ANIMAL SCIENCE 8818-B EMBRYO TRANSFER INTRODUCTION Embryo transfer* is a process by which an embryo is collected from a donor female and then transferred into a recipient female where the embryo completes its development. Embryo transfer is profitable for producers of registered purebred animals. Through the use of embryo transfer, a genetically superior female produces more offspring than she could by natural reproduction. The increased number of offspring thus maximizes the donor female s genetic abilities. Embryo transfer is used in several species of domestic animals, namely cows, horses, goats, and sheep. Research indicates its use in certain non-domestic species, such as deer, elk, bison, and wildcats. Interestingly, one country uses embryo transfer to improve the quality of animals used in the sport of camel racing. Using rabbits, a researcher at the University of Cambridge in 1890 performed the first embryo transfer. It was not until 1951 that others succeeded with the technique in cattle. As an industry, the first embryo transfers were performed in the early 1970s with beef cattle. Since then, the popularity of the procedure has increased among producers of different animal species. Breeding beef cattle interests offer large economic incentives to veterinarians and animal scientists to make embryo transfer an efficient and effective tool. The commercial embryo transfer industry has grown steadily and will continue to do so as long as embryo transfer remains a key step in many of the developing technologies, such as prenatal sex selection. DONOR, SUPEROVULATION, AND INSEMINATION Donor Female As mentioned earlier, the donor selected for embryo transfer procedures has outstanding genetics. She must also be healthy and reproductively sound. Superovulation Superovulation refers to the release of many oocytes (eggs) during a single estrus period. Superovulation of the donor can be achieved through treatment with gonadotropins. Most embryo transfer donors are treated with follicle stimulating hormone (FSH) to induce the maturation and ovulation of a larger than normal number of oocytes. For example, a single treatment of a cow results in zero to 20 or more embryos, with an average of seven normal embryos. * Underlined words are defined in the Glossary of Terms. - 1 -
When attempts are made to recover a single ovum from a non-superovulated cow at every estrus, the 12- month yield averages five calves. In contrast, when a donor is superovulated three times, the yield ranges from nine to 12 calves. Less labor is involved with use of superovulation procedures. Following embryo transfer, superovulated ova result in normal offspring with success rates similar to those achieved with normally ovulated ova. Adequate procedures exist for superovulation of laboratory and livestock species, except for the horse. A mare does not respond satisfactorily to superovulatory treatment, thus maximization of reproduction in mares must be achieved by collecting embryos at each estrus. - 2 -
Insemination Because superovulation of the donor female causes the release of a large number of eggs over a 24-hour period, many producers choose to inseminate the donor several times to achieve optimal fertilization. EMBRYO RECOVERY Success of embryo recovery depends not only on the age of the embryos but also on the technical procedure used and skill level of the technician. With superovulated donors, many follicles never ovulate; others may develop as if they had ovulated, but they never release oocytes. Furthermore, if the ovaries enlarge greatly in response to superovulatory treatment or if many ovulations occur, the fimbriae apparently do not gather all of the oocytes into the oviducts. Radically altered endocrine levels may occur as a result of superovulatory treatment. This causes the uterus to become an unfavorable environment for embryonic development. Research indicates that the incidence of degenerate embryos may increase during days 7 and 8 after estrus. Recovery rate reporting is often inaccurate because of the difficulty in counting the number of ovulations in superovulated donors. Even by examining the ovaries at surgery, the large number of ovulation sites and the frequently enlarged, abnormal appearance of the ovaries prevent accurate counts. With non-surgical recovery methods, the number of ovulations can only be estimated by palpation. Non-Surgical Recovery Advances in technology have made embryo recovery a non-surgical procedure with cattle and horses. A specially designed catheter (Foley catheter) is used when collecting embryos using non-surgical methods. - 3 -
Before the procedure, the donor s genital area is cleaned and a local anesthetic is injected in the rump or hip. After the anesthetic takes effect, the Foley catheter is inserted into the vagina and through the cervix. Fluid is forced from the catheter into the uterus to flush out the embryos. The flushed fluid is collected in a special container, and the embryos are filtered from the collected fluid. In cows and horses, non-surgical collection is repeated an unlimited number of times on an individual donor without reducing her fertility. Requirements for equipment, personnel, and time are minimal because embryos are collected at the site where the donor animal is located. This advantage is offset in the equine industry because some breed associations do not permit registration of foals resulting from embryos collected at the donor animal s site. It is difficult to pass a collection device through the cervix into the uterus of sheep, hogs, and goats. Although this anatomical barrier is not insurmountable, the economic incentive is not great enough to hasten development of nonsurgical recovery techniques for these livestock species. Surgical Recovery Unfertilized oocytes for specialized applications (such as in vitro fertilization) must be collected near the time of ovulation. This is done from the follicles, surface of the ovary, or oviduct. For most applications, embryos are collected anytime between fertilization and implantation but usually after they migrate to the uterus. In cattle and horses, embryos for commercial purposes are usually recovered 6 to 9 days after estrus. Before this time, nonsurgical recovery is ineffective. After nine days, recovery and pregnancy rates are slightly reduced, at least with surgical transfer of bovine embryos. Surgical recovery can be done in all species and is the method of choice for sheep, goats, and hogs. Techniques vary slightly with the species. EMBRYO EVALUATION After collection, the embryos are evaluated for quality using a stereoscopic microscope. Shape, color, texture, and size are some factors considered during the evaluation. Embryos are graded on a scale from one (excellent) to four (poor). Embryos are also classified on their stages of development. EMBRYO STORAGE Procedures such as embryo transfer, in vitro fertilization, sex determination, and cloning depend on maintaining the viability of embryos for hours to days outside of the reproductive tract. For many applications, the storage system must not only maintain the viability of the embryo, but must also support continued development. It may also be desirable to retard growth to a degree approaching suspended animation so development can be synchronized with later events. For example, it may be necessary to store embryos until suitable recipients become available for transfer. Donor embryos can be transferred immediately into recipients, or they can be stored for future use. - 4 -
Short-Term Storage Embryos can be stored at room temperature for one day for direct transfer from the donor to the recipients. For periods of 24 to 48 hours, the embryos must be stored at 5 o C in an appropriate medium. Most media and culture systems are adequate for maintaining the viability of the embryo between donor and recipient. Long-Term Storage If embryos are to be transported great distances or suitable recipients are not immediately available, a longterm storage system is essential. Deep-freezing embryos is storage in liquid nitrogen for an indefinite period of time. Long-term storage through freezing usually results in damage of 30% to 50% of the stored embryos. Damage is usually caused by ice crystal formation within the embryonic cell. Although the average survival rate of frozen-thawed embryos is approximately 65%, it is profitable to maintain embryos in long-term storage. TRANSFER OF EMBRYOS INTO RECIPIENT COW Synchronization of Estrus Recipients to be implanted with the collected embryos should be healthy and reproductively sound. To maximize the success rate of the transfer, the recipient s estrus should be in sync with that of the donor. Estrus synchronization is important to establish a uniform uterine environment for the embryo; therefore, only a one day or less difference in estrus between the donor and recipient is acceptable. Comparisons have been made of pregnancy rates with embryo transfer following natural versus induced estrus of recipients. Data suggest little difference in pregnancy rates whether recipients are in natural estrus in synchrony with the donor or are treated with progestogens for synchronization. Non-Surgical Transfer of Embryos For cattle and horses, technicians mainly use non-surgical techniques to recover embryos. The procedure after superovulation is to breed the donor with two doses of semen 12 hours after she comes into heat and then again 12 hours later. The fertilized eggs are recovered about a week later by flushing the uterus with a buffered solution. Flushing is essentially the reverse of the artificial insemination (AI) process. Embryos that pass inspection are loaded into an AI straw. An insemination rod is passed through the recipient s cervix and into her uterus. The embryo is expelled into the uterine horn that is on the same side as the ovulated ovary (to optimize the embryo implantation rate). Surgical Transfer of Embryos Embryos in early stages of development must be deposited in the oviducts. This procedure must be performed surgically. Surgery is also required for uterine transfers in sheep, goats, and hogs. - 5 -
Success of surgical transfer depends on interactions of a number of factors: age and quality of embryos, site of transfer, degree of estrus synchronization between the donor and recipients, number of embryos transferred, in vitro culture conditions, skill of personnel, and management techniques. Conception rates are up to five percent greater with surgical transfer. Surgical transfer of normal embryos compares to the first-service pregnancy rates using artificial insemination. The reproductive tract can be exposed for surgical transfer of embryos. Transfer occurs through a midline laparotomy with general anesthesia or through a flank incision with local anesthesia. The midline approach is less traumatic to the reproductive tract and permits better exposure in some animals. The flank approach, on the other hand, is quicker and less expensive, and it reduces risk by using a minimum of equipment. EMBRYO SURVIVAL The survival rate of the embryo from implantation to term in the recipient female ranges from 55% to 70%. Embryo quality is an important determinant of transfer success. The most accurate predictor of success is the stage of embryonic development. Recipient females are palpated within one to three months after embryo transfer to diagnose stage of pregnancy. NEW TECHNOLOGIES Embryo transfer is a composite technology that requires expertise in many areas. Embryo transfer requires technology to manage successful superovulation, embryo recovery, short-term in vitro culture of embryos, actual embryo transfer, and storage of embryos. Reproduction by embryo transfer depends on high-quality procedures in support areas. Critical to its success is employing the proper methods in the detection of estrus, synchronization of estrus, herd management, herd health, and artificial insemination. Current and emerging technologies that build upon current embryo transfer procedures include predetermination of sex, sex selection, in vitro culture, production of identical twins, and cloning. Refer to topic #8518 Cloning Bovine Embryos Using Nuclear Transfer. In addition, embryo transfer techniques proliferate a number of new industries. Such areas of research and growth include purification of gonadotropins, manufacture of micromanipulators and similar specialized equipment, international marketing of embryos, and specialized investment counseling. SELECTED WEB SITES FOR INFORMATION ON EMBRYO TRANSFER http://ars.sdstate.edu/faculty/clapper/embryo_transfer.ppt http://www.acc.com.qa/invester/embryotransfer.htm http://www.aginfo.fvsu.edu/html/publications/ GoatCenter/amoah1.htm http://www.sonic.net/~heavey/dbeckett/bovine%20embryo%20transfer.pdf www.scientecmatrix.com/seghers/tecma/scientecmatrix.nsf/_/7c1b940346356f04c1256b7400373826-6 -
Acknowledgements Holly Hutton, Graduate Assistant, Department of Agricultural Education, Texas A&M University, researched and revised this topic. Larry Ermis, Curriculum Specialist, Instructional Materials Service, Texas A&M University, reviewed and edited this topic. Vickie Marriott, Office Software Associate, Instructional Materials Service, Texas A&M University, prepared the layout and design for this topic. Christine Stetter, Artist, Instructional Materials Service, Texas A&M University, prepared the illustrations for this topic. REFERENCES Herman, H.A., Mitchell, J.R., and G.A. Doak. The Artificial Insemination and Embryo Transfer of Dairy and Beef Cattle. 8th ed., Danville, IL: Interstate Publishers, Inc., 1994. Kunkel, J. R. Embryo Transfer. Dairy Integrated Reproductive Management (IRM-26). Available: http://www.wvu.edu/~exten/infores/pubs/livepoul/dirm26.pdf [May, 2002]. Sauvé, Roger. Embryo Transfer. Available: http://www.embryobec.com/teang.html. [May, 2002]. Taylor, Robert E. and Thomas G. Field. Scientific Farm Animal Production. 6th ed., Upper Saddle River, NJ: Prentice Hall, 1998. GLOSSARY OF TERMS Donor The female from which embryos are secured to be placed in recipient females. Embryo The fertilized egg that continues to grow through cell division until it becomes attached to the uterine wall to become the fetus. Embryo transfer A technique in animal breeding in which an embryo from a superovulated female animal is transferred to the uterus of a recipient female animal; also called embryo transplant. Fimbriae Long, slender, finger-like projections at the opening of the oviduct that aid in moving the egg into the funnel. Follicle A blister-like mass on the surface of an ovary that contains a developing ovum. Gonadotropins Hormones capable of promoting gonad growth and function. Implantation Process whereby the embryonic sac attaches to uterine wall for embryo development. In vitro fertilization A laboratory technique for conception of an embryo outside the mother's body. Laparotomy A surgical procedure performed through an incision in the abdominal wall. Oocyte (ovum) The egg produced by the ovary and containing the female genes and chromosomes that join with those of the male sperm during fertilization. Progestogens Synthetic progesterone products used to control estrus. Recipient The female that carries the pregnancy to term; the surrogate mother. Superovulation The use of hormones to cause the donor female to produce more eggs than normal. Term The end of a normal gestation period. - 7 -
SELECTED STUDENT ACTIVITIES 1. Define embryo transfer. 2. What is the major purpose of embryo transfer? 3. What is the survival rate (percentage) for the embryo from implantation to term? 4. What is superovulation? What is its purpose? 5. Why should a recipient female s estrus be in sync with the donor female s estrus? 6. Describe how embryos, after evaluation, are transferred into the recipient females? 7. Name and describe each of the two types of embryo storage. a. b. 8. Discuss embryo evaluation factors and grading scale. ADVANCED ACTIVITIES 1. Research and prepare a written report on embryo transfer for an animal species of choice. 2. After reviewing the International Embryo Transfer Society s web page, http://www.iets.org/, discuss its contents with other members of the class. 3. Interview an embryo transfer technician. Orally report findings to the class. ALL RIGHTS RESERVED Reproduction prohibited without written permission. Instructional Materials Service Texas A&M University 2588 TAMUS College Station, Texas 77843-2588 http://www-ims.tamu.edu 2002-8 -