猪卵巢卵母细胞体外受精的研究

发布时间：2018-09-12 19:24

【摘要】：本研究以废弃猪卵巢为实验初始材料,用机械法抽取获得未成熟卵母细胞,体外培养成熟后用于体外受精实验的研究。本文比较了影响猪卵母细胞体外受精的影响因素,以期能够进一步完善猪胚胎的体外生产体系,为胚胎技术的进一步发展提供技术保障。本研究包括以下内容:(1)比较在m TBM中添加不同浓度的咖啡因、不同受精时间及精子上浮时间对猪体外受精的影响。(2)比较猪卵母细胞体外不同成熟时间、受精过程中颗粒细胞的存在情况及低氧环境对猪体外受精的影响。(3)研究精子获能液中添加不同浓度的Ionomycin及Ionomycin作用不同时间对猪体外成熟卵母细胞体外受精效果的影响。主要研究结果如下:1.以改良m TBM为基础液,添加咖啡因对受精胚胎的卵裂率和囊胚率有明显的促进作用,添加3mmol/L咖啡因组的受精胚胎卵裂率显著高于添加5mmol/L咖啡因组和未添加咖啡因组(P0.05),且该组的囊胚率最高;精子上浮处理1h组的卵裂率和囊胚率均显著高于其它实验组(P0.05);精卵孵育时间对受精胚胎卵裂率无显著影响(P0.05),随受精时间延长至9h,受精胚胎的囊胚率显著降低。通过各实验组比较发现,受精液中添加3mmol/L咖啡因,精子上浮1.0h,受精6h能得到较好的体外受精结果。2.体外成熟40~42h组的卵母细胞体外受精胚胎的卵裂率和囊胚率显著高于体外成熟培养36~38h组和46~48h组(P0.05);保留2-3层颗粒细胞组与不脱颗粒细胞组的受精胚胎卵裂率和囊胚率差异均不显著(P0.05),且这两组的实验结果显著高于无颗粒细胞组(P0.05);就卵裂率和囊胚率而言,5%CO2组显著高于10%CO2组和15%CO2组(P0.05)。通过对各实验组的比较,卵母细胞体外成熟培养40~42h,保留全部颗粒细胞和气相环境为5%CO2能得到较好的体外受精结果。3.获能液中添加10μmol/L的Ionomycin能显著提高受精胚胎的卵裂率和囊胚率(P0.05);用Ionomycin处理精子30min能获得较好的体外受精效果。
[Abstract]:In this study, the * oocytes from pigs were taken as experimental materials, and immature oocytes were obtained by mechanical extraction. In vitro fertilization and maturation experiments were carried out. The * * * factors affecting the in vitro fertilization of porcine oocytes were compared, in order to further improve the in vitro production system of pig embryo and further embryo technology. This research includes the following contents: (1) compare the effects of different concentrations of caffeine in M TBM, different fertilization time and sperm floating time on in vitro fertilization of pigs * * (2) compare the existence of granulosa cells in different maturation time of porcine oocytes, and the in vitro fertilization of pigs in hypoxia environment * (3) to study the effects of different concentrations of Ionomycin and Ionomycin on the in vitro fertilization of porcine in vitro maturation * oocytes. The main results are as follows: 1.. Using the modified m TBM as the base solution, adding caffeine can significantly promote the cleavage rate and blastocyst rate of fertilized embryos, and add 3mmol/L The cleavage rate of fertilized embryos in caffeine group was significantly higher than that in caffeine group (P The blastocyst rate of fertilized embryos decreased significantly after prolonging to 9 h. The results showed that the cleavage rate and blastocyst rate of oocytes in vitro fertilized embryos in 40-42 H group were significantly higher than those in 36-38 H group and in vitro matured embryos in 36-38 h group. The cleavage rate and blastocyst rate of fertilized embryos with 2-3 layers of granulosa cells and without degranulation cells were not significantly different (P 0.05), and the experimental results of these two groups were significantly higher than those of non-granulosa cells (P 0.05); the cleavage rate and blastocyst rate of 5% CO2 group were significantly higher than those of 10% CO2 group and 15% CO2 group (P 0.05). In vitro fertilization results were better when the oocytes were cultured in vitro for 40-42 hours and all granulosa cells and 5% CO2 were retained. 3. The cleavage rate and blastocyst rate of fertilized embryos were significantly increased by adding 10 micromol/L of Ionomycin to the capacitated medium (P 0.05), and the sperm treated with Ionomycin for 30 minutes had better in vitro fertilization effect.
【学位授予单位】：青海大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828

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