Current Equine Embryo Transfer Techniques

Introduction

Embryo transfer is the most widely used assisted reproductive technique for the mare. The applications of embryo transfer include: 1) obtaining foals from performance mares, 2) obtaining multiple foals from individual mares each year, 3) obtaining foals from two-year-old mares, 4) obtaining foals from reproductively unsound mares, 5) obtaining foals from mares with non-reproductive health problems, and 6) use as a research tool [1,2]. Although embryo transfer was initially proposed as a promising method for obtaining foals from aged, subfertile mares, experiments utilizing oocyte transfer [3] and embryo transfer [4] have documented that many oocytes/embryos produced by aged, subfertile mares are inherently defective and have low survival rates after transfer to recipient mares; therefore, aged, subfertile mares are not optimal candidates for embryo transfer.

The first successful equine embryo transfer was reported in 1972 [5]; however, it was not until the early 1980's that embryo transfer became an accepted clinical procedure in the equine breeding industry. At that time, widespread utilization of embryo transfer was limited by the need to maintain recipient mares at the site of embryo collection, or to ship donor mares to a centralized embryo transfer facility. In the late 1980's, a technique for cooling equine embryos was identified [6], which led to the development of a practical method of short-term (24 hr) storage and transportation of equine embryos. That breakthrough allowed embryos to be collected in the "field" and then shipped to a centralized facility for transfer to suitable recipient mares. The ability to transport cooled embryos provided veterinarians with the opportunity to offer embryo transfer service without the onerous task of maintaining recipient mares, and eliminated the need to ship donor mares to a centralized facility. This article will review current equine embryo transfer techniques.

Mare Management

Donor Mare

If indicated, a complete breeding soundness examination of the donor mare should be performed to assess her suitability for use in an embryo transfer program. If abnormalities are identified that warrant treatment (e.g., bacterial endometritis), appropriate therapy should be completed before embryo transfer procedures are performed. Breeding management of the donor involves teasing to monitor reproductive behavior, and the use of transrectal palpation and ultrasonography to monitor ovarian follicular activity during the estrous cycle. When in heat, the donor is examined daily to monitor follicular growth, which allows optimal timing of insemination with fresh, cooled or frozen semen [7]. Ovulation is routinely "timed" using human chorionic gonadotropin (hCG; 5 IU/kg IV or IM) or the gonadotropin releasing hormone (GnRH) agonist deslorelin acetate (Ovuplant®; 2.2 mg pellet SQ). The day ovulation is detected is designated as Day 0. Currently, there is no practical and efficacious method of superovulating mares, which severely limits the efficiency of embryo transfer [8].

Recipient Mares

Proper selection and management of recipient mares may be the most important factor affecting the success of an equine embryo transfer program. Recipient mares should have normal estrous cycles, and must be free of uterine and/or ovarian abnormalities. The optimum age of recipient mares is 3 to 10 years. Synchronizing estrus between donor and recipient mares can be accomplished with routine protocols using prostaglandin F2 alpha (PGF2α) alone or in combination with exogenous progesterone [9]. When in heat, recipients are examined daily with transrectal palpation and ultrasonography to monitor follicular growth and detect ovulation. The "window" of synchrony between ovulation in recipient and donor mares is +1 to -3 days (i.e., the recipient mare can ovulate one day before to three days after the donor mare) [1]. In an effort to eliminate the need for synchronizing recipient and donor mares, progestin-treated, ovariectomized mares have been used as embryo recipients [10,13], however, the success with their use has been variable, and has not been widely adopted.

Embryo Recovery

Equine embryos are selectively transported through the oviduct into the uterus on Day 5-1/2 to 6 post-ovulation [14], at which time they are at the compact morula (Fig. 1) to early blastocyst (Fig. 2) stage of development. After entering the uterine lumen, the size of the embryo increases dramatically (Table 1) as it develops into an expanded blastocyst (Fig. 3). Although embryos can be recovered on Days 6 to 9 (Table 2), the optimal time of embryo collection is Day 7 or 8. The primary indication for recovering embryos on Day 6 is for freezing embryos [1]. Embryos are not routinely collected on Day 9, because their transfer success rate is generally lower than Day 7 or 8 embryos [1].

Figure 1. Equine compact morula. The embryo consists of a compacted mass of blastomeres, with a prominent zona pellucida. The outline of individual blastomeres can be seen around the periphery of the embryo (arrow). Embryo size approximately 220 μm (bar = 100 μm).

Figure 2. Equine early blastocyst. Formation of the fluid-filled blastocoele cavity has begun, and the zona pellucida is becoming thinner. Embryo size approximately 290 μm (bar = 100 μm).

Figure 3. Equine expanded blastocyst. The blastocoele cavity is fully formed, and the inner cell mass, the future embryo-proper/fetus, can be differentiated from the outer trophoblast layer (future placenta). The zona pellucida has been replaced by the thin capsule. Embryo size approximately 560 μm (bar = 100 μm).

Embryo collection is performed using transcervical uterine lavage (Fig. 4). After placing the mare in stocks, the perineal area is cleaned with a mild detergent, rinsed thoroughly with clean water, and dried. The operator then places a sterile plastic sleeve over his or her arm, applies sterile lubricant, and introduces a sterile balloon-tipped catheter into the vagina. This author uses an 80 cm silicone catheter that has an inside diameter of 8.0 mm (French size 33; Fig. 5); other styles of flushing catheters are available. After entering the vagina, the catheter is passed through the cervix into the uterine body, and the balloon-cuff is inflated with approximately 80 cc of air or sterile saline and is pulled back against the internal cervical os to prevent loss of fluid. Once the catheter is seated appropriately, the uterus is flushed three to four times with warm (30 to 35ºC) plain or modified Dulbecco's phosphate buffered saline (DPBS) containing 1% (v/v) fetal or newborn calf serum, penicillin (100 units/ml), and streptomycin (100 ug/ml; Fig. 6a). The uterus is filled with one to two liters of DPBS during each flush (4 to 8 l used during entire procedure). After filling the uterus, the fluid is allowed to flow back out through the catheter and is passed through a 0.75 u embryo filter (Fig. 7). It is important that the embryo filter not overflow or run dry; filters are available that are designed to prevent both from occurring. The fluid passing through the filter is collected to monitor its recovery. After the first flush, the uterus is massaged per rectum during subsequent flushes, which may aid suspension of the embryo(s) in the medium and enhance fluid recovery. The majority (>90%) of fluid infused into the uterus should be recovered, and it should be free of cellular debris or blood. Recovery of "cloudy" fluid indicates the mare had an active endometritis at the time of the embryo recovery, and warrants further diagnostic evaluation. When present, blood contamination is often associated with vigorous massage of the uterus and/or manipulation of the catheter.

Figure 4. Schematic diagram of the embryo recovery procedure. From Aguilar and Woods [2] with permission.

Figure 5. Balloon-tipped embryo collection catheter.

Figure 6a. Dulbecco's phosphate buffered saline flush medium. Flush medium can be obtained commercially in prepared liquid form (a), or as a "kit" containing powdered ingredients that require reconstitution prior to use (b).

Figure 6b. Embryo flush medium in a water bath immediately prior to use.

Figure 7. Embryo collection filter. Other filter styles are also available.

At the completion of the flush, the filter cup is emptied into a sterile search dish with grid (Fig. 8), and is rinsed with DPBS (Fig. 9). The fluid is then examined for the embryo(s) using a stereo-microscope at approximately 15x magnification (Fig. 10). Large embryos (~ Day 8) are often visible with the naked eye. When an embryo(s) is identified, it is "washed" by transferring it sequentially through at least three 1 ml drops of DPBS with 10% (v/v) serum that has been sterilized using a 0.22 μ syringe-filter; after "washing" the embryo is placed into a small petri dish (35 x 10 mm) containing the same medium. The embryo is then examined at high magnification (40 to 80x) and graded on a scale of 1 (excellent) to 4 (poor) [15]. Embryos can be handled using a 0.25 or 0.5 cc semen-freezing straw (Fig. 11), 25 μl glass capillary pipette, or other suitable instrument attached to an appropriate syringe. Anytime an embryo is drawn into a handling instrument, the medium containing the embryo should be surrounded on each side by an air bubble and blank medium (Fig. 12). This prevents the embryo from accidentally being pulled out of the instrument should the tip touch something absorbent. The process of picking up and depositing an embryo should be observed under the microscope.

Figure 8. Pouring the recovered flush medium from the embryo filter into a search dish.

Figure 9. Rinsing the embryo filter with flush medium.

Figure 10. Searching for an embryo(s) using a stereo-microscope at low magnification (10 to 15x).

Figure 11. Embryo handling equipment consisting of a 0.25 or 0.5 cc semen freezing straw, adapter, and tuberculin syringe. The adapter is a urethral catheter connector®.

Figure 12. Schematic diagram showing the embryo positioned in a straw between air and fluid columns.

Once embryos are placed into the holding medium, they should either be expeditiously processed and packaged for transport or transferred into an appropriate recipient mare, since embryo viability decreases after storage in DPBS for more than 3 hours [16]. While awaiting packaging for transport or immediate transfer to a recipient, equine embryos appear to be quite tolerant of temperatures between room temperature (25°C) and body temperature (37°C). However, efforts should be made to prevent rapid or extreme changes in temperature.

Packaging Embryos for Transport

Equine embryos are cooled and transported using methods developed by Carnevale et al., [6], which utilize Ham's F-10 nutrient mixture as the holding/cooling medium. Prior to use, Ham's F-10 medium must be buffered by diffusing a mixture of 90% N2, 5% O2, and 5% CO2 gas through the medium for three to five minutes (Fig. 13), after which it is supplemented with 10% (v/v) fetal or newborn calf serum, penicillin (100 units/ml), and streptomycin (100 ug/ml; Fig. 14). Because Ham's F-10 medium must be "gassed" prior to use, it requires an appropriate compressed gas cylinder and regulator; therefore, many practitioners choose to have the embryo transfer facility that will receive the embryo provide Ham's F-10 as part of an embryo shipping "kit".

Figure 13. Diffusing a mixture of 90% nitrogen, 5% oxygen and 5% carbon dioxide through a 100-ml bottle of Ham's F-10 medium for 3 to 5 minutes.

Figure 14. Components for final preparation of Ham's F-10 medium: A) fetal or newborn calf serum, B) Ham's F-10 medium, and C) Penicillin-streptomycin solution.

To package an embryo, Ham's F-10 medium is filter-sterilized into a 5 ml "snap-cap" tube, leaving a small air gap at the top of the tube. The embryo is then carefully transferred into the medium (Fig. 15), the cap is securely snapped onto the tube, and the tube is wrapped with parafilm® (Fig. 16). A 50-ml centrifuge tube is then filled with F-10 medium (un-filtered), and the 5-ml tube containing the embryo is placed into the 50-ml centrifuge tube (Fig. 17). The cap of the 50-ml centrifuge tube is closed eliminating as much air as possible, and it is wrapped with parafilm®. The packaged embryo is then placed into an Equitainer® (Fig. 18), which passively cools the embryo to 5°C. Under those conditions, embryos can remain viable for at least 24 hours, during which time they can be transported via commercial airline or priority overnight delivery to the embryo transfer facility.

Figure 15. Placing an embryo into a 5-ml snap-cap tube containing Ham's F-10 medium.

Figure 16. The snap-cap tube containing the embryo has been capped and sealed with parafilm® (arrow).

Figure 17. The snap-cap tube containing the embryo (arrow) has been placed inside a 50-ml centrifuge tube containing Ham's F-10 medium.

Figure 18. Equitainer®. The packaged embryo is placed in an Equitainer®, for transportation to an embryo transfer facility.

Embryo Transfer

Regardless of whether embryos are transferred immediately after recovery, or cooled and transported prior to transfer, the transfer procedure can be performed surgically or non-surgically. Historically, surgical transfer has provided the highest pregnancy rates and most consistent results, generally resulting in pregnancy rates of approximately 70 to 75% one week after transfer [17]. Recent reports of non-surgical transfer have demonstrated that success rates can be equal to, or greater than, those obtained with surgical transfer [18,19].

Surgical embryo transfer is performed as a standing flank laparotomy using appropriate sedation/tranquilization in conjunction with local anesthesia. Using standard surgical techniques, the uterine horn is exteriorized through a flank incision (Fig. 19) and is punctured using a cutting-edge suture needle. The puncture wound is then enlarged by placing iris forceps through the incision into the uterine lumen. The embryo, contained within a small amount of medium (< 250 μl) in a sterile embryo handling instrument, is then deposited into the uterine lumen (Fig. 20). The puncture wound in the uterine horn is not sutured, the uterine horn is replaced into the abdomen, and the surgical incision is closed using standard technique. Because of the intra-uterine mobility of the equine embryo, it can be transferred into the uterine horn ipsilateral or contralateral to the side of ovulation.

Figure 19. Surgical embryo transfer. The uterine horn has been exteriorized through a flank incision.

Figure 20. Surgical embryo transfer. A sterile straw containing the embryo has been introduced into an incision in the uterine horn and will be advanced into the uterine lumen where the embryo is deposited.

Non-surgical embryo transfer has generally been performed using: 1) a standard artificial insemination pipette, 2) a disposable plastic "insemination gun", or 3) a reusable stainless steel "insemination gun" (Fig. 21). Regardless of which instrument is used, an outer guard is generally placed over the transfer instrument (Fig. 22). When performing a non-surgical transfer, the recipient mare is placed in stocks, sedated and the perineal area is prepared as described for the embryo recovery procedure. The operator places a sterile plastic sleeve over his or her arm, and a sterile surgeon's glove is placed over the plastic sleeve. A small amount of sterile lubricant is placed on the back of the operator's hand and applied to the vulva. The tip of the transfer instrument (covered by the outer guard) is placed in the palm of the hand and protected by placing the operator's thumb over the tip. The instrument is introduced into the vagina, and the tip of the outer guard is introduced into the external cervical os approximately 0.5 cm, at which point the instrument is advanced through the outer guard and passed through the cervix into the uterine body. The embryo can be deposited in the uterine body or in one of the uterine horns. To deposit the embryo in the uterine horn, the tip of the instrument is guided into the horn using transrectal manipulation. When the transfer instrument is positioned appropriately, the embryo is deposited as the transfer instrument is withdrawn slightly, so the tip is not pushed up against the endometrium as the embryo is expelled.

Figure 21. Non-surgical embryo transfer equipment: A) stainless steel re-useable "insemination gun", B) plastic disposable "insemination gun", C) standard insemination pipette, and D) outer protective guard.

Figure 22. Non-surgical embryo transfer equipment: A) stainless steel re-useable "insemination gun", B) plastic disposable "insemination gun", C) standard insemination pipette, and D) outer protective guard.

Summary

Embryo transfer is a valuable technique of assisted reproduction in the mare. Although the use of embryo transfer in commercial breeding programs was initially hampered by the need to provide suitable recipient mares at the site of embryo collection, or transport donor mares to a centralized embryo transfer facility, the development of methods for successfully transporting cooled embryos eliminates the need of maintaining recipients on site. Transport of embryos, coupled with the fact that the materials necessary for embryo collection and transport are well suited to a practice setting, allows more practitioners to provide embryo transfer service to their clientele who wish to utilize this technology. Although embryo transfer service is accessible to more horse owners and breeders, the efficiency of embryo transfer remains limited by the inability to reliably superovulate mares.