 days, and then they go to new home. It is important to say, a lot of clients in Brazil do not wish take their mares to a Breeding Center, then all the service has to be provide in their Ranch. No laboratory does exist in most of these places. The work has to be done in the same way, and it is true to the most of vets involved with equine embryo transfer in Brazil. Results and comparisons: Between breeding centers we manage: During 2008/09 breeding season we managed a large number of recipient mares in two different places. One was on our breeding center where we manage the mares like we think would be better. On the other place, the recipient herd was kept in a low quality pasture with no supplementation. Around 100 mares are kept together in the same pasture. Has no division between the herds until they get pregnant. In both places the recipient replacement is done with mares in bad body condition. But they take longer to get better in the second place. Also we had at this farm a lower pregnancy rate and higher EED. ( see table 2 ). It was considered by us, as a result of poor general management. Table 2. MP Reprodução 2º. Breeding Total Equina Center Center No. ( % ) No. ( % ) No. ( % ) ET 491 257 811 Preg 12 days 370 ( 75,35 ) 178 ( 69,26 ) 598 ( 73,70 ) Preg 40 days 328 ( 66,80 ) 150 ( 58,36 ) 523 ( 64,48 ) EED 42 ( 11,35 ) 28 ( 15,73 ) 75 ( 12,54 ) Lactating, cycling, non-cycling mares: When available, a lactating mare can be used as a recipient. Normally they are recipients that foaled in clients farm where we follow then. It saves money to clients, because will feed a mare pregnant and lactating and does not need introduce a new mare in their herd. Also we do not have to look for new recipients to replace our herd, increasing their use in our program. A smaller pregnant rate in lactating group was observed comparing with cycling nonlactating group, but the EED rate was similar. In all the places we had used the lactating recipient; they were kept with the breeding mares of the farm. They are kept on good pastures, are supplemented with grain, and we do not transfer embryos on them until they have ovulated twice pos-partum. Even the results are little lower than with non-lactating mares the advantages surpasses the disadvantages. Embryos are also transferred in non-cycling progesterone ( P4 ) supplemented mares. They were used in large number at our program, until 2003. Then, when the artificial lighting program were started, the number of P4 treated mares decreased a lot. However, after four years, with the increase in number of transfers, the number of non-cycling P4 treated mares increase again. Follow bellow a comparison between the results using cycling non-lactating, non-cycling P4 treated and lactating mares. (table 3) Table 3.

Cycling recipients Non-cycling recipients P4 treated Lactating recipients total No. % No. % No. % No. ( % ) ET 517 ( 63,7 ) 209 ( 25,7 ) 85 ( 10,5 ) 811 ( 100 ) Pregnant 12 days 415 ( 80,2 ) 130 ( 62,20 ) 53 ( 62,35 ) 598 ( 73,73 ) EED 48 ( 11,56 ) 21 ( 16,15 ) 6 ( 11,32 ) 75 ( 12,54 ) Pregnant 40 days 367 ( 70,98 ) 109 ( 52,15 ) 47 ( 55,29 ) 523 ( 64,48 ) Our protocol involved the use of Estradiol Benzoate ( 3mg IM, s.i.d for 2 days ) and then a shot of long-acting progesterone ( 10cc of 300mg/ml P4- Biotech ). This year, the shot of progesterone was repeated every 14 days and maintained until around 105 days of gestation. The pregnancy rate at 12 and 40 days was lower when comparing with the cycling mares, also higher was the EED in P4 treated mares. It can be explained, because on this year the most of non-cycling mares used in P4 treatment were recently bought, were in a bad body condition and had no time to adapt to our management. It can be clearly noted when we compare the pregnancy rate monthly. ( graph 2 ) Until middle of October a pregnant rate was higher than 70% then came down in November for less than 40 %. Until October, we were transferring mares which were in our management for at least 90 days, but they were not cycling. After November the pregnant rate could not sustain. At this time, the mares recently bought were introduced into the program. Those are in bad body shape, were not cycling (which denote they were not in good condition). The program got up and down. It did not decrease the importance in using this protocol, because without this we would not be able to produce these pregnancies. Graph 2. P4 supplemented mares ET number 90 80 70 60 50 40 30 20 10 0 90,00 80,00 70,00 60,00 50,00 40,00 30,00 20,00 10,00 0,00 % ET preg rate 12 d EED aug/08 sep/08 oct/08 nov/08 dec/08 jan/09 feb/09 mar/09 Fresh and cooled semen: As previously described around 40 % of donor mares are inseminated with cooled semen. This semen comes from different places of the Country. It is normally package in a insulated Styrofoam box which keeps the temperature around 16 degrees Celsius. It can be transported by mail, bus, air or Moto boys. The Moto boys are very common in Brazil. We believe the problems find out in some cooled semen are responsible for the smaller recovery rate in comparison with the fresh semen. We also work with a small amount of frozen semen.

Follow table 4 for comparison of the embryo recovery rate between fresh, cooled and frozen semen. Table 4 Fresh semen Cooled semen Frozen semen total No. of AI 637 458 57 1152 Flushs + 460 270 25 755 Recovery rate 72, 2 % 58,9 % 43,8 % 65,5% Training and non-training mares: Also is common in Brazil, transfer embryos from mares that are training and competing in shows. In our area, a lot of Ranches breeds cutting, reining and barrel racing horses. They also keep mares in training and competition. During 2008/09 breeding season 39 mares in training, including 31 mares been showed at competitions, some of then top level competitors were flushed. Those mares are riding at least 4 times a week, and the schedule for the barrel racing mares includes be showed at least once a month. All those mares produced at least one embryo and one pregnancy during this the breeding season. The results were so similar when comparing with the breeding mares worked into the breeding season. (See table 5) Table 5 Training mares ( n= 39 ) Breeding mares Total No. ( % ) No. ( % ) No. % flush 138 1014 1152 Recovery rate 96 ( 69,50 ) 661 ( 65,18 ) 757 ( 65,70) ET 105 706 811 Pregnant 12 d 82 ( 78,09 ) 516 ( 73,08 ) 598 ( 73,73 ) Pregnant 40 d 73 ( 69,52 ) 450 ( 62,74 ) 523 ( 64,48 ) EED 9 ( 10,97 ) 66 ( 12,96 ) 75 ( 12,54 ) The Brazilian Perspective: From 1986 to 2008 breeding season the embryo transfer commercial program in Brazil has increased a lot. Alvarenga and Losinno (2006) reported that approximately 6.000 ET were performed in Brazil in 2005. On last breeding season (2008-2009) we estimated that 10.000 ET were performed in our country. Since João Fleury started his program in 1985, the ET in Brazil has changed a lot. In the first 10 years the most of ET pregnancies were produced in Margalarga and Mangalarga Marchador breeds. With the time Campolina was incorporated and then the Jumping horses. From 1997 to 2004 a lot of ET pregnancies were done in Paint Horse breed using QH mares as a donor. At that time Quarter Horse was not allowing a multiple foals for the same mare in the same year. In 2005 ABQM changed the rules and allowed the register for the multiple foals

from the same mare. The number of ET in PH went down. The number of ET in QH has increased a lot from that. About 2500 ET pregnancies were produced this breeding season in QH. Below is the evolution on our commercial program since beginning. (See Table 6) Table 6 year Flush No. Recovery rate Preg. 12 days rate Preg. 40 days rate EED rate Efficiency rate 1996 13 53 % 14,3% 14,3% 0 7,6 % 1997 24 33 % 50 % 50 % 0 16,6 % 1998 32 62,5 % 75 % 65 % 13,3 % 40,6 % 1999 132 68,9 % 69,6 % 64 % 8 % 43,2 % 2001 405 64,6 % 73,1 % 60,8 % 16,75 % 38 % 2002 620 68,5 % 69,7 % 58,5 % 15,9 % 39 % 2003 652 66,8 % 74,1 % 63,7 % 14,1 % 42 % 2004 925 64 % 66 % 58 % 11,4 % 40,6 % 2005 933 62 % 72,66 % 62,68 % 13,73 % 41 % 2006 955 64% 71% 63,1% 11,6% 40,4% 2007 940 58,9% 73,2 % 67,1 % 8,3 % 39,5 % 2008 1152 65,71 % 73,7 % 64,48 % 12,54 % 45,39 % Total no. 6783 2886 42,54% Next year, with the new rules from ABQM it probably will decrease. Basically the association decided to charge more for embryos produced in mix breed mares, trying to stimulate the use of the pure breed recipient mares. Unfortunately, we are not able at this time to estimate the impact of this new rules on ET number. Anyway the Crioulo breed has just allowed the use of ET, and it is looking so promissory. Crioulo is the third breed in number of registered horses in Brasil. Also other smaller breeds, as Lusitano, are starting to use the technique.