猪卵母细胞体外成熟及单精注射制备胚胎的研究

发布时间：2019-03-15 14:03

【摘要】：将精子载体转基因法(SMGT)与单精注射(ICSI)结合制备转基因动物是一种新的转基因技术,研究历史较短,但其可行性和高效性已在多个物种得到试验证实。随着胚胎生物技术的发展,对体外成熟卵母细胞的质量要求日益增加。目前通过体外培养获得成熟卵母细胞,其胞质成熟度还是不及体内的成熟卵母细胞,影响卵母细胞体外成熟质量的因素有体外成熟培养的时间控制、体外成熟培养液中不同添加物、卵母细胞获取方法和卵母细胞质量等等。因此需对这些环节进行优化研究,本实验为研究如何在众多的卵母细胞中挑选出合适的卵母细胞用于体外成熟培养,为了能够获得更多优质的卵母细胞。以体外成熟的猪卵母细胞为材料,对猪卵母细胞体外受精和孤雌激活等相关问题进行了初步研究,同时也探索了单精子显微注射技术制备胚胎的研究。实验内容有抽取猪卵母细胞根据卵丘细胞的层数及完整性将卵母细胞分成A、B、C三组,并分别进行体外成熟培养,体外成熟44h观察卵母细胞第一极体的排出情况,试验结果:A、B、C三组卵母细胞的第一极体排出率分别为(68.94%vs47.94%vs31.65%),结果表明A组卵母细胞的的第一极体排出率明显高于B、C两组,且A组和B组差异极显著(p0.01),A组和C组pbI排出率差异极显著(p0.0]),B组和C组pbI排出率差异极显著(p0.01)。A、B、C三组卵母细胞进行孤雌激活实验,在体外培养24-48h观察卵裂情况,体外培养5-6d观察囊胚情况,实验数据结果为A、B、C三组卵母细胞培养24-48h后得到的卵裂率分别为87.72%,82.38%,52.94%。A、B组卵母细胞的卵裂率差异不显著(p0.05),A、C组卵母细胞的卵裂率差异极显著(p0.0]),B、C组卵母细胞差异极显著(p0.01)。A、B、C三组卵母细胞体外培养第5-6d的时候囊胚率分别为10.96%,6.21%,2.88%。A组卵母细胞囊胚率高于B组和C组,A组和C组差异显著(p0.05),B组囊胚率高于C组,差异显著(p0.05)。不同级别的成熟卵母细胞进行体外受精后,A组卵母细胞的囊胚率(10.61%)明显高于B组囊胚率(5.34%)和C组囊胚率(3.58%),A组和B组卵母细胞的囊胚率差异极显著(P0.01),B组和C组卵母细胞的囊胚率差异不显著(P0.05)。这一结果表明了卵丘细胞对卵母细胞体外受精受精卵胚胎的发育有影响。④进行了猪卵母细胞胞质内单精子显微注射制备胚胎的实验,并作出了改进,实验成功的制备出6枚受精卵囊胚,此试验为制备转基因猪研究奠定基础。结论:卵丘细胞对卵母细胞的体外成熟和胚胎发育有促进作用,选择3层以上包裹致密的卵丘-卵母细胞复合体体外成熟效果更好。单精子显微注射制备猪胚胎的实验成功的制备6枚受精卵囊胚。
[Abstract]:It is a new transgenic technology to prepare transgenic animals by combining (SMGT) with single sperm injection (ICSI), but its feasibility and efficiency have been proved by experiments in many species. With the development of embryo biotechnology, the quality of oocytes matured in vitro is increasing day by day. At present, the cytoplasmic maturity of mature oocytes obtained by in vitro culture is still less than that of mature oocytes in vivo, and the factors affecting the quality of in vitro maturation of oocytes are controlled by the time of in vitro maturation culture. In vitro maturation medium, different additives, methods of oocyte acquisition and oocyte quality, and so on. Therefore, these links need to be optimized. The purpose of this experiment is to study how to select suitable oocytes from a large number of oocytes for in vitro maturation culture in order to obtain more high-quality oocytes. In this paper, the in vitro fertilization and parthenogenetic activation of porcine oocytes were studied using porcine oocytes matured in vitro. At the same time, the preparation of embryos by single sperm microinjection was also explored. According to the number and integrity of cumulus cells, porcine oocytes were divided into three groups: group A, group B, and group C. The oocytes were cultured in vitro and matured in vitro for 44 hours to observe the efflux of the first polar body of oocytes. The results showed that the first polar body efflux rate of oocytes in A, B and C groups were 68.94% vs 47.94% vs 31.65%, respectively. The results showed that the first polar body efflux rate of oocytes in A group was significantly higher than that in B and C groups. There was a significant difference between group A and group B (p0.01), A group and C group; p0.0]), B group and C group had significant difference in pbI efflux rate (p0.01). A, B, A, B, P < 0.01, P < 0.01). The parthenogenetic activation test of oocytes in group C was performed. The cleavage of oocytes was observed for 24 hours in vitro, and blastocysts were observed for 5 days after culture in vitro. The results were as follows: a, B, A, B. The cleavage rate of oocytes in group C was 87.72%, 82.38% and 52.94% respectively after 24 h culture. There was no significant difference in cleavage rate between group A and group B (p0.05), A,). The cleavage rate of oocytes in group C was significantly different (p0.0), that in group B was significantly different from that in group C (p0.01). The blastocyst rate of oocytes in group A, B and C was 10.96% at the 5th to 6th day of culture, respectively, and the blastocyst rate of oocytes in group C was 10.96% after 5-6 days of culture. The oocyte blastocyst rate in group A was higher than that in group B and C, and there was significant difference between group A and group C (p0.05), B group was higher than that in group C (p0.05). The rate of blastocyst of group A (10.61%) was significantly higher than that of group B (5.34%) and group C (3.58%) after in vitro fertilization of mature oocytes of different grades, and the blastocyst rate of group A (10.61%) was significantly higher than that of group B (5.34%) and C (3.58%). There was no significant difference in blastocyst rate between group A and group B (P0.01) between), B group and C group (P0.05). The results showed that cumulus cells had an effect on the development of oocytes in vitro fertilized eggs. (4) the microinjection of intracytoplasmic sperm into porcine oocytes to prepare embryos was carried out and some improvements were made. Six fertilized blastocysts were successfully prepared, which laid a foundation for the preparation of transgenic pigs. Conclusion: cumulus cells can promote the maturation of oocytes in vitro and embryo development. It is better to select the cumulus-oocyte complex with more than three layers of density. Six fertilized oocysts were successfully prepared by single sperm microinjection of porcine embryos.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828

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