The Research Of The Correlation Between The HSP70Gene Expression And Bull Sperm Motility Before And After Freezing

With the development of science and technology, artificial insemination (AI) has been widely used in cattle breeding, so it is very important to get high quality of sperm. However, in the freezing-thawing process, about40～50%semen quality had been lost, which was not conducive to the value of bull sperm. The present study for cryopreservation of semen, mostly related to the effects of different diluents on the quality of frozen semen, exploring suitable diluent. About the mechanism of different components in dilute solutions to improving the sperm motility, related genes and proteins that affect viability of frozen sperm, the reports were relatively less.Mammalian cells could resist environmental pressure through the synthesis of heat shock proteins, the expression of heat shock protein was related to sperm motility and fertility, and sperm proteome analysis could be an effective method to evaluate sperm resistance. This study extracted total RNA from bovine sperm, discussed the RNA extraction methods, detected HSP70gene expression in sperm at every stage(fresh sperm, after equilibrium, frozen-thawed sperm) By real-time fluorescence quantitative PCR, as well as the sperm quality. And the correlation bewteen HSP70gene expression and sperm motility was analysed.The results were as follows:1. The effective method of extracting RNA was obtained, the ratio of sperm number and volume of Trizol was3.75×107/mL, and DTT could replace β-mercaptoethanol to crack sperm. Through the preliminary study we found that reference gene β-actin and HSP70gene had been expressed in fresh bovine sperm.2. HSP70gene expression level gradually decreased following the freezing process, and the expression in each period showed a significant difference, the expression in fresh sperm (0.985) was significantly higher than the sperm balanced (0.160)(P<0.05), the expression in balanced sperm was significantly higher than that in frozen-thawed sperm (0.082)(P<0.05).3. The bull sperm quality decreased following the freezing-thawing process; HSP70gene expression in sperm with high motility was high, however, HSP70gene expression in sperm with low motility was low.4. There was a strong positive correlation between the HSP70gene expression and sperm motility in each period, the correlation coefficient was0.630(fresh semen),0.785(balance of semen) and0.327(frozen-thawed semen).