Fertilization of bovine oocytes grown in vitro

Abstract

Early antral follicles 0·5–0·7 mm in diameter were dissected from bovine ovaries, and oocyte–cumulus complexes with pieces of parietal granulosa (OCCGs) were then collected from the follicles. The OCCGs containing oocytes of 90–99 m diameter (94·7 ± 2·8 µm, n = 196) were selected and embedded in collagen gels and cultured for 14 days in TCM199 containing 10% fetal calf serum and 4 mM hypoxanthine. From cultured OCCGs, 144 surviving oocytes were recovered, of which 53 were granulosa cell-enclosed oocytes and 91 were denuded oocytes. The mean diameter of the surviving oocytes was 114·2 ± 8·4 µm, significantly larger than that measured before culture (P < 0·05). The granulosa cell-enclosed oocytes and denuded oocytes were further cultured for maturation for 24 h. After culture, 72% (38/53) of the granulosa cell-enclosed oocytes and 59% (54/91) of the denuded oocytes showed normal morphology. These oocytes were then inseminated with bovine spermatozoa. After 29 h of insemination, all of the denuded oocytes had degenerated, while 32% (12/38) of the granulosa cell-enclosed oocytes showed normal morphology. Of the 12 oocytes, 5 were penetrated by spermatozoa, and 2 formed both male and female pronuclei. These results demonstrate for the first time that bovine oocytes grown in vitro acquire meiotic competence and can be penetrated by spermatozoa.