EGCG对水牛卵母细胞体外成熟和体外受精的影响

发布时间：2018-07-17 16:58

【摘要】：体外胚胎生产中,各种抗氧化剂常被添加于不同的培养液中以提高体外胚胎生产效率。实验表明,表没食子儿茶素没食子酸酯(Epigallocatechin Gallate, EGCG)能够清除健康细胞中ROS,提高配子的抗氧化能力,有利于动物卵母细胞体外成熟和精子受精。本研究主要探讨了EGCG对水牛卵子体外成熟、精子受精能力的影响以及可能的作用机理,旨在使水牛体外胚胎生产高效化。本研究包括三个试验：试验一,EGCG对水牛卵母细胞体外成熟的影响。旨在探讨EGCG对水牛卵母细胞卵丘扩展、成熟率的影响,并初步试验EGCG对水牛配子发育潜能的影响。结果发现：(1)10、20 μmol/L处理组与对照组卵丘扩展指数差异显著(2.49、2.42 vs.2.22, P0.05);5、30 μmol/L处理组卵丘扩展指数也高于对照组,但无统计学意义(2.36、2.28 vs.2.22,P0.05)；(2)10、20μmol/L处理组成熟率显著高于对照组(59.31%、50.98% vs.45.26%,P0.05)；但两组之间差异不显著；5 μmol/L和30 μmol/L处理组的成熟率与对照组差异不显著(47.04%、42.81% vs.45.26%,P0.05)；(3)在成熟液中添加EGCG,各处理组的卵裂率、囊胚率均高于对照组,其中10 μmol/L处理组效果最佳。孤雌激活后,卵裂率和囊胚率均显著高于对照组(67.07%vs.57.53%、31.69% vs.20.66%, p0.05);体外受精后,卵裂率和囊胚率也显著高于对照组(59.64% vs.44.65%、24.68% vs.15.02%, p0.05)。以上结果表明,EGCG能促进水牛卵母细胞体外成熟,并提高了卵母细胞的发育潜能。试验二,EGCG (0、5、10.20、30 μmol/L)对水牛卵母细胞体外成熟阶段抗氧化能力的影响。结果显示：(1)添加20.30 μmol/L的处理组MⅡ期ROS DCF荧光强度与对照组差异显著(18.77、25.39 vs.38.04,P0.05)；(2)各处理组MⅡ期水牛卵母细胞H202含量与对照组差异不显著(4.68、4.33、4.16、4.51 vs. 4.90, P0.05); (3) 5、10、20 μmol/L处理组GSH含量与对照组差异显著(3.0、3.21、3.25 vs.2.59, P0.05); 30 μmol/L处理组GSH含量也高于对照组,但无统计学意义(2.99 vs.2.59,P0.05)。以上结果表明：在水牛卵母细胞体外成熟过程中,EGCG能够降低卵母细胞内活性氧水平,提高GSH的合成,但不能降低H202的含量；(4)综上所述,在卵母细胞体外成熟过程中,EGCG影响氧化反应。试验三,探讨在受精液中添加10 μmol/L的EGCG对水牛精子受精能力的影响以及在成熟液和受精液中同时添加EGCG是否有协同作用。结果显示：在成熟液、受精液、成熟液和受精液中分别添加10μmol/L的EGCG后,各组卵裂率显著高于对照组(61.31%、59.14%、58.57% vs.45.71%,P0.05)；各组的囊胚率显著高于对照组(23.61%%、22.77%、23.56% vs.16.74%,P0.05)。以上结果表明,EGCG能提高水牛精子受精能力和促进早期胚胎发育,但在成熟液和受精液中同时添加EGCG没有协同作用。为了高效方便,本试验中,最佳方案为在成熟液或者受精液中添加10 μmol/L的EGCG。总之,EGCG作为一种高效的抗氧化剂,能够促进水牛卵母细胞体外成熟、体外受精,并提高了胚胎体外发育率。EGCG对提高水牛体外胚胎生产有积极影响,可提高水牛卵子的利用率。本实验室条件中,单独在成熟液或者受精液中添加10 μmol/L EGCG效果最好。
[Abstract]:In vitro embryo production, all kinds of antioxidants are often added to different cultures to improve the efficiency of in vitro embryo production. The experiment shows that epigallocatechin gallate (Epigallocatechin Gallate, EGCG) can remove ROS in healthy cells, improve the anti oxygen ability of gametes, and be beneficial to the maturation and sperm of animal oocytes in vitro. This study mainly discussed the effect of EGCG on the maturation of buffalo ovum in vitro, the effect of sperm fertilization and the possible mechanism of action to make the production of buffalo in vitro embryo efficient. This study includes three experiments: Experiment 1, the effect of EGCG on the maturation of buffalo oocytes in vitro. The purpose of this study is to explore the cumulus expansion of the oocytes of the buffalo oocytes. The effect of EGCG on the development potential of buffalo gamete was preliminarily tested. The results were as follows: (1) the cumulus expansion index of 10,20 mol/L treatment group was significantly different from that of control group (2.49,2.42 vs.2.22, P0.05), and the cumulus expansion index in 5,30 mu mol/L treatment group was also higher than that in the control group, but there was no statistical significance (2.36,2.28 vs.2.22, P0.05); (2) 1 The maturity rate of 0,20 mu mol/L treatment group was significantly higher than that of the control group (59.31%, 50.98% vs.45.26%, P0.05), but there was no significant difference between the two groups. The maturity rate of 5 and 30 micron mol/L treatment groups was not significantly different from the control group (47.04%, 42.81% vs.45.26%, P0.05), and (3) adding EGCG in the mature liquid, the cleavage rate of each treatment group and the blastocyst rate were higher than those of the control group. After the parthenogenetic activation, the cleavage rate and blastocyst rate were significantly higher than those in the control group (67.07%vs.57.53%, 31.69% vs.20.66%, P0.05), and the cleavage rate and blastocyst rate were significantly higher than those of the control group (59.64% vs.44.65%, 24.68% vs.15.02%, P0.05) after in vitro fertilization. The results showed that EGCG could promote buffalo oocyte. The effects of two, EGCG (0,5,10.20,30 mol/L) on the antioxidant capacity of buffalo oocytes in vitro were tested. The results showed that: (1) the difference in the fluorescence intensity of M II ROS DCF in the treatment group with 20.30 micron mol/L was significantly different from that of the control group (18.77,25.39 vs.38.04, P0.05); (2) each treatment group The H202 content of M II buffalo oocyte was not significantly different from that of the control group (4.68,4.33,4.16,4.51 vs. 4.90, P0.05), and (3) the GSH content in the 5,10,20 mu mol/L treatment group was significantly different from the control group (3.0,3.21,3.25 vs.2.59, P0.05), and the 30 mu mol/L treatment group was also higher than the control group, but there was no statistical significance (2.99). The above result table In the process of buffalo oocyte maturation in vitro, EGCG can reduce the level of active oxygen in oocyte and improve the synthesis of GSH, but can not reduce the content of H202. (4) in summary, EGCG affects oxidation reaction in the process of oocyte maturation in vitro. Experiment three, to study the fertilization of sperm in semen by adding 10 mu mol/L of EGCG to sperm fertilization. The results showed that the rate of cleavage in each group was significantly higher than that of the control group (61.31%, 59.14%, 58.57% vs.45.71%, P0.05), and the blastocyst rate in each group was significantly higher than that of the control group (23). The results showed that in the mature solution, the mature solution, the mature solution and the semen were added to the EGCG, the percentage of cleavage was significantly higher than that of the control group (61.31%, 59.14%, 58.57% vs.45.71%, P0.05) in the mature solution, and the mature solution and the semen respectively (23). (23 .61%%, 22.77%, 23.56% vs.16.74%, P0.05). The above results show that EGCG can improve the fertilization ability of buffalo sperm and promote the development of early embryo, but there is no synergy between the addition of EGCG in the mature liquid and the semen. In order to be efficient and convenient, the best solution for this experiment is to add 10 u mol/L EGCG. to the mature solution or the semen, EGCG, EGCG. As a highly effective antioxidant, it can promote the maturation of buffalo oocyte in vitro, in vitro fertilization, and increase the development rate of the embryo in vitro,.EGCG has a positive effect on improving the production of buffalo embryos in vitro, and can improve the utilization of buffalo eggs. In the laboratory conditions, the effect of adding 10 mu mol/L EGCG to the mature solution or the semen is the most effective. OK.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823.83

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