P-97: Optimized Combined Electrical-Chemical Parthenogenetic Activation for Zona Free Sheep Oocyte

Background: Artificial oocyte activation (AOA) has important implications in assisted reproductive technique (ART) with particular importance in somatic cell nuclear transfer (SCNT). Parthenogenesis activation using zona- free oocytes offers an alternative model that could be applied to develop protocols for the activation of reconstructed embryos for cloning. Materials and Methods: To establish the reliable combined electrical-chemical activation for zona-free oocytes,oocytes were first exposed to an electrical pulse and then treated with 18 chemical activation regimens designed through modifying duration (5, 2.5, 1 minutes) and concentration of ionomycin (5, 2.5, 1 μM) and 6-dimethyl aminopurine (6-DMAP) (2 mM for 2 or 4 hours), which is routinely used for SCNT. Results: Maximum parthenogenetic blastocyst development was obtained when oocytes were submitted to electric pulse and then to (1) 5 μM ionomycin for 5 or 2.5 minutes, both followed by 2 hours of incubation with 6-DMAP (41.7 - 1.1, and 42.4 - 1.4%, respectively), (2) 5 μM ionomycin for 1min + 6-DMAP for 4 hours (43.1 - 1.4%), and (3) 2.5 μM ionomycin for 1min + 6-DMAP for 2 hours (42.4 - 1.4%), with significant differences compared to all the other groups. Conclusion: Statistical assessment of interactions between duration and concentration of ionomycin and duration of 6-DMAP exposure revealed that (1) concentration of ionomycin may be a more important factor than its duration, (2) both a long exposure period and a low concentration of ionomycin had marked decreasing effects on parthenogenetic development of zona-free oocytes, and (3) high duration of exposure to 6-DMAP can reduce parthenogenetic development.