胚胎在體外的維持

胚胎在體外的維持第一頁(yè)，共四十頁(yè)，編輯于2023年，星期一第六章胚胎在體外的維持動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Maintaining維持embryosinvitro

Chapter6Embryosderive得自nutrientsfromthefluidinwhichtheyarebathed;moreover,ofperhapsmoreimportance,embryosdependontheambient周?chē)h(huán)境

fluidtomaintaintheirphysiologicalintegrity完整性.第二頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Todate,nochemicallydefinedmediumhasbeenformulated闡明tosupportnormaldevelopmentofbovineembryossatisfactorilyforlongerthanabout24hours.第三頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)However,severalmediaarecommerciallyavailable(orcanbepreparedeasily)thatadequatelymaintainembryosfortheusualintervalbetweencollectionandtransfer.Forlongerperiods,cryopreservation低溫貯藏

isrecommended.第四頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)STORAGECONDITIONSForshortperiodsofculture,thenutrientpropertiesofthemediumarefarlesscriticalthanpH,osmolality,temperature,sterility,andlackoftoxicity.第五頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Anembryo'srangeoftoleranceforthesepropertiesisnarrow.Inadequate不充分的controloftheseaspectsofcultureaccountsformanyfailedtransfers.Inaddition:embryosaredamagedifexposedtoexcesslight(e.g.microscopelights)forprolongedperiods,butnormallightingordaylightintheworkareaisnotharmfulifembryosarenotexposedformorethanhalfanhour.第六頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Bodytemperatureofcattleis39℃,andforcertainpurposesthistemperalureisappropriate.However,forroutine程序commercialembryotransferwedonotrecommendstorageabove37℃becausethermometers溫度計(jì),eventhoseonincubators孵卵器,arefrequentlyincorrectby1℃or2℃.Culturingembryosat41℃isextremelydamaging,whereasstoringthemforupto12hoursbetween18℃and37℃isnot.第七頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)MEDIAThesimplestmediumissterilesaline(9gmNaCl/litreofsterilized,deionized脫離子ordistilledwater).第八頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Salinewillnotsupportembryonicdevelopment,butwillkeepembryosforafewhours;itisbettertousesalineandtransferembryosquicklythantouseamorecomplexmediumwithoutaproperbuffering緩沖(作用)system.第九頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)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.

第十頁(yè)，共四十頁(yè)，編輯于2023年，星期一TABLE3Recommendedcultureconditiongs第十一頁(yè)，共四十頁(yè)，編輯于2023年，星期一第十二頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Formostapplications,werecommendamodifiedDulbecco'sphosphatebufferedsaline(PBS).Itiseasytousebecauseitdoesnothavetobeequilibratedandmaintainedinanatmosphereof5percentCO2inair.第十三頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Itcanbepreparedfromstockreagents(Table4)orpurchasedineitherready-to-useorconcentratedform,whichmustbedilutedwithsterile,deionized脫離子劑ordistilledwater.第十四頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Afrequenterrorisfailuretodiluteconcentratedmedium;embryoscontractmarkedlywhenexposedtothehighosmolality同滲重摩.CommerciallyavailableDulbecco'sPBSdoesnotusuallycontainantibiotics,sodiumpyruvate丙酮酸鹽(或酯),glucoseormacromolecules高分子.

第十五頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)

Theleastimportantingredient因素istheNapyruvate丙酮酸鈉;neitherNapyruvatenororganismsglucoseisneededformostapplications.TheCaC12andMgSO4canalsobeeliminated排除ifembryosaretobekeptinvitro在生物體外onlyashorttime.Iftheseareomitted漏檢,

osmolality同滲重摩shouldbeadjustedbyaddingextraNaC1.第十六頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)OthermediausedtoculturebovineembryosincludeTissueCultureMedium-199(TCM-199),Ham'sF-10medium,andBrinster'sMouseOvaCultureMedium-3(BMOC-3).Allofthesearecommerciallyavailable.TCM-199withHank'ssaltsdoesnotdependonCO2forbuffering,butTCM-199withEafie'ssaltsaswellasHam'sF-10andBMOC-3mustbemaintainedunderanatmosphereof5percentCO2inair.第十七頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)MACROMOLECULARSUPPLEMENTSSupplementationofmediawithalargeproteinmoleculedecreasessurfacetension張力的,whichreducesthetendencyoftheembryotofloat漂浮oradheretoplasticorglass,andhelpstoinactivate阻止活動(dòng)heavymetalsandothertoxins.第十八頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)However,macromoleculesderivedfromserumarealsoapossiblevector帶菌者forviralinfectionofembryos.BSAandbovineserumarecurrentlyrecommendedsourcesofmacromolecules.Polyvinyl乙烯聚合物alcoholandpolyvinylpyrrolidone吡咯啉arebeingstudiedaspossiblenon-biologicalmacromoleculestocircumventthedangerofinfection.第十九頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)ThesemacromoleculesdonotfunctionaswellasBSAinreducingsurfacetensionorchelating螯合toxins.BSA(FractionV)shouldbeaddedtomediumjustbeforeuseataconcentrationof0.05-0.1percentforflushingand0.4percentforculture.第二十頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Thepowdershouldbepouredverygently逐漸地onthesurfaceofthemediumandbeallowedtodissolve溶解forabout20minuteswithoutstirring活躍的orshaking搖動(dòng),(otherwiseittums小丘intoaglutinousblob粘性水滴的ormakesthemediumunusablyfrothy空洞的).第二十一頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)AftertheBSAhasdissolved,thecontainershouldbeinvertedgentlyfiveorsixtimestomixintheBSAcompletely.Themediumshouldthenbesterilizedbypassagethrougha0.22-gbacteriologicalfilter.PurertypesofBSAarealsoacceptable.BSAcanalsobepurchasedasanaqueoussolution,e.g.7percentBSAinwater.第二十二頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Ifserumisused,steer向...前進(jìn)orcalfbloodshouldbeharvestedintosterile,sealablecontainersdirectlyfromthevein(toavoidcontaminantsfromskinandhair).Aftercoagulation凝結(jié)物,clots血（凝）arecutevery2-3cmwithasterileknife,andthebloodstoredovernightat5℃.第二十三頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Clotsarethenfilteredoutandtheserumcentrifugedat2000Xgfor12-15minutes.Thesediment沉淀物isdiscarded可廢棄的andthesupernatant浮在表面的

recentrifuged換發(fā).Thisstepisrepeatedonceagain.Thefinalsupematantissterilizedbypassagethrougha0.22-gbacteriologicalfilterintosterilecontainersofconvenientsize.第二十四頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Inordertoinactivate,阻止活動(dòng)theproteincomplement,whichcanbetoxictoembryos,serummustbetreatedina56℃waterbathforexactly30minutesaftertheserumhasreached56℃.第二十五頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Serumcanbefrozenandstoredforuptoeightmonthsprovidedthatthecontainersaretightlysealed.Forqualitycontrol,onealiquotfromeachbatchofserumshouldbeincubatedovernightinasterile,sealedcontainerat37℃andexaminedthenextdayforbacterialcontamination.第二十六頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Forsomesituations,suchasinternationalmovementofembryosormaintenanceofspecific-pathogen-freeherds,additionalsterilizationofserumbygamma微克irradiation放射

isrecommended(ManualoftheInternationalEmbryoTransferSociety,1987).Heat-inactivated,sterilizedserumisaddedtomediuminsteadofBSAataconcentrationof0.5-1percentforflushingand10percentforstorage.第二十七頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)CONTAINERS容器Embryosshouldbestoredinsmall(<5ml),sterile,transparent透明的,sealable密封性能,inert惰性的container.第二十八頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Smalltesttubes,Petridishes陪替氏培養(yǎng)皿ormulti-wellplatesareconvenientforroutineuse(Figure13),butsmalltesttubesarerecommendedifembryosmustbemovedanydistancebeforeloading裝填intostraws.第二十九頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Iftheembryosaretobestoredinuncoveredcontainers/ormorethan20minutes,however,themediumshouldbecoveredwithathinlayerofnon-toxicparaffin石蠟oiltopreventevaporationandcontaminationandtoregulatetherateofgasexchangebetweenthemediumandthesurroundingatmosphere.第三十頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Paraffinoil石蠟canalsobeusedwithothertypesofcontainersandisrecommendedifsealablecontainersarenotused.第三十一頁(yè)，共四十頁(yè)，編輯于2023年，星期一FIGURE13

Left:Plasticmulti-wellplatewith16x17-mmwells(lidtoleft)第三十二頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Exceptwhiletheyarebeingmanipulated,embryosshouldbestoredinadark,dust-freeincubatororcabinet(e.g.arefrigeratororanicechestwithatightly-fittinglidsetatroomtemperatureor37℃).第三十三頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)PIPETTES吸移管Manydevices裝置,havebeenusedtomanipulateembryos.StandardPasteurpipettesaremuchtoolargeandthetipsrequirefirepolishingtopreventdamagetoembryos.第三十四頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)WeprefertomakeourownpipettesfromPyrex耐熱玻璃glasstubingwitha4-mmoutsidediameter.Glass,in15-cmlengths,isheatedinthecentrewithaBunsenbumer本生燈andpulled拉tomakeanoutsidediameteroflessthan1mm.第三十五頁(yè)，共四十頁(yè)，編輯于2023年，星期一動(dòng)物繁殖學(xué)雙語(yǔ)教學(xué)Thisisthenscoredwithadiamondpencilandbrokentomaketwopipet