Embryo Transfer in Horses

Embryo Transfer in Horses : Introduction

In mares, embryo transfer is used to increase productivity of a given mare or to obtain a foal from a mare that is not able to carry a foal to term. Superovulation is of variable efficacy in mares. Recent work with bid administration of equine FSH resulted in an increased ovulation rate (3-4 ovulations/mare) and increased embryo recovery (>1.5 embryos/treated mare, compared with 0.5 embryos/control mare). Equine FSH is now commercially available in the USA. For fertile, naturally ovulating donors, an embryo recovery rate of 60-70% and a pregnancy rate after transfer of 70% can be expected; these rates are 30% and 50% for subfertile donors. Breeding of the donor mare is done as for a conventional pregnancy; however, ultrasonography is essential to determine the exact day of ovulation, on which the day of embryo recovery is based.

The recipient mare should ovulate from 1 day before to 3 days after the donor mare ovulates. If a large recipient mare herd is available, mares that have ovulated at the appropriate time may be selected from the herd. If small numbers of recipients are used, the time of ovulation of the donor and recipient mares must be synchronized. This may be done using a progesterone and estrogen regimen (see hormonal control of estrus, Hormonal Control of Estrus: Introduction). The recipient mares should be started on the regimen 2 days after the donor to help ensure that they do not ovulate before the donor. Mares should be examined by ultrasonography daily to detect ovulation. To aid synchronization, ovulation may be speeded in the donor or recipient by administration of human chorionic gonadotropin (hCG) or deslorelin when a mature follicle is present. Two recipients should be synchronized for each donor to enable the recipient with the best synchronization to be chosen at the time of transfer; in addition, both recipients may be needed if the mare ovulates 2 follicles. Alternatively, ovariectomized or follicle-suppressed progesterone-treated mares may be used as recipients, eliminating the need for donor synchronization and for examination of recipient mares to detect ovulation. Recipients should be ovariectomized no more than 6 mo before transfer or should be treated with estrogen and then progesterone for a period (followed by at least 1 wk of no treatment) before being used as a recipient. Follicular suppression may be induced by administration of 2 deslorelin implants (2.2 mg, IM) to induce ovulation of a mature follicle, followed by prostaglandin administration when the resulting corpus luteum is 5-7 days old. This may result in up to 30 days of follicular suppression. The ovariectomized or suppressed recipient is untreated until donor ovulation is detected (day 0), or may be given estradiol (eg, 3.3 mg estradiol 17b, IM, sid) during this time. Starting on day 2, the recipient is given progesterone in oil, 300 mg, IM, sid. After embryo transfer, the recipient is continued on a progestagen daily until day 100 of gestation, after which placental progesterone is sufficient to maintain pregnancy. Recipients with intact ovaries may be able to discontinue progesterone sooner if secondary ovulations are seen after day 40 of pregnancy. Ovariectomized recipient mares have normal pregnancy rates after transfer, as well as normal parturition, lactation, and maternal behavior; they may also be used successfully again as embryo recipients after foaling. Fewer data are currently available on follicle-suppressed, hormone-treated mares, but no abnormalities in pregnancy or maternal function have been noted.

Embryos are usually recovered on day 7 or 8 after donor mare ovulation. Modified Dulbecco’s phosphate buffered saline with 1% fetal calf serum, or a commercially available equine embryo flush solution, is used for embryo recovery. Three 1-L flushes are performed. The mare is restrained in stocks with the tail wrapped and tied to the side, and the perineum is scrubbed and dried. A Foley-type catheter is passed manually through the vagina and cervix and into the uterine body. The cuff is then inflated with 30-40 mL of air and is pulled caudally to rest against the internal cervical os. The flush medium (1 L) is infused by gravity flow into the uterus. The fluid is then drained from the uterus by gravity flow. A wait of 3 min before draining may increase embryo recovery rates; alternatively, the fluid may be massaged throughout the uterus by palpation per rectum before it is drained. The fluid may be collected into a 1-L container and then poured through an embryo filter, or the filter may be placed directly in the outflow line. The latter may limit the speed of the outflow, potentially reducing embryo recovery. After the 3 uterine flushes have been performed, the donor mare is given PGF_2α, 10 mg, IM, to shorten the cycle and to reduce the chance of endometritis due to organisms introduced into the uterus during flushing.

The embryo is then located within the filter contents. Early embryos are similar to those of cattle, being still enclosed by the zona pellucida, appearing somewhat like a parasite egg ~200 µm in diameter. Later embryos have a hollow center and cellular rim or, if completely expanded, appear as a gray, semitranslucent sphere of up to 0.5 mm diameter on day 7 and 1 mm diameter on day 8. The embryo is transferred into a small (35 mm) Petri dish containing fresh medium with 10-20% neonatal calf serum or commercial handling medium, and is washed by transferring it at least 2 more times to droplets of fresh medium. As soon as the embryo is washed, it may be transferred, which should be done within 3 hr of collection.

For transcervical transfer, the recipient should be restrained and the perineum scrubbed as described above for the donor. Tranquilization of the recipient with xylazine or acepromazine, if necessary, does not appear to lower pregnancy rates. Tranquilization may relax the cervix and aid in a smooth transfer. The embryo is loaded into an insemination pipette or an embryo straw in the following sequence: medium, air, medium, air, medium containing the embryo, air, medium, air. If a straw is used, it is then loaded into an insemination gun. The pipette or insemination gun is passed manually into the vagina. The external os of the cervix is located but not cannulated with the finger. By stabilizing the cervix with the fingers, the pipette is passed into the external os, and the hand is withdrawn from the vagina. The pipette is manipulated through the cervix by palpation per rectum, and the embryo is expelled into the uterus. Surgical transfer may be done via flank incision as described for cattle (see above) but has no benefit over transcervical transfer.

Embryo freezing is still not widely used in horses because larger embryos (>6.5 days old) have poor viability after thawing. However, embryos in commercial handling media may be successfully cooled and stored for up to 24 hr in semen transport containers, allowing time for shipment to an embryo transfer facility, with no decrease in pregnancy rates after transfer.