Advances in Assisted Reproductive Techniques in the Horse

The uterus is imaged with ultrasound to determine whether it is fully distended with fluid.

Several developments in assisted reproductive techniques in horses have increased the ability of subfertile mares and stallions to produce offspring. These procedures include embryo transfer and embryo cryopreservation, oocyte harvest from live and postmortem mares, and uterine tube hydrotubation.

Embryo Transfer and Cryopreservation

Embryo Transfer

Embryo transfer, which allows the acquisition of embryos from mares that cannot carry their own foals to term, has been available for decades. Donor mares may be athletes or suffer from reproductive pathologies or orthopedic conditions that inhibit them from successfully carrying or delivering a foal.

Unlike in cattle, superovulation in horses is unreliable and the medications necessary to induce multiple ovulations are not available commercially. Therefore, often only 1 or 2 embryos can be recovered from a mare with conventional breeding and embryo harvest.¹

RELATED:

• Welfare Quality and Stress in Working, Breeding Horses
• Factors That Adversely Affect Reproductive Success in Mares

Typically, mares are monitored with palpation and ultrasound per rectum during estrus until a follicle larger than 35 mm and signs of estrus (ie, endometrial edema and cervical relaxation) are present. An ovulation induction agent—most commonly human chorionic gonadotropin or gonadotropin-releasing hormone ana-log (deslorelin)—is administered, and insemination is performed 24 hours later. Ovulation is usually documented 24 hours after insemination. The embryo flush is performed 6.5 to 9 days after the mare has ovulated. Older mares (>14 years) or mares that have been bred with frozen semen usually are flushed on day 8 because embryo development may be slower in these cases.² The recovered embryo is washed several times prior to transcervical transfer into a recipient mare, which has been synchronized to ovulate on the same day, 1 day before, or up to 3 days after the donor mare.³

Practitioners who perform embryo transfer use different anti-biotic and progesterone regimens after the procedure. Palpation and ultrasound per rectum are typically performed 4 to 5 days after the transfer to check for positive signs of pregnancy, and the pregnancy is monitored continuously at the discretion of the practitioner. Although pregnancy rates of 90% per transfer are possible, they are typically closer to 75%.² Success with this procedure depends on several factors, including mare synchronization, availability of recipients, uterine or ovarian pathology, embryo quality, successful breeding management, and embryo flush and transfer techniques.

Cryopreservation

Occasionally, instead of transferring the embryo into a recipient, the embryo is cryopreserved (frozen). This is performed in cases for which no recipient is available or when genetic disease screening, exportation or importation of embryos, or embryo sexing is desired. For the cryopreservation of embryos, 2 procedures exist: slow cooling and vitrification.

Slow cooling, or “conventional freezing,” was the first proce-dure used for this purpose. Glycerol, a cryoprotectant, is added to the embryos, which are usually packaged in ampules or 0.5-mL plastic straws and frozen in a programmable freezer at a very specific rate. Due to the time and expense involved, another method, vitrification, was developed.

Vitrification is an extremely rapid cooling method that changes a liquid into a solid, glass-like state that prevents ice crystal formation.^4,5 A programmable freezer is unnecessary and commercial vitrification kits are available, easing and speeding the procedure. However, there are disadvantages with cryopreserving equine embryos. During vitrification, the embryo is exposed to high concentrations of cryoprotectants, which may be harmful. Damage to the embryo is variable based on the vitrification media used and exposure time. Additionally, pregnancy rates are usually lower with cryopreserved embryos than with embryos transferred fresh or after cooling to 5°C, especially with embryos larger than 300 μm.⁴ Acceptable pregnancy rates of 55% after cryopreservation have been obtained with embryos recovered at day 6 to 6.5 and smaller than 300 μm.^5,6

Unfortunately, performing an embryo flush at day 6 to 6.5 postovulation may not offer a solution. The equine embryo enters the uterus 5.5 to 6 days after ovulation.^2,7 If ovulation is not documented within a couple of hours of occurrence, the embryo flush may not be successful because the embryo may not yet be present in the uterus. Despite some disadvantages, cryopreserving embryos is a viable option when recipient mares are unavailable, transfer early in the following year is desired, genetic testing or embryo sexing is conducted, or domestic or international shipping is desired. Oocyte Harvest

The catheter is removed from the uterus following the procedure and flushed with media to ensure that the embryo is recovered.

In this procedure, the unfertilized egg (oocyte) is harvested, and then fertilization is performed in vitro using a specialized technique. Oocytes are harvested from ovarian follicles in live mares using ultrasound-guided transvaginal oocyte aspiration (TVA) or directly from the ovaries of mares post-mortem. TVA is performed under standing sedation and can be done every 14 days, or when appropriate follicle size and numbers are present for aspiration.² The oocytes are shipped to a laboratory that performs intracytoplasmic sperm injection (ICSI). In vitro fertilization is unpredictable and difficult to repeat in the horse; therefore, ICSI is used for in vitro production of embryos.

Two types of oocytes can be aspirated from either large preovulatory follicles (mature oocytes) after the mare has been given an ovulation-inducing agent or small to medium follicles (immature oocytes). Oocyte recovery rates vary but are generally 75% or greater for dominant stimulated follicles and 50% to 60% for small to medium follicles.^8-10 This difference in recovery rate is due to a tight adherence of the immature oocyte to the cumulus cells within the follicle, which loosens in dominant stimulated follicles as the oocyte prepares to be released at ovulation.

Once oocytes arrive at an ICSI laboratory, they are placed into culture media and incubated until the procedure can be performed. One sperm is injected into a mature oocyte using a micromanipulator, and this oocyte is placed into a culture medium and incubated. If an embryo develops, it can either be transferred into a recipient mare or cryopreserved.

Transvaginal ultrasound-guided follicular aspiration and ICSI allow for the production of embryos from subfertile mares that are unsuccessful embryo donors along with subfertile stallions or those in which semen reserves are low. Disadvantages of TVA and ICSI include the increased costs and potential problems that can occur with a more invasive procedure. The few complications reported from TVA include intra-abdominal hemorrhage, ovarian abscess, rectal tear, peritonitis, and colic.¹¹ The potential complications should be communicated to owners prior to performing the procedure. No effects on fertility have been reported in mares that have undergone repeated TVA procedures.^12,13

Postmortem oocyte harvest also can be performed after an untimely death to preserve the genetics of a mare. Ovaries should be harvested as soon as possible and within 6 to 8 hours after death to ensure the most satisfactory outcome.¹⁴ Removal of ovaries prior to euthanasia with barbiturates is recommended, as the drugs’ effect on oocytes is unknown. Ovaries can be processed immediately and oocytes shipped to an ICSI laboratory, or the ovaries themselves can be shipped to an ICSI laboratory. If ovaries are processed immediately, oocytes can be placed in room temperature media without detrimental effects on maturation or blastocyst rate and shipped overnight for ease of transport.²

If shipping ovaries, there are different protocols depending on transport time. If it is less than 2 hours, ovaries should be transported at 37°C. Ovaries should be transported at around room temperature (15°C-20°C) if transport time will exceed 2 hours.² Maturation, blastocyst, and pregnancy rates from oocytes harvested from ovaries postmortem depend on many factors, including transport time and method, mare age, illness, and duration of illness. To ensure the best results, oocytes should be recovered immediately and shipped; if this is not an option, the ovaries should be shipped immediately to a laboratory to be processed.

Uterine Tube Hydrotubation

During uterine tube hydrotubation, the uterine tube is catheterized and flushed in a retrograde direction via hysteroscopy.¹⁵ This procedure is performed under standing sedation and uses a video endoscope to visualize the endometrium and uterotubal junction. The uterus is insufflated with air or sterile fluid (physiologic saline or lactated Ringer’s solution) to allow visualization of the uterine tube papilla.^15,16 Uterine tube hydrotubation can be performed in either estrus or diestrus, but the degree of cervical relaxation during estrus may make insufflation of the uterus difficult; therefore, it may be best performed during mid-diestrus when the cervix is tightly closed.^15,16 Ten to 20 mL of sterile physiologic saline is injected into each uterine tube via a catheter placed into the papilla.^15,16

In a study by Inoue et al, 26 of 28 subfertile mares became pregnant after undergoing this procedure. Sixty-four percent (9/14) of treated mares became pregnant in the same breeding season when previously they had been bred unsuccessfully.¹⁵ Uterine tube hydrotubation is a viable option for mares in which a cause of infertility has not been identified by routine diagnostic techniques, stallion factors, or chromosomal abnormalities.

Conclusion

Each of these techniques has allowed for the production of foals from subfertile mares and stallions that were not able to produce offspring with conventional breeding. Assisted reproductive techniques will continue to be used and improved in equine clinical practice as demand dictates their need in the equine community.

Born in Rio de Janeiro, Brazil, Dr. Walbornn obtained her DVM from the University of Florida College of Veterinary Medicine in 2014, then completed an internship at the Equine Medical Center of Ocala in Florida and a residency in equine reproduction at Texas A&M University. She joined Rood & Riddle Equine Hospital in Wellington, Florida, in 2017.

References:

• McCue PM, LeBlanc MM, Squires EL. eFSH in clinical equine practice. Theriogenology. 2007;68(3):429-433. doi: 10.1016/j.theriogenology.2007.04.027.
• Hinrichs K. Assisted reproduction techniques in the horse. Reprod Fertil Dev. 2012;25(1):80-93. doi: 10.1071/RD12263.
• Squires EL, McCue PM, Vanderwall D. The current status of equine embryo transfer. Theriogenology. 1999;51(1):91-104.
• Squires EL, McCue PM. Cryopreservation of equine embryos. J Equine Vet Sci. 2016;41:7-12. doi: 10.1016/j.jevs.2016.03.009.
• Stout TA. Cryopreservation of equine embryos: current state-of-the-art. Reprod Domest Anim. 2012;47(suppl 3):84-89. doi: 10.1111/j.1439-0531.2012.02030.x.
• Czlonkowska M, Boyle MS, Allen WR. Deep freezing of horse embryos. J Reprod Fertil. 1985;75:485-490.
• Freeman DA, Weber JA, Geary RT, Woods GL. Time of embryo transport through the mare oviduct. Theriogenology. 1991;36:823-830.
• Carnevale EM, Coutinho da Silva MA, Panzani D, Stokes JE, Squires EL. Factors affecting the success of oocyte transfer in a clinical program for subfertile mares. Theriogenology. 2005;64(3):519-527. doi: 10.1016/j.theriogenology.2005.05.008.
• Colleoni S, Barbacini S, Necchi D, Duchi R, Lazzari G, Galli C. Application of ovum pick-up, intracytoplasmic sperm injection and embryo culture in equine practice. Proc Am Assoc Equine Prac. 2007;554-559.
• Jacobson CC, Choi YH, Hayden SS, Hinrichs K. Recovery of mare oocytes on a fixed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection. Theriogenology. 2010;73(8):1116-1126. doi: 10.1016/j.theriogenology.2010.01.013.
• Carnevale EM. Clinical considerations regarding assisted reproductive procedures in horses. J Equine Vet Sci. 2008;28(11):686-690. doi: 10.1016/j.jevs.2008.10.005.
• Mari G, Barbara M, Eleonora I, Stefano B. Fertility in the mare after repeated transvaginal ultrasound-guided aspirations. Anim Reprod Sci. 2005;88(3-4):299-308. doi: 10.1016/j.anireprosci.2005.01.002.
• Vanderwall DK, Hyde KJ, Woods GL. Effect of repeated transvaginal ultrasound-guided follicle aspiration on fertility in mares. JAVMA. 2006;228(2):248-250. doi: 10.2460/javma.228.2.248.
• Ribeiro BI, Love LB, Choi YH, Hinrichs K. Transport of equine ovaries for assisted reproduction. Anim Reprod Sci. 2008;108(1-2):171-179. doi: 10.1016/j.anireprosci.2007.08.001.
• Inoue Y, Sekiguchi M. Clinical application of hysteroscopic hydrotubation for unexplained infertility in the mare [published online November 7, 2017]. Equine Vet J. doi: 10.1111/evj.12781.
• Schnobrich M. How to use endoscopy (hysteroscopy and laparoscopy) to manage the sub-fertile mare. Proc Am Assoc Equine Prac. 2016;169-174.