Luis Losinno
Embryo Transfer
Embryo transfer (ET) involves scheduled artificial insemination (AI) of a donor mare, recovery of embryos by transvaginal uterine lavage 7 to 9 days after ovulation, and nonsurgical embryo transfer to a synchronized recipient mare. Embryos produced in vitro by intracytoplasmic sperm injection (ICSI) or somatic cell nuclear transfer (SCNT) also can be transferred. In both cases, embryos can be cryopreserved (chilled, frozen, or vitrified) before transfer. Some commercial programs have transfer efficiency higher than 65%, but a 50% rate is considered acceptable, as this value is based on a 70% per-cycle recovery rate (RR) and a 70% pregnancy rate (PR) after transfer (RR × PR = 0.7 × 0.7 = 0.49). In practical terms, this means that two flushings (cycles) are needed to achieve one pregnancy. Initial resistance to ET by some breed organizations has largely been resolved (with the exception of Thoroughbreds), and now a significant percentage of embryos in the Americas are transferred (Brazil, 43%; Argentina, 29%; and the United States, 18%).
Indications, Applications, and Limitations
Embryo transfer can increase reproductive efficiency, but it also has limitations that can lead to unfounded expectations. The main indications for ET include obtaining multiple foals per year from selected mares; production of foals from mares in competition and those incapable of maintaining a pregnancy; shortening of the generation interval; elimination of the risks of pregnancy and parturition for valuable mares; and production of numerous related offspring for genetic investigations.
On average, broodmares produce 5 to10 foals during their reproductive life. In continuous programs, ET can increase production as much as 10-fold. Some mares have produced more than 80 offspring by ET, but sport horse donor mares, particularly polo and jumping horses on intensive training programs, produce fewer embryos per season because of competitions and travel. Controlled experiments suggest that ET efficiency rates are decreased in sport horses because of the stress of exercise, but this is unsupported by results achieved in commercial ET programs in competition polo and jumping mares. Embryo transfer is ideally suited for mares that cannot maintain a pregnancy because of either reproductive (embryonic death, recurrent abortions, or cervical incompetence) or nonreproductive (e.g., musculoskeletal injuries or abdominal hernias) problems. Use of ET can extend the reproductive life of a mare by enabling the harvesting of embryos from 1- or 2-year-old fillies and from aged mares. In many breeds, this technique allows “rescue” of mares older than 20 years with high breeding value. Although the expected efficiency of aged mares is lower than average, it is still commercially viable, especially when follicle stimulation protocols are used. Embryo transfer is an exceptional tool for genetic programs evaluating certain crosses. For example, 5 to 10 pregnancies can be obtained from the same donor to the same or all different stallions in the same season. Although controlled genetic programs in horses are uncommon, this biotechnology multiplier tool can have an enormous positive impact on genetic evaluation.
Embryo Transfer Programs
Embryo transfer programs have evolved in different countries, driven by breed society needs and geographic, economic, and marketing factors. Programs can be closed (restricted to one farm), open (providing commercial embryos), or both and can vary greatly in size. The scale determines the degree of logistic complexity. Some commercial programs produce more than 1500 pregnancies per season. Embryo transfer centers have consistent efficiency because they control many variables, such as stallions, donors, recipients, facilities, feed, health, and personnel, but the fixed costs are high. Ambulatory programs have lower operating costs but less control over the variables that determine success.
Embryo Transfer Procedures
The technique is simple and easy to learn but involves a complexity of multiple variables acting simultaneously. To maximize success, ET programs should use fertile mares and stallions, but that usually is not the veterinarian’s decision. However, the author conducts an exhaustive donor breeding soundness examination to estimate potential reproductive ability and to determine (1) if the mare or stallion is suitable (and to what extent) to enter an ET program, and (2) a program’s potential output, based on statistical data that consider breed, age, health status, and ET efficiency.
Donor Mare Management
Ideally, donors should be in excellent health and body condition and be cycling normally. A breeding soundness examination should reveal no endometritis, sexually transmitted diseases, or structural or functional reproductive tract problems. Donors should be inseminated with semen from fertile stallions. The timing and number of ovulatory cycles per year will vary with geographic region, but in subequatorial countries, a young donor should have 5 to 8 months of regular cyclic activity. In a typical ET program ([ovulation/AI] → [flushing/prostaglandin F2α] → [ovulation/AI]), with an interovulatory interval of 15 to 17 days, a mare could be flushed 8 to 14 times per season. Based on this number and expected ET efficiency, one can calculate the potential number of embryos that can be obtained per year. Regular reproductive examination by palpation and ultrasonography is imperative to monitor follicular development and ovulation, with the day on which ovulation is detected considered day 0. Spontaneous multiple ovulations increase in frequency toward the longest days of the year and are more frequent in mares older than 15 years, in certain breeds (up to 35% in Polo Argentino mares, a breed with a strong Thoroughbred influence), and when ovulation is induced. It is therefore advisable to check donors 48 hours after ovulation to both verify the number of corpora lutea, and to check for the presence of intrauterine fluid or postovulatory uterine edema.
Every donor in an ET program should be artificially inseminated, except in exceptional cases in which controlled natural mating may be recommended. Examples would include stallions with low in vitro sperm survival, in which natural breeding, for reasons unknown, improves recovery rates, and stallions that are refractory to semen collection. Artificial insemination allows control of semen quality. Natural mating is suboptimal because there is no control of either the concentration or progressive motility of sperm cells or the presence of urine, blood, or pus in the ejaculate. There are many important variables that affect the success of an ET program, and it cannot be assumed that a stallion is fertile because it ejaculates.
A donor can be flushed, on average, every 14 days during the physiologic breeding season. Depending on a mare’s follicular status and the veterinarian’s clinical judgment, ovulation inducers such as administration of 1500 IU human chorionic gonadotropin (hCG) intravenously or 1 mg of gonadotropin-releasing hormone controlled release analog1 given intramuscularly can be used to increase the number of ovulations per cycle. Results from the author’s laboratory indicate that in commercial embryo production, ovulation, and embryo recovery rates differ significantly with donor age and type of induction (Table 167-1).
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