Efficient Method For Combined Electrical-Chemical Parthenogenetic Activation of Bovine Oocytes

Purpose: Parthenogenetic activation is among the crucial steps determining successful development of mammalian cloned embryos. This study, therefore, was conducted to evaluate the efficiency of a novel combined electrical-chemical artificial activation method for bovine cloning. Materials and Methods: In vitro matured bovine oocytes than were initially exposed to electrical pulse, used for cell fusion during cloning, and then treated with temporal sequential combinations of 3 chemical activators [calcium ionophore (CI), strontium (SR) and ethanol (ET)], followed by exposure to a protein kinase inhibitor or used for in vitro fertilization as control group. Treated and naturally fertilized oocytes were further cultured for up to 8 days. Embryo development was scored daily and blastocyst cell counting carried out using differential staining at day 8 of culture. Results: Among 15 temporal sequential combinations of three chemical activators, the best cleavage rates were associated with double (SR CI, 84.4%), triple (CI SR ET, 79.4%) and single (CI, 73.7%) compounds, respectively, which were not significantly different with each other and with in vitro fertilized (IVF) (85.5%). The highest blastocyst rates were gained with ET SR (25%), SR CI ET (21.7%) and CI (20.3%) accordingly which were not significantly different with each other but significantly lower than IVF (46%).Embryo cell counting further confirmed reasonably better quality of blastocysts produced using double, triple and single compounds. Conclusion: Although most of the sequential artificial activation compounds induced high cleavage rate close to IVF, but this did not assure comparable further embryo development to the blastocyst stage.Nevertheless, the results suggest exposure of in vitro matured bovine oocytes to electrical pulse, followed by exposure to CI 6- dimethylaminopurine (6-DMAP) or ET SR 6-DMAP could be regarded as the optimal artificial activation.