驴卵母细胞成熟及GV期玻璃化冷冻的研究

发布时间：2018-01-06 13:09

  本文关键词：驴卵母细胞成熟及GV期玻璃化冷冻的研究　出处：《内蒙古农业大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 驴 GV期卵母细胞 玻璃化冷冻 微丝 线粒体

【摘要】：本实验主要研究不同采集方法回收卵母细胞的效率及不同浓度的FSH对卵母细胞成熟率的影响,同时对GV期的卵母细胞进行玻璃化冷冻,并对其显微结构进行观察,研究结果如下：1.切割法获得卵母细胞成功率为81.57%,综合法获得卵母细胞成功率为79.01%,两者差异不显著(P0.05),而利用抽吸法获得卵母细胞成功率为48.09%,显著低于抽吸法和综合法(P0.05)。2.卵母细胞成熟培养体系中分别添加不同浓度的FSH,根据成熟率的不同来分析FSH的最适浓度,结果表明60μg/mL、80μg/mL的FSH的成熟率(21.43%、38.98%)显著低于添加100μg/mL、120μg/mLFSH的成熟率(51.77%、47.29%)(P0.05),添加100μg/mLFSH成熟体系的成熟率略高于120μg/mL的成熟体系,但差异不显著(P0.05)。3.本试验比较了不同培养时间对卵母细胞成熟率的影响,结果表明随着培养时间的延长,成熟率稳步提高,到达30～32 h后成熟率趋于稳定。4.采用四个浓度梯度(Ⅰ、Ⅱ、Ⅲ、Ⅳ)分别对GV期卵母细胞进行玻璃化冷冻,解冻后体外成熟对其形态正常率和成熟率进行统计,结果表明四种冷冻保护剂成熟培养后卵母细胞的形态正常率分别为84.62%、87.50%、71.43%、68.63%,差异不显著(P0.05),其中冷冻液Ⅱ的成熟率(28.13%)显著高于其他三组的成熟率,但都显著低于对照组。由此可知最佳的冷冻剂为II组：20%DMSO+20%EG+60%M199+0.25M Sucrose。5.分别选用普通玻璃细管、OPS管、GMP管作为冷冻载体冷冻GV期驴卵母细胞,解冻后成熟培养并对其形态正常率和成熟率进行统计,发现卵母细胞形态正常率分别为81.67%、87.50%、76.47%,差异不显著(P0.05)；OPS管组卵母细胞成熟率(28.13%)显著高于普通玻璃细管组(11.67%),但与GMP管组(23.53%)差异不显著,因此OPS管是最合适的冷冻载体。6.本试验还对正常卵母细胞及解冻后的卵母细胞进行微丝及线粒体的免疫荧光染色,免疫荧光结果显示冷冻组卵母细胞微丝在皮质区的分布明显减少,部分微丝杂乱分布于胞质中央,冷冻组卵母细胞的线粒体数量明显低于正常组,由此可以说明冷冻对卵母细胞微观结构有损伤。
[Abstract]:The effect of different collection methods on oocyte recovery efficiency and FSH concentration on the maturation rate of oocytes was studied. At the same time, vitrification of GV phase oocytes was carried out. The results were as follows: 1. The success rate of oocytes obtained by cutting method was 81.57, and the success rate of oocytes obtained by comprehensive method was 79.01%. There was no significant difference between the two groups (P 0.05), but the success rate of oocytes obtained by aspiration was 48.09%. It was significantly lower than that of suction method and comprehensive method (P0.05 路2.The optimum concentration of FSH was analyzed according to the different maturation rate of FSH). The results showed that the maturation rate of 60 渭 g / mL 80 渭 g / mL FSH was significantly lower than that of adding 100 渭 g / mL. The maturation rate of 120 渭 g / mLFSH was 51.77 and 47.29, respectively. The maturation rate of the matured system added 100 渭 g / mLFSH was slightly higher than that of 120 渭 g / mL maturation system. The effect of different culture time on the maturation rate of oocytes was compared. The results showed that the maturation rate increased steadily with the extension of culture time. The maturation rate of GV oocytes was stable at 30 h. 4. Four concentration gradients (鈪，

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