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KMID : 0900920030270030215
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Korean journal of Animal Reproduction
2003 Volume.27 No. 3 p.215 ~ p.223
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Effects of Fertilization Time and Culture Medium of Pig Oocytes Matured In Vitro by liquid Boar Sperm Stored at 4^{circ}C
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Park Chang-Sik
Lee Young-Joo
Kim Moon-Young
Chang Young-Jun
Jin Dong-Il
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Abstract
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This study was to investigate the effects of fertilization time and culture medium of pig oocytes matured in-vitro by liquid boar sperm. The sperm rich fraction (30∼60 ml) was slowly cooled to room temperature (20∼23^{circ}C) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min 800 {times} g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of the LEN diluent to provide 1.0{times}10^{9} sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4^{circ}C. The medium used for oocyte maturation was TCM-199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 mutextrm{g}/ml insulin, 2 mutextrm{g}/ml vitamin B_{12} , 25 mM HEPES, 10 mutextrm{g}/ml bovine apotransferrin, 150 muM cysteamine, 10 IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75 mutextrm{g}/ml sodium penicillin G, 50 mutextrm{g}/ml streptomycin sulfate and 10% pFF. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5^{circ}C, 5% CO_2 in air. Oocytes were inseminated with liquid boar sperm stored at 4^{circ}C for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9 h in 500 {mu}ell mTBM fertilization media with 1.0{times}10^{6} sperm/ml concentration, respectively. Thereafter, oocytes were transferred into 500 {mu}ell NCSU-23, HEPES buffered NCSU-23, PZM-3 and PZM-4 culture media, respectively, for further culture of 6, 48 and 144 h. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times for 6 and 9 h than in those for 1 and 3 h. The rates of cleaved oocytes were higher in the fertilization times for 6 and 9 h (85.0 and 84.6%) than in those for 1 and 3 h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time for 6 h (33.6%) than in that for 1, 3 and 9 h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9, 27.6, 26.3 and 24.4 in the fertilization times for 6, 9, 3 and 1 h, respectively. The rate of blastocyst from the cleaved oocytes and the number of cells per blastocyst were higher in HEPES buffered NCSU-23 culture medium than in NCSU-23, PZM-3 and PZM-4 culture media. In conclusion, we found out that liquid boar sperm stored at 4^{circ}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 {mu}ell TBM fertilization medium with 1{times}10^{6} sperm/ml concentration and the HEPES buffered NCSU-23 culture medium for in-vitro fertilization of pig oocytes matured in-vitro.
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KEYWORD
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In-vitro fertilization, Pig oocyte, Liquid boar sperm, Culture medium
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