牛胚胎体外生产及玻璃化冷冻影响因素的探究

发布时间：2018-05-27 22:45

本文选题：牛胚胎 + 体外受精　；参考：《吉林大学》2017年硕士论文

【摘要】：随着牛畜产业的现代化发展,牛胚胎体外生产、胚胎冷冻、胚胎移植,已经成为牛生产繁殖的重要技术手段。然而在胚胎体外生产与胚胎冷冻过程中,一些问题仍然不明确或存在争议,从而导致体外胚胎生产效率低、胚胎冷冻成功率低等现象。活体采卵技术(Ovum Pick-up,OPU)的出现,为牛胚胎体外生产提供了源源不断的卵母细胞,更加促进了这一产业的发展。但是,由于这一技术的引入以及现场实际条件的制约,使得传统的实验室胚胎生产体系不再适用,需要进行优化调整。ε-聚赖氨酸(ε-Poly-L-lysine,ε-PL)具有抗菌性良好、有效浓度低、安全无毒、可生物降解、水溶性好、热稳定性好等优点,且一些研究表明,ε-PL对细胞膜以及核酸具有一定的保护作用,因此预计它可在胚胎冷冻中起到保护作用。1.本实验对牛胚胎体外生产体系进行了适当的优化与调整,使其更加适用于现场胚胎生产,研究表明:(1)与5%CO2、5%O2、90%N2的培养体系相比,5%CO2、95%空气的培养体系可以取得更高的囊胚率(29.28%,7.29%);(2)当培养液用量为每个胚胎5 uL时,其囊胚率要高于每个胚胎10 uL与3 uL(43.52%,28.57%,40.11%);(3)当胚胎培养数量为10个时,其囊胚率要高于培养数量为5个与3个,采用共培养体系可以提高囊胚率(43.52%,22.83%,16.09%,32.04%);(4)在IVM前保留全部卵丘细胞,IVF前去除适当的卵丘细胞,可以取得更高的囊胚率。2.实验探讨了ε-PL在牛胚胎玻璃化冷冻中的保护作用,研究表明:(1)ε-PL不可单独作为冷冻保护剂,进行胚胎冷冻;(2)传统玻璃化冷冻保护剂在添加ε-PL后,可以取得更加的保护效果,在0 g/L、10 g/L、20 g/L、30 g/L四个浓度中,20 g/L的冷冻效果最佳。(3)当冷冻液缓冲时间为7.5 min时,取得的胚胎冷冻效果要优于5 min、10 min。实验结果表明,ε-PL对牛胚胎玻璃化冷冻具有保护作用.
[Abstract]:With the modernization of cattle and livestock industry, bovine embryo production in vitro, embryo freezing and embryo transfer has become an important technical means for cattle production and reproduction. However, in the process of in vitro embryo production and embryo freezing, some problems are still unclear or controversial, which leads to the low efficiency of in vitro embryo production and the low success rate of embryo freezing. The emergence of Ovum Pick-upopu (Ovum Pick-upOPU) provides a continuous stream of oocytes for in vitro production of bovine embryos and promotes the development of the industry. However, due to the introduction of this technique and the restriction of the field conditions, the traditional laboratory embryo production system is no longer applicable and needs to be optimized and adjusted. 蔚 -Poly-L-lysine (蔚 -PL) has good antibacterial properties, low effective concentration, safe and non-toxic. Some studies have shown that 蔚 -PL has some protective effects on cell membrane and nucleic acid, so it is expected that 蔚 -PL can play a protective role in embryo freezing. The system of bovine embryo production in vitro was optimized and adjusted to make it more suitable for field embryo production. The results showed that the blastocyst rate was higher than that of 5CO _ 2N _ 2 and 5CO _ 2O _ 290N _ 2. A higher blastocyst rate could be obtained when the amount of culture medium was 5uL, and the blastocyst rate was higher than that of each embryo (10uL and 3uL43.52g 28.577.57) when the number of embryo culture was 10, the blastocyst rate was higher than that of 5uL) when the number of embryos cultured was 10, the blastocyst rate was higher. The blastocyst rate of the blastocyst was higher than the number of 5 and 3. The blastocyst rate could be increased by using co-culture system. The blastocyst rate was increased by 22.83% and 16.09% and 32.04% respectively) the proper cumulus cells could be removed before IVM was retained, and a higher blastocyst rate of 0.2 could be obtained. The protective effect of 蔚 -PL on vitrification of bovine embryos was studied. The results showed that 蔚 -PL could not be used as cryopreservation agent alone. At 0 g / L 10 g / L 20 g / L 30 g / L, 20 g / L, the best freezing effect is 20 g / L) when the buffer time of the solution is 7.5 min, the embryo freezing effect is better than that of 5 min or 10 min. The results showed that 蔚-PL had protective effect on vitrification of bovine embryos.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823

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