Embryo Transfer in Mares
Dr. Sylvia J. Bedford-Guaus
The chosen recipient is prepared for transfer by wrapping and tying her tail. Her uterus must be checked for tone and evidence of fluid. The embryo should never be transferred to a mare that has fluid in the uterus.
Synchronization Protocol for Donor and Recipients
Many synchronization protocols for donor and recipients are used, depending on personal preferences and experience. Hormones are given to the donor and recipients to manipulate their estrus cycle with the objective that they all come into heat at the same time. These treatments can vary from giving mares a single shot of prostaglandin, to placing the mares in a full treatment schedule of progesterone and estrogen shots.
Although cumbersome, a series of progesterone/estrogen shots provide the best synchrony between donor and recipients. Briefly, the protocol consists of a daily injection of a combination of progesterone and estrogen for 8 to 10 days (150 mg of progesterone plus 10 mg Estradiol-17b per shot). This suppresses the mare's own hormones, and when the treatment is discontinued most mares should come into heat in 3 to 5 days. Because some mares may ovulate while in treatment, a prostaglandin shot is usually given at the end of the progesterone-estrogen series for better synchrony.
Oral progestagens marketed to keep mares out of heat do not work as well for synchronization protocols.
Once donor and recipients are in heat and the donor mare is bred to the appropriate stallion, ovulation may be induced in all mares by giving them a shot of human chorionic gonadotropin. This will ensure a tighter ovulation synchrony between donor and recipient. A recipient is ideal if she ovulated on the same day or one day after the donor. Less ideal recipients are those ovulating one day before or farther from the ovulation date of the donor.
Unfortunately, even the best synchronization protocols can fail and donor and recipients may not always come into synchronized heat and ovulate as expected. This is a good reason to have always more than one recipient available for each donor mare. At least two recipients per donor is recommended, three would be ideal.
Breeding Management of the Donor Mare
The first limiting factor for the success of the embryo transfer program is that the donor mare must become pregnant. It is therefore imperative to practice good breeding management of the donor mare.
The donor mare may be bred with fresh or transported-cooled semen. It is important that the semen is of good quality and that the stallion has good established pregnancy rates. Obviously, the ideal stallion for a mare is not always chosen in regards to his fertility potential. However, old and subfertile stallions should be avoided to prevent introducing another factor that may preclude success of the embryo transfer program. For this same reason, the use of frozen-thawed semen should be avoided.
Optimal breeding management consists of breeding a mare during estrus as close to ovulation as possible. Ideally, the reproductive tract of donor and recipients should be examined daily by palpation and ultrasonography per rectum to ascertain the optimal time for breeding the donor and to assess synchrony with the recipients.
Around breeding, the donor must be given an injection of human chorionic gonadotropin to induce ovulation, and the recipients are treated in a similar manner (but not bred) for tighter ovulation synchrony.
Ovulation should always be confirmed by ultrasound examination. The day of ovulation is considered 'day 0' and the donor mare's uterus is usually flushed on day 7 after ovulation.
Embryo Collection Procedure
Embryo recovery from the donor mare is usually performed on day 7 postovulation. First, the mare is checked by palpation and ultrasonography per rectum to ascertain tone and appearance of the uterus. A mare that is pregnant will most likely have a very toned, homogeneous uterus, a closed cervix, and there will be no fluid in the uterus as visualized with ultrasound. At this time, the embryo is too small to be seen on ultrasound.
The mare is prepared for embryo recovery by cleaning the perineal area (vulva, anus and buttocks) with povidone iodine (Betadine) scrub three times, and rinsing thoroughly with water after each scrub. After generous rinsing, the area is dried with clean paper towels.
The person performing embryo transfer recovery should wear clean palpation sleeves and sterile gloves, and a sterile, water based jelly should be used for lubrication. One hand is introduced in the mare's vagina to pass the Foley catheter through the cervix and into the body of the uterus. Then, the catheter's balloon is inflated with air, using a small syringe, and thus is securely lodged within the cervix to avoid losing fluid during the uterine flush.
It takes at least two people to perform the embryo collection procedure, one holding the catheter within the mare's tract, and an assistant helping with fluid insertion and recovery. One liter of fluid is run into the mare's uterus by gravity, then recovered into a large glass cylinder, or directly passed through a filter to trap the embryo. This is repeated three times.
Some fluid should be kept within the recovery filter, to avoid drying out of the embryo. Once the recovery procedure is finished, fluid within the filter is decanted into a Petri dish. Searching for the embryo is performed under magnification with a dissecting microscope.
Embryo Transfer Procedure
If recovery is done on day 7 postovulation, the embryo should be at the blastocyst stage and should be easy to visualize under a dissecting microscope by an experienced operator.
Once found, the embryo is washed in sterile DPBS with 10 percent fetal calf serum. Embryos can be handled with 0.25 ml French semen freezing straws or glass pipettes connected to a rubber bulb or to a small tuberculin syringe.
The embryo is commonly transferred transcervically, that is, through the cervix of the mare as if performing artificial insemination. For this purpose:
Then, the perineal area of the recipient mare is thoroughly scrubbed as explained for the donor.
The embryo can be loaded into an insemination pipette connected to a 5-ml Air-Tite syringe. About 1-ml of air is loaded into the syringe first, then the pipette is loaded with three small pockets of medium separated by pockets of air, with the fluid pocket in the middle containing the embryo.
The person performing the transfer procedure should wear a surgical sterile glove over a clean shoulder length plastic sleeve. The pipette is inserted in the vagina and carefully passed through the cervix. Once advanced into the uterine body the syringe plunger is pushed forward and the embryo injected into the uterus.
The recipient mare should be checked for pregnancy around 7 days after the transfer.
Another possibility is to perform surgical embryo transfer. This is done with the sedated recipient in the standing position through a flank incision that allows exteriorization of the uterine horn. There is no obvious advantage in transferring embryos surgically, and the procedure is more traumatic for the recipient mare.