Study On Differential Protein Of Freezing Semen In Sheep Breeds

Sperm is a very complex and highly differentiated cell.The sperm freezing and thawing process will lead to irreversible damage to sperm organelle structure and physiological process of cells.As a result,sperm motility,locomotor performance,acrosome and plasma membrane integrity are reduced.And lead to reduces sperm function and semen quality.In this study,four kind of sheep breeds semen were frozen and iTRAQ technology was used to explore the Differential Protein of semen among sheep breeds.The main findings are as follows:The fresh qualified semen of Australian White,Whitehead dorper,Hu sheep and Blackhead dorper were freezed.The post-freezing results showed that the sperms' overall vitality,the advancement of vitality and rapid advance vitality of Hu sheep were significantly higher than those of Australian White sheep,Whitehead dorper and Blackhead dorper(P<0.05).The sperms' overall vitality,advancement of vitality and rapid advance vitality of Whitehead dorper were significantly lower than Australian White,Hu sheep and Blackhead dorper(P<0.05).It can be seen that the semen frost resistance of Hu sheep is best among the four sheep breeds,and the Whitehead dorper is the worst.According to the above results,the semen of rams were collected and mixed as semen pool of Hu sheep and Whitehead dorper.And the samples of Hu sheep fresh sperm(hx),Hu sheep live sperm(hh),Hu sheep frozen sperm(hd),and Whitehead fresh sperm(bx),Whitehead live sperm(bh),Whitehead frozen sperm(bd),were collected respestively.The technology of iTRAQ was used to analysis the relative and absolute quantitative of total protein in each sample.As a result,there are 56,54,121,and 205 differential proteins respectively for hh.vs.bh,hd.vs.bd,hd.vs.hx,and bd.vs.bx(The differential proteins were FC?1.5,FC?0.67,and p-value?0.05).When the absolute value of log2 FC was greater than 0.8,the difference protein in the group of hh.vs.bh group were ENO1?S100A12 and HK3,that in the hd.vs.bd group were KRT4,SYNE1,KRT73,that in the hd.vs.hx group was PRDX2,KRT5,AHSG,that in the bd.vs.bx group were ENO1,ALB,NPPC,Hyaluronidase,Alpha-1-acid glycoprotein,Beta-defensin,SPACA3,SPA17,AHSG,TSGA10,AFP,SPATA19,SPEM1.As a result of GO enrichment,the differential proteins in the hh.vs.bh group were mainly enriched in intracellular non-membrane-bound organelle,biological regulation,metal ion binding,etc;the differential proteins in the hd.vs.bd group were mainly enriched in intracellular,lipoprotein metabolic process,structural molecule activity,etc;the differential proteins in the hd.vs.hx group were mainly enriched in organelle,response to oxidative stress,nucleic acid binding,etc;the differential proteins in the bd.vs.bx group were mainly enriched in extracellular regions,carbohydrate metabolic process,enzyme inhibitor activity,etc.As a result of KEGG enrichment,differential protein accumulation in hh.vs.bh mainly involves Morphine addiction,Arachidonic acid metabolism,etc;differential protein accumulation in hd.vs.bd mainly involves Spliceosome,MAPK signaling pathway,etc;differential protein accumulation in hd.vs.hx mainly involves N-Glycan biosynthesis,Hepatitis B,etc;bd.vs.bx differential protein accumulation mainly involves Vitamin digestion and absorption,Parkinson's disease,etc.In summary,that the semen frost resistance of Hu sheep is best and that of Whitehead dorper is the worst among the four sheep breeds.With absolute value of log2 FC more than 0.8,there are 3 difference proteins in which one is up-regulation and two are down-regulation in hh.vs.bh group,there are 3 difference proteins which are all down-regulation in the hd.vs.bd group,there are 3 difference proteins which one is up-regulation and two are down-regulation in the hd.vs.hx group,there are 13 difference proteins which three are up-regulation and ten are down-regulation in the bd.vs.bx group.