猪分级卵母细胞体外成熟时间规律的研究

试验根据猪卵丘－卵母细胞复合体（COCs）的颗粒细胞层数，把COCs分成A、B、C和D级，A、B级用于体外分别培养24、32、38、44、48和54 h，C级用于体外分别培养24、32、38、44、48、54和60 h，观察它们不同时间排出极体数，然后将排出极体的卵母细胞孤雌激活。结果发现，A和B级COCs培养48 h时成熟率最高（83.2%和78.0%），明显高于同级水平培养24、32和38 h的成熟率；不同级别COCs体外培养相同时间，A与B级成熟率差异不显著（P＞0.05），但二者与C级的成熟率差异显著（P＜0.05）；体外培养48 h COCs卵裂率最高（81.7%），与培养38 h前的卵裂率差异显著（P＜0.05）。结果表明，A、B级卵母细胞更适于体外培养，能获得高的成熟率和卵裂率，COCs周围颗粒细胞对卵母细胞的成熟有显著影响；COCs培养38 h之前，部分虽能看到极体，但激活后卵裂率极低，说明卵母细胞并未真正成熟，通常通过排出极体判断卵母细胞成熟是不够准确的，COCs体外培养44～48 h是成熟的最佳时期，能为体细胞核移植提供大量优质的MⅡ期卵母细胞。

Research of Pig Oocytes in vitro Maturation Time

The objectives of the present study were first to carry out the cumulus-oocyte complexes (COCs) divided into levels A, B, C and D according to cumulus cells, which levels A and B were cultured in vitro for 24, 32, 38, 44, 48 and 54 h, and C was cultured in vitro for 24, 32, 38, 44, 48, 54 and 60 h, respectively. Subsequently, the oocytes with extruded polar body at different time intervals were induced by application of an AC pulse of 10 V for 5 s followed by 1.6 kV/cm for 60 μs using an Electro Cell Manipulator 2001. These results showed that the maturation rate of COCs of levels A and B arrived at the highest period was no significant difference (83.2% and 78.0%, P＞0.05) in vitro for 48 h, but both were significantly higher than the same level COCs cultured for 24, 32 and 38 h. Different levels of COCs culture same time in vitro, the maturation rate of levels A and B were not significant (P＞0.05), but both were significantly higher than the levels C. The rate of cleavage (78.2%) in vitro culture for 44 h was significantly higher(P＜0.05) than COCs cultured for 24 to 38 h. In conclusion, COCs of levels A and B were more suitable for oocytes cultured in vitro, obtaining a high maturation rate and cleavage, granule cells had significant influence on oocyte maturation. COCs cultured in vitro before 38 h, some oocytes may be observed the polar body, but the developmental rate to cleavage were extremely lower, indicating that oocytes were no really mature. At the same time，oocytes of the developmental maturation could not judge only by excluding the polar body. Oocytes cultured in vitro 44 to 48 h was the best period of in vitro maturation(IVM) and provide a number of high-quality metaphase-II (MⅡ) oocytes for production of parthenotes and somatic cell nuclear transfer(SCNT) embryos.