胚胎在體外的維持

胚胎在體外的維持演示文稿本文檔共38頁；當(dāng)前第1頁；編輯于星期一\0點3分（優(yōu)選）胚胎在體外的維持本文檔共38頁；當(dāng)前第2頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Todate,nochemicallydefinedmediumhasbeenformulated闡明tosupportnormaldevelopmentofbovineembryossatisfactorilyforlongerthanabout24hours.本文檔共38頁；當(dāng)前第3頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)However,severalmediaarecommerciallyavailable(orcanbepreparedeasily)thatadequatelymaintainembryosfortheusualintervalbetweencollectionandtransfer.Forlongerperiods,cryopreservation低溫貯藏

isrecommended.本文檔共38頁；當(dāng)前第4頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)STORAGECONDITIONSForshortperiodsofculture,thenutrientpropertiesofthemediumarefarlesscriticalthanpH,osmolality,temperature,sterility,andlackoftoxicity.本文檔共38頁；當(dāng)前第5頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Anembryo'srangeoftoleranceforthesepropertiesisnarrow.Inadequate不充分的controloftheseaspectsofcultureaccountsformanyfailedtransfers.Inaddition:embryosaredamagedifexposedtoexcesslight(e.g.microscopelights)forprolongedperiods,butnormallightingordaylightintheworkareaisnotharmfulifembryosarenotexposedformorethanhalfanhour.本文檔共38頁；當(dāng)前第6頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Bodytemperatureofcattleis39℃,andforcertainpurposesthistemperalureisappropriate.However,forroutine程序commercialembryotransferwedonotrecommendstorageabove37℃becausethermometers溫度計,eventhoseonincubators孵卵器,arefrequentlyincorrectby1℃or2℃.Culturingembryosat41℃isextremelydamaging,whereasstoringthemforupto12hoursbetween18℃and37℃isnot.本文檔共38頁；當(dāng)前第7頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)MEDIAThesimplestmediumissterilesaline(9gmNaCl/litreofsterilized,deionized脫離子ordistilledwater).本文檔共38頁；當(dāng)前第8頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Salinewillnotsupportembryonicdevelopment,butwillkeepembryosforafewhours;itisbettertousesalineandtransferembryosquicklythantouseamorecomplexmediumwithoutaproperbuffering緩沖(作用)system.本文檔共38頁；當(dāng)前第9頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(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.

本文檔共38頁；當(dāng)前第10頁；編輯于星期一\0點3分TABLE3Recommendedcultureconditiongs本文檔共38頁；當(dāng)前第11頁；編輯于星期一\0點3分本文檔共38頁；當(dāng)前第12頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Formostapplications,werecommendamodifiedDulbecco'sphosphatebufferedsaline(PBS).Itiseasytousebecauseitdoesnothavetobeequilibratedandmaintainedinanatmosphereof5percentCO2inair.本文檔共38頁；當(dāng)前第13頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Itcanbepreparedfromstockreagents(Table4)orpurchasedineitherready-to-useorconcentratedform,whichmustbedilutedwithsterile,deionized脫離子劑ordistilledwater.本文檔共38頁；當(dāng)前第14頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Afrequenterrorisfailuretodiluteconcentratedmedium;embryoscontractmarkedlywhenexposedtothehighosmolality同滲重摩.CommerciallyavailableDulbecco'sPBSdoesnotusuallycontainantibiotics,sodiumpyruvate丙酮酸鹽(或酯),glucoseormacromolecules高分子.

本文檔共38頁；當(dāng)前第15頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(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.本文檔共38頁；當(dāng)前第16頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)OthermediausedtoculturebovineembryosincludeTissueCultureMedium-199(TCM-199),Ham'sF-10medium,andBrinster'sMouseOvaCultureMedium-3(BMOC-3).Allofthesearecommerciallyavailable.TCM-199withHank'ssaltsdoesnotdependonCO2forbuffering,butTCM-199withEafie'ssaltsaswellasHam'sF-10andBMOC-3mustbemaintainedunderanatmosphereof5percentCO2inair.本文檔共38頁；當(dāng)前第17頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)MACROMOLECULARSUPPLEMENTSSupplementationofmediawithalargeproteinmoleculedecreasessurfacetension張力的,whichreducesthetendencyoftheembryotofloat漂浮oradheretoplasticorglass,andhelpstoinactivate阻止活動heavymetalsandothertoxins.本文檔共38頁；當(dāng)前第18頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)However,macromoleculesderivedfromserumarealsoapossiblevector帶菌者forviralinfectionofembryos.BSAandbovineserumarecurrentlyrecommendedsourcesofmacromolecules.Polyvinyl乙烯聚合物alcoholandpolyvinylpyrrolidone吡咯啉arebeingstudiedaspossiblenon-biologicalmacromoleculestocircumventthedangerofinfection.本文檔共38頁；當(dāng)前第19頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)ThesemacromoleculesdonotfunctionaswellasBSAinreducingsurfacetensionorchelating螯合toxins.BSA(FractionV)shouldbeaddedtomediumjustbeforeuseataconcentrationof0.05-0.1percentforflushingand0.4percentforculture.本文檔共38頁；當(dāng)前第20頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Thepowdershouldbepouredverygently逐漸地onthesurfaceofthemediumandbeallowedtodissolve溶解forabout20minuteswithoutstirring活躍的orshaking搖動,(otherwiseittums小丘intoaglutinousblob粘性水滴的ormakesthemediumunusablyfrothy空洞的).本文檔共38頁；當(dāng)前第21頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)AftertheBSAhasdissolved,thecontainershouldbeinvertedgentlyfiveorsixtimestomixintheBSAcompletely.Themediumshouldthenbesterilizedbypassagethrougha0.22-gbacteriologicalfilter.PurertypesofBSAarealsoacceptable.BSAcanalsobepurchasedasanaqueoussolution,e.g.7percentBSAinwater.本文檔共38頁；當(dāng)前第22頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Ifserumisused,steer向...前進(jìn)orcalfbloodshouldbeharvestedintosterile,sealablecontainersdirectlyfromthevein(toavoidcontaminantsfromskinandhair).Aftercoagulation凝結(jié)物,clots血（凝）arecutevery2-3cmwithasterileknife,andthebloodstoredovernightat5℃.本文檔共38頁；當(dāng)前第23頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(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.本文檔共38頁；當(dāng)前第24頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Inordertoinactivate,阻止活動theproteincomplement,whichcanbetoxictoembryos,serummustbetreatedina56℃waterbathforexactly30minutesaftertheserumhasreached56℃.本文檔共38頁；當(dāng)前第25頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Serumcanbefrozenandstoredforuptoeightmonthsprovidedthatthecontainersaretightlysealed.Forqualitycontrol,onealiquotfromeachbatchofserumshouldbeincubatedovernightinasterile,sealedcontainerat37℃andexaminedthenextdayforbacterialcontamination.本文檔共38頁；當(dāng)前第26頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Forsomesituations,suchasinternationalmovementofembryosormaintenanceofspecific-pathogen-freeherds,additionalsterilizationofserumbygamma微克irradiation放射

isrecommended(ManualoftheInternationalEmbryoTransferSociety,1987).Heat-inactivated,sterilizedserumisaddedtomediuminsteadofBSAataconcentrationof0.5-1percentforflushingand10percentforstorage.本文檔共38頁；當(dāng)前第27頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)CONTAINERS容器Embryosshouldbestoredinsmall(<5ml),sterile,transparent透明的,sealable密封性能,inert惰性的container.本文檔共38頁；當(dāng)前第28頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Smalltesttubes,Petridishes陪替氏培養(yǎng)皿ormulti-wellplatesareconvenientforroutineuse(Figure13),butsmalltesttubesarerecommendedifembryosmustbemovedanydistancebeforeloading裝填intostraws.本文檔共38頁；當(dāng)前第29頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Iftheembryosaretobestoredinuncoveredcontainers/ormorethan20minutes,however,themediumshouldbecoveredwithathinlayerofnon-toxicparaffin石蠟oiltopreventevaporationandcontaminationandtoregulatetherateofgasexchangebetweenthemediumandthesurroundingatmosphere.本文檔共38頁；當(dāng)前第30頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Paraffinoil石蠟canalsobeusedwithothertypesofcontainersandisrecommendedifsealablecontainersarenotused.本文檔共38頁；當(dāng)前第31頁；編輯于星期一\0點3分FIGURE13

Left:Plasticmulti-wellplatewith16x17-mmwells(lidtoleft)本文檔共38頁；當(dāng)前第32頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)Exceptwhiletheyarebeingmanipulated,embryosshouldbestoredinadark,dust-freeincubatororcabinet(e.g.arefrigeratororanicechestwithatightly-fittinglidsetatroomtemperatureor37℃).本文檔共38頁；當(dāng)前第33頁；編輯于星期一\0點3分動物繁殖學(xué)雙語教學(xué)PIPETTES吸移管Manydevices裝置,havebeenusedtomanipulateembryos.StandardPasteurpipettesaremuchtoolargeandthetipsrequirefirepolishingtopreventdamagetoembryos.本文檔共38頁；當(dāng)前第34頁；編輯于