Assisted Reproductive Techniques

Take Home Message: Assisted reproductive technologies (ART) are used in clinical practice for 2 main reasons—to increase the number of desired offspring from breeding stock beyond that which is physiologically feasible, or due to compromised fertility of the mare or stallion. Throughout the next decade, the client demand for many ART services will likely increase so that desired offspring are created. This will likely include a broader demand by breeders for preimplantation genetic diagnosis (PGD), which identifies not only gender of an embryo, but the carrier state of many heritable equine diseases, as well as preservation of valued genetic lines.

I. EMBRYO TRANSFER (ET)

Equine embryo transfer has grown to become a common procedure that is offered in many veterinary practices. The procedure consists of flushing a donor mare’s uterus with a buffered solution, 6-10 days following insemination and ovulation, in an attempt to recover at least one embryo, which is then transferred transcervically into a surrogate mare. Success rates of the procedure depend on a number of factors that include the age and endometrial health of the donor (and recipient), fertility of the semen, embryo quality, degree of synchrony between the donor and recipient mares, the day of embryo recovery as well as technical expertise, to name a few.

Hundreds of equine embryos are shipped yearly via airlines and courier companies to veterinarians that manage large recipient herds. Intensive management of the shipment and media are crucial for success of the embryo transfer. The number of engaged people required to coordinate the time-sensitive events of embryo production and transfer can be surprising, and a single mishap can lead to failure. Coordination of events and between all involved parties is critical to the likelihood of success.

II. EMBRYO VITRIFICATION

Until recently, only morula and early blastocyst-stage embryos less than 300 microns in diameter were capable of surviving cryopreservation. It was presumed that the blastocoel fluid of later-stage, more developed and expanded blastocyts, disrupted the blastocoel fluid concentration of the cryopreservative agents used in freezing, or that the capsule unique to the equine embryo impeded their penet ration. The precise timing of embryo recovery by flushing a donor mare 6.5 days after ovulation represents a time frame that is not always optimal for completion of transit time through the oviduct and into the uterus. Hence, embryo yield can be low. Moreover, recovered embryos do not always reflect the time frame since ovulation; embryos that should have been 7-8 day expanded blastocysts and were found to be at an earlier blastocyst or morula stage were vitrified in early studies as well as early embryos of an appropriate age.

An early study reported 16-day pregnancy rates of 67% after day 6 embryos were vitrified in 0.25 ml straws, warmed and then transferred into the uterus of recipient mares^1,2 The initial results were set to revolutionize the efficient cryopreservation of equine genetic lines, for vitrification provides an attractive option for many in the breeding industry under the following circumstances:

1. A synchronized recipient mare is unavailable.
2. To hold an embryo while awaiting PGD results.
3. Allows multiple embryos to be collected (via embryo flush or oocyte collection) without the expense of a synchronized recipient mare.
4. Sale of the vitrified embryo.
5. International distribution of genetics.
6. Enhance genetic variability and hybrid vigor.
7. Assess offspring phenotype—“temporary genetic banking”
8. Late or offseason cycling mares can produce embryos for a more desirable transfer date.
9. Unexpected double or triple ovulation.
10. Preserve genetics (in euthanized mares).
11. Production of embryonic stem cells.

Commercial vitrification kits and a user-friendly protocol were developed. However, several follow-up studies found that pregnancy rates of these warmed early embryos were highly variable,^3,4 and foaling rates at times disappointing, causing researchers to explore different protocols and media including those used successfully in other species for vitrification of embryos. But it was the trophectoderm biopsy of equine expanded b lastocysts that ultimately in spired the successful vitrification and thawing of embryos greater than 300 microns in diameter, due to the concommitant collapse of the blastocoel that occurred with capsular puncture.

Choi et al⁴ reported an 86% pregnancy rate (6/7) from expanded blastocysts 407 t o 565 um in diameter that were collapsed, vitrified and warmed before transfer. This group of researchers prepared the collapsed embryo using a method described by Campos-Chillon⁵ and loaded it into a fine-diameter microloader tip^a as described by Sun et al⁶ before plunging the pipette into liquid nitrogen. When warming the embryo, the tip of the microloader was immersed in a warmed drop of thawing media, following the protocol described by Sun et al,⁶ thereby releasing the embryo.

This author has found similar pregnancy rates using the protocol described by Choi et al for the vitrification of collapsed blastocysts as well as earlier stage embryos. However, instead of utilizing a microloader tip , the embryos are loaded onto a beveled trough of a device called the Cryolock®.^b The warming process is similar to that described above.

III. PGD/ BLASTOMERE/TROPHOECTODERM BIOPSY

One of the most exciting developments over the past few years lies in the successful biopsy of the equine embryo. Presently, the Veterinary Genetic Laboratory (VGL) at the University of California-Davis (https://www.vgl.ucdavis.edu/services/horse.php) provides a service to detect not only gender, but also several lethal or crippling inherited diseases from biopsied TE cells with >97% accuracy. Through a process of whole gene amplification, specific gene loci for HERDA, HYPP, lethal white, cerebellar abiotrophy, etc., are reliably identified. Furthermore, prediction of coat color of the resultant offspring is also possible.

Choi et al⁷ reported normal pregnancy rates following biopsy and transfer using a piezo drill and aspiration needle biopsy technique in embryos that ranged in size from the morula stage to the expanded blastocyst stage (up to 1 300um in diameter). Herrera et al⁸ recently reported similar results; this group found no significant difference in 25 day pregnancy rates between transferred biopsied embryos versus nonbiopsied embryos (59% versus 62% respectively), nor any difference based on embryo size in either group. Furthermore, in this latter study, the capsule was breached without the use of a piezo drill. From a commercial viewpoint, it is important to note that embryos can be held overnight in warmed holding media prior to biopsy⁷ as well as held for as much as 7-10 hours after biopsy without affecting viability⁸. Therefore, embryos can be recovered at one farm, shipped overnight for biopsy at a laboratory, and then returned or shipped elsewhere for transfer into a surrogate mare.

IV. OOCYTE RETRIEVAL

Transvaginal oocyte aspiration from donor mares represents another means of increasing the number of offspring from valued mare lines, and often from mares that cannot support embryo development in utero. Oocytes may be aspirated via ultrasound-guided needle puncture through either the flank or the vagina. Although it is easier to recover mature oocytes from large preovulatory follicles, the recovery of multiple immature oocytes is also possible, especial using a curvileanear probe and transvaginal aspiration. It is also possible to recover immature oocytes from ovariectomized mares, and even postmortem mares. Oocytes recovered from a dominant follicle should be maintained at 38°C for the best chance of survivability; however immature oocytes can be maintained at 20°C en route to the laboratory, and even over a 48-hour period⁹. Thus, postmortem ovarian dissection, using a bone curette to scrape small follicles for adhered oocytes, can occur prior to shipment to an IVF laboratory.

V. OOCYTE TRANFER (OT)/GAMETE INTRAFALLOPIAN TRANSFER (GIFT)

These terms are often used interchangeably but the techniques are distinguished from each other depending on the location of sperm de position. In both procedures, oocytes are further matured in the laboratory in specific media under tightly controlled atmospheric conditions. The mature oocyte may be then transferred via flank laparotomy to the oviduct of a recipient mare. For OT, the recipient mare is usually inseminated both pre- and postoviductal transfer, depositing the spermatozoa into the recipient mare’s uterus. With the GIFT procedure, both the spermatozoa and oocyte are transferred into the oviduct of the recipient mare. High pregnancy rates can be achieved in this manner, especially when the oocytes arise from younger donor mares.

The transoviductal transfer of early embryos (2-3 day) has also been successful in the mare, even at the 4-cell stage just following cleavage. In fact, in some cases, aspirated immature oocytes that have undergone in vitro maturation an d fertilization are believed to result in higher pregnancy rates than those permitted to mature in vitro to the blastocyst stage before transfer.

VI. IN VITRO MATURATION (IVM) OF OOCYTES

As stated above, immature oocytes can be recovered via transvaginal ultrasound-guided follicular aspiration, even during winter anestrus, and matured in vitro. It is estimated that this technique may yield up to 50% recovery rates. Many donor mares may undergo repeated follicular aspiration every 2 weeks without detrimental effect during a single breeding season. However, when multiple immature follicles have been aspirated on repeated sessions, thickening of the serosa surrounding the ovary has been noted. [...]