牛细胞核移植研究简报

A Brief Report of Study on Bovine Nuclear Transfer

本研究探讨了用体外受精胚胎作供体、体外成熟卵母细胞作受体进行核移植的可能性，比较了两种不同核移植方法的效果。本研究结果在我国首次获得牛核移植的成功。从屠宰场回收的黄牛卵巢中抽取未成熟的卵母细胞，采用以前报道的方法（Ｓｈｉｅｔａｌ，Ｔｈｅｎ－ｏｙｅｎｏｌｏｙ，１９９１，３５；２７１）进行体外成熟（ＩＶＭ）和体外受精（ＩＶＦ），获得ＩＶＭ如母细胞和ＩＶＦ胚胎。体外受精用精液为奶牛精液。在ＩＶＭ２３～２４ｈ＄母细胞去核后用作受体卵，体外发育到８～３２细胞期的ＩＶＦ胚胎用作核供体．去核后的卵母细胞分两组：！．在Ｗ２６ｈ（或３０ｈ）用８０Ｖ／ｍｍ４０μｓ电激活两次，然后注入供体胚胎卵裂球，ＩＶＭ３２ｈ（或３６ｈ）进行电融合；Ⅱ．注卵裂球前不激活，在ＩＶＭ３１ｈ进行电融合。两组融合条件相同（８０Ｖ／ｍｍ４０μｓ×２，间隔１ｓ），融合液为０．３Ｍ甘露醇＋０．０５ｍＭＣａＣｌ２＋０．１ｍＭＭｇＳＯ４＋１０ｍＭ组氨酸。融合印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５～８ｄ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奶牛子直角内。２个月后直检妊娠情况。本研究结果如下：Ⅰ组操作后存活率（９０．?

The possibility of bovine nuclear transfer (NT) using in vitro matured (IVM) oocytes and in vitro fertilized (IVF) embryo was investigated, and the efficiencies of two methods of nuclear transfer are compared in this paper. The first successful bovine nuclear transfer in China is reported here. Oocytes taken from yellow cattle ovaries were matured in vitro and fertilized in vitro using cow semen as the previous report (Shi et al., Therigenology, 1991, 35: 271). IVM oocytes were enucleated after culture for 23～24 h. IVM/IVF embryos at 8～32-cell stage were used as the nuclear donors. The enucleated oocytes were divided into two groups for two treatments. (GⅠ), the enucleated oocyte was activated with two DC electric pulses (80 V/mm 40 μs) at IVM 26 h (or 30 h), then a single blastomere was microinjected into the perivitelline space of the enucleated oocyte. The fusion between blastomere and enucleated oocyte was carried out at IVM 32 h (or 36 h). (G Ⅱ), the nucleated oocyte was non-activated. The fusion was induced at IVM 31 h. The conditions of two groups were the same (80 V/mm 40 μs×2, 1 sec interval). The fusion solution was composed of o.3 M mannitol, 0.05 mM CaCl2, 0.1 mM MgSO4 and 10 mM histidine. Fused embryos were co-cultured with granulosa cell monolayers. The cleavage of NT embryos was evaluated after culture for 24 hours. The morulae and blastocyst embryos (M-BL) developed from NT were transferred to the horn of synchronous dairy recipients. The pregnancy of recipients was diagnosed 2 months later. The rates of survival (90. 5%, 389/430) and fusion (55. 0%, 193/351) of G Ⅰ were lower than those of G Ⅱ significantly (95. 9%, 186/194 and 65. 0%, 121/186, respectively) (P<0. 05), which indicates that the extra electric activation has disadvantageous effects on the survival and fusion of the reconstructed embryos. Although the cleavage rate and the developmental rate of M-BL embryos were not different significantly between two groups, G I (63. 0%, 87/138 and 15. 2%, 21/138, respectively) had better results than G Ⅱ(50. 0%, 52/ 103 and 7. 8%, 8/103). Sevellteen M-BL embryos from G Ⅰwere transferred to 12 synchronous recipients. Out of 4 recipients diagnosed, one was pregnant and aborted 4 months later. Another 8 recipients were in observation. Fight M-BL embryos from G Ⅱ were transferred to 4 recipients. One of the reed)ients was pregnant and gave birth to a male calf after pregnancy of 284 days. The phenotype of calf was F, hybrid (yellow cattle×dairy cow),which accorded with the genotype of the donor embryo. These indicate that the calf is developed from the NT embryo. It's the first nuclear transfer calf ill China.In conclusion, basing on on r established IVM--IVF and in vitro culture (IVC) system, the successful pregnallcy and living NT calf can be achieved by using IVM oocytes and IVF embryos.