303 PARTHENOGENETIC DEVELOPMENT OF PIG OOCYTES BY CHEMICAL ACTIVATION

Abstract

Efficient activation is essential for the success of animal cloning by nuclear transfer. The aim of this study was to investigate the effects of chemical activation agents on parthenogenetic development of pig oocytes matured in vitro. 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 showing first polar body were selected for activation. Oocytes were activated as follows. First, all oocytes were activated with 25 mM HEPES buffered NCSU-23 medium containing 8% ethanol for 10 min. After that, in treatment 1, oocytes were incubated in the NCSU-23 medium supplemented with 7.5 μg mL⁻¹ cytochalasin B for 3 h. In treatment 2, oocytes were incubated in the NCSU-23 medium supplemented with 10 μg mL⁻¹ cycloheximide for 3 h. In treatment 3, oocytes were incubated in the NCSU-23 medium supplemented with 7.5 μg mL⁻¹ cytochalasin B for 1.5 h, and then were incubated in the NCSU-23 medium supplemented with 10 μg mL⁻¹ cycloheximide for 1.5 h. In treatment 4, oocytes were incubated in the NCSU-23 medium supplemented with 7.5 μg mL⁻¹ cytochalasin B plus 10 μg mL⁻¹ cycloheximide for 3 h. Following activation, oocytes were transferred into 500 μL NCSU-23 culture medium containing 0.4% BSA for further culture for 20 and 144 h. Activated oocytes were fixed and stained for evaluation of activation rate, cleaved oocytes, blastocyst formation rate and cell numbers per blastocyst. Data were analysed by ANOVA and Duncan’s multiple range test using the SAS program. The rate of oocyte activation was higher in treatment 4 (62.1%) than in treatment 1, 2 and 3 (52.0, 49.6 and 58.0%, respectively). The percentage of cleaved oocytes was lower in treatment 1 and 2 (56.9 and 55.2%) than in treatment 3 and 4 (68.8 and 68.5%). The rate of blastocyst formation from the cleaved oocytes was higher in treatment 3 and 4 (19.8 and 22.0%) than in treatment 1 and 2 (12.1 and 11.7%). Mean cells per blastocyst were lowest in treatment 2 (21.2 ± 0.9) compared to treatment 1, 3 and 4 (27.3 ± 2.2, 30.4 ± 3.8 and 30.9 ± 3.4, respectively). In conclusion, cytochalasin B combined with cycloheximide was more efficient for parthenogenetic development of pig oocytes matured in vitro.