Production of live foals from sperm-injected oocytes harvested from pregnant mares

In vitro fertilization in horses has been less successful than anticipated owing to: (i) the inability to collect large numbers of good quality oocytes; (ii) alterations in the zona pellucida that occur during in vitro maturation of equine oocytes; and (iii) inadequate preparation of equine sperm cells. In addition, studies in humans, mice and cattle have indicated that high concentrations of glucose in culture media may inhibit embryonic development in vitro and this may also be a problem for development of equine embryos in vitro. The aims of the present study were: (i) to achieve fertilization of equine oocytes by sperm injection; and (ii) to determine whether culture media containing low concentrations of glucose are beneficial for the development of early stage equine IVF-derived embryos. In Expt 1, in vitro matured oocytes obtained from pregnant mares were subjected to intracytoplasmic sperm injection (ICSI), subzonal sperm injection (SUZI) or one of three control treatments. The cleavage rates were greater for oocytes subjected to ICSI (39%) than for oocytes subjected to SUZI (6%) (P < 0.05). The transfer of two embryos into one recipient mare resulted in the presence of an embryonic vesicle in the uterine body at day 14 after ICSI, but it was lost subsequently between days 16 and 18 after ICSI. In Expt 2, oocytes were subjected to ICSI and cultured for 48 h in either TCM-199 or P-1(TM) medium (glucose- and phosphate-free) supplemented with 15% fetal bovine serum. The cleavage rates for embryos cultured in the two culture media were different (47% and 63% in TCM-199 and P-1(TM), respectively; P < 0.10). In addition, four grade 1 embryos were transferred surgically into the oviducts of four recipient mares 48 h after ICSI. Three pregnancies were identified by ultrasonography by the presence of an embryonic vesicle in the uterine body by day 16 after ICSI. Two of these pregnancies proceeded to term, resulting in the birth of two healthy fillies, one at day 319 and the other at day 328 of gestation.