Comparative Analysis Of Quality Traits And Proteomics Between Summer And Winter Duroc Boar Semen

Since the introduction of Duroc boar into china,it has played a major role in promoting the development of lean pigs in China,and has become a high quality breed.However,the semen quality of Duroc boar showed significant difference in summer and winter,and the semen volume and sperm motility was obvious reduced in summer.Nearly 70-80% of the boar sperm motility is below 0.5,and the reproductive capacity of semen was lost.The litter size in sows after artificial insemination with available semen was also significantly lower than that in winter,which seriously affects the breeding efficiency.Traditional researchers always focused their attention on the semen basic quality parameters affected by season,however,the intrinsic molecular mechanism that season affecting the boar semen quality are not well studied.Therefore,this study is the first report to explore the mechanism underlying season-related reduction of sperm quality in Duroc boar from the perspective of energy metabolism,oxidative stress,and protein post-translational modifications.This study mainly includes the following parts:1.We explored the exact nature of the correlation between protein post-translational modifications(PTMs)and sperm motility.Results of the present study indicated that the amount of the P-PKAs,PTP,Pan-Kac and Tub-Kac was greater in sperm of fresh semen samples with relatively greater motility than in sperm of fresh semen samples with relatively lesser motility.Similarly,the amounts of phosphorylation and acetylation gradually decreased with the reduction in the motility of sperm in liquid stored semen samples.Interestingly,the P-PKAs(r = 0.634,P < 0.01)and Pan-Kac(r = 0.380,P < 0.05)were positively correlated with sperm motility in fresh semen,whereas only P-PKAs(r = 0.607,P < 0.01)were positively correlated with sperm motility during liquid storage.2.We measured the quality parameters of semen collected in summer and winter.Our results showed that the volume of semen collected in summer was significantly lower than that in winter,and the sperm concentration was significantly higher than that in winter.Meanwhile,the motility,curvilinear velocity and straight-line velocity of sperm obtained in summer were significantly lower than that in winter.In addition,the membrane and acrosome integrity of sperm in summer were also lower than that in winter.3.We used Label-free quantitative analysis to compare the proteome differences in spermatozoa,seminal plasma and exosomes obtained in summer and winter.As results,95,97 and 88 differentially expressed proteins were detected in sperm,seminal plasma and exosomes respectively.Bioinformatics analysis indicated that these up-regulated proteins(e.g.HSPA2,GSTP1,CLIC4,GAPDH,ALPL,HSPA1 L and ZPBP)identified in summer semen were mainly involved in heat stress,oxidative stress,energy metabolism process and protein post-translational modifications;and these down-regulated proteins(e.g.ARSA,MUC20 and CDH1)were mainly related to fertilization process,cell adhesion and protein phosphorylation process.4.In addition,to verify these biological processes speculated from the bioinformatics analysis,we evaluated the ATP level,ROS level,MDA level,total antioxidant activity and protein modifications level of sperm collected in summer and winter.As expected,compared with winter,the sperm collected in summer had significantly lower ATP level and antioxidant activity.Additionally,the ROS and MDA level of sperm collected in summer were markedly higher than that in winter.Furthermore,results of Western-blot showed that the level of protein phosphorylation and acetylation of sperm harvested in summer were significantly lower than that in winter.The immunofluorescence signal of protein phosphorylation and acetylation in summer sperm were weaker than that in winter sperm,which consistent with the results of Western-blot.Taken together,our results indicated that the mechanisms that season affecting sperm quality are as follows.On the one hand,the sperm are under oxidative stress in summer,generating more ROS,and the excessive ROS has negative impacts on the structure and energy metabolism of sperm.On the other hand,the reduction of protein phosphorylation and acetylation in summer sperm are the other major reasons for the decrease of sperm motility.