In Vitro Fertilization and Development of In Vitro-Matured Oocytes Aspirated from Pregnant Mares1

Abstract

Oocytes were collected from 12 mixed breed pregnant mares during early gestation (Days 33 through 146) via transvaginal ultrasound-guided aspiration. Oocytes harvested from experimental mares were matured for 37 to 48 h at 37°C in an atmosphere of 5% C0 2 in air. After maturation of the oocytes, the cumulus cells were removed and a small hole (2-3 um in diameter) was created in the zona pellucida of the oocyte with use of an acidic Tyrode's solution. The oocytes (n = 32) were then coincubated with fresh equine sperm cells treated with Ca 2+ ion-ophore at three concentrations (0.1, 1.0, or 3.0 uM) at 39°C in an atmosphere of 5% C0 2 in air for 2 h. Ca 2+ ionophore at these three concentrations did not noticeably affect sperm motility. After fertilization, the oocytes were cocultured with ten 8-cell mouse embryos on a bovine ovi-duct cell monolayer in droplets of 100 ja. 1 of culture medium at 39°C in an atmosphere of 5% C0 2 in air. There was a significant difference in the cleavage rate among the zona-drilled oocytes fertilized with sperm cells treated with the three different concentrations of Ca 2+ ionophore. Significantly more oocytes cleaved in the 1.0 uM sperm cell treatment (79%) than in the control (0%), 0.1 uM (0%) and 3.0 |xM (33%) treatment groups. In addition, 45.5% of the cleaved oocytes in the 1.0 uM treatment group developed to morula stage and 18.2% reached blastocyst stage during an 8-day in vitro culture period. The concentration of Ca 2+ ionophore, the zona drilling, and the coculture with bovine oviduct cells and mouse embryos may all have contributed to the improved fertilization rate and the subsequent development of in vitro fertilization (IVF)-derived equine embryos in this study.