冷冻载体优化制备及对牛卵母细胞玻璃化冷冻效果的影响

发布时间：2018-06-26 21:56

本文选题：牛 + 卵母细胞　；参考：《河南科技大学》2017年硕士论文

【摘要】：哺乳动物的卵子冷冻技术在珍稀动物品种资源保存、人类辅助生殖领域及胚胎研究中一直是重要的研究内容。卵母细胞玻璃化冷冻过程中,冷冻体积、冷冻保护剂浓度、冷冻降温-解冻速率等因素是影响冷冻效果的关键,合适的冷冻载体可以减小冷冻体积,从而提高降温-解冻的速率。近年来,虽有多种载体的报道研究,但不同研究者报道的制作方法和效果不尽相同。因此本研究旨在探索不同载体的研究方法,以卵母细胞冷冻后的发育能力为评定指标,通过4种冷冻载体的对比试验,筛选出在我们现有实验条件下的最佳玻璃化冷冻载体并探讨冷冻不同发育时期卵母细胞的冷冻效果,从而为提高卵母细胞的冷冻保存效果奠定技术基础。1.冷冻载体制备方法研究试验1:OPS载体的优化。将细管一分为二,分别以常规酒精灯和简易酒精灯为热源,对细管一端旋转加热,待前端待细管前端膨大时,迅速用小镊子将膨大部分向另一端平稳拉伸,经冷却固定后,剪去前端膨大部获得OPS冷冻载体。结果表明,只有以简易小酒精灯为热源可对OPS载体成功优化,且此方法不仅能增加细管的受热均匀度,易于操作,而且也能减少材料损耗。试验2:GMP载体制备方法研究。以卵针前端细部(内径约为1 mm,管壁厚度约为0.09 mm,长度约为6 cm)为原材料,分别以PC-10型拉针仪、常规酒精灯和简易酒精灯为热源,探究GMP载体的制备方法;以玻璃管(外径0.8cm)为原材料,以酒精喷灯为热源,探究GMP载体的制备方法。结果显示:以酒精喷灯为热源,以细玻璃管为原材料可以成功制备出GMP载体。试验3:GMP冷冻载体的改进。以酒精喷灯和常规酒精灯为热源,分别对外径为0.8 cm、0.4 cm和0.3 cm玻璃管旋转加热。结果显示,外径为0.8 cm的玻璃管制备的GMP载体与外径为0.5 cm的制备效果并无差异;外径为0.4 cm和0.3 cm的细玻璃管以酒精喷灯为热源,制备出的载体外径在0.43~0.5 mm间,保留其后端的载杆经测量发现距前端载体约4 cm可以作为GMP载体使用,但该方法材料损耗较大;以常规酒精灯为热源时,制备的载体经测量外径为0.4~0.5 mm,管壁厚度约为0.03 mm,距后端载体约3 cm可作为GMP载体,并且在制备的过程中载体和载杆间会有一小玻璃气泡产生,冷冻时对虹吸作用也有一定促进作用。结论:用常规酒精灯,外径为0.3 cm和0.4 cm玻璃管制备的GMP载体效果最佳,将塑料细管一分为二,使其一端加热可在原有OPS制备方法基础上极大降低了损耗。2.不同载体对牛未成熟卵母细胞玻璃冷冻效果的比较将抽取的COCs随机分为5组,OPS组、GMP组、冷冻叶片组、自制叶片组以及对照组。4组冷冻组细胞经液氮冷冻解冻后分别进行IVM、IVF和IVC,新鲜对照组不做冷冻处理,直接进行IVM、IVF和IVC,统计成熟率、卵裂率和囊胚率。结果显示:冷冻组的形态正常率、成熟率、卵裂率以及囊胚率与新鲜对照组各项指标(100%、78.1%±3.1%、64.4%±2.8%、34.7%±2.5%)均显著差异(P㩳0.05),OPS组和GMP组形态正常率(74.3%±1.8%,72.5%±2.6%)无显著差异(P㧐0.05),上述二组均显著低于冷冻叶片组(82.1%±1.3%,P㩳0.05),但三组间成熟率、卵裂率和囊胚率均无显著差异(P㧐0.05),自制叶片组则与其他冷冻组的各项发育指标差异均显著(P㩳0.05)。结论:自制OPS、GMP冷冻载体均可成功地玻璃化冷冻牛未成熟卵母细胞。3.不同发育时期卵母细胞玻璃化冷冻效果的影响将抽取的COCs随机分为4组,体外培养0 h、8 h、16 h、以及22 h,采用GMP冷冻法分别冷冻培养不同时间的卵母细胞,解冻后继续依此培养24 h、16h、8 h、和2 h,之后分别进行IVM、IVF、IVC,统计各组的形态正常率、成熟率、卵裂率以及囊胚率。结果显示:经解冻后培养0 h、8 h、16 h和22 h的形态正常率分别为72.7%、75.3%、77.7%和78.0%,四组间差异均不显著(P㧐0.05),培养0 h的卵母细胞同培养8h的各项发育指标差异均不显著(P㧐0.05),但培养16 h的卵母细胞与前两者各项发育指标差异均显著(P㩳0.05),而培养22 h的卵母细胞各项发育指标均高于其他发育时期组。结论:卵母细胞的抗冻性随其成熟程度增加而增强。
[Abstract]:The technology of mammalian oocyte cryopreservation is an important research content in the conservation of rare animal species resources, in the field of human assisted reproduction and in the study of embryos. In the process of vitrification of oocytes, the factors such as freezing volume, concentration of cryopreservation agent, freezing cooling rate and thawing rate are the key factors affecting the freezing effect and suitable freezing carrier. The freezing volume can be reduced to increase the rate of cooling and thawing. In recent years, although there have been reports of various carriers, the methods and effects of different researchers have been different. Therefore, this study aims to explore the research methods of different carriers. The development ability of oocyte after cryosurgery is evaluated by 4 kinds of cryosurgery. The optimal vitrification carrier under the existing experimental conditions was screened and the cryopreservation effect of the oocyte in different cryopreservation period was investigated, thus the technical basis for improving the cryopreservation of oocytes was established, and the optimization of the study on the preparation of the.1. cryopreservation method was established. The tubes were divided into two parts. Do not take the conventional alcohol lamp and the simple alcohol lamp as the heat source, and heat the end of the pipe. When the front end of the tube is expanded, the bulk is stretched quickly with the small tweezers to the other end. After the cooling is fixed, the OPS freezer is cut to the front end. The result shows that only the simple small alcohol lamp can be used as the heat source for the OPS carrier. Work optimization, and this method can not only increase the heat uniformity of the tube, easy to operate, but also reduce the loss of material. Study on the preparation method of 2:GMP carrier. The raw material is the front end of the egg needle (the inner diameter is about 1 mm, the thickness of the tube wall is about 0.09 mm, the length is about 6 cm) as the raw material, with the PC-10 type drawing needle instrument, the conventional alcohol lamp and the simple alcohol lamp respectively. For the heat source, the preparation method of GMP carrier is explored. Using the glass tube (outer diameter 0.8cm) as the raw material and the alcohol spray lamp as the heat source, the preparation method of the GMP carrier is explored. The results show that the GMP carrier can be successfully prepared with the alcohol spray lamp as the heat source and the fine glass tube is used as the raw material. The improvement of the 3: GMP freezing carrier is tested. The alcohol spray lamp and the conventional alcohol lamp are used. For the heat source, the external diameter of 0.8 cm, 0.4 cm and 0.3 cm glass tubes was rotated. The results showed that the GMP carrier with the outer diameter of 0.8 cm was not different from the preparation effect of the outer diameter of 0.5 cm; the outer diameter of the thin glass tube with the diameter of 0.4 cm and 0.3 cm was the heat source of the alcohol spray lamp, and the outer diameter of the prepared carrier was between the 0.43~0.5 mm and the back end. It is found that about 4 cm from the front end carrier can be used as a GMP carrier, but the material loss is great. When the conventional alcohol lamp is used as the heat source, the measured outer diameter of the carrier is 0.4~0.5 mm, the thickness of the tube wall is about 0.03 mm and the back end carrier is about 3 cm as the GMP carrier, and there will be a carrier and the carrier in the process of preparation. A small glass bubble is produced and freezing has a certain effect on the siphon effect. Conclusion: the GMP carrier with 0.3 cm and 0.4 cm glass tubes with the conventional alcohol lamp is the best, and the plastic pipe is divided into two, and the heat can be heated on the basis of the original OPS preparation method, and the loss of.2. is greatly reduced to the immature cattle. The comparison of the effect of vitreous cryopreservation of oocyte was divided into 5 groups: OPS, GMP, frozen leaves, homemade leaves and.4 groups of the control group, IVM, IVF and IVC were frozen after freezing and thawing. IVM, IVF and IVC, statistical maturity, cleavage rate and blastocyst rate were carried out in the fresh control group without freezing. The results showed that the morphological normal rate, maturity, cleavage rate and blastocyst rate of the cryopreservation group were significantly different (100%, 78.1% + 3.1%, 64.4% + 2.8%, 34.7% + 2.5%) of the fresh control group (P? 0.05). There was no significant difference in the normal rate (74.3% + 1.8%, 72.5% + 2.6%) in group OPS and GMP group (P? 0.05), and all of these groups were significantly lower than those of the frozen leaves group. 1.3%, P? 0.05), but there was no significant difference between the three groups of maturation rates, cleavage rate and blastocyst rate (P? 0.05). The difference between the self-made leaf group and the other freezing groups was significant (P? 0.05). Conclusion: homemade OPS, GMP cryopreservation can be successfully vitrified oocytes of cold frozen bovine oocytes at different developmental stages of vitrification. The effect of frozen effect was randomly divided into 4 groups of COCs, which were cultured in vitro 0 h, 8 h, 16 h, and 22 h. The oocytes were frozen for different time by GMP freezing method. After thawing, 24 h, 16h, 8 h, and 2 h were continued, then IVM, IVF, and 2 h respectively. The results showed that the normal morphological rates of 0 h, 8 h, 16 h and 22 h after thawing were 72.7%, 75.3%, 77.7% and 78%, respectively, and there was no significant difference between the four groups (P? 0.05). The differences in the development indexes of the oocytes with the culture of 0 h were not significant (P? 0.05), but the differences in the development indices of the oocytes in the cultured 16 h were both significant (P? P). However, the developmental indexes of oocytes cultured at 22 h were higher than those of other developmental stages. Conclusion: the antifreeze ability of oocytes increases with the maturity of oocytes.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823

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