作　　者： ; ; ; ;

机构地区： 华南师范大学信息光电子科技学院激光生命科学教育部重点实验室

出　　处： 《华南师范大学学报（自然科学版）》 1995年第3期76-77,共2页

摘　　要： 本研究探讨了用体外成熟卵母细胞和体外受精胚胎进行核移植的可能性。本研究结果在我国首次获得牛核移植的成功。从屠宰场回收的卵巢中抽取卵球－卵母细胞复合体，采用以前报道的方法（石德顺等，广西农业大学学报’１９９４：１３：１－５）进行体外成熟（ＩＶＭ）和体外受精（ＩＶＦ），获得ＩＶＭ卵母细胞和ＩＶＦ胚胎，体外成熟２３－２４ｈ后，在含０．１％透明质酸酶的无钙镁ＰＢＳ（ＰＢＳ）内，用细管反复吹打去掉卵丘细胞。选择具有明显第一极体的卵母细胞通过显微操作移去第一极体及其附近约一半的细胞质进行去核，去核的卵细胞在成熟液内继续培养到ＩＶＭ３０ｈ．体外发育到８－３２细胞期的ＩＶＦ胚胎用作核供体．供体胚胎用含０．２５％Ｐｒｏｎａｓｅ（溶于ＰＢＳ￣－）溶解透明带，并用细管吹打将其分散成单个卵裂球，在ＩＶＭ３０ｈ将单个卵裂球显微注入去核卵母细胞的卵周隙内，并在ＩＶＭ３１ｈ用８０Ｖ／ｍｍ４０μｓ两次电脉冲（间隔１秒）来诱导卵裂球与去核卵母细胞的融合。融合卵在颗粒细胞单层培养滴内共培养，体外培养２４ｈ后观察卵裂结果，经体外培养５－８天后发育到桑椹和囊胚的核移植胚胎移植到同期发情的受体．２个月后直检妊娠情况．本实验中共操作１９４枚卵母细胞，? he possibility of bovine nuclear transfer using in vitro matured axytes and in vitro fertil-ized embryo was investigated. The first successful bovine nuclear transfer in China is reportedhere.Bovine cumulus-oocyte complexes aspirated from ovaries were matured in vitro and fertil-ized in vitro(IVM ,IVF)as the previous report(Shi et al.,J. Guangxi Agric, Univ.)After cul-ture for 23-24h, the complexes were stripped of cumulus by pipetting in Ca￣(+ +)-Mg￣(+ +)-freePBS(PBS￣-)with 0.1%Hyaluronidase. Those oocytes with the obvious first polar fory(FPB)were selected and enucleated by removing the FPB and about half of cytoplasm underneath theFPB. The enucleated oocytes were cultured in mature medium until IVM 30h. Donor embrvoswere IVM/IVF embryos at 8-32-cell stage,and were separated into individual blastomeres bypipetting after removing off the zona pellucida in PBS￣- with 0. 25%Pronase, Then a single blas-tomere was microinjected into the perivitelline space of the enucleated oocyte at IVM 30h. The fu-sion between blastomere and enucleated oocyte was induced by two 80V/mm 40μs,1 sec interval,DC electric pulses at IVM 31h. Fused embryos were co-cultuerd with granulosa cell manolayers.The cleavage of NT embryos was evaluated after cultuer for 24 hours, After culture for 5-8 days, NT embryos which developed to morulae and blastocyst stages were transferred to synchronousrecipients. The pregntincy of reciplents was diagnosed 2 months later.In this experiment, 194 oocytes were manipulated, 186 survived the manipulation,resultingin 96%of stirvial rate, Attempts of electrofusion of the enucleated oocytes with blastomeres re-sulted in 65%(121/186) successftil fusion, Althotigh 50%(52/103)of the fused embryos cleav-aged to 2-cell or more after culture for 24h, the developmental rate of morulae and blastocysts(M-BL) was only 8%(8/103).Eight M-BL embryos were transferred to 4 recipients. One ofthe recipients was pregnant and gave birth to a male calf at full-term, It is the first nucleartransfer calf in China.In conc

关 键 词： 体外成熟 体外受精 核移植 牛犊 牛

领　　域： [农业科学] [农业科学]