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 (30–60mL) was slowly cooled to room temperature (20–23°C) by 2h after collection. Semen was transferred into 15-mL tubes, centrifuged at room temperature for 10min at 800g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5mL of the LEN (11.0g lactose hydrate, 20mL egg yolk, 0.05g N-acetyl-D-glucosamine and 100mL distilled water) diluent to provide 1.0×109 spermmL−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 26.19mM 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. Oocytes were inseminated with boar sperm stored at 4°C for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9h in 500μL TBM fertilization media with 1×106mL−1 sperm concentration. Thereafter, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA for further culture of 6, 48 and 144h, 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 9h than in those of 1 and 3h. The percentage of polyspermic oocytes was highest in fertilization time of 9h compared with other incubation times. The rates of cleaved oocytes were higher in the fertilization times of 6 and 9h (85.0 and 84.6%) compared with those of 1 and 3h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time of 6h (33.6%) than in that of 1, 3 and 9h (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 1h, 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 6h in 500μL TBM fertilization medium with 1×106mL−1 sperm concentration for in vitro fertilization of pig oocytes matured in vitro.
In vitro Fertilization and Development of Pig Oocytes Inseminated with Boar Sperm by Different Sperm Washing Media after Thawing of the Frozen Straws
