Differentiation Of Cow Deficient Semen And The Development Of Eradication Technology As Well As Its Application In Sex-sorted Frozen Semen Production

The breeding technology of dairy cow sex-sorted frozen semen and artificial insemination can quickly increase the number of dairy cows with high quality and improve the economic benefits of dairy farming.It is the most successful and most effective means of breeding biotechnology in dairy farming industry.Defected sperm of dairy cows include three types : morphological deformities,acrosome damage,DNA defects and dead sperm.Defected sperm in dairy cows semen have a direct bad influence on the utilization of genetic resources of dairy cows,the production efficiency of sex-sorted frozen semen and the conception rate of artificial insemination.The objective of this study was to identify three types of defected sperm from dairy cows semen by specific proteins and remove defected sperm by combing with the immune magnetic beads before the X / Y sperm separation.The purpose ofthese operations is to improve the production efficiency of frozen semen of dairy cows and the proportion of normal sperm to promote the industrialization of sex-sorted frozen semen.At the same time,we want to provide some experimental basis for the research and application of artificial assisted reproduction and other related aspects.This study includes two parts: First,the study on the identification of dairy cows' sperm with abnormalities,damaged acrosome and DNA defect and removal technology of defected sperm of dairy cows semen in combination with immune magnetic bead method.First,the correlation between the level of ubiquitination of sperm and sperm abnormalities and DNA defect was analyzed.The results showed that the ubiquitination level of sperm was significantly correlated with sperm abnormalities by means of western blotting and single-channel flow cytometry(p<0.05).The results of TUNEL assay and ubiquitination level of sperm by two-channel flow cytometry were further demonstrated that the ubiquitination level of sperm has a significant correlation with sperm DNA defect level(p <0.05).On the basis of these results,the anti-ubiquitination protein was selected as the specific antibody in combination with immune magnetic beads for removing the abnormalities and damaged acrosome sperm in the semen of the dairy cows.The results showed that the technical process can effectively remove the abnormalities and damaged acrosome sperm in the semen of the dairy cows(p <0.05).We conclude that the combination of specific anti-ubiquitination protein and immune magnetic beads can remove the abnormalities and damaged acrosome sperm of dairy cowssemen before X / Y sperm separation to improve the quality of sex-sorted frozen semen.Secondly,the study on the specific binding of dairy cows' dead sperm to Annexin V was confirmed and the application of immune magnetic beads to remove dead sperm from dairy cows semen.The results showed that Annexin V can bind specifically to dead sperm of dairy cows.According to this,Annexin V was selected as the specific marker of dead sperm of dairy cows,and then the technical process of removing dead sperm from dairy cows semen was designed by using immune magnetic beads combined with Annexin V.The results showed that the sperm motility,membrane integrity,mitochondrial activity and X/Y sperm separation effectiveness were improved with different amplitudes after sorting,indicating that the technical process can effectively remove part of the dead sperm.But it had no significant impact on the quality of sex-sorted frozen semen(p>0.05).We conclude that the use of Annexin V combined with immune magnetic beads method can effectively remove dead sperm from dairy cows sperm and significantly reduce the rate of dead sperm during X/Y sperm separation(p<0.05).This technical process can effectively improve the sex-sorted frozen semen of dairy cows production efficiency,reduce production costs,and promote the industrialization of the product applications.