The Identification Of Marker Proteins Affecting Sperm Freezing Resilience Rate In Dorper Sheep

In the field of in vitro fertilization and artificial assisted reproduction,how semen is better preserved has always received much attention.Sperm resuscitation rate is an important indicator to judge sperm tolerance,it is affected by many factors,such as different seasons,individual differences and physiological conditions of male animals,all of which affect sperm protein concentration and composition.Proteins are the undertaker of all life activities.It has become a research hotspot to study the mechanism of sperm resuscitation from protein.Through freezing and resuscitation tests,the sperm resuscitation rate of 16 Dorper sheep was tested.Finally,three groups of sperm with the high resuscitation rate and three groups of sperm with the lowest resuscitation rate were selected.Using TMT combined with LCMS/MS method to identify differential proteins,and to analyze the expression of differential proteins by GO,PPI and KEGG,to screen the proteins that affect sperm recovery,and to analyze the function of differential proteins,revealing Mechanisms that affect sperm cryopreservation.Our results were listed as follows:1.The test of freezing resuscitation found that the average resuscitation rate of sperm in the three groups with high resuscitation rate was 68.78%;the average recovery rate of sperm in the three groups with low resuscitation rate was 28.21%.2.A total of 2464 differential proteins were identified,of which 2144 were quantified.The differentially expressed conditions were set as multiple>1.2,score>20,and the unique peptides not less than 2.There were significant differences in 184 proteins,of which 112 proteins were up-regulated and 72 proteins were down-regulated.Analysis of GO functional annotations,pathway enrichment and protein interaction revealed that sperm proteins in different resuscitation groups were different,which involved multiple biological processes.3.In high resuscitation group,S100A8,S100A9,S100A12,S100A14,and S100A16 plasma binding proteins regulate sperm viability by controlling Ca2+;HYOU1 participates in anti-sperm apoptosis;PRDX1 protects against antioxidation and cell death;ENOI participates in glycolysis pathway to provide nutrition for sperm.4.In Low resuscitation group,ME3 is involved in sperm metabolism;HSPA9 is lethal to sperm;SOD1 catalyzes oxygen free radicals;VDACs are involved in ATP and Ca2+transport;ADAM20 inhibits sperm-egg binding.These proteins are enriched in low resuscitation group and participate in cell aging,apoptosis,immune damage and oxidative stress.The oxidative phosphorylation and thermogenesis pathway were enriched in the low resuscitation group,which reduced freezing sperm resuscitation rate.In this study,proteomic analysis of sperm from high resuscitation rate group and low resuscitation rate group of Dorper sheep was carried out.The results showed that the factors affecting sperm cryopreservation resuscitation rate of Dupo sheep might be related to the difference of sperm protein expression profiles,which provided a reference for markers of sperm Freezing Resilience Rate.