绵羊卵母细胞体外成熟、孤雌激活及核移植胚胎发育的研究

发布时间：2018-05-21 02:21

本文选题：绵羊卵母细胞 + 超数排卵　；参考：《河南农业大学》2015年硕士论文

【摘要】：本试验研究了绵羊幼羔超数排卵和成年羊卵母细胞的体外成熟、孤雌激活以及体细胞核移植胚胎发育的特点,以期优化绵羊超排、卵成熟、卵活化、胚胎发育条件,为开展发育机理研究及提高绵羊克隆效率提供进一步的技术支持。本试验研究结果主要包括以下几个方面:(1)本试验采用宁波、中科院和加拿大激素分别对幼羔进行超排处理,对比不同激素对幼羔超排效果的影响。其可用COCs百分比分别为69.6%、87.9%、88.4%,平均可用卵数分别为15.1枚、25.1枚、27.1枚,差异显著(P0.05),说明宁波产激素超排效果不如中科院和加拿大激素,而中科院和加拿大之间的可用COCs百分比和平均可用卵数差异不显著(P0.05),说明中科院和加拿大激素使用效果相差不大,但是加拿大激素可用COCs百分比和平均可用卵数比中科院激素分别高0.5%和2枚,说明加拿大激素效果最好。(2)添加FBS培养的卵母细胞,幼羔组和成年羊组的成熟率分别为51.1%和77.3%,两者差异显著(P0.05),添加ESS培养的卵母细胞,幼羔组和成年羊组分别为59.0%和78.9%,两者差异显著(P0.05),说明成年羊卵母细胞的体外成熟效果要优于幼羔。添加ESS的幼羔组成熟率比添加FBS组高7.9%,两者差异显著(P0.05),说明添加ESS能够提高超排羔羊卵母细胞的体外成熟率。(3)比较Ionomycin+6-DMAP和A23187+(CHX和CB)两种激活方式,幼羔一和幼羔二组获得的卵裂率和囊胚率分别为50.7%、13.4%和48.0%、11.1%,两组的卵裂率和囊胚率差异皆不显著(P0.05),成年羊一和成年羊二组的卵裂率和囊胚率分别为69.5%、29.5%和67.6%、28.6%,两组间卵裂率和囊胚率差异不显著(P0.05),但是,成年羊一组的卵裂率和囊胚率比成年羊二组分别高出1.9%和0.9%,幼羔一组的卵裂率和囊胚率比幼羔二组分别高出2.7%和2.3%,而成年羊组卵裂率和囊胚率皆显著高于幼羔组(P0.05),说明两种激活方式都能有效激活绵羊卵母细胞,而成年羊卵母细胞激活后发育效果要比幼羔卵母细胞好。(4)比较体外培养22~24h后幼羔卵母细胞和成年羊卵母细胞去核效果,其去核个数、去核成功率分别为72、75、60.0%、50.3%,幼羔卵母细胞去核成功率比成年羊卵母细胞高9.7%,两者差异显著(P0.05),说明体外成熟22~24h的幼羔卵母细胞去核效果优于成年羊卵母细胞。(5)幼羔组和成年羊组重构胚的卵裂率和囊胚率分别为29.0%、5.0%、58.8%、17.0%,成年羊组卵裂率和囊胚率比幼羔组分别高出29.8%和12%,差异显著(P0.05),说明幼羔超排卵母细胞核移植重构胚发育效果不如成年羊卵母细胞。
[Abstract]:The characteristics of ovulation, parthenogenetic activation and somatic nuclear transfer embryo development of ovulation and adult sheep oocytes were studied in order to optimize the conditions of ovulation, ovum maturation, oocyte activation and embryo development. It provides further technical support for the study of development mechanism and the improvement of sheep cloning efficiency. The results of this study mainly include the following aspects: 1) the effects of different hormones on the superovulation of young lambs were studied by using Ningbo, Chinese Academy of Sciences and Canadian hormones respectively. The percentages of available COCs were 69.6% and 87.9%, respectively, and the average number of available eggs was 15.1 eggs. The difference was significant (P 0.05), which indicated that the effect of hormone superovulation in Ningbo was not as good as that of Chinese Academy of Sciences and Canadian hormone. However, there was no significant difference in the percentage of available COCs and the average number of available eggs between the Chinese Academy of Sciences and Canada, indicating that there was no significant difference in the effect of hormone use between the Chinese Academy of Sciences and Canada. However, the percentage of available COCs and the average number of available eggs in Canada were 0.5% and 2 higher than those in the Chinese Academy of Sciences, respectively, indicating that Canadian hormone had the best effect on oocytes cultured with FBS. The maturation rates of young lambs and adult sheep were 51.1% and 77.3%, respectively. The difference between the two groups was significant (P 0.05). 59.0% and 78.9% of young lambs and adult lambs had significant difference (P0.05%, respectively), which indicated that the maturation effect of adult sheep oocytes was better than that of young lambs. The maturation rate of lambs supplemented with ESS was 7.9 higher than that with FBS. The difference between the two groups was significant (P 0.05), which indicated that the addition of ESS could increase the maturation rate of oocytes of superovulation lambs in vitro.) compared with Ionomycin 6-DMAP, A23187 CHX and CB3, the in vitro maturation rate of oocytes in superovulation lambs could be increased by adding ESS. The cleavage rate and blastocyst rate were 50.7% and 48.0%, respectively. There was no significant difference in cleavage rate and blastocyst rate between the two groups. The cleavage rate and blastocyst rate of adult sheep group 1 and adult sheep group 2 were 69.529.5% and 67.67.628%, respectively. There was no significant difference between blastocyst rate and blastocyst rate, however, The cleavage rate and blastocyst rate of the adult sheep group were 1.9% and 0.9% higher than those of the adult sheep group, respectively. The cleavage rate and blastocyst rate of the lambs group 1 were 2.7% and 2.3% higher than those of the young lambs group, respectively, while the cleavage rate and blastocyst rate of the adult sheep group were significantly higher than those of the young lambs group. P0. 05, which shows that both activation modes can activate ovine oocytes effectively. The developmental effect of adult sheep oocytes after activation was better than that of young lamb oocytes. The success rate of oocyte denucleation in young lambs was higher than that in adult sheep oocytes by 9.7%, and the difference was significant (P0.05), indicating that the oocyte denucleation effect in vitro was better than that in adult sheep oocyte group and adult goat oocyte group (P < 0.05), indicating that the effect of oocyte denucleation in vitro was better than that in adult sheep oocyte group and adult goat oocyte group (P < 0.05). The cleavage rate and blastocyst rate of reconstructed embryos were 29.0% and 5.0%, respectively. The cleavage rate and blastocyst rate of adult sheep group were 29.8% and 12.1% higher than that of young lambs group, respectively. The difference was significant (P 0.05), which indicated that the effect of nuclear transfer of superovulation mother cell of young lambs on embryo reconstruction was not as good as that of adult goat oocytes.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

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