Posted on 25 Nov 2019 by Nathalie De Martin
Photo © Fotobron: Bridge Clinic
While the racehorse industry chose to ban ET and ICSI, both techniques are commonly used in jumping and dressage sport. Already in 1972 embryo transplants were performed in the US. and Argentina, and while the first human ICSI was established in Belgium in 1991, it took until 1996 before the first ICSI equine pregnancy was announced.
Embryo transfer (ET), that is, recovery of an embryo from the uterus of a donor mare and transfer of the embryo to the uterus of a recipient mare, is a well‐established equine assisted reproductive technique (ART) (Squires, McCue, & Vanderwall, 1999). The standard protocol for equine ET is to monitor the preovulatory follicle of the donor mare via ultrasonography per rectum. The mare is inseminated when the follicle appears to be ready to ovulate, or in coordination with induction of ovulation by administration of a GnRH or LH analog. The preovulatory follicle is monitored daily after insemination so that the day of ovulation (Day 0) can be determined. The embryo is flushed from the donor mare’s uterus, typically on Day 7 or 8 after ovulation. Flushing is performed by filling the uterus with ~1 L of flush solution, then draining the fluid through a filter and repeating this multiple times. The embryo is located in the fluid retained in the filter, and is washed and transferred transcervically to the uterus of a recipient mare that ovulated from a day before to 3 days after the donor mare. With a fertile donor mare, the expected embryo recovery is about 75% and the expected pregnancy rate after transfer about 75%, for a ~50% recipient pregnancy rate per cycle (Squires et al., 1999; Vogelsang, Bondioli, & Massey, 1985), although experienced practitioners may report higher rates than this.
Embryo transfer can be used to obtain more than one foal per donor mare per year, or to obtain foals from donor mares that the owner does not want to be pregnant (because the mare is in competition, the mare is too valuable to risk the potential dangers of pregnancy or foaling, or the mare has other issues, such as lameness, pelvic damage, or even maternal behavior). Embryo transfer can also be used to recover embryos from donor mares that are capable of conception but which suffer from repeated early embryonic death associated with factors such as uterine periglandular fibrosis or other age‐related uterine changes (Kenney, 1978).
The main limitations of embryo transfer as an equine ART are that (a) recovery of embryos requires that the donor mare have a functional tract, at least to the point that sperm can reach the oviduct, the mare ovulates normally, and the oviduct and uterus can support fertilization and development of the embryo until Day 7 or 8 after ovulation; (b) breeding the donor mare requires a full insemination dose of good‐quality semen; and (c) superovulation of mares is problematic (Squires & McCue, 2007), so only the potential embryo resulting from ovulation of the one dominant follicle (or sometimes two, if the donor mare spontaneously double‐ovulates) is available. Use of ET in mares in competition can be complicated by the effects of stress (Campbell, 2014), and repeated manipulation of the mare’s uterus (breeding, uterine flush) can be associated with induction of endometritis (Campbell, 2014).1
Oocyte recovery and intracytoplasmic sperm injection (ICSI) can be used for production of foals from mares that cannot become pregnant or provide an embryo under standard reproductive management. This includes mares with cervical, uterine or oviductal abnormalities or disease. However, ICSI will NOT be helpful if the cause of the mare’s subfertility is due to her oocyte quality. In the clinical ICSI program at Texas A&M, the main driver for ICSI is on the part of the stallion owner, to allow breeding of mares by top stallions that have aged or died and so have only limited supplies of frozen semen available. The efficiency of frozen semen is greatly increased by ICSI, as only a few sperm are needed, from which one is selected to fertilize each oocyte. Frozen semen can be thawed, diluted up to 1:200, and refrozen in a large number of straws of “ICSI doses” (Choi, Love, Varner, & Hinrichs, 2006a) and used for ICSI successfully. If motility is good, a portion of this straw (a “cut”) can be used at a time, for fertilization of numerous oocytes, thus allowing an existing store of frozen semen to produce a large number of embryos.
Because of the higher costs associated with ICSI, it is therefore recommended to first try embryo transplants before working with ICSI. Another advantage of ICSI is also that it can be performed on a mare that has just died, also known as post-mortem.
The ICSI procedure
The first step of the ICSI procedure is to recover oocytes from your mare. Oocytes may be recovered from small (immature) follicles, or from the one large preovulatory follicle. The oocytes are then cultured to induce maturation. This maturation mimics the developmental changes that would occur naturally in an oocyte within the mare, during the day or so immediately before ovulation. For oocytes recovered from small (immature) follicles, the maturation process generally takes 30 hours. An oocyte recovered from the one preovulatory follicle after hormone stimulation is already maturing at the time it is recovered, and is cultured for 12 hours or less prior to ICSI.
Those oocytes that mature are then subjected to ICSI, that is, injection of each oocyte with an individual sperm from the desired stallion. For this procedure, the sperm sample (fresh or frozen-thawed) is washed and prepared, and one sperm is injected into the cytoplasm of each oocyte under a high-power microscope. The resulting fertilized oocytes are cultured in the laboratory for 7 to 10 days, to allow development into blastocysts, that is, embryos suitable for transfer to a recipient mare. If you do not wish to transfer the embryos right away, they may be vitrified for later transfer.
Let’s talk numbers: results
You have undoubtedly heard about various results of the success of ICSI and so we decided to request the findings from Texas M&A University. They published the results below in January 2017.2
- About 65% of oocytes collected from immature (small) follicles are expected to mature in the laboratory. If your veterinarian collects the oocyte from the one dominant preovulatory follicle, after administration of an ovulation-inducing hormone, essentially 100% of these oocytes are maturing at the time of collection. Only mature oocytes can be fertilized by ICSI.
- After ICSI of oocytes recovered from small follicles, we anticipate that about 20% of the injected oocytes will develop to blastocysts, if the sperm is from a normally fertile stallion. If the oocyte is from a preovulatory follicle, the chance of the oocyte developing to a blastocyst is about 40%.
- While overall there is an average of one blastocyst produced per aspiration session, individual sessions produce variable results: at the EELab in 2016, 39% of aspiration sessions (oocytes recovered on aspiration from one mare) resulted in NO BLASTOCYST being produced; 24% resulted in one blastocyst being produced, 17% resulted in two blastocysts being produced, and 20% resulted in 3 or more blastocysts being produced.
- The average pregnancy rate per transferred embryo in 2016 was 67%; however, this can very depending upon the conditions at the embryo transfer center to which the embryo is shipped. For example, in 2016, pregnancy rates at centers transferring 8 or more embryos varied from 50% to 87%.
- In 2016, 13% of the pregnancies established with ICSI embryos resulted in pregnancy loss, typically before 30 days.
- The ongoing pregnancy rate (over 90 days) after transfer of an ICSI embryo is expected to be about 60%. That means that after the second pregnancy check there is still a big chance that there is no foal to be born!
In Europe, on the other hand, we are still a little behind on our American colleagues. In a study conducted by Avantea, Utrecht University and James Cook University in March 20193, it is cited that 18 to 36% of the injected oocytes develop into a blastocyst. It should be noted that these low figures often have to do with the quality of the oocytes (which is lower in older mares) and the marginal fertility of certain stallions.
At the end of July 2018, another study was conducted by Avantea and the University of Utrecht looking into the gestational percentage for ICSI transfers.4 They found that the percentage of pregnant mares after 7-10, 23 and 37 days of the transfer was 56% respectively (147 / 261), 49% (129/261) and 48% (124/261). The likelihood of pregnancy tended to be lower for IVP embryos from infertile mares or when embryos were transferred into recipient mares on day 6 after ovulation rather than on day 4 or 5. Finally, the diameter of the embryonic vesicle 7 days post transfer was lower for pregnancies that were lost compared to those that were maintained.
Finally: at the Zangersheide Quality Auction held on 20 and 21 September, no fewer than 17 of the 104 foals were born via Ovum Pick Up-ICSI at Avantea5. They raised an average price of 48,470 euros, compared to the overall average of 22,340 euros. With all this information, you can calculate how interesting ICSI is for you!