Screening And Expression Of Related Proteins Before And After Freezing Of Sheep Sperm

As a high-quality meat breed,Dober sheep is.favored by the breeding industry,but because of the generally low fertility rate of sheep frozen semen in daily production,the promotion and popularization of excellent breeds such as Dober and their frozen semen products are restricted.The freezing process will bring a series of effects and damage to sperm.Some studies have shown that freezing can change the structure and function of sperm,damage the plasma membrane of the head and the structure of the acrosome,flagellum and axillary wire at the tail.These effects and damage This may be part of the reason for the low fertility rate of frozen sperm.However,most of the current research on frozen semen quality has focused on morphological changes of freeze-thaw,improvement of external storage conditions,and optimization of protective agent ratios.The internal mechanism of the decline in fertility rate after sperm freezing has not been studied in depth.With the continuous progress of proteomics research methods,new opportunities have been brought to further study the internal mechanism of the decrease in fertility rate after sperm freezing.In this experiment,the extracted fresh and frozen sperm proteins were subjected to SDS-PAGE electrophoresis,and the distinct protein bands were identified by liquid chromatography-tandem mass spectrometry(LC-MS/MS).A total of 489 proteins were obtained.Relevant literature,biochemical analysis(mass spectrometry analysis,GO analysis,KEGG analysis)and other methods screened several proteins that may be related to sheep frozen sperm quality,and determined the relative expression of their genes.The purpose of this experiment is to provide some theoretical basis for further revealing the internal mechanism of sheep sperm cryo-apoptosis or injury.1.The sperm motility of frozen sperm and fresh sperm were measured,and the plasma membrane integrity of sperm of both groups was tested by using eosin biostaining combined with hypotonic swelling test.The results showed that the sperm motility and plasma membrane integrity of the frozen sperm decreased significantly(P<0.01)compared to fresh sperm.2.SDS-PAGE electrophoresis was performed on the extracted frozen sperm and fresh sperm total proteins.The electrophoresis results showed that the frozen sperm and fresh sperm groups had significantly different positions at 100KDa,20KDa,and 14KDa.Liquid chromatography tandem mass spectrometry(LC-MS/MS)was used to identify the 489 proteins obtained by mass spectrometry analysis,bioinformatics analysis,etc.,and finally screened out five proteins that may be related to frozen sperm quality(Tubulin alpha chain,Tubulin beta chain,Tektin 5,Outer dense fiber of sperm tails 2,Hexokinase 1).3.Using GAPDH as an internal reference,the relative expression levels of the genes corresponding to the above five proteins were measured by fluorescent quantitative PCR.The results showed that the relative expression levels of the five genes after freezing were differently down-regulated compared to fresh sperm.Conclusion:The sperm motility,plasma membrane integrity and comprehensive quality are positively correlated with the expression levels of the corresponding genes of the above proteins.