锌对牦牛卵母细胞减数分裂成熟及其发育的影响

发布时间：2018-06-14 19:42

本文选题：牦牛 + 硫酸锌　；参考：《西南民族大学》2017年硕士论文

【摘要】：采用体外成熟(IVM)方法获得大批量卵母细胞是现代繁殖育种技术顺利开展的重要环节,卵母细胞的成熟质量直接影响后续受精卵的发育能力。与黄牛相比,体外成熟的牦牛卵母细胞进行体外受精的效率普遍较低。卵母细胞氧化损伤等原因都影响牦牛的受精效率,因此,如何提高牦牛卵母细胞的体外成熟质量是当下急需解决的关键问题之一。锌是动物机体必不可少的微量元素,对细胞的生长、增殖、分裂及免疫功能方面具有重要的作用,迄今为止,锌对牦牛卵母细胞体外成熟的影响仍未见报道。此外,研究发现,生存在青藏高原地区的牦牛血液中锌元素出现季节性缺乏。本研究旨在探究硫酸锌对牦牛卵母细胞体外成熟及其后期发育的影响,结果如下:1.经检测发现,40头牦牛血液和相对应卵泡液中锌的浓度分别是1.03±0.01μg/m L和0.20±0.03μg/m L,IVM和IVF培养基中未检测到锌。2.添加不同浓度(0、0.5、1.0、1.5和2.0μg/m L)的硫酸锌,对牦牛卵丘细胞的扩增程度和卵母细胞核的成熟率均无显著影响(P0.05)。3.IVM培养基中添加锌(1.5和2.0μg/m L),成熟的牦牛卵母细胞中的GSH含量显著的高于对照组和其他低浓度组(P0.05);与对照组相比,牦牛卵母细胞中的ROS含量随锌浓度的增加(1.0、1.5和2.0μg/m L)而显著降低,且差异极显著(P0.01);此外,添加2.0μg/m L Zn显著提高了牦牛卵母细胞复合体中SOD的含量(P0.05)。4.用添加了不同浓度锌的成熟牦牛卵母细胞进行体外受精,结果发现0.5、1.0、1.5和2.0μg/m L锌组的卵裂率显著高于对照组(72.67%、75.31%、81.17%和80.07%VS 60.74%)(P0.05);添加2.0μg/m L硫酸锌能显著提高囊胚率(P0.05)。5.运用qRT-PCR检测不同浓度锌对牦牛卵母细胞中锌转运体蛋白基因mRNA的表达变化。结果显示,经不同锌浓度处理的牦牛成熟卵母细胞中的锌转运蛋白Zn T(SLC30A3、SLC306、SLC30A9)和ZIP(SLC39A6、SLC39A14)家族成员的m RNA均表达,随锌离子浓度的增加均表现为上调趋势,但上升幅度各有差异,且随发育至囊胚阶段,表达量均表现降低。综上所述,在体外培养体系中锌的添加可以通过调节细胞内GSH、ROS和SOD水平和相关转运基因的表达来改善牦牛卵母细胞和胚胎的发育潜力。为此,在体外培养牦牛卵母细胞时,适量的添加锌有利于牦牛胚胎的体外生产效率。
[Abstract]:In vitro maturation IVM method is an important step in the development of modern breeding technology. The maturation quality of oocytes directly affects the development ability of subsequent fertilized eggs. The efficiency of in vitro fertilization of yak oocytes was generally lower than that of yellow cattle. The oxidative damage of oocytes affects the fertilization efficiency of yaks. Therefore, how to improve the quality of yak oocytes maturation in vitro is one of the key problems that need to be solved. Zinc is an essential trace element in animal body, which plays an important role in cell growth, proliferation, division and immune function. So far, the effect of zinc on yak oocyte maturation in vitro has not been reported. In addition, it was found that there was seasonal deficiency of zinc in the blood of yaks living in Qinghai-Tibet Plateau. The aim of this study was to investigate the effect of zinc sulfate on in vitro maturation and anaphase development of yak oocytes. The results are as follows: 1. Zinc concentrations in blood and corresponding follicular fluid of 40 yaks were found to be 1.03 卤0.01 渭 g / mL and 0.20 卤0.03 渭 g / mL IVM and IVF medium respectively. Zinc sulfate with different concentrations of 0. 5, 0. 5, 1. 0, 1. 5 and 2. 0 渭 g / mL, There was no significant effect on the expansion degree of yak cumulus cells and the maturation rate of oocyte nucleus. 3. The content of GSH in the mature yak oocytes was significantly higher than that in the control group and other low concentration groups compared with the control group, and the addition of zinc to 1.5 and 2.0 渭 g / mL of zinc in IVM medium had no significant effect on the proliferation of yak cumulus cells and the maturation rate of oocyte nucleus, and compared with the control group, the content of GSH in mature yak oocytes was significantly higher than that in the control group. The Ros content in yak oocytes decreased significantly with the increase of zinc concentration (1.0 渭 g / mL and 2.0 渭 g / mL), and the difference was very significant (P 0.01), in addition, the addition of 2.0 渭 g / mL Zn significantly increased the content of SOD in yak oocyte complex (P0.05 路4). Mature yak oocytes with different concentrations of zinc were fertilized in vitro. The results showed that the cleavage rate of 0.5 渭 g / mL zinc group was significantly higher than that of the control group (72.67% and 80.07% vs 60.74%), and the blastocyst rate was significantly increased by adding 2.0 渭 g / mL zinc sulfate. The expression of zinc transporter gene mRNA in yak oocytes was detected by qRT-PCR. The results showed that the mRNA expression of zinc transporter Zn TsSLC30A3 (SLC30A3) and ZIPASLC39A6 (SLC39A14) in yak matured oocytes were all up-regulated with the increase of zinc concentration, but there were differences between them. And with the development of blastocyst stage, the expression level decreased. In conclusion, zinc supplementation in vitro culture system can improve the developmental potential of yak oocytes and embryos by regulating the levels of GSH Ros and SOD and the expression of related transporter genes. Therefore, when yak oocytes were cultured in vitro, zinc supplementation was beneficial to the in vitro production efficiency of yak embryos.
【学位授予单位】：西南民族大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823.85

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