Study On Protein Expression Differences Of The Sheep Sperm Before And After Cryopreservation

Dorper sheep as a high-quality mutton sheep,has seriously restricted the popularization and application of sheep semen in production due to the low fertilization rate of frozen semen.At present,the research and evaluation of frozen damage of sperm mainly focus on commercial cryoprotectants,while researching on mechanism of frozen damage of sperm is relatively lacking.The freezing and thawing process of sperm will damage and miss some sperm proteins,and the changes of these proteins have a qualitative impact on the function of sperm.The development of proteomics technologies such as two-dimensional electrophoresis and mass spectrometry provides technical support for comprehensively revealing the mechanism of sperm frostbite from the protein level.Therefore,this experiment studies on protein differences of sheep sperm before and after freezing in deep.1)Compared with fresh semen,frozen semen has significantly lower vitality,acrosome integrity rate and sodium hyaluronate reaction rate.2)Using PDQuest to analyze frozen fresh semen two-dimensional electrophoresis map,165 protein spots were detected,the expression difference multiple was set to 2 times,and 75 difference spots were detected,of which 48 protein spots were up-regulated and 27 protein spots were down-regulated.3)DIA mass spectrometry results showed that the abundance ratio?1.5 or ?0.67 and P_value?0.05 were used as screening conditions for differential protein screening between fresh semen group and frozen semen group.A total of 151 differential proteins were screened out,including 58 up-regulated proteins and 93 down-regulated proteins.The GO analysis was used to analyze the identified differential proteins,indicating that the protein changes caused by freezing on sperm are mainly related to stress response.4)According to the mass spectrometry results,differential proteins were screened with a bidirectional range of molecular weight of 15-300kDa,pH3-10 and an expression differential multiple of 2.A total of 78 differential proteins were screened,32 proteins were up-regulated and 46 proteins were down-regulated.The comparison between the two research methods shows that there is no difference in the number of differential proteins detected,and there is difference in the number of up-down proteins.Studies have shown that both two-dimensional electrophoresis and mass spectrometry can identify proteins and compare differential proteins.What's more,the comparison results laid a foundation for exploring the mechanism of sperm freezing.