本文選題：牦牛 + 硫酸鋅��； 參考：《西南民族大學》2017年碩士論文

【摘要】：采用體外成熟(IVM)方法獲得大批量卵母細胞是現(xiàn)代繁殖育種技術順利開展的重要環(huán)節(jié),卵母細胞的成熟質量直接影響后續(xù)受精卵的發(fā)育能力。與黃牛相比,體外成熟的牦牛卵母細胞進行體外受精的效率普遍較低。卵母細胞氧化損傷等原因都影響牦牛的受精效率,因此,如何提高牦牛卵母細胞的體外成熟質量是當下急需解決的關鍵問題之一。鋅是動物機體必不可少的微量元素,對細胞的生長、增殖、分裂及免疫功能方面具有重要的作用,迄今為止,鋅對牦牛卵母細胞體外成熟的影響仍未見報道。此外,研究發(fā)現(xiàn),生存在青藏高原地區(qū)的牦牛血液中鋅元素出現(xiàn)季節(jié)性缺乏。本研究旨在探究硫酸鋅對牦牛卵母細胞體外成熟及其后期發(fā)育的影響,結果如下:1.經檢測發(fā)現(xiàn),40頭牦牛血液和相對應卵泡液中鋅的濃度分別是1.03±0.01μg/m L和0.20±0.03μg/m L,IVM和IVF培養(yǎng)基中未檢測到鋅。2.添加不同濃度(0、0.5、1.0、1.5和2.0μg/m L)的硫酸鋅,對牦牛卵丘細胞的擴增程度和卵母細胞核的成熟率均無顯著影響(P0.05)。3.IVM培養(yǎng)基中添加鋅(1.5和2.0μg/m L),成熟的牦牛卵母細胞中的GSH含量顯著的高于對照組和其他低濃度組(P0.05);與對照組相比,牦牛卵母細胞中的ROS含量隨鋅濃度的增加(1.0、1.5和2.0μg/m L)而顯著降低,且差異極顯著(P0.01);此外,添加2.0μg/m L Zn顯著提高了牦牛卵母細胞復合體中SOD的含量(P0.05)。4.用添加了不同濃度鋅的成熟牦牛卵母細胞進行體外受精,結果發(fā)現(xiàn)0.5、1.0、1.5和2.0μg/m L鋅組的卵裂率顯著高于對照組(72.67%、75.31%、81.17%和80.07%VS 60.74%)(P0.05);添加2.0μg/m L硫酸鋅能顯著提高囊胚率(P0.05)。5.運用qRT-PCR檢測不同濃度鋅對牦牛卵母細胞中鋅轉運體蛋白基因mRNA的表達變化。結果顯示,經不同鋅濃度處理的牦牛成熟卵母細胞中的鋅轉運蛋白Zn T(SLC30A3、SLC306、SLC30A9)和ZIP(SLC39A6、SLC39A14)家族成員的m RNA均表達,隨鋅離子濃度的增加均表現(xiàn)為上調趨勢,但上升幅度各有差異,且隨發(fā)育至囊胚階段,表達量均表現(xiàn)降低。綜上所述,在體外培養(yǎng)體系中鋅的添加可以通過調節(jié)細胞內GSH、ROS和SOD水平和相關轉運基因的表達來改善牦牛卵母細胞和胚胎的發(fā)育潛力。為此,在體外培養(yǎng)牦牛卵母細胞時,適量的添加鋅有利于牦牛胚胎的體外生產效率。
[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 鹵0.01 渭 g / mL and 0.20 鹵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.
【學位授予單位】：西南民族大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S823.85

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