Parthenogenesis of rabbit oocytes activated by different stimuli.

Oocyte activation is one of the essential elements determining the success of nuclear transfer and the subsequent development of cloned embryos both in vitro and in vivo. Experiments were conducted to optimize the protocol for oocyte activation in a regular nuclear transfer study. In vivo derived oocytes were collected at 14-15 h from New Zealand white rabbits after ovulation treatment and were activated +18 h post-ovulation treatment. Single activation agents including calcium ionophore (A23187, 5 microM, 5 min), ethanol (Eth, 7%, 7 min), and thimerosal (200 microM, 10 min) were tested. Cleavage rates were highest in the ethanol-treated group (37%) compared to other treatments (19-25%). Very low blastocyst rates (2-3%) resulted which were not significantly different among treatments (P>0.05). Combined single agent treatment (calcium stimulators) with protein kinase inhibitor, 6-DMAP were used to achieve a full oocyte activation. Both pronuclear and blastocyst formation rates were significantly higher (P<0.05) in the Eth+6-DMAP treatment group (38 and 27%) than in the other groups (16-21 and 7-9%, respectively, P<0.05). Low (0.2mM) and high (2.5mM) concentrations of 6-DMAP treatments with different treatment lengths (1.5 and 3.5h) in the combined groups were also compared. Blastocyst formation and cleavage rates were greater in the high concentration with less treatment time groups (36% versus 4-20%, P<0.05). In conclusion, single activation agents, either Ca2+ stimulators or protein kinase inhibitors, could not fully activate mature rabbit oocytes. The best activation procedure obtained in this study was the Eth+6-DMAP combined treatment, which may be incorporated into regular nuclear transfer or cloning protocols.