304 INFLUENCE 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 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 μg mL⁻¹ insulin, 2 μg mL⁻¹ vitamin B₁₂, 25 mM HEPES, 10 μg mL⁻¹ bovine apotransferrin, 150 μM cysteamine, 10 IU mL⁻¹ PMSG, 10 IU mL⁻¹ hCG, 10 ng mL⁻¹ EGF, 0.4% BSA, 75 μg mL⁻¹ sodium penicillin G, 50 μg mL⁻¹ streptomycin sulfate and 10% pFF. After about 22 h of maturation, oocytes were cultured without cysteamine and hormones for 22 h 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 0.3 M mannitol solution supplemented with 0.1 mM CaCl₂, 0.2 mM MgCl₂, 0.5 mM HEPES and 0.01% BSA, and transferred to a chamber consisting of two electrodes 1 mm 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 2.5 kV cm⁻¹ for 30, 60 and 90 μs, 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 1.0 kV cm⁻¹ for 60 μs. After activation, oocytes were transferred into 500 μL NCSU-23 culture medium containing 0.4% BSA and cultured for 20 h. Activated oocytes were fixed for 48 h 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 1.0 kV cm⁻¹ for 60 μs (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 1.0 kV cm⁻¹ for 60 μs might be more efficient than other strengths and durations for activation of pig oocytes.