Yale University Policy for Aseptic Amphibian Survival Surgery

Background:Asepticsurgerybydefinitionissurgeryperformedwithoutcontaminationorexposuretopathogens.AccordingtotheAWR"sandtheGuidefortheCareandUseofLaboratoryAnimals,allsurvivalsurgeryshouldbeperformedusingasepticprocedures,includingtheuseofsterilesurgicalgloves,masks,sterileinstruments,andaseptictechniques.

Policy:Aseptictechniquemustbeappliedtoallsurvivalsurgeriesinwhichamphibiansareinvolved.Understanding,itmaybeimpossIBLetoachieveanasepticsiteontheanimal,a"cleancontaminated"sitecanbeachievedbyapplyingthesameasepticstandardtoamphibians.Whereverpossible,thesamestandardsshouldapplytoallcold-bloodedspecies.

Guidelines:Theseguidelinesareprovidedtohelpensureoptimalresultsforasepticsurvivalsurgeryusinglaboratoryamphibians.PleaserefertotheFrogOocyteSurgerySOPformoredetailsonthatprocedure.

Spacerequirements:Separatespacesshouldbeusedforthefollowingactivities:

1. animalpreparation,
2. surgicalarea,
3. postoperativerecovery

Acleanspaceshouldbededicatedtosurgeryandmaintainedassuchduringthesurgicalprocedure.Surfacesshouldbewipedwithdisinfectantbeforeandaftersurgery.Thesurgicalareaalsoshouldbecoveredwithacleandrapeorbenchcoatpaper.

1. Steam:250°F.,15p.s.i.,for30minutes.
2. Gas-Ethyleneoxide:Exposurerequires8-10hoursinaspecialcABInet(gasautoclave).Plasticimplantablematerialsmustbeaeratedfortwotosevendays,dependingonequipmentrecommendations.
3. Liquid-Immersionin:
   • 2%glutaraldehydeplus7.05%phenol(Sporocidin,Cidex,Sonacide)Instrumentsmustbeimmersedfortenhoursina1:10solution.Theymustberinsedinsterilewaterorsalinepriortouse.
   • Chlorinedioxide(Clidox)-Instrumentsmustbeimmersedforsixhoursina1:5:1solutionandrequirerinsinginsterilewaterorsalinepriortouse.
4. Physical-Hotbeads:Insertioninabedofhotbeadsfor10-15secondsworkswellforre-sterilizationofinstrumentswhenperformingmultiplesurvivalsurgeries.However,thismethodonlysterilizestheinstrumenttips.Theinstrumentsmustbefreeoforganicmaterialpriortocontactingthebeads.Instrumentsalsomustbeallowedtocooltoavoidburningtissue.Sterilizedinstrumentsmustbeplacedonasteriledrapetoreducethechancesofcontamination.Anewsterileinstrumentpackmustbeusedaftereveryfourtofiveindividualanimals.

Animalpreparation:Animalsshouldbeanesthetizedawayfromthesurgicalarea.AnesthesiaisinducedbyplacingtheanimalinMS222-treatedwateratroomtemperature.Beforebeginninganyprocedure,confirmthattheMS-222iswithintheexpirationdate.Contacttheon-callVCSveterinarianforthemostappropriateformulationfortheparticularamphibianspecies(Xenopusformula:0.2%MS222,5mMHepes,pH7.5;Axolotls:0.1%MS222).Lackofresponsetotoepinchorrightingreflexshouldbeusedtodeterminedepthofanesthesia.OnceananestheticdepthhasbeenachievedwithMS222,continuedanesthesiacanbeachievedbyloweringthebodytemperaturebyplacingonfoilorsaranwrapovercrushedice.Neverplacetheanimaldirectlyoncrushedice.Theanimal"sskinshouldbekeptmoistthroughouttheprocedurebywettingwithfreshtankwater.

SurgicalProcedures:Amphibiansurgeriesareconsideredclean-contaminatedsurgeries.Thesurgicaltechniquerequiresasepsis,gentletissuehandling,minimaldissectionoftissue,appropriateuseofinstrumentsandcorrectuseofsuturematerial.Thesurgeonmustscrubhandsandarmswithadisinfectantsoapanddonamaskandsterilizedglovesandusesterileinstruments.Drapingofthepatientandfullgowningofthesurgeonmayhelpreducethecontaminationofthesurgicalfield,butaredeemedoflittlepracticalbenefitinmostinstances.

PostoperativeCare:Recoveryismonitoredbywatchingforpurposefulmovements.Allanimalsmustbereturnedtotheanimalfacilitywithin12hours.IACUCapprovalisrequiredforlabhousing,anyperiodbeyond12hours,andisbasedonscientificjustification.Checktheanimaldailyfor3consecutivedayspost-operativelytomonitorforwounddehiscenceandothersignsofcomplications(i.e.,lethargy,wounddehiscence,inflammationetc).ReportanycomplicationstoVCS.

Currently,thereisanexceptionforfish/amphibianstothePost-operativeAnalgesicPolicy,becausetheefficacyandimpactofanalgesicsarenotwelldefinedinthelatterspecies.Thereison-goingreviewofthispolicyasscientificinformationisgatheredsupportingtheefficacyorlackofimpactofanalgesics.Atsuchtime,thisexceptionwillbereassessedandbroughtbeforetheIACUC.

Recordkeeping:Therecordsmaybekeptintheanimalroominanotebookthroughtherecoveryperiodandreturnedtoyourlabforstorageafterwards.Therecordsmustcontainthefollowinginformation:

1. Dateofsurgery
2. Species,animalidentification
3. Protocolnumber
4. Procedure
5. Anesthetic,includingdose
6. Analgesic,ifgiven,includingdose
7. Antibiotic,ifgiven,includingdoseandroute
8. Postoperativemonitoring(dailyforthefirst3daysunlesstherearecomplications)
9. Complications,contactVCSimmediately
10. Thecagecardshouldalsoindicatethedateofsurgeryandprocedureperformed.
11. Dateandmethodofeuthanasia.

References:O"Roukeetal.2002.BIOLOGyandDiseasesofAmphibians.In:LaboratoryAnimalMedicine.Foxetal.,eds.AcademicPress,London.WrightDM.2001.SurgicalTechniques.In:AmphibianMedicineandCaptiveHusbandry.WrightandWhitaker,eds.KriegerPublishingCompany,Malabar,Florida