【作者】 金仁桃；

【导师】 章孝荣；

【作者基本信息】 安徽农业大学 ， 动物遗传育种与繁殖， 2004， 硕士

【摘要】 本试验以波尔山羊耳成纤维细胞为供体，分别以牛、羊、兔的卵母细胞为受体进行体细胞核移植，构建了山羊-兔、山羊-牛和山羊-山羊三种重构胚。研究了不同的胞质环境对重构胚体外发育的影响与核移植中的胞质效应，并对异种克隆胚胎采用了去透明带移植和配种后再移植的方法，探讨移植方法和妊娠因子对异种克隆胚胎在受体动物体内发育的影响，旨在为解决异种克隆胚移植不易着床发育的难题和为提高动物克隆效率提供理论依据。试验获得了如下结果：1．以山羊耳成纤维细胞为供体，以牛、羊、兔卵母细胞做为胞质受体构建重构胚，并经体外培养，发现不同的胞质受体均能支持重构胚的早期发育，但受体胞质对重构卵的重构胚早期发育速度和卵裂率均有显著影响。其中山羊-牛和山羊-兔重构卵的融合率分别为59.91%和74.10%，重构胚的卵裂率分别为57.48%和72.57%，囊胚率分别为9.59%和18.06%；山羊-兔和山羊-山羊重构卵的融合率分别是60.01%和74.10%，重构胚的卵裂率分别是56.68%和72.57%，囊胚率分别是11.76%和 18.06%。各组数据间差异显著（p<0.05）。2.山羊-兔和山羊-牛两种异种克隆胚的体外早期发育速度与山羊-山羊同种克隆胚及山羊的超排胚胎相比，以山羊-兔异种克隆胚的发育速度最快，牛的最慢，山羊-山羊同种克隆胚及山羊超排胚胎的次之。此外，山羊-兔异种克隆胚、山羊-山羊同种克隆胚和正常受精胚的囊胚期卵裂球数目也存在显著差异（86±4.09 vs 105±3.26 vs120±4.05，p<0.05）。3．经对超排获得的山羊受精胚、山羊同种克隆胚和羊-兔异种克隆胚的囊胚期胚胎染色体分析表明，三种来源的胚胎其染色体数目均为60条，且所有常染色体均为端部着丝点染色体，X染色体为第二大的端部着丝点染色体，说明山羊同种克隆胚和山羊-兔异种克隆胚均为山羊的核型，是供体细胞核在不同胞质中的重新编程的结果。4.不同融合操作及培养密度对于羊-兔重构胚的早期发育速度及卵裂率均有影响。其中再融合卵（RFO）与融合激活卵（FAO）共培养与分滴培养其融合率分别为74.88和60.44%，重构胚的卵裂率分别为72.04%和56.79%，囊胚率分别为20.69%和8.70%，各组数据间差异显著（P<0.05）。而且共培养重构胚的发育速度也要明显快于分滴培养。而不同的培养密度（15枚/100μl和5枚/100μl）对于重构胚的卵裂率没有显著影响。其融合率分别为75.00%和74.04%，卵裂率分别为72.22%和71.43%，囊胚率分别为20.00%和18.18%，各组数据间差异不显著（P>0.05）。但密度大组重构<WP=7>胚发育速度则明显要快一些。5. 以山羊为受体，采用配种后再移植、去透明带移植和常规移植三种移植方法，共移植山羊-牛异种克隆胚62枚（配种后移植12枚，常规移植50枚）、山羊-兔异克隆胚158枚（配种后移植47枚，去透明带移植42枚，常规移植69枚）、山羊-山羊同种克隆胚2枚（常规方法移植），但均未产生克隆后代。综上所述，以山羊体细胞为供体，以兔、牛和山羊的卵母细胞的胞质为受体进行体细胞核移植，表明不同的受体胞质均能支持克隆胚早期发育，并且这些克隆胚均表现为供体的核型，但受体胞质以及电融合和培养方案对克隆胚早期发育都有显著的影响。这些研究结果，不仅对完善动物克隆技术，而且对促进动物繁殖学、发育生物学的发展均具有重要的科学意义。更多还原

【Abstract】 The three types of reconstructed embryos(reconstructed embryo of goat-rabbit, goat-bovine and goat-goat) were produced respectively by nuclear transfer using goat ear firbroblast cells as donors and rabbit, bovine and goat ooctyes as recipients, and the factors of cytoplast environments influencing cloned embryo development in vitro, cytoplast effect in nuclear transfer, the methods of transferring cloned embryo and the effects of pregnancy factor on implantation of cloned embryo were studied in this experiment, in order to supply basis for solving the difficulty of implantation of inter-species cloned embryo in recipient and improving the efficiency of cloning. The experiment results were as following:All cytoplast recipients derived from oocytes of rabbit, bovine and goat can support early development of cloned embryo, the fusion rate of reconstructed embryos of goat-bovine and goat-rabbit were 59.91% and 74.10%, their cleaved rate were 57.48% and 72.57%, blastocyst rate were 9.59% and18.06% respectively, and the fusion of reconstructed embryos of goat-rabbit and goat-goat were 60.01% and 74.10%, their cleaved rate were 56.68% and 72.57%, blastocyst rate were 11.76% and18.06% respectively. These results showed that the type of cytoplast recipient influencing significantly the development of reconstructed embryos (P<0.05). The development speed was different in different type of cloned embryos, in those, development speed of cloned embryos of goat-rabbit was fast, and cloned embryos of goat-bovine was slowest in the comparison of cloned embryos of goat-rabbit, goat-bovine, goat-goat and embryos got by superovulation, but the development speed had no significant difference between cloned embryos of goat-goat and superovulization embryos. In addition, siganificant differences were found in the number of blostomers of blastocysts of goat-rabbit, goat-goat and superovulation embryos (86±4.09 vs 105±3.26 vs124.05，P<0.05).Three kinds of embryos’ chromosome, including goat fertilization embryo, goat-goat cloned embryo and goat-rabbit cloned embryo were analyzed, The results showed that all of them have 60 chromosomes with same character, all were telocentric chromosome, besides the X-chromosome is the second larger telocentric chromosome. These results proved that all chromosome of cloned embryos of goat-rabbit or goat-goat derived from goat, that is to say, the nuclei of goat somatic cell were <WP=9>reprogrammed in the different recipient cytoplast.The development speed and the cleaved rate of cloned embryos were influenced by fusion protocols and culture density. The fusion rates of RFO (re-fusion oocyte) and FAO (fusion-activation ooctye) were 74.88% and 60.44%(P<0.05), and the cleaved rates of them were 72.04% and 56.79%(P<0.05), the blastocyst rate is 20.69% and 8.70% respectively (P<0.05). But the significant differences were not found in the development of cloned embryos between with 15 embryos/100μl and with 5 embryos/100μl density culture protocol, in which, fusion rate, cleaved rate and blastocyst rate were respectively 75.00% and 74.04%, 72.22% and 71.43% and 20.00% and 18.18% (P>0.05). However, the development speed of cloned embryos was quicker in high density group than that in low density group.Cloned embryos were transferred with 3 methods (after mating transfer, zona free transfer and traditional transfer) and female goats as recipients. With these method, 62 cloned embryos of goat-bovine (12 embryos with after mating transfer method, 50 embryos with traditional transfer method), 158 cloned embryos of goat-rabbit (47 embryos with after mating transfer method, 42 embryos with zona free transfer method and 69 embryos with traditional transfer method ) and 2 cloned embryos of goat-goat (with traditional transfer method ) were transferred. But, Up to date, offspring had been not produced. In summary, that results suggested that the cytoplast of rabbit, bovine or goat oocyte can support reprogramming of nuclei of goat somatic cells and early development in vitro of reconstructed embryos, a更多还原