Analysis Of The Impact Of Hydrogen Peroxide On The Functional Integrity Of Goat Spermatozoa: Sperm Membrance Integrity, Acrosome Reaction, Acrosin And DNA Integrity

Objective: Hydrogen peroxide(H₂O₂) are known to regulate spem capacitation, spermfunction and DNA integrity. However, the mechanisms underlying this regulation remainunclear. To supply some arguments to this mechanisms, In the present work we show that theimpact of hydrogen peroxide on the acrosome reaction(AR),sperm membrance integrity,acrosin activity and DNA fragmentation.Methods: There are five parts in this trial. in the first part, high-qulity sperms acquired byup-stream method were co-cultured with hydrogen peroxide of different doses,and theabove-mentioned five parameters were observed by microscope at same point. In the secondpart,treatment of spermatozoa with H₂O₂(20μM) were co-cultured,all the parameters wereobserved by microscope at different time point. In the third part, catalase was detected ingoat semen. Effects of H2O2 on sperm function were indirectly confirmed by the removalof plasma and addition of catalase(CAT).In the forth part,the treatment of spermatozoa withH₂O₂(20μM) from different goats were co-cultured,the effects of H₂O₂ on sperm functionwere observed.In the fifth part,we investigated the effects of H2O2 of different doses andlow temperature conservation on sperm DNA integrity sperm DNA integrity was determinedusing a sigle cell gel electrophoresis(comet) assay.Result: 1,H₂O₂ of low concentration(10μM～20μM) had no obvious effect on spermmembrance integrity, acrosin positive rate and motility rate(P＞0.05);That ofconcentration(H₂O₂ 20μM)increased the acrosome reaction obviously(P＜0.01).However, atmuch higher concentrations of H₂O₂(50μM～100μM),there is significant reduction indate(P＜0.01). 2,After incubation at 37℃, there was no significant effect on spermmembrance integrity between 1h and 2h. As to acrosome reaction,maximal effect was seenafter 3h of incubation with 20μM H₂O₂. However, the maxmal effect of the other parameterswas seen after 2h. 3,we found that goat semen contains substantial amounts of catase. Theseminal plasma and spermatozoa contain respectively 216.25±3.9455U/(10⁸/ml) and12.12±2.3293U/(10⁸/ml). After removal of plasma, all the date had more significantlyreducted (P＜0.01). Comparing with normal treatment,the date increased significantly afteraddition of CAT (200IU)(P＜0.01).However,addition of a much higher concentration(CAT400IU) hadno significant effect on sperm function. 4,After 1h incubation with a concentration ofH₂O₂(20μM),all the parameters from different goats had no significant difference. 5,Treatment of spermatozoa with H₂O₂ caused a dose-dependent increase in the positive rateof DNA fragmentation.After a low temperature conservation in refrigeratory, the positiverate of DNA fragmentation had a increase, but slowly(P＞0.05).Conelution: this report suggests that relatively low concentrations of H₂O₂ are beneficialfor sperm function and DNA integrity, but that too high a concentration effects them. A rangeof doses,CAT can protect sperm function.The low temperature conservation does not affectDNA integrity of undamaged sperm.