MORPHOLOGICAL EVENTS DURING IN-VITRO FERTILIZATION OF PREPUBERTAL GOAT OOCYTES MATURED IN-VITRO

Experiments were carried out to study morphological changes temporally associated with in vitro fertilization (IVF) of prepubertal goat oocy tes and to elucidate some of the abnormalities occurring during this p rocess. The effects of different intervals of insemination on subseque nt embryonic development were also studied. Prepubertal goat oocytes c ollected at slaughter were matured in TCM199 supplemented with estrous goat serum (20%), FSH (10 mu g/ml), LH (10 mu g/ml) and estradiol-17 beta (1 mu g/ml) for 27 h at 38.5 degrees C. Matured oocytes were inse minated with freshly ejaculated spermatozoa following capacitation as described by Younis et al. (37) but with 100 mu g/ml heparin. Represen tative oocytes were fixed every 2 to 4 h from 2 to 28 h after insemina tion for a study of sperm penetration, sperm head decondensation, meio tic activation, female chromosome decondensation, and male and female pronuclear formation. At the same intervals after insemination, some o f the ova were co-cultured on granulosa cell monolayers for up to 9 d. Sperm penetration into the ooplasm was first observed at 4 h post ins emination; decondensation of male and female chromatin and formation o f male and female pronuclei occurred at 6 to 8 and 10 to 16 h after in semination, respectively. Highest proportions of oocytes were penetrat ed after exposure to spermatozoa for 8 h. There were no significant di fferences in ovum penetration after longer insemination intervals. Cle avage was first observed 24 h after insemination. Three types of abnor malities were observed. These were polyspermy, polygyny and asynchrony in the development of the female and male pronuclei, apparently due t o a delay in the decondensation of the male pronucleus. Significantly higher proportions of oocytes cleaved (31.2 to 45.5%) after 20, 24 or 28 h insemination intervals than following shorter intervals of exposu re to spermatozoa. However, the sperm exposure interval did not signif icantly affect subsequent embryonic development to the blastocyst stag e. Embryos resulting from oocytes exposed to sperm cells for at least 12 h developed further than the 8-cell stage. (C) 1997 by Elsevier Sci ence Inc.