Transvaginal Ultrasound-Guided Oocyte Retrieval from Cyclic and Pregnant Horse and Pony Mares for In Vitro Fertilization1

Owners of valuable, clinically infertile mares often request that scientists develop assisted reproductive technologies to help obtain viable foals. Successful in vitro fertilization (IVF) in horses may provide one way of obtaining offspring. The objectives of this study were to develop a simple and effective, repeatable, ultrasound-guided, transvaginal procedure to recover oocytes from developing follicles in cycling mares and to determine the safety and feasibility of this approach to oocyte recovery for IVF in early-gestating mares. In experiment 1, follicular aspiration was performed on developing follicles from 25 cycling mares and from 5 early-gestating mares. The estrous cycles of mares were shortened by the i.m. injection of prostaglandin F2" during early diestrus, with a follicular aspiration procedure performed during each induced estrus. This allowed each cycling mare to be aspirated one to three times during the 32-day experimental period. Pony and horse mares were then administered hCG when preovulatory follicles reached 32-34 mm and 35-38 mm in diameter, respectively. Developing follicles were aspirated just before the expected time of ovulation with use of transvaginal ultrasonography. Follicles of pregnant mares were aspirated when the largest follicle of each mare reached a diameter of ≥ 25 mm either one, two, or three times between Days 22 and 66 of pregnancy. A mean of 2.28 follicles was punctured per collection procedure for cycling mares, resulting in a mean of 1.76 follicles that could be flushed. When only the apparent pre-ovulatory follicles were considered, the recovery rate was 42.9%, which was less than that of pregnant mares at 75.8%. Most oocytes from cyclic mares were classified as good quality, and those of pregnant donors appeared also to be of acceptable quality. The objectives of experiment 2 were to retrieve adequate numbers of viable oocytes from pregnant mares during the first half of gestation by repeated transvaginal ultrasound-guided aspiration and to confirm the viability of recovered oocytes by IVF. The follicular waves of 14 mares were monitored by ultrasound between Days 21 and 150 of pregnancy. Two aspiration treatments were applied to this group of pregnant mares. Only follicles with a diameter ≥ 20 mm were aspirated in 7 of these mares (treatment 1), whereas all follicles ≥ 4 mm were aspirated in the other 7 oocyte-donor mares (treatment 2). During the 129-day interval that mares were considered for aspiration, the mean number of aspiration procedures performed per mare was 7.6 and a mean of 18.9 oocytes were recovered per mare. The mean number of oocytes recovered during a single aspiration procedure was 2.5. A random group of 47 oocytes were in vitro-matured and in vitro-fertilized with fresh sperm cells using Ca2+ ionophore A23187 aided by zona drilling with acidic Tyrode's solution. In summary, the 20 oocytes that were classified as good-to-excellent-quality oocytes had a cleavage rate of 65%, and 38.4% of those cleaving developed to morula and blastocyst stages after 192 h of coculture. This oocyte collection procedure proved to be reliable and repeatable up to eleven times in both cyclic and early-pregnant mares without any detectable adverse effects to donor females. Furthermore, an acceptable number of oocytes can be collected in this manner without risking the pregnancy of donor mares. Results indicate that ≈143% of the oocytes retrieved from early-pregnant mares could be considered as viable when cleavage was used as a criterion in this study. To our knowledge, this is the first report of successful IVF of in vitro-matured equine oocytes collected from pregnant donors.