Comparison of chemical, electrical, and combined activation methods for in vitro matured porcine oocytes.

Abstract

Factors influencing porcine oocyte activation were systematically studied. This study included (1) the effect of ionomycin plus various chemical agents on activation, (2) comparison of different electrical activation parameters, (3) optimization of combined activation, and (4) evaluation of the optimized protocols. The results showed that (1) blastocyst rates of ionomycin (Ion) + 6-dimethylaminopurine (6-DMAP) (29.7 ± 1.1%), Ion + cytochalasin B (CB) + cycloheximide (CHX) (29.8 ± 1.2%), Ion + CB + 6-DMAP (30.4 ± 1.6%), and Ion + CB + CHX + 6-DMAP (30.2 ± 2.7%) were significantly higher than Ion + CHX (15.8 ± 1.5%, p < 0.05); (2) the parthenogenetic blastocyst formation of electrical activation was optimal when oocytes were activated by three direct current (DC) pulses of 1.00 kV cm(-1) for 80 μs (39.5 ± 1.1%); (3) blastocyst rates of DC + CB + CHX (55.4 ± 1.2%) and DC + CB + 6-DMAP (50.4 ± 2.9%) were significantly higher than DC + 6-DMAP, DC + CB + CHX + 6-DMAP, electrical activation, and chemical activation alone (p < 0.05); and (4) approximately 84% of parthenogenetic blastocysts yielded by the optimized protocol were diploid, which was significantly higher than that of electrical activation blastocysts (40%). Using the optimized electrical and combined activation protocol, high blastocyst rates were generated by intracytoplasmic sperm injection (ICSI) (34.6 ± 1.1%), cytoplasmic microinjection (CI) (52.3 ± 2.2%), and handmade cloning (HMC) (31.2 ± 1.0%), respectively. This study concludes that the optimal activation protocol of in vitro matured porcine oocytes was combined activation with parameter as three DC pulses of 1.00 kV cm(-1) for 80 μs plus CB and CHX treatment.