Embryo Transfer in Horses
Embryo transfer in the equine industry has been primarily used to obtain offspring from mares with restricted reproductive potential (mares with undiagnosed subfertility, uterine pathologies, or simply older mares) or from performance mares that must remain nonpregnant to continue to compete. Most breed associations allow the registration of foals born by embryo transfer, and an increasing number now allow the registration of multiple foals born in the same year. Because of this recent increased acceptance by breed registries, many horse breeders have obtained several foals in one breeding season from a single donor mare.
Superovulating mares with a purified form of equine follicle-stimulating hormone extract preparation (eFSH) to increase ovulation and embryo recovery rates is no longer commonly done commercially. Difficulties with manufacturing suitable gonadotropins and the variable response of mares to FSH stimulation have precluded widespread commercial use of this approach to produce equine embryos. It was thought that the low embryo recovery rate in mares ovulating >4 follicles/ovary could be attributed to an inability of the ovarian fossa to accommodate multiple ovulations in that area, or alternatively, excessive response to the FSH stimulation could result in dysmatured oocytes.
A recent advance in embryo transfer technologies in commercial settings has been the possibility of producing in vitro equine embryos using intracytoplasmic sperm injection (ICSI). Frozen semen from deceased stallions can be efficiently used to produce in vitro embryos by using only a portion of a frozen semen straw to provide sperm for the ICSI procedure. The number of ICSI-derived equine embryos is expected to continue to increase sharply during the next few years.
The horse embryo is notoriously challenging to cryopreserve, probably owing to its relatively large diameter and the presence of an embryonic capsule that limits interaction between cryoprotectant agents and the embryo. Embryos of morula or early blastocyst stage (<6.5 days after ovulation) are preferable for cryopreserving, because after thawing, older embryos have invariably low morphologic quality that results in poor pregnancy rates. A vitrification media for horse embryos is commercially available, and acceptable pregnancy rates varying from 40%–60% have been reported. Successful cryopreservation of expanded equine blastocysts also has been reported for embryos that had their blastoceles collapsed after embryo biopsy procedures.
Because one of two embryos are typically recovered from the donor mare, it is important that breeding soundness evaluations are done for both donor and recipients. Daily ultrasonography of the uterus and ovaries of donor and potential recipient mares during estrus provide critical information about the time of ovulation, which is essential for determining the day of ovulation and day of embryo collection and to assist in the selection of qualified recipient candidates for embryo transfer.
Nonsurgical techniques are currently used to collect and transfer horse embryos. Embryo collection is usually performed on day 7 or 8 (day 0 = ovulation). The use of good standard techniques generally yields a recovery rate of ~75%; this rate can be as high as 90% for young maiden or fertile mares or as low as 10%–20% in subfertile mares. Before embryo collection, palpation per rectum and ultrasonography are performed mainly to document the presence of the corpus luteum, the degree of cervical tone, and absence of any fluid in the uterus. Mild soap or povidone-iodine scrub can be used to wash the perineal region, followed by a thorough rinsing with clean water.
Sedation is not used in most cases, but it may be needed when working with uncooperative mares. Acepromazine should be avoided, because it may cause relaxation of the uterus and potentially impair fluid recovery or manipulation of the reproductive tract during flushing of the uterus; sedation with an α2-agonist (eg, xylazine, detomidine) is preferred. Embryo collection is performed using a routine transcervical uterine lavage procedure, using a silicone catheter with an inflatable cuff. Using sterile technique, the silicone catheter (28–34 in. long, French size 28–37, depending on the size of the reproductive tract of the mare) is introduced through the cervix into the uterus, and then the balloon is inflated (typically 60–80 mL) with air or collection medium; the catheter is then gently pulled back so the cuff seals the internal cervical os. Complete flush media with antibiotics, surfactant, and bovine serum albumin is available commercially and ready to use. Alternatively, as with cattle, flush media can be prepared with Dulbecco’s phosphate-buffered saline and 1%–5% fetal bovine serum; 1–2 L are infused into the uterus and then allowed to flow back into a graduated cylinder or plastic bucket to measure the recovered volume. Once the uterus is filled, it can be gently manipulated per rectum to aid the recovery of fluid. An embryo filter equipped with a 75-micron mesh is usually connected to the end of the outflow tubing to yield ~40–60 mL of fluid. In some cases, an injection of oxytocin (10 IU) may be given to minimize the retention of a large amount of flush media in the uterus. The recovered fluid should be clear and free of significant amount of debris and blood. Nearly 100% of the fluid used to lavage the uterus should be recovered. A total of 4–8 L of flush media is used to complete the embryo collection procedure (flush). If retention of fluid is suspected or if the outflow of media is interrupted, transrectal ultrasonography may be used to assess the presence of intraluminal fluid. A common cause of interrupted outflow drainage of flush media results from placing the tip of the catheter intracervically and not in the body of the uterus: the media easily flow into the uterus but not outward.
Once the flush is completed, the remaining fluid in the filter is searched in a sterile dish with grid under a magnification of 15× using a stereomicroscope. Once the embryo is found, it is transferred to a well in a sterile tissue culture dish containing holding media (commercial, ready-to-use with 0.4% albumin or made with Dulbecco’s phosphate-buffered saline plus 10%–20% fetal bovine serum). The embryo should be washed at least 3 times by transferring into two other wells previously filled with holding media. Embryo manipulation is done with a 0.25-mL straw connected to a tuberculin syringe with a 16-gauge needle, a 0.5-mL straw directly connected to a tuberculin syringe, or a capillary pipette (20 μL) attached to a tuberculin syringe. After being washed, the embryo should be transferred to a recipient mare (within 1 hr) or prepared for short storage and transportation. The stage of the embryo (morula, early, or expanded blastocyst) and quality (1, best; 5, dead) should be recorded.
Although surgical transfer of equine embryos was once thought to yield higher pregnancy rates than nonsurgical transfer techniques, the latter are now the preferred method to transfer horse embryos. Recipient mares should be reproductively sound and in good body condition and health. Synchronization of ovulation should be maximized by hormonal manipulation of the estrous cycle and daily transrectal ultrasonography. Good pregnancy rates result from using recipients that have ovulated from 1 day before the donor to 3 days after the donor. In addition, progestagens such as altrenogest may be used until the day of pregnancy examination 4–5 days after the transfer (~12–13 days of gestation).
The nonsurgical embryo transfer is performed using transcervical catheterization. Plastic straws, 0.25 or 0.5 mL, are used to load the embryo; the column of medium containing the embryo should be surrounded by two small columns of air, which are in turn surrounded by two columns of medium. The straw loaded with the embryo is fitted into an embryo transfer “gun.” Embryo transfer sheaths with side delivery can minimize embryo damage during the transfer procedure.
Horse breeders without an adequate number of recipients and veterinarians with modest investment in equipment can collect embryos and prepare them for short storage and transport to a centralized embryo transfer facility. Pregnancy rates do not appear to differ from those obtained with fresh embryos, especially if embryos are shipped counter-to-counter on the same day they are collected. Embryos are routinely shipped in commercially available holding media that are ready-to-use preparations containing 0.4% bovine serum albumin and antibiotics. The embryo is placed into a 2-mL polypropylene cryotube vial or a 5-mL snap-cap tube that contains the holding media. The tube with the embryo is then placed into a semen shipping container designed to cool and transport horse semen. The container is preferably shipped to an embryo transfer facility on the same day the collection is performed using an airline carrier (counter-to-counter) or processed for overnight delivery using a commercial carrier.