Study On Genetic Polymorphism Of HSP70 Gene And Its Protein Expression Level And Their Relationship With Semen Quality In Buffalo

In this study,effect of breed,temperature and age on semen quality was explored with analysis of production data in 44 Nili-Ravi and 33 Murrah buffalo in breeding station of Guangxi.The relationship between SNP locus of HSP70 gene and semen quality was detected using DNA pool sequencing and HRM genotyping in Nili-Ravi and Murrah buffalo..The effect of HSP60 and HSP70 protein expression level on semen quality was investigated through examining the activities of acrosin,acid phosphatase and alpha glucosidase,and protein content of HSP60 and HSP70.These results provides theoretical basis for study on the mechanism of HSP70 in semen and breeding on river buffalo bull.The main results are as following:1.There was a significant difference betweensperm motility and semen volume(P<0.05)in Nili-Ravi and Murrah buffalo.The sperm motility in Nili-Ravi buffalo was higher than that in Murrah buffalo.The sperm density and frost resistance 3-5 year-old bulls in Nili-Ravi buffalo were significantly higher than that of 6-9 and 10-16 year-old bull semen(P<0.05),sperm frost resistance of 3-5 year-old bull semen was significantly higher than that of 10-16 year-old bull(P<0.05),higher than the 6-9 year old bull(P>0.05).The results of age factor effect on the semen quality in Murrah buffalo showed that there was no significant difference among 3 age groups in sperm motility,semen volume and sperm motility after freezing,sperm density and living sperm count of 6-9 year-old bull semen was significantly higher than that of 10-16 year-old bull semen(P<0.05).In respect of frost resistance of sperm,,10-16 year-old bull was the lowest(0.44),3-5 year-old bull was the highest(0.47).,and there was significant difference(P<0.05)between them;semen quality in winter(January to March)was the worst,the sperm motility was significantly lower than in summer(June to October),spring(April,May),and winter(November,December)(P<0.05).2.There was three SNP locus in the CDS region of HSP70 gene(602,708and 1284),and among these SNP locus,the mutation at 602 was nonsynonymous.The results of statistical analysis showed that different genotypes of 602 loci had significant effect on sperm motility,living sperm count and sperm freezing tolerance in Murrah and Nivi-Ravi buffalo(P<0.05),but no significant effect on semen volume,708 sites and 1284 sites had no significant effect on the bull semen quality(P>0.05).3.In respect of acrosin activity,acid phosphatase,alpha glucosidase activity in semen of different sperm motility,acrosin and alpha glucosidase activity in high sperm motility group was significantly higher than that in low sperm motility group(P<0.05),but there was no significant relationship between the activity of acid phosphatase and sperm motility(P>0.05).4.Through examining HSP60 and HSP70 protein expression levelusing Western blotting,the results showed that the HSP70 protein expression level in high sperm motility group was significantly higher than that in low sperm motilitygroup(P<0.05),there was no significant difference between the HSP60 protein expression level and sperm motility(P>0.05).