牛-牛及山羊-牛克隆胚胎体外培养条件的优化

Optimization of Culture Measure for Bovine-bovine and Goat-bovine Cloned Embryos in vitro

通过胞质内注射法将牛和山羊胎儿耳朵成纤维细胞分别注入去核牛卵母细胞中构建同种胚胎和异种胚胎。采用mCR2aa和mSOF分别培养,然后在mSOF中按不同培养时间添加8mg/mLBSA或者10%FBS,培养前3d和培养3d后添加的补充物质及次序为:(1)BSA+FBS;(2)BSA+BSA;(3)FBS+BSA;(4)FBS+FBS。根据培养胚胎的卵裂率、8/16-cell发育率、囊胚发育率及囊胚细胞数筛选出最好的培养方法。结果:(1)mSOF中培养同种胚胎和异种胚胎的卵裂率,8/16-cell发育率以及囊胚发育率均明显高于在mCR2aa中的培养结果(P<0.05)。(2)添加BSA+FBS组的mSOF培养胚胎的卵裂率、8/16-cell发育率、囊胚发育率和囊胚细胞数同种依次为79.8%±7.1%、49.7%±3.5%、21.5%±1.8%和115.2±4.3,异种依次为40.1%±6.3%、29.2%±2.0%、13.4%±2.1%和100.1±3.0,均明显高于其他培养组(P<0.05)。结论:山羊-牛异种克隆胚胎可以用优化的牛胚胎培养体系进行培养。同种胚胎和异种胚胎的最佳培养方法均为前3d用mSOF+BSA培养液,3d后用mSOF+FBS培养液。

This study is conducted to explore an effective culture method for supporting the embryo development. The cattle fetal ear fibroblasts and the goat fetal ear fibroblasts are transplanted into the enucleated cattle oocytes separately by oocyte intraplasmic nuclear injection method to construct bovine cloned embryos and goat-bovine cloned embryos. The embryos are first cultivated in modified charles rosenkrans 2 amino acid medium （mCR2aa） and modified synthetic oviduct fluid medium （mSOF） separately. Then BSA （8mg/mL） or FBS（10%） can be added to mSOF according to the different culture period. The supplements and orders, added during the first three days and after three days are as follow： BSA and BSA, BSA and FBS, FBS and BSA, FBS and FBS. On the basis of the cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, the best culture way can be screened out. Result： First, cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, cultivated in mSOF solution are all higher than those cultivated in mCR2aa（ P 〈 0.05）. Second, the cleavage rate and 8/16-cell rate, adding BSA and FBS into mSOF, are in turn 79.8% ± 7.1%, 49.7% ± 3.5%, 21.5% ± 1.8%, and 115.2 ± 4.3 in bovine cloned embryo, and 40.1% ± 6.3% ,29.2% ± 2.0% ,13.4% ± 2.1% and 100.1 ± 3.0 in goat-bovine cloned embryo, which are significant higher than other culture groups（ P 〈 0.05）. Conclusion： The goat-bovine cloned embryo can be cultivated by the optimized culture measure of bovine cloned embryo. The best culture ways of bovine cloned embryo and goat-bovine cloned embryo are all to use mSOF supplemented BSA in the first three days and then use mSOF supplemented FBS in the next five days.