獭兔体外成熟卵母细胞孤雌激活及体细胞核移植

[目的] 建立利用獭兔体外成熟卵母细胞进行孤雌激活及体细胞核移植的技术体系,促进獭兔现代育种技术的发展。[方法] 以獭兔屠宰场废弃卵巢为试验材料,利用体外成熟、孤雌激活、体细胞核移植等方法,研究了獭兔体外成熟卵母细胞支持胚胎发育的能力。[结果] 1)直径大于1 mm卵泡内的卵母细胞孤雌激活后的发育能力显著强于0.7~1.0 mm卵泡内的卵母细胞。2)卵母细胞体外培养(IVC)15~21 h后孤雌激活,卵裂率显著高于IVC 12 h组。3)2.5 μmol？L-1 Ionomycin处理5 min,再用2 mmol？L-1 6-二甲基氨基嘌呤(6-DMAP)与5 μg？mL-1亚胺环己酮(CHX)联合处理1 h,孤雌激活效果最好。4)IVC 14 h后,78%的卵母细胞极体与核偏移角度小于10°;IVC 18 h后,34.6%的极体偏离核的角度大于45°。5)体外成熟的獭兔卵母细胞可以较好支持体细胞核移植的重构胚,核质作用期间采用MG132处理,降低了重构胚的囊胚率。[结论] 从大于1 mm卵泡内回收卵母细胞并IVC 18 h后,采用2.5 μmol？L-1 Ionomycin处理5 min,再用2 mmol？L-1 6-DMAP与5 μg？mL-1 CHX联合处理1 h,是獭兔孤雌激活和体细胞核移植的最佳方案。
[Objectives] The aim of this paper was to establish the methods of parthenogenetic activation and somatic cell nuclear transfer used in Rex rabbit mature oocytes in vitro, and to promote the development of modern breeding techniques in Rex rabbit.[Methods] Wasting ovaries obtained from the rabbit slaughterhouse were used as experimental materials. We made use of several methods in this research, such as in vitro maturation, parthenogenetic activation, somatic cell nuclear transfer, etc. We studied the ability of embryonic development in detail.[Results] 1)The growth ability of parthenogenetic activation oocytes which were greater than 1 mm follicle group was stronger than the 0.7-1.0 mm follicle group. 2)The cleavage rate was significantly higher in the in vitro cultured(IVC)15-21 h group than in the IVC 12 h group. 3)The best parthenogenetic activation method was using 2.5 μmol？L-1 Ionomycin to treat it for 5 min, and then using 2 mmol？L-1 6-Dimethylaminopurine(6-DMAP)and 5 μg？mL-1 cycloheximide(CHX)to incubate for 1 h. 4)The angle between polar body and nuclear was smaller than 10° in 78% oocytes which were IVC for 14 hours. But only 34.6% polar body moved more than 45° from nuclear when the oocytes were IVC for 18 hours. 5)Incubating the reconstructed embryo with MG132 during nucleus-cytoplasmic interaction reduced the blastocyst rate.[Conclusions] The best method of parthenogenetic activation and somatic cell nuclear transfer was that obtaining the oocyte from greater than 1 mm follicle and applying IVC to it for 18 h, then using 2.5 μmol？L-1 Ionomycin to treat it for 5 min, and then using 2 mmol？L-1 6-DMAP and 5 μg？mL-1 CHX to incubate for 1 h