The following topics are provided by Dr. W. B. Ley, author of Broodmare Reproduction, horse-repro.com and rectortownequine.com
- Embryo Transfer
- Follicle Aspiration and Oocyte Collection for Cryopreservation
- GIFT or Gamete Intra-Fallopian Transfer
- ICSI or Intracytoplasmic Sperm Injection
Embryo Transfer
Embryo transfer is a common form of assisted reproductive technology, or ART. It can be very successful and rewarding, but has the potential to be frustrating as well. This is why you should not go into this procedure without first knowing all the facts and costs involved, and all of the inherent risks. Some of the reasons for doing an embryo transfer include: using a donor mare that may already be performing in a show or race career and you do not wish to interrupt this while she carries a foal; you wish to lease a mare for embryo donor purposes; your mare is physically unable to carry her own pregnancy; or you wish to produce two or more foals from the same donor mare in the same breeding season. These are but a few. However, the main reason many look to ET as an alternative breeding plan is for their older broodmare who has difficulty conceiving or carrying her own pregnancy to term. This is not a good plan! Older mares have lesser quality eggs, and as a result often have lesser quality embryos that do not transfer well, or fail to develop at all. You must consider this fact, before venturing down the ET path. This is not an impossible situation, but many attempts may be required before obtaining an embryo of sufficient quality for a successful transfer and resultant pregnancy in the recipient mare. That can be frustrating, and frankly, expensive!
The steps involved are first to decide on the donor mare and why you wish to do this. Once that hurdle is cleared, you must then decide how you will breed her, whether by live cover, fresh AI, transported semen, or frozen semen. All are possible, but realize that conception rates with frozen semen can be lower. You may have to breed her on more than one cycle to get an embryo for transfer. Simultaneously, you will need to decide on a recipient mare. Do you have one or more of your own to volunteer? That would be the least costly. Alternatively, we can provide a recipient, or you could contract with a third party to have the embryo shipped to their facility for transfer to a recipient. If you are not using your own mare as a recipient, then you will have to decide whether you are going to purchase or lease the recipient mare provided by the alternate source. We can arrange for either. Having crossed these fences we get down to the details of synchronization, breeding, and the transfer itself.
Both the donor mare and the recipient mare (or mares) will need to be in synchrony with respect to their stage of estrous cycle and timed to ovulate within 2 or 3 days of each other. This can take some manipulation, a lot of rectal and ultrasound examinations, and good communication. If the donor and recipients are not on the same farm or under the care of the same veterinarian, the opportunity for delays and lack of synchrony can be frustrating, but not impossible. The donor mare should be inseminated or bred according to her follicle development just prior to ovulation as with any other breeding. The recipient(s) need to have ovulated 1-1.5 days beforehand, or within 2 days afterward.
The flushing procedure to obtain an embryo is then set for day 7, 8 or 9 after the donor mare ovulates.
On the day of the embryo flush the donor mare is prepared similar to a standard AI, and an embryo flushing catheter is placed into and through her cervix. A balloon is inflated to seal the internal opening of the cervix to prevent any flushing fluid from escaping. One to five liters of warmed electrolyte solution is lavaged into and allowed to drain from her uterus to dislodge and recover any embryo (or embryos) that may be present. The return fluid is passed through a sterile filter cup that traps anything the size of an equine embryo. This cup is then searched for one or more embryos. Once an embryo is identified it is evaluated for quality and stage of development. If of sufficient quality. it is then washed several times in special media to prevent carrying bacteria from the donor mare to the recipient mare.
At this point one of three options exist: transfer the embryo directly to a recipient mare within an hour of its recovery; package the embryo in special shipping media and send it to a distant farm or reproductive center for transfer into one of their recipient mares; cryopreserve (or freeze) the embryo if it is not too advanced in its development for thawing and transfer to a recipient mare at a later point in time.
Management of the recipient mare involves administering an anti-inflammatory agent such as flunixin just prior to the embryo transfer. The embryo transfer itself is accomplished in a manner very similar to a standard insemination via the cervix. The recipient mare will be started on daily progesterone supplementation (e.g., oral altrenogest) to be continued for the next week. She is examined seven days after the transfer to determine if she held the embryo and is indeed pregnant. If she is pregnant, her management would be similar to any other pregnant mare. If she is not pregnant, the whole process must be repeated, should all involved be willing to do so.
THERE are no guarantees, stated or implied. Many factors have to be taken into consideration: age of the donor mare; quality of the semen used to breed the donor mare; quality of any embryo recovered; skill, competence, and experience of the professionals involved; synchrony of donor and recipient mares; and the chance that the embryo will survive the transfer procedure, and continue to develop and establish pregnancy in the recipient.
For these reasons, I have elected to separate out fees for each individual part of the process, rather than state one fee for the entire package. So many factors come into play. One is dependent upon the preceding event for its own success. If the semen does not arrive or is of poor quality, there is little need to attempt an embryo flush. If the recipient mare(s) do not ovulate in synchrony with the donor, what options are left for a transfer? If the donor mare does not conceive and no embryo is recovered, there is no need to have the recipient mare(s). For these reasons I have set up a progressive fee scale, the client only pays for the services rendered without an obligation for events that might not happen.
Follicle Aspiration and Oocyte Collection for Cryopreservation
Follicle aspiration or egg retrieval is common in women undergoing an in vitro fertilization ( IVF) procedure for infertility. It is also a technique used in mares on a research basis to obtain ova (aka: oocytes or eggs) for IVF or intracytoplasmic sperm injection ( ICSI) studies. The introduction of a better method to cryopreserve (freeze) ova using the process of vitrification has now enabled this procedure to become a commercially viable option for breeding management. As many follicles as can be visualized using transvaginal ultrasound are punctured and their contents aspirated. The aspirated fluids are search for oocytes. Recovered oocytes are graded and good ones are transferred to either a holding or a maturation medium. If the oocytes are to be used within a short period of time, they would be transferred along with viable sperm into the oviduct of a recipient mare (gamete intrafallopian transfer, GIFT), allowing fertilization to occur in the natural setting. Alternatively, one or more oocytes could be matured for a period of 18-24 hours and then subjected to ICSI for direct fertilization by one sperm. This fertilized oocyte would then be cultured for a day or two and then transferred into the oviduct of the recipient mare. If neither of these options were selected, the oocytes could be cryopreserved for use at a later time (weeks, months, or years). The advantages to this are preserving the young mare’s oocytes for later use, after she has had a successful and productive career as an athlete. Or to preserve her oocytes for use in a GIFT or ICSI procedure at a later date allowing a recipient mare to produce her foals while she continues an active show or performance career.
GIFT or Gamete Intra-Fallopian Transfer
GIFT stands for gamete intrafallopian transfer. A gamete is a male or female sex cell (a sperm, or an egg). During GIFT, sperm and eggs are mixed and injected into one or both fallopian tubes of the female. She can either be the donor of her own egg or may be an unrelated recipient. After the gametes have been transferred, fertilization can take place in the fallopian tube as it does with natural, unassisted reproduction. Once fertilized, the embryo travels to the uterus by natural processes . The basic steps of GIFT are ovulation egg harvest ( oocyte aspiration ) and surgical gamete transfer. The eggs can be harvested during a laparoscopy procedure or more commonly by an ultrasound-guided follicle aspiration procedure . The transfer is performed either by standing surgical flank laparotomy or laparoscopy procedure, eggs are mixed with sperm, and the gametes are transferred into the recipient’s oviduct.
ICSI or Intracytoplasmic Sperm Injection
Intracytoplasmic sperm injection (ICSI) is when a single sperm is injected into an egg (oocyte) using micromanipulation under a specialized microscopic system. ICSI is used when the sperm can not penetrate the egg on its own, or as a means of assisted fertilization, or when sperm are of a limited supply. It is commonly used when the diagnosis for infertility is on the male side.
