HSP70多态性及其蛋白表达量与水牛精液品质相关性的研究

发布时间：2018-05-11 17:24

本文选题：水牛 + 精液品质　；参考：《广西大学》2017年硕士论文

【摘要】：本研究通过对广西品改站种公牛站44头尼里-拉菲和33头摩拉水牛近年的精液生产数据的分析,探讨品种、气温和年龄对公牛精液品质的影响。采用DNA池PCR的方法筛选尼里-拉菲和摩拉水牛HSP70基因的SNP位点,通过HRM技术进行基因分型,研究碱基突变和公牛精液品质的相关性。进一步对摩拉和尼里-拉菲水牛中不同精子活力的精液进行顶体酶、酸性磷酸酶、α-葡萄糖苷酶活力测定和HSP60和HSP70蛋白含量定性分析,研究了 HSP70对公牛精液品质的影响,为研究HSP70在精子中的作用机制和河流型公水牛的选育提供了一定的理论基础。试验结果如下:1、结果表明,摩拉和尼里-拉菲水牛种公牛的精液在精子活力和采精量方面有显著性的差异(P0.05),尼里-拉菲水牛的精子活力比摩拉水牛高,其采精量要低于摩拉水牛;在尼里-拉菲水牛中,3-5岁种公牛的精子密度和抗冻性能都要显著的高于6-9岁和10-16岁组公牛(P0.05),3-5岁的种公牛的精子抗冻活力显著高于10-16岁的公牛(P0.05),高于6-9岁的公牛但不显著(P0.05)。摩拉水牛精液品质的年龄因素影响分析结果显示:3个年龄段水牛种公牛的精子活力、精子冻后活力和采精量都没有显著性的差异,6-9岁的种公牛精子密度和活精子数要显著高于10-16岁组(P0.05)。从精子抗冻性方面看,10-16岁组的公牛是最低的,只有0.44,3-5岁的公牛最高,达到了 0.47,有显著性的差异(P0.05);莫拉和尼里-拉菲水牛的精液品质在冬季(1-3月)是最差的,精子活力显著低于夏季(6-10月)和春秋季(4-5 月、11-12 月)(P0.05)。2、在HSP70基因CDS区检测到3个SNP位点,分别是CDs区602bp位点、708bp和1284bp位点,其中602位点为有义突变,导致了甘氨酸→谷氨酸突变,另2处为无义突变。统计分析结果表明:602位点的不同基因型显著影响了摩拉和尼里-拉菲水牛的精子活力,活精子数以及精子抗冻能力(P0.05),但对公水牛采精量和精子密度没有显著性的影响,708位点和1284位点对公牛精液品质无显著性影响(P0.05)。3、在不同活力的摩拉和尼里-拉菲水牛精液的顶体酶、酸性磷酸酶、α-葡萄糖苷酶三种酶活力测定中,高活力组的顶体酶和α-葡萄糖苷酶显著高于低活力组的精液(P0.05),但酸性磷酸酶与精子活力相关性不显著(P0.05)。4、通过HSP60和HSP70蛋白的Western blotting分析,发现与低活力组相比,高活力组的HSP70蛋白要显著高于低活力组(P0.05),其HSP60蛋白含量无显著性差异(P0.05)。
[Abstract]:In this study, the semen production data of 44 Niri-Lafite and 33 Mora buffaloes were analyzed to explore the effects of breed, temperature and age on the semen quality of bulls. The SNP loci of HSP70 gene of Niri-Lafite and Mora buffalo were screened by DNA pool PCR, and the relationship between base mutation and bull semen quality was studied by HRM technique. Furthermore, acrosome enzyme, acid phosphatase, 伪 -glucosidase activity and HSP60 and HSP70 protein content in semen of different sperm motility of Mora and Nili-Lafite buffalo were determined, and the effect of HSP70 on the semen quality of bull was studied. It provides a theoretical basis for the study of the mechanism of HSP70 in spermatozoa and the breeding of river male buffalo. The results were as follows: 1. The results showed that there was a significant difference in sperm motility and sperm yield between Mora and Near-Lafite buffalo, and the sperm motility of Neri Lafite Buffalo was higher than that of Moray Buffalo. The amount of semen collected was lower than that of Mora buffalo. The spermatozoa density and antifreeze activity of 3-5-year-old bull were significantly higher than those of 6-9 year old and 10-16 year old group in Nili Lafite buffalo. The spermatozoa antifreeze activity of breed bull was significantly higher than that of 10-16 years old and 6-9 years old, but it was not significantly different from that of 6-9 years old and 10-16 years old. The age factors influencing semen quality of buffalo showed that the sperm motility of three age buffalo bulls. There was no significant difference in spermatozoa motility and spermatozoa collection after freezing. The sperm density and sperm count of the bull aged 6-9 years were significantly higher than that of the 10-16 years old group (P0.05). From the point of view of sperm antifreeze, the bull of 10-16 years old group was the lowest, and only 0.445-year-old bull had the highest value (0.47, P 0.05), and the semen quality of Mora and Nili-Lafite buffalo was the worst in the winter (from January to March). The motility of spermatozoa was significantly lower than that in summer (June to October) and in spring and autumn (April to December). Three SNP loci were detected in the CDS region of HSP70 gene, namely CDs 602bp locus 708bp and 1284bp locus. Among them, 602 locus was a seminal mutation, which led to glycine glutamate mutation. The other two were nonsense mutation. The results of statistical analysis showed that different genotypes at the 1: 602 locus significantly affected the sperm motility of Mora and Nili Lafite buffalo. The number of live spermatozoa and the antifreeze ability of spermatozoa were P0.05, but there was no significant effect on the amount of spermatozoa and sperm density in male buffalo. There was no significant effect on the semen quality of bull at locus 708 and locus 1284. There was no significant effect on the semen quality of bull. There was no significant effect on the semen quality of bull at different motility of Morrah and Nili-Lafite. The acrosome enzyme of bovine semen, The activity of acid phosphatase and 伪 -glucosidase, The levels of acrosin and 伪 -glucosidase in high activity group were significantly higher than those in low activity group, but the correlation between acid phosphatase and sperm motility was not significant. Western blotting analysis of HSP60 and HSP70 protein showed that compared with low activity group, there was no significant correlation between ACP and sperm motility. The HSP70 protein in the high activity group was significantly higher than that in the low activity group, and the content of HSP60 protein in the high activity group was not significantly different from that in the low activity group.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823.83

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