INTRODUCTION TO EMBRYO TRANSFER

Transcription

1 INTRODUCTION TO EMBRYO TRANSFER Embryo transfer is widely practiced in cattle by animal scientists and veterinarians throughout the world. Its main purpose is to propagate valuable genetic potential as an adjunct to artificial insemination, but embryo transfer has also been used to diagnose the functional integrity of the reproductive system and to study such things as disease transmission through embryos, the physiology of twinning, the cloning of embryos and various genetic phenomena. Even when herds are infected with some diseases, disease-free embryos can be obtained. EMBRYO TRANSFER IN COWS Synchronization of estrous cycles The estrous cycles of the donor and recipients should ideally be synchronized to within 24 hours of one another. This is usually achieved by estrous cycle synchronization with prostaglandins or progestogen implants. The superovulated donor normally shows estrus sooner than the recipients. Therefore, when prostaglandins are being used for estrous synchronization, the donor receives her second injection of prostaglandins 24 hours after the recipients. Similarly, progestogen implants are removed from the donor one day after they are removed from the recipients. Superovulation of the donor There are two hormones that are used to induce superovulation in the cow, i.e. ecg or FSH (usually of porcine origin). Because of the relatively short half-life of FSH, it must be administered much more frequently (twice daily vs only a single injection) than ecg to induce 319 superovulation but results with FSH are more predictable than those with ecg and ecg is seldom used for superovulation anymore. Treatment with FSH is usually begun three days before the second prostaglandin injection or withdrawal of the progestogen implant and it is continued for 4 days. When the donor shows estrus, she is inseminated 12 and 24 hours after the onset of estrus. The ovulatory response usually varies from 3 to 20 ovulations with a median number of 7 to 8 fertilized ova. For various reasons, up to 25% of cows do not respond to superovulatory treatment at all. Collecting of the embryos The original method of collecting embryos in all species was surgical but postoperative adhesions limited the productive life of valuable donor animals. At present, the vast majority of embryos are collected non-surgically, at 7 or 8 days after ovulation, by flushing the uterus with a solution such as Dulbeccos phosphate buffer containing 1% heat-treated steer serum, to protect the embryos and prevent them from sticking to the glassware. A filtration device is usually used to separate the embryos from most of the flushing medium to facilitate embryo searching. A small volume of fluid containing the embryos is placed in a petri dish under a stereo dissecting microscope. The embryos are then located with small pipettes and a grid locator marked into the base of the petri dish. The embryos are examined for signs of normal development and those that are considered to be viable are transferred to the surrogate dams. The donor is then treated with prostaglandins and 2 to 3 cycles are allowed to occur before her next superovulation. Notes

2 Notes If required, the process is repeated 60 to 70 days later. Transferring the embryos Embryos are transferred both surgically and non-surgically but non -surgical transfer is now more popular. In surgical transfer, the uterine horn ipsilateral to the corpus luteum is exposed through a flank incision and the embryo is deposited into the lumen of the uterus at the ovarian end of the horn. A capillary tube carrying the embryo is inserted through a small puncture in the uterine wall made with the back of a suture needle. A single embryo is deposited into the uterine lumen. If the embryo is to be non-surgically transferred, it is drawn into a 0.25 ml Cassou insemination straw and deposited as far up the uterine horn as possible without causing trauma. A special, long, double-sleeved Cassou gun is used. As a general rule, 40% to 65% of all embryos transferred are maintained as normal pregnancies until term. One should remember that approximately 50 percent of transferred embryos will produce bulls which are almost useless to the farmer. There will also be a range in the quality of the heifers that are born; in fact, some will be inferior to heifers that were not produced by embryo transfer. Embryo transfer is also expensive. EMBRYO TRANSFER IN ANIMALS OTHER THAN COWS In mares, embryo transfer is allowed in all breeds except Thoroughbreds. It is usually performed because of conditions which prevent the maintenance of pregnancy or because pregnancy would be hazardous to the mare or foal. For example the mare may have pelvic or abdominal injuries or her previous foal 320 may have had neonatal isoerythrolysis. Also, an owner may wish to use a mare for competition while a surrogate dam carries her foal. Mares are difficult to superovulate with conventional methods and collection normally involves obtaining just a single embryo. Recently however, an Equine FSH extract has become available, virtually doubling the chances of collecting two embryos per flush. Its availability from Bioniche Animal Health is limited. The mean ovulation rate and embryo rate can be as high as six or seven oocytes with some treatments but the highest percentage of ovulations (about 3.9 on average) and embryos (1.9 on average) may be obtained using 12.5 mg efsh q 12 h. Treatment are best when there are no dominant follicles (>25mm) in the ovaries at the time when treatment is started. This is usually at under day 6 of the cycle. The cost of superovulation in 2004 was estimated at approximately $(US)500 for the efsh alone. Owing to the ability of the mare s uterine tube (fallopian tube) to distinguish between fertilized and non-fertilized embryos, any embryo collected from the uterus can be taken to be fertilized; the close inspection of embryos to separate live, dead, or unfertilized embryos is usually not necessary. As in cattle, transfer of the embryos to recipients can be surgical or non-surgical. Surgical transfer is still more successful in mares than non-surgical transfer but owners are not inclined to allow surgical transfer, as it is far more expensive and traumatic. When the donor is not superovulated, the estrous cycle of the donor and recipient are synchronized with P&E. However, at this time (2004) a reliable regime for super-ovulation that uses

3 P&E as its basis, has not been established. This is regarded as a severe impediment to the adoption of E.T. on a wide scale in the equine industry. In some cases, ovariectomized mares and even mules have been used as recipients. In those cases the surrogates are usually treated with progestogens to maintain pregnancy until about 120 days of gestation. After that the placenta maintains pregnancy. Flushing of mares is different from cattle in that the whole uterus is flushed for embryos and when one is obtained, it is put into the uterus without regard for which ovary contains the corpus luteum (Cf cattle). Remember that the embryo normally migrates through the uterus up until 16 days of gestation anyway! Success rates are usually about 30 percent overall, i.e. from breeding to the maintenance of pregnancy in a recipient. When owners are informed that there is approximately a 70% chance that they will be wasting the $1, fee involved in transfer, they often refuse the procedure. been frozen and thawed successfully but less (and only sporadic success in some cases) has been obtained in species other than cattle. Human embryos have also been frozen. The usual method for freezing cattle embryos is described below. After high-quality embryos have been selected for freezing, they are placed in special media containing either glycerol or ethylene glycol. Recently, ethylene glycol has become the most common cryoprotectant used. Cryoprotectants stabilize electrolytes around the embryos and protect their cell membranes. Held within the freezing medium in a 0.25 ml Cassou straw (an AI straw), the embryos are cooled to approximately C and then seeded by contact with a supercooled piece of metal. The seeding process starts the growth of very fine crystals in the embryo and causes a more gradual release of latent energy than if the embryo froze suddenly and spontaneously. If that were to happen, large ice crystals would form, destroying the embryo. Notes Embryo transfer is also practiced in pigs, sheep and goats using similar principles to those described for cows. All of these species can be superovulated for maximum embryo production and, in all cases, transfer of the embryos is usually surgical. Embryo transfer has been described in the cat and dog, wild felids and many other wild animals. It is currently used in zoos and wild animal parks for propagation of threatened species. In such cases, both intra-and extra-species surrogate dams have been used and occasionally the embryos are frozen as well, creating the concept of a frozen zoo. FREEZING EMBRYOS Embryos from all domestic species have 321 A f t e r s e e d i n g, g ra d u a l c o o l i n g (0.3 to 0.8 C/minute) continues to approximately -35 C, at which time the embryo is plunged into liquid nitrogen at -196 C. This stops the dehydration process that is essential to embryo survival (water crystals act like daggers and can damage cell organelles and membranes. However, excessive dehydration will also kill the embryo!). It also brings metabolic processes within the embryo to a virtual standstill. Embryos can be stored at this temperature indefinitely. If embryos have been frozen using ethylene glycol as a cryoprotectant, they are thawed in air for 5 to 7 seconds, then in a 30 C water bath for 30 seconds. They are then transferred to the recipient directly, without removing the cryoprotectant. The straw is dried off, loaded into a Cassou gun and transferred into the uterine horn ipsilateral

4 Notes to the CL. Many AI technicians are now certified to transfer embryos as well as to perform AI. Embryos frozen using glycerol are usually thawed at air temperature for 10 to 15 seconds, then in a water bath at 35 C for 20 to 30 seconds. Glycerol is removed with descending concentrations of the cryoprotectant or with sucrose. After this, the embryo is put in culture medium and transferred to the surrogate dam using normal transfer procedures. Pregnancy rates with frozen embryos are slightly lower than those obtained with non-frozen embryos. 8. Lindner, G.M. and Wright, R.W. Bovine embryo morphology and evaluation. Theriogenology, 20: , Hinrichs, K. Embryo transfer in the mare; a status report. Animal Reproduction Science, 33: , McKinnon, A. and Squires, E.L. Equine embryo transfer. Veterinary Clinics of North America; Equine Practice 4: , Squires, E.L. Embryo Transfer. In: McKinnon and Voss (eds.), Equine Reproduction, Lea and Febiger, pp , SUGGESTED READING ON EMBRYO TRANSFER 1. Drost, M. Embryo Transfer. In: Roberts, Veterinary 0bstetrics and Genital Diseases, (Theriogenology) 3rd ed., Published by the author, pp , Hasler, J.F. Commercial applications of in vitro fertilization in cattle. Compendium on Continuing Education for the Practicing Veterinarian, 16: , Mapletoft, R.J. et al. Embryo Transfer and Genetic Engineering. In: Morrow (ed.), Current Therapy in Theriogenology 2, W.B. Saunders Co., pp , Nash, J.G. Embryo transfer for the bovine practitioner. Symposium on herd health managemnt - dairy cow. Veterinary Clinics of North America: Large Animal Practice 3: , Seidel, G.E. and Elsden, R.P. Embryo Transfer in Dairy Cattle, W.D. Hoard & Sons, Co., Shea, B.F. Evaluating the bovine embryo. Theriogenology, 15:31-42, Blanchard, T., Varner, D.D. and Schumacher et al. J. An Overview of Embryo Transfer in Horses. In: Manual of Equine Reproduction, 2nd edition, Mosby, pp , Riera, F.L. Equine Embryo Transfer. In: Samper, J.C., (ed.), Equine Breeding Management and Artificial Insemination, W.B. Saunders Co., pp , McCue, P.M., Squires, E.L., Bruemmer, J.E. and Niswender, K.D. Equine embryo transfer: technique, trends and anecdotes. Proceedings for the Annual Meeting. Society for the Theriogenology, pp , McKinnon, A.O. and Squires, E.L. Morphologic assessment of the equine embryo. Journal of the American Veterinary Medical Association, 192: , Buckrell, B. Embryo transfer for sheep and goats. Proceedings, American College of Theriogenologists, Society for Theriogenology Small ruminant Short Course, pp , Current Therapy in Large Animal Theriogenology, edited by R.S. Youngquist, W.B. Saunders Co, various chapters, 1997.

5 18. Proceedings for Annual Meeting, Society for Theriogenology, various papers, Notes 19. Proceedings of the Annual Conferences, International Embryo Transfer Society, Theriogenology, January issues. 323

6 Notes 324