Equine Embryo Transfer
Patrick M. McCue, DVM, PHD, Diplomate ACT
Equine Reproduction Laboratory
Colorado State University
Ft. Collins, CO 80523
Embryo transfer (ET) involves the removal of an embryo from the uterus of one mare and the transfer of that embryo into the uterus of another mare. The first successful equine embryo transfer was reported in 1974. Embryo transfer can be very beneficial in the valuable recipients, sub-fertile mares can donate embryos to reproductively healthy mares in athletic competition can donate embryos and remain in training. Consequently, embryo transfer has become a common assured reproductive technique in many breeds. The use of embryo transfer has increased in the past decade as technology that allows for short-term transportation of equine embryos has become available. Cooled-transported embryo programs have made embryo transfer available to horse owners that do not want to ship a donor mare to a referral center.
Breed Restrictions
A majority of equine breed registries in the United States, with the exception on the Jockey Club (Thoroughbreds) allow the registration of foals produced by embryo transfer. Breed registries should be contacted before initiating embryo transfer procedures. Some breeds will not register foals born from embryo transfer. Breeds that will allow ET often require advanced notification and blood typing of the stallion, donor, recipient and foal to prove parentage. In addition, the number of foals registered per year for a given mare may be restricted.
Donor Breeding Management
The best embryo donors are mares, reproductively sound mares. Collection and transfer is less successful in older mares, immature mares (<3 years old), barren mares and mares with an inflamed, infected or fibrotic uterus. The vast majority of donor mares are bred by artificial insemination. The choice of fresh, cooled-transported or frozen-thawed semen is dictated by breed, location of the stallion and mare, stallion availability and owner preference. The donor mare is examined by transrectal ultrasonography daily during late estrici to determine the exact day of ovulation (day 0).
Embryo Collection
Embryo recovery is usually attempted 7 or 8 days post ovulation. We prefer to collect embryos on day 8 for the following reasons: 1_ embryo recovery rate is high. 2) a majority of embryos are expanded blastocysts on day 8 that are easily observed under the microscope, 3)occasionally, small morula-stage embryos are recovered on day 8 that may not be in the uterus on day 6 or 7, and 4) embryos recovered later (i.e. day 9) are often too large to be handled without damaging the embryo.
The most common method of embryo collection in the mare is nonsurgical transcervical uterine lavage. A sterile silicone catheter with an inflatable cuff is inserted through the cervix and the cuff inflated with air. The uterus is lavaged three to four times with 1-2 liters of prewarmed embryo flush media the flush media is then allowed to flow back out through the catheter and is passed through an embryo filter. Recovery of the flush media is aided by massage of the uterus per rectum and, occasionally, administration of oxytocin. Recovery of the uterine lavage fluid is monitored by collecting the fluid in 1 liter graduated cylinders. Contents of the lilter are poured into a search dish and examined for the presence of an embryo. Recovered embryos are washed by transferring them sequentially through several drops of holding media and are held in this medium until transfer.
Embryo recovery rate is influenced by many factors, such as age and fertility of the donor mare, quality of the sire’s semen, day of recovery, number of ovulations and clinical expertise. Embryo recovery rates using sub fertile donors are only about 15 to 20%; whereas embryo recovery rates for healthy, reproductively sound mares may approach 75%.
Cooled Transported Embryos
Embryo transfer can still be a part of your breeding management program if your mare is maintained at home by shipping the embryo to a facility for transfer into a synchronized recipient. Equine embryos can remain viable at 5*C for at least 24 hours when stored in Han’s F-10 media that has been buffered by diffusing a mixture 90%N2, 5 %O2, and 5% Co2, through the medium for three to five minutes. The medium is supplemented with 10 %calf serum and antibiotics prior to cooling. The embryo is placed in a small snap or screw cap test tube filled with the buffered Ham’m. The tube is packaged in a passive cooling system (Equitainer*, Hamilton Thorne Research, South Hamilton, MA) and transported by commercial airline or priority overnight delivery service. Alternative complete embryo transport media that do not need to be buffered with the gas mixture are currently being tested for clinical use.
In 2001, 83.9% of Grade 1 and 2 embryos shipped to CSU from donor mares housed off-site resulted in a pregnant recipient mare after transfer. Pregnancy rates are not significantly different for embryos transferred immediately after collection (i.e. embryos collected from donor mares on-site) versus embryos cooled and transported by commercial airline (counter-to-counter) or overnight courier service.
Recipient Management
Mares intended to be embryo recipients should be young (3 to 10 years of age) and reproductively healthy. Since owners of donor mares often wish to begin embryo transfers in February or March, ET recipient mares should be placed under lights in early December to advance the onset of the first ovulation of the year. If cycling embryo recipients are not available in the early spring, transitional mares or ovariectomized mares can be administered exogenous progesterone and used as a recipient.
A donor embryo can be successfully transferred into a recipient mare that ovulates the day prior (_1), the same day (0) or up to 3 days (-3) after the donor mare. If a limited number of recipients are available, an individual recipient must be synchronized with each donor mare. Synchronization of ovulations in horses is challenging. The most commonly used techniques include two injections of prostaglandins approximately 14 days apart and administration of progesterone or progesterone plus estradidol for 10-14 days, with prostaglandins at the end of steroid treatment. Response to exogenous hormone administration varies greatly between mares, and no synchronization scheme is ideal or completely predictable. In most instances it is recommended that multiple recipients be synchronized along with the donor in order to have at least one mare ovulating during the critical time window. Ideally, the recipient mare would have ovulated 1-2 days after the donor mare.
Success of an embryo transfer program is highly dependent on the quality of recipient mare. Potential recipients are evaluated 5 days after ovulation to determine if they qualify to receive an embryo that cycle. Criteria used for evaluation include palpation and ultrasonography of the reproductive tract per rectum and occasionally analysis of blood of a corpus luteum on ultrasonography and an absence of endometrial folds (edema) or free flux, within the uterine lumen. Mares that “pass” this examination are available for use as recipients for the next 5 days. Mares are rejected as potential recipients if poor uterine or cervical tone, a small (or absent) corpus luteum, endometrial edema or uterine fluid are detected. Serum progesterone concentrations can be measured if corpus luteum function is in doubt.
Non-surgical Embryo Transfer Technique
Transfer of embryos into synchronized recipient mares at CSU is performed nonsurgically. Recipient mares are administered acepromazine and flunixin meglumine (Banamine) immediately prior to transfer and a single dose of antibiotics immediately after transfer. Embryos are transferred transcervically into the uterus of the recipient mare using either a 0.25 ml. Straw and transfer instrument or an insemination pipette, depending on the size of the embryo.
Currently all recipient mares are maintained on the synthetic progestagen Regumate after receiving an embryo. Blood samples are collected periodically from the recipient mares to confirm if endogenous progesterone levels are high enough to maintain the pregnancy. Regumate treatment can be discontinued if progesterone levels are > 4.0 ng/ml. It is recommended that supplementation is continued if levels are < 4.0 ng/ml.
The pregnancy check on the recipient mare is performed 5 days after transfer. A definitive positive or negative pregnancy status is known by 7 to 9 days after transfer, which is approximately day 15 to 17 days after ovulation of the donor mare. Pregnancy examinations are subsequently performed on days 25, 35 and 50 after ovulation of the donor mare.
Pregnancy Rates
Factors affecting transfer success include embryo quality, age of the donor mare, transfer technique, recipient quality and synchrony of the recipient. Pregnancy rates after transfer decline as the grade of embryo recovered declines.
Prior to the 2000 breeding season, a majority of the embryos at CSU were transferred surgically. Pregnancy rate for grade 1 embryos transferred surgically in 1999 was 71.8%. In the 2000 and 2001 breeding seasons, all embryo transfers were performed using a nonsurgical technique and pregnancy rates of 74.1% and 74.4%, respectively, were achieved after transfer of Grade 1 or 2 embryos.
Embryo Freezing
The first successful term pregnancy from a frozen-thawed equine embryo was reported in 1982. Several studies have since shown that the technique is possible, but pregnancy rates are still close to those obtained utilizing fresh or cooled embryos. Embryos collected six days after ovulation survive the freezing and thawing procedures better than day 7 or day 8 embryos.
Summary
Embryo transfer has become a common technique to enhance reproduction in sub fertile and performance mares in many breeds. The ability to collect and ship embryos to a commercial facility for transfer has made embryo transfer much more widely available to horse owners.
Updated: Jan. 2002