Hu　Shan,　Zhang　Xiao-gang,　Han　Cong,　Wei　Shuai-yi,　Xie　Dong-qi,　Du　Ren-rang,　and　Hu　Jian-hong

College　of　Animal　Science　and　Technology,　Northwest　A&F　University,　Yangling　712100,　Shaanxi,　China

Effects of Three Different Diluents on Quality of Boar Semen Stored at 17℃

HuShan,ZhangXiao-gang,HanCong,WeiShuai-yi,XieDong-qi,DuRen-rang,andHuJian-hong*

CollegeofAnimalScienceandTechnology,NorthwestA&FUniversity,Yangling712100,Shaanxi,China

Toinvestigatetheeffectsofdifferentdiluentsonthequalityoftheboarsemenstoredat17℃,andassesstherelationship betweenspermmotilityandtherelativelevelsofenzymes,threecommercialdiluents(DiluentⅠ,DiluentⅡ andDiluentⅢ)andthree boarbreedsemens(Yorkshire,LandraceandDuroc)wereutilized.Thespermmotility,effectivesurvivaltime,survivalindex,catalase (CAT),thetotalanti-oxidativecapacity(T-AOC)andmalondialdehyde(MDA)levelswereevaluated.Theresultsshowedthatthere weresignificantinteractioneffectsbetweendiluentsandbreedsontheboarspermmotility(P＜0.001),survivaltime(P＜0.001),CAT levels(P＜0.001)andT-AOClevels(P＜0.001),butneithereffectsnorinteractioneffectsbetweendiluentsandbreedsonsurvival index(P＞0.05).Alloftheparametersvariedsignificantlywiththeincreaseofthestoragetime(P＜0.001).Thesurvivaltimeincreased 12.9%inYorkshireboarsemendilutedwithDiluentⅢthanwithDiluentⅡ,whilethesurvivaltimeincreased6.6%inLandraceboar semendilutedwithDiluentⅡthanwithDiluentⅢ.BothCATandT-AOClevelsweresignificantlypositivecorrelatedwithsperm motilityinallthethreeboarbreeds(P＜0.001),whileMDAlevelsweresignificantlynegativecorrelatedwithspermmotility(P＜0.001). TheseresultsindicatedthatDiluentⅢandDiluentⅡ weretheoptimalcommercialdiluentsforYorkshireandLandraceboarsemen storedat17℃,respectively.

boarsemen,storage,differentdiluents,survivalindex,enzymeactivity

Introduction

Artificialinsemination(AI)isamomentoustoolin pigindustrytoimprovethegeneticgenewithfrozen semen(Castellanoet al.,2010).Comparedwithother animalspecies,boarspermisfragiletocoldshockfor differentphospholipids,suchasunsaturatedfattyacids (UFAs),compositioninspermmembranes(Fraczek et al.,2001;Zhanget al.,2012;Silvaet al.,2015). Frozenboarsemenhasbeenperformedsince1975,but thecommercialdiluenthasnostablebreakthroughfor thesusceptibilityofboarspermatozoa(Zhanget al., 2012).Asthebarrieroflowfecundityratescausedby AIusingfreeze-thawsemen,extensiveworkhasbeen doneonpreservationofboarsemenwithdiluentat normaltemperature(15-20℃)(Althouseet al.,1998; ZouandYang,2000;Vytet al.,2007;Perez-Llano et al.,2009).Ithasbeenconsideredthattheliquid boarsemenwouldmaintainhigherspermfertility comparedwithfrozensemen(ClarkeandJohnson, 1987).Therefore,storageofboarsemeninnormaltemperaturewasincreasinglyexploitedinAI.

However,therearestillmanydeficienciesinnormal temperaturestorage.Duringthestorageprocessof boarsemen,boarspermissubjectedtodecrease motilityandviability.Inthemeantime,thesperm membranepermeabilitywouldbealteredpartly byoxidativedamagethatcausedbyinappropriate formationofreactiveoxygenspecies(ROS)(Wang et al.,1997;GuthrieandWelch,2010).Thesystems ofprotectiveanti-oxidantinspermatozoa,stem fromthecytoplasm,areconsistedofglutathione peroxidase(GSH-Px),catalase(CAT)andsuperoxide dismutase(SOD)(Bilodeauet al.,2001).Ithasbeen authenticatedthatthissystemswhichareessentialto maintainspermmotilityandviabilitydefendagainst thelipidperoxidation(Huet al.,2009).

Keepingpacewiththedevelopmentofthe preservationboarsemenfrom15℃to17℃,many researchersareinterestedinthediluentbreakthrough. ThediluentwillfacilitatetobalancepH,maintain spermmembranesintegrity,inhibitspermcapacitation anddefendtheoxidativedamage(Watson,1990; Weitze,1991;Huet al.,2009).Theeffectsofdiluent onthespermqualityparameters,duringthestorage processwerewelldocumented(Estienneet al., 2007;KusterandAlthouse,1999;Vytet al.,2004; Waterhouseet al.,2004;Martín-Hidalgoet al., 2013).Asthediluentneedtobeadaptedtoindividual differencesamonganimals(Gadea,2003;Levis, 2000),Martín-Hidalgoet al.(2013)studiedtheeffects ofdifferentdiluentsonspermmotilityandviability, reactiveoxygenspecieswerethoughttobethe importantreasonthatproducedanddamagedsperm cellmotilityandgenomicintegrity(Clarkson,1988; Storey,1997;Aitkenet al.,1998;Bilodeauet al., 2000;Footeet al.,2002),andtheresultsrequired abetterunderstandingthatdifferentdiluentshad differenteffectsonsemenfromdifferentboarbreeds.

Toinvestigatetheeffectsofdifferentdiluentson thespermmotilityandthelevelsofenzymesfrom differentboarbreedsstoredat17℃,andassessedthe relationshipbetweenspermmotilityandtherelative levelsofenzymes,threedifferentdiluentsandthree boarbreedsemens(Yorkshire,LandraceandDuroc) wereutilizedinthepresentstudy.Thespermmotility, ROSrelatedenzymelevelsofcatalase(CAT),thetotal anti-oxidativecapacity(T-AOC)andmalondialdehyde (MDA)duringthestorageweremeasured,the correlationbetweenspermmotilityandeachenzyme levelwasalsoanalyzed.

Materials and Methods

Thecareandproceduresofboarthatusedtocollect semenwereapprovedbytheAnimalCareand UseCommitteeofFujianAgricultureandForestry University.

Semen collection and diluent prepare

Thesexuallymatureboars(1.5-2years)ofYorkshire (n=5),Landrace(n=5)andDuroc(n=5)fromShanjia Co.,Ltd.wereemployedinthisstudyfromSeptember of2012toSeptemberof2013.Total150-200mL semenwascollectedfromeachboarineachtimeby glovedhandtechniqueandfilteredthroughfourlayers ofsterilecottongauze.Subsequently,thesemenwas transferredintoasteriletube.Thevolume,sperm concentrationandpercentmotilespermatozoawere assessedinsperm-richfractions.Whensemenhad morethan80%ofspermmotilityandnormalsperm morphologywouldbeused.Toeliminatevariability betweentheevaluated,theejaculateswerepooledfor eachbreedboarinthepresentstudy,respectively.All theexperimentswererepeatedthreetimes.

Diluentswerepurchasedfromthreecommercial companiesandthecompositionswerenotclearforthe protectionofintellectualproperty.Theyweredesigned asthegroupsofDiluentⅠ,DiluentⅡandDiluentⅢanddissolvedinthedouble-distilledwaterwiththe samevolume.

Semen processing

Afterthesemenqualitytomeettherequirements, thecollectedboarsemenwasequallydividedintothreeparts.Subsequently,theratioofsemendiluted byDiluentⅠ,DiluentⅡandDiluentⅢwas2:1.The temperatureofdiluentsweresamewithboarsemen. Thedilutedsemenwasgentlymixed,andallthetubes werewrappedby10layersofsterilegauze.They wereslowlycooledto17℃.Finally,theywerestored inthe17℃thermostat.Weshooksemenonceevery 12handdetectedtheparametersevery24huntil spermmotilitydecreasedto50%.

Assessment of sperm motility, effectively survival time and survival index

Spermmotilityreferstotheabilityorintensityof spermactivity.Phasecontrastmicroscopywasrecommended for motility evaluation. Five μL semen sampleswereplacedonglassandvisuallyexamined thepercentageoflinearmotilespermbylight microscopeat400×.Thepercentageofspermmotility washigherthan50%,whichwasconsideredasthe effectivelysurvivaltime.Thesurvivalindexofsperm wascalculatedasthefollowingequation:I=(MA+MB)× 2/D(Iindicatedthesurvivalindex,MAandMBdemonstratedthespermmotilityinAdayandBday,respectively,andDwasconsideredastheintervaltime betweenAdayandBday.

Biochemical assay

Semenwaspouredinto0.5mLcentrifugetubes andcentrifugedat1000r?min-1for10min.The supernatantwasaspirated,thenadded1mLof1% Triton,mixedwellandplacedafter20min,then centrifugedat4000r?min-1for30min.Thenthe supernatantwasextractedfordetectingenzyme activity.TheenzymaticactivitiesofCAT,T-AOCand MDAweredetectedbythekit(NanjingJianchengCo., Ltd.,China)followingthedescription,respectively.

CAT

CATactivityassaywasperformedaccordingtothe methodofGoth(1991).Total0.3mLofpretreated semenwasincubatedin1.7mLofsubstrate(pH7.0, 65 μmol ?L-1hydrogenperoxidein50mmol?L-1phosphatebuffer)at37℃for60s.CATdecomposition ofhydrogenperoxidereactioncanberapidlysuspendedbytheadditionof1.0mLof32.4mmol?L-1ammoniummolybdate.Theremaininghydrogenperoxide couldreactwithammoniummolybdateandproduce akindoflightyellowcomplexcompound.Then, measuredtheproductionamountat405nmagainst blankcontainingallthecomponentsexcepttheenzyme onapectrophotometer.subsequently,calculatedCAT concentration.ThevalueofCATwasexpressedin U?mL-1.

T-AOC

T-AOCactivityassaywasperformedaccordingto themethodofdoubleantibodysandwich.Wesetthe standardhole,thesampleholeandblankholeinthe microtiterplatecoatedbyT-AOCantibody.Added 50 μL standard solution in standard hole, added 10 μL sample and 40 μL sample diluent in sample hole,withoutinblankhole.Inadditiontotheblank hole, each hole added 100 μL detection antibody thatmarkedbyHRP,sealedtheholesandincubatedat37℃for60min.Thenwashedtoremove unboundcomponents.Eachwellweresequentially added the substrates of A and B, each 50 μL. Gently shook30sandincubatedat37℃for15min. Subsequently,ODvalueofeachholethatwasadded 50 μL terminated liquid was measured at 520 nm. Finally,calculatedT-AOCconcentrationbythe standardcurve.

MDA

MDAactivityassaywasperformedaccordingtothe methodofdoubleantibodysandwich.Afteradded thesampletowellofmicrotiterplatecoatedbyMDA antibody,reactionformacomplex.Thenwashed completely.Subsequently,addedTBAsubstrate solution,andmeasuredODvalueat532nm.Finally, calculatedMDAconcentrationbythestandardcurve.

Statistical analyses

Allresultswereexpressedasmeanvalues±SD.andP＜0.05wasusedasthelevelofstatisticalsignificance. SPSSStatistics17.0wasusedforthedataanalyses. Theeffectsofthediluentonsurvivaltimeandsurvival indexinsemenfromdifferentboarbreedswere analyzedwithatwo-wayanalysisofthevariance (ANOVA).Theeffectsofthediluentandstoragetime onspermmotility,CAT,T-AOCandMDAlevelsin semenfromdifferentboarbreedswereanalyzedwith athree-wayANOVA.ANOVAwasfollowedbya Duncanmultiple-comparisontestifitwasnecessary toevaluatethedifferencesbetweenthevaluesof differentexperimentalgroups.APearsoncorrelation wasusedtodeterminetherelationshipbetweensperm motilityandeachenzymelevel.

Results

Effect of different diluents on sperm motility and autioxidat enzyme level

Theeffectsofthepreservationtime,diluenton motilityandrelativeenzymaticlevelsofsemenare showninTable1.

Table 1　Effects of preservation time and diluents on motility and relative enzymatic levle of semen from different boar breeds at 17℃

Continued

ItwasobviouslyobservedfromTable1thatthere weresignificantinteractioneffectsbetweenbreeddiluentandsemenbreed-timeonspermmotility (P＜0.001,P=0.039),CAT(P＜0.001,P＜0.001)and T-AOC(P＜0.001,P＜0.001)levels.Intheprocessof preservation,thespermmotility,CATandT-AOC contentsdecreasedsignificantly,butMDAcontent increased.However,therewereneithersignificant effectsnorsignificantinteractioneffectsbetween breed-diluentandsemenbreed-timeonMDAlevels. Furtherone-wayANOVAindicatedthatthestorage timesignificantlyaffected(P＜0.001)thespermmotility,CAT,T-AOCandMDAlevelsineachbreed.

InYorkshireboarsemen,thespermmotilityand CATlevelsofdilutedwithDiluentⅢweresignificant higherthanthosewithDiluentⅡafter3daysand during0-3days,respectively(P＜0.05),andT-AOC levelsofboarsemendilutedwithDiluentⅢwere significanthigherthanthosewithDiluentⅠduring3-4 days.However,therewerenosignificantdifferences ofMDAlevelsamongthediluents.

InLandraceboarsemen,thespermmotilityand CATlevelsdilutedwithDiluentⅡweresignificant higherthanthosewithDiluentⅢ(P＜0.05)after 3daysandduring2-3days,respectively.CATand T-AOClevelsofLandraceboarsemendilutedwith DiluentⅡweresignificanthigherthanthosewith DiluentⅠ during0-3days(P＜0.05),butMDAlevels ofLandraceboarsemendilutedwithDiluentⅠwere significanthigherthanthosewithDiluentⅢafter 2days(P＜0.05).InDurocboarsemen,therewereno significanteffectsofdiluentonspermmotility,CAT, T-AOCandMDAlevels.

Effect of different diluents on sperm survival index and survival time

Thesurvivaltimeandsurvivalindexofspermare showninTable2.

ItwasclearlyseenfromTable2thatthediluents andbreedshadnoeffectsandnointeractioneffects onsurvivalindex.Thereweresignificantinteraction effectsbetweendiluentsandbreedsonsurvivaltime. Furtherone-wayANOVAindicatedtheeffective survivaltimeofYorkshireboarsemendilutedwith DiluentⅢwassignificantgreaterthanthatwith DiluentⅡ(P=0.038,Table2),andLandraceboar semendilutedwithDiluentⅡwassignificantgreater thanDiluentⅢ(P=0.040,Table2),butnosignificant effectswereobservedinothergroups.

Relationship of sperm motility and autioxidant enzyme level

BothCATandT-AOClevelsweresignificantly positivecorrelatedwithspermmotilityinallthethree boarbreeds(Fig.1),PearsoncorrelationofCAT andspermmotilityofYorkshire,Landrace,Duroc were0.988,0.962,and0.988,respectively,Pearson correlationofT-AOCandspermmotilityofYorkshire, Landrace,Durocwere0.987,0.993and0.998, respectively.However,MDAlevelweresignificant negativecorrelatedwithspermmotility(Fig.1), PearsoncorrelationofMDAandspermmotilityof Yorkshire,LandraceandDurocwere–0.932,–0.941 and–0.958,respectively.

Table 2　Survival time and index of sperm from three boar breeds in diluents at 17℃

Fig. 1　Relationship of sperm motility and enzyme level

Discussion

Thecommercialdiluentswereproducedtomaintain andprotectthefertilityofspermatozoaduringstorage process(Watson,1990).Dilutedpowderthatcould increasethevolumeofsementomakereasonable andeffectiveuseofsemencouldprovidespermwith metabolicnutrientsneededforthesurvivaltoextend thespermsurvivaltimeandimprovethefertilization abilityofsperm.Diluentcommonlycontainsnutrients, bufferingagent,antioxidants,enzymes,essentialmetalionsandantibacterialsubstancesandsoon.Diluent composition,eachcompositionconcentrationand theratioamongthecompositionshadveryimportant influencesonsemenpreservationeffect.Forexample, thecombinationofamides(3%)andglycerol(2%) wasmoreefficientatcryo-protectionthanglycerol aloneforsemenfreezingfromboarsofthePiaubreed (Pinhoet al.,2014).Someresearchershadaddeda certainamountofenergysubstancesindilutesolution suchasglucose,fructose,lactose,sucrose,etc.They couldimprovespermmotilityandextendthesperm tostoragetime.Glucosewasacommondiluent nutrients,whichcouldprovidetherequiredenergy forthesurvivalofspermandadjustsemenosmotic pressure.Bovineserumalbumin(BSA)couldstabilize thestructureofspermcellsandneutralizesperm metabolitestoprolongthesurvivaltime,maintaining spermmotilityandreducespermagglutination.K+couldmaintainthetransportofNa+-K+pumpthat couldproduceATPtomaintainthesurvivaland movementofspermonspermmembrane.Inaddition, bothVAandVBthatwereaddedinthediluentcould protectthespermfromdifferentfunction.

Itwaswelldocumentedthattheeffectofstorage timeonsemenqualityvariedamongboars(Waberski et al.,1994;Kommisrudet al.,2002;Waterhouse et al.,2004).Thisvariationhadalsobeenobservedby Martín-Hidalgoet al.(2013)whoreportedthatsemen toleranceduringpreservationwasmoreinfluenced bythediluentsinIberianthaninDurocbreed.Inthe presentstudy,thediluentsandbreedshadnoeffects andinteractioneffectsonsurvivalindex,butthere weresignificantinteractioneffectsbetweendiluents andbreedsonspermmotilityandsurvivaltime.The preservationeffectofYorkshireboarsementhat wasdilutedwithdiluentⅢwhichspermmotility in5daysremainedat0.54andsurvivaltimewas higherthanthatwithdiluentⅡ,wasthebest.The effectofLandraceboarsementhatwasdilutedwith diluentⅡthatspermmotilityandsurvivaltime werehigherthanthosewithdiluentⅢafter3days (P＜0.05)wasthebest.Buttheeffectsofthreediluents onDurocboarsemenwerenotsignificantlydifferent. Comparingwithotherbreeds,thisresultindicatedthat thesemenofDurocwaslessersensitivetodiluents andfurtherstudyareneeded.Cometothetrialresults, ontheonehand,maybeduetodifferentproportions ofspermandseminalplasmaindifferentbreedsemen thatcouldleadtodifferencesaftersemendilution. Studiesfoundthatsemenvolumehadsignificant differencethatLandrace,YorkshireandDurocreduced inturnbetweendifferentbreeds.Sincethespermof smallvolume,thedifferencesbetweensemenvolume mainlyreflectedonthecontentoftheseminalplasma. Theelectrolytecompositioninseminalplasmacould promotespermprematureaging.Atthesametime, theseminalplasmacontainsvariousionsthatcould maintainspermsurvival.Inaddition,theseminal plasmahadantioxidants,proteinandotherbeneficial ingredientsthatcouldmaintainmembranestabilityand fertility.Forexample,themeanpercentageofsperm motilityofHampshireboarswassignificantlylowerin washedthaninunwashedseminalplasmairrespective ofholdingduringpreservation(Chutiaet al.,2014). Extendingcryopreservedboarspermin50%seminal plasmawouldsignificantlyimprovespermmotility andviability,withthepossibleneteffectofpositively impactingsowfertilityandextendingthesperm's lifespan(Garciaet al.,2010).Ontheotherhand,these threediluentswereimportedproducts,anddidn't indicatethespecificcomponentsofthediluents. Thisresearchwasbasedontheinstructiontodiluteandpreserveboarsemen;eachdiluentmightcontain thesamecomponents,suchasglucose,EDTA, sodiumcitrate,etc.,buttheconcentrationofeach componentmightbedifferent.Atthesametime, eachdilutionmightalsoaddothersubstances,such asBAS,antibiotics,etc.,toprotectthespermbytheir ownresearches.Sotheresultsofthistestmightbe causedbyonereasonalone,mightalsobecausedby combinationoftworeasons.Thereasonneedstobe carefullyanalyzedinfuturestudies.

Undernormalphysiologicalconditions,ROSproducedbyrespiratorymetabolismofspermwasalways inadynamicequilibriumwhichproducedconstantly andclearedbyantioxidantenzymesatthesametime in the body (Strze?ek et al.,2012).However,after ejaculation,spermthatwereinevitablyexposedtoair, contactthelargeamountsofoxygenanddestroythe redoxsystemsproducedalargenumberofROSand wereaffectedseverely;then,sucheffectscouldplaya majorroleonspermmembrane(Vascoet al.,2007). CATthatcouldinduceH2O2todecomposeintowater andmolecularoxygencouldcleanH2O2in vivoand avoidthatthatcellswerepoisonedbyH2O2(Huet al.,2009).ThelowerCATcontent,thelowercellular antioxidantcapacity.T-AOClevelcouldreflectthe organismdefenseoxidativedamageabilitystrong orweak.WhenT-AOCcontentwasstronger,thecontentofbiologicalantioxidantsinthebodywashigher andtheabilitytoresisttheoxidativedamageis stronger.Duringthesemenextending,T-AOClevel andantioxidantcapacityofboarsemendecreased (Brezezinska-Slevbodzinskaet al.,1995).Thelevelof MDAthatwastheendproductoflipidperoxidation metabolismrepresentedthedegreeofspermoxidative damage.ThehigherMDAcontent,thedeeperthe degreeofspermlipidperoxidation(Kumaresanet al., 2009).Inthepresentstudy,duringtheprocessof preservation,CATandT-AOClevelsofspermatozoa decreasedsignificantlywhileMDAlevelincreased significantly.Inearlystageofthepreservationprocess, thespermhadstrongantioxidantcapacityandthe lowlevelofoxidativedamage.Withtheextensionof storagetime,unsaturatedfattyacidsoccurredlipid peroxidationinthespermthatdamagedorinhibited theactivityofantioxidantenzymesanddestroyed thespermprotein,whichcausedspermstructure damage,brokeoriginalequilibriumstateandproduced moreoftheperoxide,whichledtotheexacerbation ofthedegreeofoxidativedamageofspermfurther, thedeclineofthetotalantioxidantcapacityandthe increaseofMDAlevelsgradually.

Inthepresentstudy,thereweredifferenteffects ofdifferentdiluentsonthelevelsofCAT,T-AOC andMDAinYorkshireandLandraceboarsemen. ButInDurocboarsemen,therewerenosignificant effectsofdiluentonCAT,T-AOCandMDAlevels. Thetrialresults,ontheonehand,mightbeduetothe differencesamongdifferentvarietiesofpigsperm,on theotherhand,mightbebecausethesethreediluents haddifferencesinantioxidantsandtheirproportion.

Reactiveoxygenspeciesweregenerallyconsidered tobecytotoxicagentsduetotheirabilitytoinduce lipidperoxidationwithinthespermplasmamembrane andmodifyDNAstructures(Sanockaet al.,2005).It waswellestablishedthatROScoulddamagesperm plasmamembraneandhencereducespermmotility (Guthrie&Welch,2005;Chanapiwatet al.,2009). AsthearisingofROSformedthroughtheunivalent reductionofoxygen,whichledtotheimpairmentof spermmotility,functionalmembraneintegrityand fertilitythroughoxidativestressandtheproduction ofcytotoxicaldehydes(AlvarezandStorey,1989).In thisstudy,thefactthatbothCATandT-AOClevels werepositivecorrelatedwithspermmotilityinall threeboarbreedswhileMDAlevelwassignificant negativecorrelatedwiththespermmotilityindicated thatduringtheprolongingpreservationofsperm,the decreaseofspermactivitypartlycausedbythedecline ofantioxidantcapacity.

Conclusions

Inconclusion,thediluentshouldadapttoeach particularsemenbreed.DiluentⅢwastheoptimalcommercialdiluentforthestorageofYorkshireboar semen.ButDiluentⅡwastheoptimalcommercial diluentforLandraceboarsemenstoredat17℃. Withtheextensionofstoragetime,thefactthat CAT,T-AOCcontentdecreased,butMDAcontent increasedwasbecausethatspermweredestroyedby ROS,whichcausedthedecreaseofthelevelsofthe antioxidantenzymesandthetotalantioxidantcapacity andtheincreaseoftheoxidativedamage.Inaddition, thedecreaseofspermactivityduringthestoragepartly dependedonthedeclineoftheantioxidantcapacity.

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S814.8 Document code: A Article ID: 1006-8104(2015)-02-0036-11

1April2015

SupportedbytheNationalSwineIndustryTechnologySystem(CARS-36);theScientifcandTechnologicalProjectofYanglingDemonstrationZone (2014NY-22)

HuShan(1992-),female,engagedintheresearchofanimalreproductivephysiology.E-mail:1243860951@qq.com

*.HuJian-hong,professor,engagedintheresearchofanimalreproductivephysiology.E-mail:hjh19732008@126.com