The Research Of The Relationship About The Express Of Related Protein/Gene And Bull Sperm Vitality After Freezing

Extensive use of artificial insemination has already speeded up the animal’s geneticchoice and improved the productivity, followed sperm cryopreservation of domestic animaldeveloped to reduce or inhibit sperm metabolism in order to prolong the life of sperm in vitro.Althougth the artificial insemination and sperm cryopreservation were extensive used inbovine industry, the fertility were lower with the insemination of cryopreservation spermwhich resulted the less value of herdsire. Most researchs looked for another bettercryopretectant and appropriate proportion for application, yet they were lacking in theoreticalstudy. The present study researched the effect of HSP90in sperm cryopreservation usingwestern blot and the changes of CSP.HSP60.HSP10and CIRBP gene expression usingrealtime fluorescence quantitative PCR in freezing process. The experiment demonstrated thatHSP90is important to maintain sperm motility after frozen,and CSP may also participate inregulation of sperm resistance to cold stress.The results were shown following:1. Different methods of protein extration from bull cryopreservation sperm were used toget high concentration of sperm protein. Bull sperm was treated by lysis solution withdifferent proportions to extract total protein.The results were shown that w.e could get enoughnumbers of proteins for western blot on only used four tubules in200μl lysis solution. Theresults suggested HSP90in frozen-thawed spermatozoa had significantly decreased,compared with flesh spermatozoa before cryopreservation and HSP90wa s not found in theseminal plasma before or after cryopreservation. That HSP90in bull spermatozoa wasdecreased substantially after cryopreservation may result from protein degradation rather thanleakage into the seminal plasma.2. The present study was conducted to investigate fertility-associated proteins in bullsperm and the correlation between90ku heat-shock protein (HSP90) and semencharacteristics. Semen of9Holstein cow were collected by a bovine artificial vagina. Thefresh semen characteristics of sperm motility, morphology, viability and concentration wereevaluated. After semen was freeze-thawed, sperm progressive motility, membrane integrityand acrosome integrity were assessed. Then the9ejaculates were clustered for freezingresistance on the basis of their sperm quality, sperm protein was subjected to SDS- polyacrylamide gel electrophoresis (SDS-PAGE) analysis and Western blot. Sperm qualityassessment proved that individual differences of sperm quality after frozen are large. TheSDS-PAGE results revealed that there was a substantial diverse of90ku protein in frozen-thawed spermatozoa. Western blot analysis demonstrated that this protein was HSP90. Thus,this study indicated that the expression of HSP90was associated with semen characteristics.Related coefficient between HSP90expression and sperm motility, membrane integrity,acrosome integrity respectively are0.364、0.447and0.402. The expression of HSP90couldbe utilized in developing a reliable and simple method to determine the bull sperm freezingresistance.3. The relative genes expression were detected by quantitive real-time PCR. The resultsshowed that the expression of CSP.HSP60and HSP10were higher after frozen, but nosignificant difference were found in HSP60and HSP10(P＞0.05),only CSP increasedsignificant(P﹤0.05).The expression of CIRBP reduced after frozen, yet also no significantdifference was found(P＞0.05).