胚胎在体外的维持

胚胎在体外的维持第一页，共四十页，编辑于2023年，星期一第六章胚胎在体外的维持动物繁殖学双语教学Maintaining维持embryosinvitro

Chapter6Embryosderive得自nutrientsfromthefluidinwhichtheyarebathed;moreover,ofperhapsmoreimportance,embryosdependontheambient周围环境

fluidtomaintaintheirphysiologicalintegrity完整性.第二页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Todate,nochemicallydefinedmediumhasbeenformulated阐明tosupportnormaldevelopmentofbovineembryossatisfactorilyforlongerthanabout24hours.第三页，共四十页，编辑于2023年，星期一动物繁殖学双语教学However,severalmediaarecommerciallyavailable(orcanbepreparedeasily)thatadequatelymaintainembryosfortheusualintervalbetweencollectionandtransfer.Forlongerperiods,cryopreservation低温贮藏

isrecommended.第四页，共四十页，编辑于2023年，星期一动物繁殖学双语教学STORAGECONDITIONSForshortperiodsofculture,thenutrientpropertiesofthemediumarefarlesscriticalthanpH,osmolality,temperature,sterility,andlackoftoxicity.第五页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Anembryo'srangeoftoleranceforthesepropertiesisnarrow.Inadequate不充分的controloftheseaspectsofcultureaccountsformanyfailedtransfers.Inaddition:embryosaredamagedifexposedtoexcesslight(e.g.microscopelights)forprolongedperiods,butnormallightingordaylightintheworkareaisnotharmfulifembryosarenotexposedformorethanhalfanhour.第六页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Bodytemperatureofcattleis39℃,andforcertainpurposesthistemperalureisappropriate.However,forroutine程序commercialembryotransferwedonotrecommendstorageabove37℃becausethermometers温度计,eventhoseonincubators孵卵器,arefrequentlyincorrectby1℃or2℃.Culturingembryosat41℃isextremelydamaging,whereasstoringthemforupto12hoursbetween18℃and37℃isnot.第七页，共四十页，编辑于2023年，星期一动物繁殖学双语教学MEDIAThesimplestmediumissterilesaline(9gmNaCl/litreofsterilized,deionized脱离子ordistilledwater).第八页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Salinewillnotsupportembryonicdevelopment,butwillkeepembryosforafewhours;itisbettertousesalineandtransferembryosquicklythantouseamorecomplexmediumwithoutaproperbuffering缓冲(作用)system.第九页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Moreover,salinecanbesteam-sterilized(atleast30minutesat121℃underapressureof104kilopascals千克力,)ifa0.22-gmbiologicalfiltrationsystemisnotavailable.Aseriousproblemwithsaline,however,isthatembryoswillfloat漂浮orsticktoplasticorglassunlessamacromolecule高分子suchasbovineserumalbumin(BSA)orserumisadded,bothofwhichrequiremembranefiltrationforsterilization.BSAandserumaresterilewhenpurchased.

第十页，共四十页，编辑于2023年，星期一TABLE3Recommendedcultureconditiongs第十一页，共四十页，编辑于2023年，星期一第十二页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Formostapplications,werecommendamodifiedDulbecco'sphosphatebufferedsaline(PBS).Itiseasytousebecauseitdoesnothavetobeequilibratedandmaintainedinanatmosphereof5percentCO2inair.第十三页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Itcanbepreparedfromstockreagents(Table4)orpurchasedineitherready-to-useorconcentratedform,whichmustbedilutedwithsterile,deionized脱离子剂ordistilledwater.第十四页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Afrequenterrorisfailuretodiluteconcentratedmedium;embryoscontractmarkedlywhenexposedtothehighosmolality同渗重摩.CommerciallyavailableDulbecco'sPBSdoesnotusuallycontainantibiotics,sodiumpyruvate丙酮酸盐(或酯),glucoseormacromolecules高分子.

第十五页，共四十页，编辑于2023年，星期一动物繁殖学双语教学

Theleastimportantingredient因素istheNapyruvate丙酮酸钠;neitherNapyruvatenororganismsglucoseisneededformostapplications.TheCaC12andMgSO4canalsobeeliminated排除ifembryosaretobekeptinvitro在生物体外onlyashorttime.Iftheseareomitted漏检,

osmolality同渗重摩shouldbeadjustedbyaddingextraNaC1.第十六页，共四十页，编辑于2023年，星期一动物繁殖学双语教学OthermediausedtoculturebovineembryosincludeTissueCultureMedium-199(TCM-199),Ham'sF-10medium,andBrinster'sMouseOvaCultureMedium-3(BMOC-3).Allofthesearecommerciallyavailable.TCM-199withHank'ssaltsdoesnotdependonCO2forbuffering,butTCM-199withEafie'ssaltsaswellasHam'sF-10andBMOC-3mustbemaintainedunderanatmosphereof5percentCO2inair.第十七页，共四十页，编辑于2023年，星期一动物繁殖学双语教学MACROMOLECULARSUPPLEMENTSSupplementationofmediawithalargeproteinmoleculedecreasessurfacetension张力的,whichreducesthetendencyoftheembryotofloat漂浮oradheretoplasticorglass,andhelpstoinactivate阻止活动heavymetalsandothertoxins.第十八页，共四十页，编辑于2023年，星期一动物繁殖学双语教学However,macromoleculesderivedfromserumarealsoapossiblevector带菌者forviralinfectionofembryos.BSAandbovineserumarecurrentlyrecommendedsourcesofmacromolecules.Polyvinyl乙烯聚合物alcoholandpolyvinylpyrrolidone吡咯啉arebeingstudiedaspossiblenon-biologicalmacromoleculestocircumventthedangerofinfection.第十九页，共四十页，编辑于2023年，星期一动物繁殖学双语教学ThesemacromoleculesdonotfunctionaswellasBSAinreducingsurfacetensionorchelating螯合toxins.BSA(FractionV)shouldbeaddedtomediumjustbeforeuseataconcentrationof0.05-0.1percentforflushingand0.4percentforculture.第二十页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Thepowdershouldbepouredverygently逐渐地onthesurfaceofthemediumandbeallowedtodissolve溶解forabout20minuteswithoutstirring活跃的orshaking摇动,(otherwiseittums小丘intoaglutinousblob粘性水滴的ormakesthemediumunusablyfrothy空洞的).第二十一页，共四十页，编辑于2023年，星期一动物繁殖学双语教学AftertheBSAhasdissolved,thecontainershouldbeinvertedgentlyfiveorsixtimestomixintheBSAcompletely.Themediumshouldthenbesterilizedbypassagethrougha0.22-gbacteriologicalfilter.PurertypesofBSAarealsoacceptable.BSAcanalsobepurchasedasanaqueoussolution,e.g.7percentBSAinwater.第二十二页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Ifserumisused,steer向...前进orcalfbloodshouldbeharvestedintosterile,sealablecontainersdirectlyfromthevein(toavoidcontaminantsfromskinandhair).Aftercoagulation凝结物,clots血（凝）arecutevery2-3cmwithasterileknife,andthebloodstoredovernightat5℃.第二十三页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Clotsarethenfilteredoutandtheserumcentrifugedat2000Xgfor12-15minutes.Thesediment沉淀物isdiscarded可废弃的andthesupernatant浮在表面的

recentrifuged换发.Thisstepisrepeatedonceagain.Thefinalsupematantissterilizedbypassagethrougha0.22-gbacteriologicalfilterintosterilecontainersofconvenientsize.第二十四页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Inordertoinactivate,阻止活动theproteincomplement,whichcanbetoxictoembryos,serummustbetreatedina56℃waterbathforexactly30minutesaftertheserumhasreached56℃.第二十五页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Serumcanbefrozenandstoredforuptoeightmonthsprovidedthatthecontainersaretightlysealed.Forqualitycontrol,onealiquotfromeachbatchofserumshouldbeincubatedovernightinasterile,sealedcontainerat37℃andexaminedthenextdayforbacterialcontamination.第二十六页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Forsomesituations,suchasinternationalmovementofembryosormaintenanceofspecific-pathogen-freeherds,additionalsterilizationofserumbygamma微克irradiation放射

isrecommended(ManualoftheInternationalEmbryoTransferSociety,1987).Heat-inactivated,sterilizedserumisaddedtomediuminsteadofBSAataconcentrationof0.5-1percentforflushingand10percentforstorage.第二十七页，共四十页，编辑于2023年，星期一动物繁殖学双语教学CONTAINERS容器Embryosshouldbestoredinsmall(<5ml),sterile,transparent透明的,sealable密封性能,inert惰性的container.第二十八页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Smalltesttubes,Petridishes陪替氏培养皿ormulti-wellplatesareconvenientforroutineuse(Figure13),butsmalltesttubesarerecommendedifembryosmustbemovedanydistancebeforeloading装填intostraws.第二十九页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Iftheembryosaretobestoredinuncoveredcontainers/ormorethan20minutes,however,themediumshouldbecoveredwithathinlayerofnon-toxicparaffin石蜡oiltopreventevaporationandcontaminationandtoregulatetherateofgasexchangebetweenthemediumandthesurroundingatmosphere.第三十页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Paraffinoil石蜡canalsobeusedwithothertypesofcontainersandisrecommendedifsealablecontainersarenotused.第三十一页，共四十页，编辑于2023年，星期一FIGURE13

Left:Plasticmulti-wellplatewith16x17-mmwells(lidtoleft)第三十二页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Exceptwhiletheyarebeingmanipulated,embryosshouldbestoredinadark,dust-freeincubatororcabinet(e.g.arefrigeratororanicechestwithatightly-fittinglidsetatroomtemperatureor37℃).第三十三页，共四十页，编辑于2023年，星期一动物繁殖学双语教学PIPETTES吸移管Manydevices装置,havebeenusedtomanipulateembryos.StandardPasteurpipettesaremuchtoolargeandthetipsrequirefirepolishingtopreventdamagetoembryos.第三十四页，共四十页，编辑于2023年，星期一动物繁殖学双语教学WeprefertomakeourownpipettesfromPyrex耐热玻璃glasstubingwitha4-mmoutsidediameter.Glass,in15-cmlengths,isheatedinthecentrewithaBunsenbumer本生灯andpulled拉tomakeanoutsidediameteroflessthan1mm.第三十五页，共四十页，编辑于2023年，星期一动物繁殖学双语教学Thisisthenscoredwithadiamondpencilandbrokentomaketwopipet