Influence of cumulus cells and sperm concentration on cleavage rate and subsequent embryonic development of buffalo (Bubalus bubalis) oocytes maturedand fertilized in vitro

The objective of the present study was to investigate the effects of sperm concentration and presence or absence of cumulus cells on fertilization, cl eavage rate and subsequent embryonic development upto the blastocyst stage in buffalo. Cumulus-oocyte-complexes (COCs) obtained from slaughterhouse ov aries were matured in vitro in TCM-199+10% FBS+5 mu g/mL FSH-P for 24 h. Af ter maturation the COCs were either used as such (cumulus-intact) or freed from attached cumulus cells by repeated pipetting (cumulus-free). Frozen-th awed buffalo spermatozoa were treated with IO mu g/mL heparin and 2.5 mM ca ffeine for sperm capacitation. Oocytes were fertilized in vitro with 1 to 2 , 4 to 5 or 9 to 10 million sperm/mL and the cleavage rate was recorded 42 to 44 h post insemination. The cleaved embryos were co-cultured with buffal o oviductal epithelial cells for 10 d post insemination and the uncleaved o ocytes were fixed and stained with aceto-orcein for determination of the pe netration rate. The cleavage rate and the proportion of cleaved embryos tha t developed to morula and blastocyst stages were significantly higher (P<0. 05) whereas the proportion of degenerated oocytes and those that became arr ested at the 2 to 16-cell stage were significantly lower (P<0.05) with cumu lus-intact than with cumulus-free oocytes at the 3 sperm concentrations. In creasing the sperm concentration increased the cleavage rate significantly (P<0.05) from 1 to 2 million-through 9 to 10 million sperm/mL but had no ef fect on the proportion of cleaved embryos that developed to morula and blas tocyst stages. In conclusion, the results of the present study suggest that cumulus cells have a positive influence on fertilization, cleavage and sub sequent embryonic development. Increase in sperm concentration increases cl eavage rate without affecting subsequent embryonic development. (C) 1998 by Elsevier science Inc.