绵羊胞质内单精子注射（ICSI）的研究

发布时间：2018-03-11 09:16

本文选题：绵羊卵母细胞　切入点：体外成熟　出处：《河南农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：本实验重点研究了影响绵羊胞质内单精子注射(ICSI)中的因素,以期优化绵羊ICSI的操作技术和体外成熟体系,为开展发育机理研究及提高绵羊ICSI效率提供进一步的技术支持。研究结果主要包括如下几个方面:(1)不同季节对卵母细胞体外培养的影响。结果表明季节对卵母细胞的总数量没有影响。春季和秋季获得的可用卵母细胞数显著高于夏季和冬季。就成熟率而言,春季、秋季和冬季差异不显著。综合回收率和成熟率,秋季是绵羊卵母细胞体外培养的最佳时节,而夏季不适绵羊卵母细胞的采集培养。(2)不同成熟时间(20~22h、22~24h、24~26h、26~28h)对ICSI的影响。结果显示卵母细胞的卵裂率随成熟时间的增加而逐渐提高;卵母细胞成熟22~24 h桑囊率最高,之后开始降低。因此,卵母细胞成熟22~24h是进行ICSI的最佳时机。(3)高渗操作液对ICSI的影响,结果显示高渗操作液能显著提高操作成功率,但对卵裂率(43.9%、36.5%;P0.05)和桑囊率(41.4%、38.7%;P0.05)差异不显著。(4)第一极体位置(6点和12点)对ICSI的影响,结果发现极体位置对ICSI卵裂率(32.5%vs 34.5%,P0.05)和桑囊率(33.6%、33.3%;P0.05)都没有显著影响。(5)超声波处理不同状态精子(鲜精和冻精)对ICSI的影响。结果表明超声波处理鲜精和冻精对卵母细胞的卵裂率(39.1%vs 37.4%,P0.05)和桑囊率(33.7%vs 35.1%,P0.05)均差异不显著。(6)精子不同处理方式(NaOH和超声波处理)对ICSI的影响,结果表明两种处理方式对卵母细胞的卵裂率(37.4%vs 46.6%,P0.05)差异显著;NaOH处理组的桑囊率(52.1%)差异显著高于超声波处理组(35.1%)。(7)精子共孵育对ICSI卵发育的影响。以NaOH处理精子ICSI作为对照,NaOH处理精子与质粒共孵育后对ICSI胚的卵裂率(37.9%vs 46.6%,P0.05)和桑囊率(45.3%vs52.1%,P0.05)的影响差异不显著。(8)非共培养和卵丘细胞共培养系统对ICSI胚的影响。结果显示共培养和非共培养对卵裂率(41.7%vs 44.0%,P0.05)和桑囊率(41.8%vs 35.7%,P0.05)影响不显著。
[Abstract]:In order to optimize the operation technology and in vitro maturation system of sheep ICSI, the factors affecting ICSI were studied. The results include the following aspects: 1) the effect of different seasons on the culture of oocytes in vitro. The results show that seasons affect oocytes fine. The number of available oocytes obtained in spring and autumn was significantly higher than that in summer and winter. There was no significant difference in spring, autumn and winter. The results showed that the cleavage rate of oocytes increased gradually with the increase of maturation time, and the rate of mulberry sac was the highest at 22 h maturation, and the effect of different maturation time on ICSI was observed in different maturation time of unwell sheep oocytes. The results showed that the cleavage rate of oocytes increased gradually with the increase of maturation time, and the mulberry sac rate of oocytes matured at 22 hours after maturation was the highest. The effect of hyperosmotic solution on ICSI was found to be the best time for ICSI. The results showed that hyperosmotic solution could significantly improve the successful rate of operation. But there was no significant difference in cleavage rate (43.9%) and mulberry sac rate (41.4%, 38.7%, P0.05). The effect of the first polar body position (6 and 12:00) on ICSI was not significant. The results showed that polar body position had no significant effect on ICSI cleavage rate (32.5 vs 34.5p 0.05) and mulberry sac rate (33.6% and 33.33.3% P0.05). (P 0.05) Ultrasonic treatment of different state sperm (fresh and frozen sperm) on ICSI. The results showed that ultrasonic treatment of fresh and frozen spermatozoa on oocyte fine oocyte. The cell cleavage rate (39.1 vs 37.4P0.05) and mulberry sac rate (33.7 vs 35.1and P0.05) had no significant difference (P 0.05).) the effects of different treatments of sperm (NaOH and ultrasound) on ICSI were not significant. The results showed that the effect of two treatments on oocyte cleavage rate 37.4 vs 46.6% (P0.05) the difference of mulberry sac rate in NaOH treatment group was significantly higher than that in ultrasonic treatment group (35.1g / L). The effect of co-incubation of spermatozoa on the oocyte development of ICSI was studied by using ICSI treated with NaOH as an example. There was no significant difference between the cleavage rate of ICSI embryos treated with NaOH and plasmids (37.9 vs 46.6% P0.05) and the rate of mulberry sac (45.3 vs 52.1% P0.05). The effect of non-co-culture and cumulus cell co-culture system on ICSI embryos was not significant. The results showed that the effects of co-culture and non-co-culture on ICSI embryos were not significant. There was no significant effect on cleavage rate 41.7% vs 44.0% P0.05) and mulberry sac rate 41.8% vs 35.7P0.05).
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S826

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