289 EFFECT OF FERTILIZATION TIME OF PIG OOCYTES MATURED IN-VITRO BY BOAR SPERM STORED AT 4°C

Abstract

The use of boar sperm stored at 4°C may be a useful tool for in vitro production of pig embryos. Therefore, this study was undertaken to investigate the effect of fertilization time of pig oocytes matured in-vitro by boar sperm. The sperm-rich fraction (30–60 mL) was slowly cooled to room temperature (20–23°C) by 2 h after collection. Semen was transferred into 15-mL tubes, centrifuged at room temperature for 10 min at 800g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 mL of the LEN (11.0 g lactose hydrate, 20 mL egg yolk, 0.05 g N-acetyl-D-glucosamine and 100 mL distilled water) diluent to provide 1.0 × 10⁹ sperm mL⁻¹ at room temperature. The resuspended semen was cooled in a refrigerator to 4°C. 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. Oocytes were inseminated with boar sperm stored at 4°C for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9 h in 500 μL TBM fertilization media with 1 × 10⁶ mL⁻¹ sperm concentration. Thereafter, oocytes were transferred into 500 μL NCSU-23 culture medium containing 0.4% BSA for further culture of 6, 48 and 144 h, fixed and stained for the evaluation of fertilization parameters and developmental ability. Data were analysed by ANOVA and Duncan’s multiple range test using the SAS program. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times of 6 and 9 h than in those of 1 and 3 h. The percentage of polyspermic oocytes was highest in fertilization time of 9 h compared with other incubation times. The rates of cleaved oocytes were higher in the fertilization times of 6 and 9 h (85.0 and 84.6%) compared with those of 1 and 3 h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time of 6 h (33.6%) than in that of 1, 3 and 9 h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9 ± 3.3, 27.6 ± 2.7, 26.3 ± 2.2 and 24.4 ± 1.8 in the fertilization times of 6, 9, 3 and 1 h, respectively. In conclusion, we found out that boar sperm stored at 4°C could be used for in vitro fertilization of pig oocytes matured in vitro. Also, we recommend the coincubation time of 6 h in 500 μL TBM fertilization medium with 1 × 10⁶ mL⁻¹ sperm concentration for in vitro fertilization of pig oocytes matured in vitro.