Embryo flushing and transfer (ET) has become increasingly popular with non-Thoroughbred breeders over the last five to 10 years. The availability of centres maintaining a herd of recipient mares, which receive embryos for transfer, has led to an increased interest among practitioners in providing embryo flushing and shipment services to their own clients. This article reviews the factors and techniques which affect the success of this assisted reproductive technique.
Choice of donor
Before commencing, mare owners should check that the stud book in which they wish to register the foal permits the use of ET. The major application of ET is in enabling
Some sub-fertile mares with specific problems, e.g. cervical injuries, may be good candidates for embryo transfer. However, research has shown that old mares who have failed to conceive themselves are poor candidates for ET
top-class sports horse mares to produce foals while still competing, rather than having to defer their stud careers. Embryo transfer can also increase the number of foals produced per mare per year. ET may be appropriate for valuable mares that are unable to carry a foal safely to full term themselves, e.g. due to previous colic surgery or a ventral rupture. Some sub-fertile mares with specific problems, e.g. cervical injuries, may be good candidates for embryo transfer. However, research has shown that old mares who have failed to conceive themselves are poor candidates for ET, since the fundamental problem is at the level of their aged oocytes.
Breeding the donor mare
Donor mares should be bred using good quality semen/a stallion of known fertility, following conventional protocols (McCue, 2010). Despite research interest in the development of a technique for superovulating mares, this is not currently commercially available. Donor mares should be assessed for post-breeding endometritis, and treated if necessary.
Embryo flushing
Embryo flushing in the mare is a non-surgical trans-cervical technique (Hartman, 2011) which is minimally invasive and generally well-tolerated. The risks associated with any transrectal examination in the mare also apply during embryo flushing. Mares should be restrained or sedated as necessary to minimise these risks.
All embryo flushing and holding media and equipment are now commercially available.
The flush is usually performed at day seven or eight post-ovulation for immediate transfer or chilling of embryos. Embryos are recovered more efficiently later, but are more robust earlier. Recovery rates from aged mares may be improved by flushing on day eight rather than day seven.
In brief, the technique may be described as follows:
- Restrain donor in stocks, wrap and elevate tail, aseptically prepare hindquarters. All residues must be rinsed off.
- Using a sterile plastic sleeve-covered hand and arm, delicately introduce a uterine flushing catheter (80 or 150cm long, balloon-tipped, silicone catheter; Figure 1) through the cervical opening and into the posterior uterine body.
- Fill the balloon cuff with 60-80ml of air and pull the catheter caudally to establish a seal at the internal cervical os.
- Use 1,000-3,000ml of commercially prepared equine embryo flushing media for each flush (depending on mare size and tolerance). n Run the flush into the uterine lumen by gravity using Y-tubing attached to the foley catheter (Figure 2).
- Depending on the parity of the mare and the size of the mare’s uterus, it may help to massage the filled uterus per-rectum before retrieval, to ensure that the fluid has reached all of the uterus.
- Run the fluid from the uterus back through the Y-tubing and into an in-line embryo filter/cup (Figure 3). The filter must always be bathed in fluid to prevent embryo dessication and must not be allowed to overfill.
- Depending on the parity of the mare and the size of the mare’s uterus, an i.v. injection of oxytocin may be given prior to the final flush to aid uterine evacuation.
- After the final flush, rinse the tubing before disconnecting it, in case the embryo is trapped between the cuff and the cup.
Embryo processing and assessment
Whether embryos are to be transferred directly or chilled for shipment to a recipient mare, the initial processing and assessment is the same. Briefly, the technique can be described as follows:
- The fluid retained in the embryo cup/filter is placed in sterile Petri dishes by gently swirling the fluid in the cup and tipping it quickly into the Petri dish.
- Rinse the cup using some of the flush media which has been retained for the purpose.
- Examine the contents of the Petri dishes using a dissecting microscope (x 10-25) to locate the embryo(s). Gentle swirling of the Petri dish brings the embryo and any endometrial cells to the centre; embryos are heavy and so sink to the bottom of the Petri dish. Push any cellular debris out of the way during searching with a sterile semen straw.
- Using a sterile semen straw, transfer embryo(s) into a small Petri dish containing commercial embryo holding medium.
- Wash up to six times by transfer between small Petri dishes containing commercial embryo holding medium.
- If the embryo is to be shipped chilled to a specialist facility for transfer, it should be loaded into a shipper as per the receiving facility’s instructions.
Vets should be aware that the fact that an embryo was not recovered from the uterus does not necessarily mean that the mare did not conceive. It is therefore normal practice to treat a donor mare with a luteolytic dose of prostaglandin F2α after flushing, to avoid the possibility of the donor mare remaining pregnant herself.
Conclusion
The ability to ship embryos to centres maintaining recipient herds, for transfer, has reduced some of the logistical issues previously associated with using ET in the UK. The commercial availability of embryo flushing materials and equipment has also made it easier for veterinarians to offer an embryo flushing service in practice. Nonetheless, veterinary expertise in equine reproduction and appropriate selection of embryo donors are necessary prerequisites for a successful embryo flushing programme.
