Effects of caffeine, cumulus cell removal and aging on polyspermy and embryo development on in vitro matured and fertilized ovine oocytes.

The objectives of these studies were to determine the effects of cumulus cell removal and caffeine treatment on the development of in vitro matured ovine oocytes aged in vitro until until fertilization. Oocytes were denuded (DO) at 24h post-onset of maturation (hpm), control cumulus oocyte complexes (COC's) and DO groups were fertilized at 24 hpm or returned to culture in the presence or absence of 10mM caffeine and fertilized at 30 hpm. Removal of cumulus cells and aging both increased polyspermy, caffeine reduced this increase, however, with the exception of DO's (30 hpm) vs. COC's (24 hpm) the differences were not statistically significant. Aging significantly decreased cleavage between COC groups at 24 hpm and 30 hpm and caffeine did not affect this (68.4%, 73.4%, 74.0% respectively). In contrast, the frequency of cleavage was significantly reduced in the DO (24 hpm) group as compared to COC controls (45.6% vs. 68.4% (P<0.05)), however, cleavage increased in the DO group on aging (73.4%) and this was not affected by caffeine (73.0%). The percentage of COC's and DO's developing to the blastocyst stage significantly decreased on aging, caffeine treatment of DO's prevented this (31.3%, 12.7% and 29.4% respectively (P<0.05)) but had no effect on COC's (4.2% vs. 3.9%). Total cell numbers in blastocysts were not statistically different (92.4+/-5.2, 84.7+/-3.7 and 80.4+/-5.8 (P>0.05)). In summary caffeine treatment of aged COC's had no significant effect on the frequency of development, however, in aged DO's caffeine treatment statistically increased development to blastocyst and lowered the frequency of polyspermy.