Embryo Transfer

The benefits of embryo transfer are great. Older mares, competition mares and reproductively challenged mares can now produce foals by transferring their embryos to donor mares, which will continue the pregnancy and deliver the foal. Before performing an embryo transfer check the regulations of your breed registry first!

Two physiological issues that limit the practicality of embryo transfer in mares:

1. Mares cannot easily be superovulated because their ovaries are unresponsive to eCG (Equine Chorionic Gonadotrophin) and Follicle Stimulating Hormone (FSH), possibly because they are already present in the mare’s system. Until agents are found which will cause superovulation in mares, as eCG and FSH do in other animal species, this will remain a problem. 2) Variations in mares’ estrus cycles and pinpointing ovulation times makes synchronization between donor and recipient difficult when using fresh embryos.
2. An embryo enters the uterus around day five or six from conception. Non-surgical flushing is best done at day six to eight, when there is a 75% recovery rate. Because the embryo is very active until around day 14, a body flush is the best method of retrieving embryos.

Embryo transfer basics:

• It is ideal if the recipient mare ovulates 24 hours after the donor mare. Implantation can still be successful if recipient ovulates 24 hours ahead to 60 hours behind the donor.
• There are two methods of obtaining embryos: surgical or non-surgical recovery. The preferred method of surgical recovery is through the flank while the donor mare is standing, and is sedated and given a mild local anesthetic. The non-surgical technique is performed between day six and eight, and results in a 75% recovery rate.
• Transfer of the embryo should take place as soon as possible following collection, preferably within two to four hours. Pregnancy rates drop dramatically if an embryo is out of the donor mare for more than one to two hours.
• Transfer can be surgical or non-surgical. The preferred surgical transfer is through the flank while mare is standing under mild sedation and local anesthesia. Pregnancy rates with this method are 75-80%. With non-surgical transfer the conception rate is 35-40%.
• Freezing of equine embryos is difficult because of the high lipid content and large size of the embryo. Best results with frozen embryos are with using are with the pre-six day embryos (morulas) obtained by surgical oviduct flushing through the flank.

Non-surgical Equine Embryo Transfer--embryo collected six to eight days after ovulation:

• Prepare donor mare secured in stocks, wrap tail, palpate rectally to determine follicular development
• Palpate ovary on which follicle was found previously to determine if there is a corpus luteum
• Backrake to remove all feces
• Wash perineal area three times with surgical scrub, rinse well and dry with surgical cotton

Embryo collection:

• Wearing a lubricated sterile palpation sleeve lubricated with non-embryocidal lubricant, introduce extra long disposable Foley Cather size 28 or 30 French Gauge with and 80 ml. balloon. Run manually through cervix until tip is approx. 5 cms into unterine body. The balloon of the catheter is then filled with 80 mls. of air or 60 mls of PBS and seated into the internal os of cervix. Both inlet and outlet tubing then attached to the Foley and both clamped
• Flush uterus by allowing a half liter of flushing medium to run into uterus by gravity or until mare shows signs of discomfort due to stretching. When uturus is full clamp off inlet tube and release outlet tube, allowing fluid to flow back into an in-line filter. Repeat procedure until three liters of fluid have been used. 95% of fluid is recovered. If not, gently massage uterus via rectum until this has occurred
• After flushing, iIrrigate uterus with 50 ml. Of Furacin solution to prevent infection and to be sure pregnancy does not result if an embryo is left behind

Embryo handling and Identification:

• Usually the embryo(s) can be seen in the fluid in the filter. If this is not the case, pour contents into a Quebec petrie dish and flush filter several times with PBS. A stereo microscope is used to identify and locate the embryo. Overal recovery rate is 75-80%

Preparation for transfer:

• Embryo is washed at least twice in PBS supplemented with 10% serum maintained at 37 degrees Centigrade

Loading transfer pipette:

• A sterile 55 cm pipette. The embryo is positioned between two air pockets on either side of it, to minimize movement within the pipette

Recipient preparation:

• Same as donor

Transferring Embryo:

• Technician, wearing two sterile gloves, holds a.i. rod with embryo between hands. Introduce hand into vagina, “feather” cervix and guide rod gently into uterus. Insert embryo into uterus and carefully remove rod, while holding lips of vulva together to prevent aspiration of air into vagina. Rinse pipette with PBS into pert dish, and carefully examine contents under microscope to be sure the embryo was transferred

Flushing medium used:

• Dulbecco’s Modified Phosphate Buffer Saline (PBS) with 1% of heat inactivated bovine serum albumin or heat inactivated gelding serum added, and 1% of penicillin or Gentamycin also added to the solution

Timetable:

• Day 0 prostaglandin 8-10 mg. to donor and recipient
  Day 13 8-10 mg prostaglandin to donor and recipient
  Day 16 Beginning of estrus ??
  Day 17 Palpate donor and recipient, monitor follicle size and ovulation. Breed donor mare if using artificial insemination.
  Day 18 Palpate (day 3 of estrus?) 3000 IU HCG given IV to donor and recipient.
  Day 19 Palpate both mares, Breed donor
  Day 20 Palpate both mares
  Day 21 Palpate both mares Breed donor
  Day 7 from ovulation Collect embryo. Prostaglandin to donar and new recipients,. Donor returns to day 13 of schedule.
  Day 16 from ovulation—US pregnancy examination
  Day 28 from ovulation—Pregnancy examination by rectal palpaption and US