304INFLUENCE OF ELECTRICAL STIMULUS ON ACTIVATION OF PIG OOCYTES MATURED IN VITRO

Abstract

Electrically induced activation of pig oocytes deserves particular attention for research on parthenogenesis. The aim of this study was to improve electrical activation of in vitro matured pig oocyte. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Cumulus-free oocytes involving first polar body were selected for activation. For electrical activation, oocytes were rinsed twice in 0.3M mannitol solution supplemented with 0.1mM CaCl2, 0.2mMMgCl2, 0.5mM HEPES and 0.01% BSA, and transferred to a chamber consisting of two electrodes 1mm apart which were overlaid with the same activation solution. Experiment 1 was conducted to investigate the effect of electrical pulse on oocyte activation. Oocytes were activated with DC pulses of 1.0, 1.5, 2.0 and 2.5kVcm−1 for 30, 60 and 90μs, respectively. Experiment 2 was carried out to investigate the effect of electrical stimulus frequency on oocyte activation. Oocytes were activated one, two and three times, with a DC pulse of 1.0kVcm−1 for 60μs. After activation, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA and cultured for 20h. Activated oocytes were fixed for 48h in 25% acetic acid (v:v) in ethanol at room temperature, and stained with 1% orcein (w:v) in 45% acetic acid (v:v) to examine pronucleus formation. Data were analyzed by ANOVA and Duncan’s multiple range test using the SAS program. The rate of activation was highest in the DC pulse of 1.0kVcm−1 for 60μs (75.1%) compared with the other durations and strengths (62.5–63.1%). Activation rate by electrical stimulus frequency was highest (76.0%) when oocytes were activated by a one-time pulse. In conclusion, the results suggested that electrical stimulus with a single DC pulse of 1.0kVcm−1 for 60μs might be more efficient than other strengths and durations for activation of pig oocytes.