白藜芦醇对牛精液冷冻保存效果的影响

发布时间：2018-06-27 23:01

本文选题：白藜芦醇 + 牛精液　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：随着人工授精技术的快速发展,精液冷冻保存技术也不断得以完善。但是在冷冻保存及解冻过程中,由于诸多因素的存在,解冻后的牛精子的活率、质膜完整率、顶体完整率与鲜精相比仍然会大幅度降低,精子受精能力也会大幅度减弱。导致这一问题的原因不仅仅与诸如温度、渗透压和氧化应激等因素有关,而且可能与精子本身结构及功能的改变有关。白藜芦醇是一种天然的非黄酮类多酚类复合物,在抗炎、抗病毒、抗肿瘤和调节氧化还原信号等方面发挥重要作用,并且能够与多种细胞内信号通路的靶分子相互作用。目前,关于不同浓度白藜芦醇对牛精液冷冻保存效果的影响未见相关报道。本研究拟在牛精液冷冻稀释液中添加不同浓度的白藜芦醇,测定冷冻后牛精子的活率、顶体完整率、质膜完整率及抗氧化指标包括总抗氧化能力、丙二醛含量和谷胱甘肽过氧化物酶活力,最后筛选适用于牛精液冷冻保存的白藜芦醇的最佳添加浓度。本研究主要获得以下结果:1.在一定浓度范围内(15~150μM),随着白藜芦醇浓度的升高,冻融精子的活率、顶体完整率和质膜完整率也随之升高。但是,当白藜芦醇浓度超过一定水平时,冷冻精子质量显著下降。当白藜芦醇浓度为150μM时,冻融精子的活率、顶体完整率和质膜完整率均显著高于对照组和其他组(P0.05),分别为55.7%、72.5%和53.5%;当白藜芦醇浓度达到200μM时,精子活率、顶体完整率和质膜完整率相对于150μM处理组均有所下降,精子活率显著低于对照组(P0.05),但质膜完整率与对照组相比差异不显著(P0.05);200μM处理组的顶体完整率与100μM处理组的顶体完整率差异不显著(P0.05)。表明150μM白藜芦醇为最适添加浓度。2.在牛精液冷冻保存的过程中,随着白藜芦醇添加浓度的升高(15~150μM),精子的T-AOC和GSH-Px活力逐渐增强,MDA含量逐渐降低,说明精子的抗氧化能力与白藜芦醇浓度有一定的剂量依赖性。稀释液中添加150μM白藜芦醇,精子的GSH-Px活力最强,MDA含量最低,分别达到113.3μmol/L和96.14 nmol/mL,与对照组差异显著(P0.05);100μM组精子的T-AOC最高,但与150μM组差异不显著(P0.05);当白藜芦醇浓度达到200μM时,其总抗氧化能力(T-AOC)及MDA含量显著低于对照组(P0.05),GSH-Px活力低于对照组,但差异不显著(P0.05)。因此,添加150μM白藜芦醇时,冻融精子的抗氧化能力最高。3.相关性分析结果表明,精子活率与T-AOC、MDA含量之间为显著相关(P0.05),相关系数分别为0.85和-0.76;顶体完整率、MDA含量与T-AOC、GSH-Px的相关性均不显著(P0.05);GSH-Px活力与精子活率、质膜完整率的相关性达到极显著水平(P0.01),相关系数分别为0.99和0.98,这为评估精子膜受损程度提供了一个新的检测指标,该方法更加准确。综上所述,在牛精液冷冻稀释液中添加一定浓度水平的白藜芦醇能够提高冻融牛精子的质量和抗氧化能力,150μM白藜芦醇的保护效果最佳。
[Abstract]:With the rapid development of artificial insemination technology, semen cryopreservation technology has been improved. However, during freezing and thawing, the sperm motility, plasma membrane integrity, acrosome integrity and fertilization ability of the thawed bovine sperm will still be significantly decreased compared with fresh spermatozoa due to the existence of many factors. The causes of this problem are not only related to factors such as temperature, osmotic pressure and oxidative stress, but also to changes in the structure and function of sperm itself. Resveratrol is a natural non-flavonoid polyphenol complex, which plays an important role in anti-inflammatory, anti-virus, anti-tumor and redox signal regulation, and can interact with target molecules of various intracellular signaling pathways. At present, there is no report on the effect of resveratrol at different concentrations on freezing preservation of bovine semen. In this study, different concentrations of resveratrol were added to frozen bovine semen to determine the sperm motility, acrosome integrity, plasma membrane integrity and antioxidant indexes including total antioxidant capacity. Malondialdehyde (MDA) content and glutathione peroxidase (GSH) activity were determined. Finally, the optimal concentration of resveratrol for frozen preservation of bovine semen was selected. In this study, the following results were obtained: 1: 1. With the increase of resveratrol concentration, the motility, acrosome integrity and plasma membrane integrity of freeze-thawed spermatozoa increased within a certain concentration range (15 ~ 150 渭 M). However, when the concentration of resveratrol exceeded a certain level, the quality of frozen sperm decreased significantly. When the concentration of resveratrol was 150 渭 M, the viability rate, acrosome integrity rate and plasma membrane integrity rate of freeze-thawed sperm were significantly higher than those of the control group and other groups (P0.05), which were 55.7% and 53.5%, respectively, when the concentration of resveratrol reached 200 渭 M, the sperm motility rate was significantly higher than that of the control group and other groups. The acrosome integrity rate and plasma membrane integrity rate decreased compared with 150 渭 M treatment group. Sperm motility was significantly lower than the control group (P0.05), but the plasma membrane integrity rate was not significantly different from the control group (P0.05). The acrosome integrity rate of 200 渭 M treatment group was not significantly different from that of 100 渭 M treatment group (P0.05). The optimum concentration of resveratrol was 150 渭 M. During the cryopreservation of bovine semen, with the increase of resveratrol concentration (15 ~ 150 渭 M), the activity of T-AOC and GSH-Px decreased gradually, indicating that the antioxidant ability of sperm was dependent on the concentration of resveratrol in a dose-dependent manner. The concentration of GSH-Px in sperm was the lowest, reaching 113.3 渭 mol / L and 96.14 nmol / mL, respectively. The T-AOC of spermatozoa was the highest in 100 渭 M group (P0.05), but not significantly different from that in 150 渭 M group (P0.05), and when the concentration of resveratrol reached 113.3 渭 M, the concentration of resveratrol reached 200 渭 M. The total antioxidant capacity (T-AOC) and MDA content were significantly lower than the control group (P0.05) GSH-Px activity was lower than the control group, but the difference was not significant (P0.05). Therefore, when 150 渭 M resveratrol was added, the antioxidation ability of freeze-thawed spermatozoa was the highest. 3. The results of correlation analysis showed that there was a significant correlation between sperm motility and T-AOCX MDA content (P0.05), the correlation coefficients were 0.85 and -0.76, respectively, and the correlation between acrosome integrity rate and T-AOCnGSH-Px activity was not significant (P0.05), and there was no significant correlation between sperm motility and sperm motility. The correlation of plasma membrane integrity rate was very significant (P0.01), the correlation coefficients were 0.99 and 0.98, respectively, which provided a new index for evaluating the degree of sperm membrane damage, and the method was more accurate. In conclusion, adding a certain concentration of resveratrol to the frozen diluent of bovine semen can improve the quality of frozen and thawed bovine spermatozoa and the antioxidant ability of 150 渭 M resveratrol.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823

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