| 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 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... |
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