guidelines for the use of animals in research and …
TRANSCRIPT
GUIDELINES
FORTHEUSEOFANIMALS
INRESEARCHANDTEACHING
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TableofContents
I)OVERVIEW..........................................................................................................................................................4A)ANIMALSCOVEREDUNDERTHESAINTMARY’SCOLLEGEOFCALIFORNIATHEIACUCPROTOCOL....................4B)POLICIES................................................................................................................................................................................4C)COMPLIANCE.........................................................................................................................................................................5D)REFERENCES:.......................................................................................................................................................................6
II)INSTITUTIONALANIMALCAREANDUSECOMMITTEE(IACUC).....................................................7A)RESPONSIBILITIES...............................................................................................................................................................7B)PRINCIPALINVESTIGATOR(PI):RESPONSIBILIES.......................................................................................................8C)SAINTMARY’SCOLLEGEADMINISTRATION:RESPONSIBILITIES................................................................................8
III)IACUCREVIEWPROCESS............................................................................................................................9A)APPROVALPERIOD..............................................................................................................................................................9B)TYPESOFREVIEW...............................................................................................................................................................9C)TYPESOFMODIFICATIONS..............................................................................................................................................10D)APPROVALPROCESS........................................................................................................................................................10E)TYPESOFREVIEWOUTCOMES.......................................................................................................................................11
IV)NEWAPPLICATIONFORIACUCAPPROVAL.......................................................................................12A)SCIENTIFICMERIT............................................................................................................................................................12B)ANIMALCATEGORIES.......................................................................................................................................................12
V)IACUCMANDATEDTRAININGFORRESEARCHERSUSINGANIMALSINTHEIRRESEARCH.13VI)IACUCPOLICYONRESEARCHERNON-COMPLIANCE......................................................................14A)PURPOSE.........................................................................................................................................................................14B)AUTHORITY....................................................................................................................................................................14C)POLICY..............................................................................................................................................................................15i)DeterminationofNon-Compliance........................................................................................................................15ii)ClassificationofNon-Compliance.........................................................................................................................15iv)ConsequencesandResolutionofSignificantNon-ComplianceIncidents............................................16v)FederalReportingofNon-Compliance................................................................................................................16
D)REFERENCES......................................................................................................................................................................18VII)IACUCGUIDELINES...................................................................................................................................19A)ZEBRAFISHINCLUSIONINIACUCPROTOCOLS.............................................................................................19B)ANESTHESIAGUIDELINES-RODENTS...............................................................................................................20C)SURGERYGUIDELINES–RODENTS.....................................................................................................................22D)MAINTAININGANIMALSTUDYAREAS(RODENTS)....................................................................................25E)EUTHANASIAGUIDELINESFORRODENTS......................................................................................................27F)GUIDELINESFOREUTHANASIAOFZEBRAFISH............................................................................................31G)COHOUSING....................................................................................................................................................................32H)RATCAGEDENSITY....................................................................................................................................................33I)MOUSECAGEDENSITY...............................................................................................................................................34
VIII)IACUCSTANDARDPROCEDURES.......................................................................................................35A)OVARIECTOMYINRATSANDMICE....................................................................................................................35B)VASCULARPERFUSIONWITHFIXATIVESFORTISSUECOLLECTION........................................................................37C)BARHOLDINGFORGRIPSTRENGTH..................................................................................................................38D)FORCEDEXERCISE/WALKINGWHEELSYSTEM...........................................................................................39E)CROSSINGZEBRAFISH...............................................................................................................................................40F)OOCYTECOLLECTIONFROMXENOPUSLAEVISFROGS.............................................................................41
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ResearchandInstructionusingAnimalSubjects
I) OVERVIEW
Animaluseinresearchandteachingisessentialtoprovidestudentswiththeexpertisetheyneedtocontinueintocareersandfurthereducationinagriculture,veterinarymedicineandother biological sciences. The use of animals in research has led to many significantdiscoveriesinthefieldofbiologyandmedicine.Therefore,itisimportanttoexposestudentstoanimalresearchwhileensuringthehumanetreatmentanduseofanimalsforresearchandteaching.Each facultymember,studentandstaffmember involved in thecareof theanimals is responsible for thewelfare and humane treatment of the animals during theresearchorinstructionaluseatSaintMary’sCollegeofCalifornia.Therefore,SaintMary’sCollegeofCaliforniahasestablishedpoliciesandguidelinesforthehumanetreatmentanduse of animals in research and teaching. These guidelines are in compliance with theapplicablefederalandstateregulations.TheanimalcareanduseguidelinesareadaptedfromtheAnimalCareandUseGuidelinesatUCSF,withtheirpermission.
A) Animals covered under the Saint Mary’s College of California the IACUC protocol Rodents(Rats,mice,Hamsters,Guineapigs)Chick(afterbirth)Amphibians(Frogs(Xenopus,Rana,Buffo),toads,Salamanders,Mudpuppies),Zebrafish
B) Policies 1)Theuseofanimalsinteachingandresearchshouldbelimitedtoexperimentsthatdemonstrateprinciples,obtainnewresearchinformationthatwillultimatelybenefitthesociety.Theuseofanimalsshouldbesupplementedwithothermethodswheneverfeasible–suchasmodels,invitrosystems.Theinvestigatororinstructorshouldexplorepossiblemethodstoreduceandreplacetheanimalmodel,ifpossible.2)AllresearchandteachingactivitiesusingvertebrateanimalswillbereviewedbytheInstitutionalAnimalCareandUseCommittee(IACUC).TheIACUCcommitteewillapplytheguidelinesintheAnimalWelfareActandtheHealthResearchExtensionActof1985(andtherevisions)toreviewandapproveorwithholdapprovaloftheproposeduseofanimals.3)Theprocurement,careanduseofanimalswillbeinaccordancetotheguidelinesintheAnimalWelfareActandtheHealthResearchExtensionActof1985(andtherevisions).
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4)Theexperimentalproceduresusinganimalswillbeplannedandconductedinamannerastominimizepainanddistresstotheanimals.Theseprocedureswillbeperformedbytrainedpersonnel(typicallyafacultymember).Thestudentsparticipatingintheproceduremustbeadequatelytrainedintheprocedureandsupervised.Ifanyexperimentalordemonstrativeprocedure,oritsconsequences,havethepotentialtoinducesignificantand/orlastingpain,distressorsuffering,appropriatemethodsoftranquilization,anesthesiaandanalgesiamustbeused.Anypainfulordistressfulprocedure,regardlessofwhetheritcanorcannotbeobviated,mustbereviewedandapprovedinadvancebytheInstitutionalAnimalCareandUseCommittee.5)EuthanasiaontheanimalsshouldbeperformedinamannerconsistentwiththelatestrecommendationsoftheAmericanVeterinaryMedicalAssociationPanelonEuthanasia.AlltheproposedmethodsmustbeapprovedinadvancebytheInstitutionalAnimalCareandUseCommittee(IACUC).6)Anyfacultymember,staffmemberorstudentwhobelievesorknowsanyviolationtothispolicycansubmitawrittencomplainttotheChairpersonoftheInstitutionalAnimalCareandUseCommitteeforreviewoftheparticularprocedure.TheCommitteewillreviewthepertinentfactsandreporttheirfindingstotheappropriateadministrativeofficial.
C) Compliance Inordertoensurecompliance,labtechnicianswillbetrainedtoseeandcarefortheanimalshousedinthefacility.Thelaboratoryprotocolswillbeauditedrandomlybycompliancepersonnel.Also,theIACUCwithinspecttheanimalfacilitiesatleastonceayear.Onceaprotocolisapproved,allpersonnelwithlessthanoneyearworkingwiththespeciesarerequiredtoparticipateinamandatorytrainingthatmustbecompletedbeforetheworkbegins.IfadeviationfromaresearchprotocolordiscomfortinananimalisnotedbytheIACUCcommitteemembers,theyareresponsibleforensuringthatanyissuesregardinganimalwell-beingisaddressed.Itisalsodocumentedintheanimal’smedicalrecord.TheincidentisinvestigatedbytheIACUC.ThedirectoroftheIACUCthensubmitsaformallettertothedirectoroftheOfficeofLaboratoryAnimalWelfare,signedbytheSaintMary’sCollegeofCaliforniaofficial,whichispartoftheNationalInstitutesofHealth(NIH).Theprocessistransparent,asreflectedinthe79U.S.OfficeofLaboratoryAnimalWelfare(OLAW)reports,whichweremadeoveraperiodoffiveyears.
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D) References: 1)USDAAnimalCarePolicyhttp://www.aphis.usda.gov/wps/portal/aphis/ourfocus/animalwelfare2)PublicHealthServicePolicyonHumaneCareandUseofLaboratoryAnimalshttp://grants.nih.gov/grants/olaw/references/phspol.htm3)GuidefortheCareandUseofLaboratoryAnimalshttp://www.nap.edu/catalog.php?record_id=129104)HealthResearchExtensionActof1985http://grants.nih.gov/grants/olaw/references/hrea1985.htm5)AVMAEuthanasiaGuidelineshttps://www.avma.org/KB/Policies/Documents/euthanasia.pdf6)IACUC.org(http://www.iacuc.org)
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II)InstitutionalAnimalCareandUseCommittee(IACUC)
Toensurethecomplianceoftheguidelines,SaintMary’sCollegeofCAhassetupacommittee–TheinstitutionalAnimalCareandUsecommittee(IACUC)tooverseethecompliancewiththefederalandstateguidelines.ThecommitteeatSaintMary’sCollegeshallconsistofnotlessthan4membersandshallincludeatleast:●aChairperson;●oneDoctorofVeterinaryMedicine●onepracticingscientistexperiencedinresearchinvolvinganimals;●onememberwhoseprimaryconcernsareinanonscientificarea;and●oneindividualwhoisnotacurrentemployeeorstudentattheCollegeThisCommitteewillbeappointedbytheDeanoftheSchoolofScience.
A) Responsibilities 1)Reviewalltheapplicationsforproposeduseoflivevertebratesforresearchandteaching.2)NotifyinvestigatorsandtheDeanoftheSchoolofScienceinwritingitsdecisiontoapproveorwithholdapprovalofproposedanimaluseprocedures.3)Inspecttheanimalcarefacilitiesonceinsixmonths.4)Ensurethatthepersonnelconductinganimalproceduresaretrainedinanimaluseandintheprocedures.5)Reviewtheinstitutionalprogramforhumanecareanduseofanimalsinresearchandteachingevery6months.6)Reviewconcernsinvolvingcareanduseofanimals.7)Suspendactivitiesthatarefoundtobenotincompliancewiththefederalorstateguidelines.8)ProvideinformationandrecommendationstotheDeanoftheSchoolofScience,ViceProvostforUndergraduateAffairsandtheProvostregardingdifferentaspectofanimalprogram,facilitiesortrainingofpersonnel.9)SubmitreportsonanimalutilizationactivitytofederaloversightregulatoryagenciesincludingtheUSDAandtheNationalInstituteofHealth’sOfficeofLaboratoryAnimalWelfare(OLAW).
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B) Principal Investigator (PI) : Responsibilities
Beforeinitiating,modifying,orrenewinganyresearchprojectthatusesanimalsubjects,PrincipalInvestigatorsmustsubmitanapplicationtotheCommitteeonAnimalsResearch(IACUC)forreviewandapproval.PrincipalinvestigatorsarealsoresponsibleforrenewingtheirIACUCapprovalsonayearlybasis
Principalinvestigatorsareresponsibleforassuringthateveryoneundertheirsupervisioninvolvedinthecareanduseoflaboratoryanimalsisproperlytrained,isthoroughlyknowledgeableaboutthedetailsoftheapprovedprotocol,understandshisorherobligationtocomplywithallapplicableregulationsandfederalandinstitutionalpolicies.
Anyonewhowitnessesorhasreliableinformationaboutnon-compliancewithanIACUC-approvedprotocoloranincidentofmistreatmentofanimalsshouldnotifytheChairoftheInstitutionalAnimalCare&UseCommittee.
C) Saint Mary’s College Administration: Responsibilities
TheDeanoftheSchoolofSciencemustassurethefederalgovernmentonanannualbasisthatSaintMary’sCollegeofCAisincompliancewithallprovisionsoftheAnimalWelfareAct,theNIHGuidefortheCareandUseofLaboratoryAnimals,andthePHSPolicyontheHumaneCareandUseofLaboratoryAnimals.
ThecollegeprovidestheresourcestosupporttheactivitiesoftheIACUC,maintainnecessaryrecords,andreportasrequiredonthestatusoftheanimalcareanduseprogramandthemembershipoftheInstitutionalAnimalCare&UseCommittee.
ThecollegeprovideslegalprotectionformembersoftheInstitutionalAnimalCare&UseCommitteeandtoprincipalinvestigatorsgrantedapprovaltoconductsuchresearchwhohavemettheirobligationsingoodfaith.
TheInstitutionalOfficial(IO)mustreporttotheNIH'sOfficeofLaboratoryAnimalWelfare(OLAW)andtheAnimalCaresectionoftheUnitedStatesDepartmentofAgricultureonthecircumstancesandactionstakeninrespectto:
•anyseriousorcontinuingprotocolviolation;•anyseriousdeviationsfromtheprovisionsintheGuidefortheCareandUseofLaboratoryAnimals;or•suspensionofanyactivitybytheInstitutionalAnimalCare&UseCommittee.
UndertheprovisionsofFreedomofInformationActandtheCaliforniaPublicRecordsAct,thecollegeisrequired,uponrequest,toreleasetothepublicdetailsofanyspecific,fundedresearchproject.
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III)IACUCReviewProcess
A) Approval Period
Theproposalsforusinganimalsforaresearchstudywillbeapprovedforamaximumperiodof5years.TheIACUCwillsendcourtesyreminderstoPIsapproximately75and45daysbeforetheexpirationdateofanapprovedstudytoallowsufficienttimeforrenewalprocessing.Note:Ifrenewalapplicationsarenotrenewedandapprovedbytheexpirationdateandthereareanimalsbeingusedforthestudy,thePrincipalInvestigatorandhis/herstaffwillnothaveaccesstotheanimalsuntiltheapplicationhasbeenapproved.
B) Types of Review
SMC-IACUChasadoptedformalizedprocedures,knownas“DesignatedReviewer”,approvedbytheUSDAandPHS.Thisprocessallowsforreviewofproposedactivities(protocols)bylessthanafullCommittee.
FullCommitteeReview:Initialapplications,andmajorchanges(seebelow)inapprovedprotocolsarereviewedbythefullcommitteeandrequirecompletionoftheFullCommitteeApplication.
DesignatedSubcommitteeReview:Applicationsforrenewalafterinitialreviewandapproval,arereviewedbytheIACUCchairwhocanrequireasub-committeetoreviewthechangesifnecessary.Forrenewalapplication,thePIwillneedtocompletetherenewalformifthechangestotheproposalareminorsuchaspersonnelchanges,updatestoanimalnumbersandminoreditstoproposal.Ifamajormodificationtotheprotocolisinvolved,thePIneedstosubmitacompleteapplication(seebelowunderModificationreview).
UrgentReviews:UndercertainrarecircumstancestheIACUCChairmaydesignateareviewerforurgentreviews.Theinvestigatorsmustprovidecompellingreasonsforthesetypesofreviews.
TissueCollection:TheIACUCChairmaydesignateareviewerforprotocolsproposingtissuecollectiononly.
ModificationReview:IfthePIwishestomakemodificationstotheapprovedprotocolduringitsapprovalperiodorduringrenewalperiod,thePIneedstosubmitacompleteapplicationformforaFullcommitteereview.Thesechangesmustbeapprovedbeforetheyareinitiated.Approvalforamodificationdoesnotchangetheexistingexpirationdateifwithinanapprovalperiod.
LossofQuorum:IfaquorumislostduringaconvenedmeetingoftheFullCommitteeduetotheearlydepartureofoneormoreCommitteemembers,theIACUCChairmaycallforavotebytheFullCommittee(beforeanymembersareexcused)toreviewtheremainingprotocolsontheagendaviathedesignatedmemberreviewprocess.
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C) Types of Modifications
AdministrativeModificationssuchasadditionordeletionofpersonnel,changeinstudytitle,changeinlocation,arereviewedandapprovedbythechairoftheIACUC.
OtherModificationssuchaschangesintheoriginalobjectives,experimentalprocedures,animalnumbersorspecies,experimentalagents,therapeuticagents,oranesthetic/analgesicregimenarereviewedbyDesignatedMemberReviewortheFullCommitteedependingontheextentofthemodificationasdeterminedbythechairoftheIACUCoradesignatedpre-reviewer.
ModificationstotheprotocolmaynotbeimplementeduntilreviewedandapprovedbytheIACUC.
D) Approval Process
TheIACUCapprovalprocessforanewapplicationtakesfromfour–sixweeksandincludesthefollowingsteps:
1. Onceyousubmityourapplication,itwillbepre-reviewedbythechairoftheIACUCoradesignatedpre-reviewertomakesurethatitisclearandcompleteandthatalladministrativerequirementsaremet.Youmayreceiveane-mailrequestingrevisions.
2. Onceyoureviseyourapplicationaccordingtothepre-reviewcomments,youmustresubmitit.Ifitisnotacceptedbythepre-reviewersforFullCommitteereview,youwillbeaskedforfurtherrevisions.IfitisacceptedforFullCommitteereview,itwillbesenttoCommitteemembersoneweekpriortothenextscheduledmeeting.
3. TheCommitteewillreviewyourapplication,andyouwillreceiveanapprovalletter,oraletteraskingforfurtherrevisionstotheapplicationandyourwrittenresponse.
4. Onceyouhavesubmittedyourrevisedapplication,yourresponseletter,andanyadditionalrequireddocuments(e.g.P.I.certificationpage,evidenceofscientificmeritreview)yourresponsewillbereferredtotheCommitteeChair(unlesstheFullCommitteewishestoreviewitagain)forapproval.
Toavoidproblemswithfundingagenciesorexpirationofpreviousapprovals,pleasebesuretoallowenoughtimeforalltheseprocedures.
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E) Types of Review Outcomes
AfterIACUCreview,oneofthefollowingdeterminationsismadeforeachapplication:
FullApprovalisgrantedwhentheCommitteehasnoconcernsabouttheapplication.TheinvestigatorissentaletterwithaIACUCapprovalnumber,validforoneyear,andmaybegintheproject.
RevisionsRequestedpriortoapprovalisgivenwhenthemembersrequireawrittenresponsefromtheinvestigator.Themembersmayasktheinvestigatortoclarifyapoint,providefurtherinformation,ormakerevisionsintheprotocol.Theinvestigator'sresponseisnormallyreviewedbytheIACUCChair.Noapprovalisgivenuntilthequestionsand/orconcernsoftheCommitteehavebeensatisfactorilyaddressedandapprovedbytheChair.
ReturnedforAdditionalInformationpriortocommitteere-reviewisrequestedwhenseriousconcernsareraisedandthemembersagreethatadditionalinformationand/orjustificationisneededbeforeapprovalcanbereconsidered.Theinvestigator'sresponsemustincludeapoint-by-pointletteraddressingallconcerns,aswellasarevisedapplication.TheresponseisthenreviewedbytheFullCommittee.
DisapprovalisgivenwhentheCommitteecompletelyrefusestoapproveaprotocol.
Onrareoccasions,theCommitteemayencountermajordifficultyinmakinganassessmentandanoutsidereviewermaybeaskedtoconsidertheprotocol.
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IV) New Application for IACUC Approval
A) Scientific Merit TheIACUCwillnotreviewproposalsforScientificMeritbutrequiresthateachPIsubmitastatementthataddressesthefollowingareaspertainingtoScientificMeritoftheproposal.Theseareasinclude(adaptedfromtheNIHpeerreviewprocess):•Significance:doestheprojectaddressanimportantprobleminthefield?•Investigator:arethePIandotherresearcherscapableofcarryingouttheproject?•Innovation:dotheexperimentschallengecurrentscientificparadigms?•Approach: are theoverall strategy,methodologies,andanalysesappropriate toaccomplish thespecificaimsoftheproject?•Environment:isthescientificenvironmentadequateforsuccessfulcompletionofthework?
B) Animal categories
Perfederalregulations,experimentalproceduresarecategorizedaccordingtotheirpotentialforcausingpainordistresstoanimalsubjects.Youmustdesignatecategoriesforallstudyanimals.
USDACategory Definition
C Animalsuponwhichteaching,research,experiments,ortestwereconductedinvolvingnopain,distress,oruseofpain-relievingdrugs.
DAnimalsuponwhichexperiments,teaching,research,surgery,ortestswereconductedinvolvingaccompanyingpainordistresstotheanimalsandforwhichappropriateanesthetic,analgesic,ortranquilizingdrugswereused.
E
Animalsuponwhichteaching,experiments,research,surgeryortestswereconductedinvolvingaccompanyingpainordistresstotheanimalsandforwhichtheuseofappropriateanesthetic,analgesic,ortranquilizingdrugswouldhaveadverselyaffectedtheprocedures,results,orinterpretationoftheteaching,research,experiments,surgery,ortests.(AnexplanationoftheproceduresproducingpainordistressintheseanimalsandthereasonssuchdrugswerenotusedmustbeattachedtotheUSDAAnnualReport).
Ananimaliscounted:*onlyonceperyear,evenifitwasusedinmorethanoneProtocol*inthemostpainful/distressfulCategory,ifusedinmorethanonecategory
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V)IACUCMANDATEDTRAININGFORRESEARCHERSUSINGANIMALSINTHEIRRESEARCH
IACUCatSaintMary’sCollegeofCArequiresanin-person/onlinetrainingsessionforanyresearcherorpersonnelinvolvedinanimalcareanduse.Trainingtopicsinclude:
1. Policy/GuidelinesReview:• Policy:ProtocolRequirementsforAnimalHousingandStudyAreas• GuidelinesfortheLaboratoryHousingofResearchAnimals• Guidelines:MaintainingAnimalStudyAreas
2. Documentationrequirements:• Dailytasksaredocumentedeverydayanimalsarepresent.• Intheeventofintermittentanimalhousing,researchersareinstructedon
howtocompletetheformsothatitindicatestherearenoanimalshoused.• Informationisprovidedonwhatisexpectedoflaboratorycarepersonnelif
temperature/humidity/lightcyclevaluesfalloutsideofthespecificationswithinTheGuide.
• DailychecksheetretentionforareasonabletimetofacilitateIACUCreview3. Feed,water,drugs,andmaterials:labeling,storageandinventorymanagement
practices,includingestablishingaprogramforensuringitemsarewithindate.4. Caging
• Enrichment,socialhousing,identification,cagespacerequirements5. CleaningandSanitization
• ACleaning/sanitizationSOPisinplaceorwillbedevelopedbythelabafterreviewinganysite-specificneedsduringthistrainingsession.
• Cagechange/waterbottlechangeintervals6. Healthmonitoring
• Reviewgeneralclinicalsignsofpain/distress/disease• Contactingemergencyveterinarycarewhenneeded
7. Proficiencydemonstration• Theoperationandresettingofathermometer/hygrometer• Maintenanceoftheappropriatelightcycle
8. ThefollowingrequiredpostingswillbeprovidedbyIACUC:• emergencyon-callveterinarycareinformation• SignforreportingAnimalCareandUseConcerns• IACUCPolicyonExpiredDrugsandMaterialsUsedinAnimalResearch• Ensurethatapestcontrolprogramisinplace
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VI)IACUCPOLICYONRESEARCHERNON-COMPLIANCE
A) PURPOSE
TheIACUCisrequiredtoensurehumanecareanduseofvertebrateanimalsusedforresearch,instruction,andtesting,andtoadheretotheapplicablefederalandstateregulationsandinstitutionalpoliciesaffectingtheuseofanimalsatSaintMary’sCollegeofCA.Asaresult,theIACUCmonitorstheanimalcareanduseprogramanditsvariouscomponentsforcompliancewithalltheappropriateregulationsandpolicies.TheIACUChasdevelopedthispolicyforevaluatingissuesofnon-compliancewiththeIACUCprotocols,policiesandregulatoryguidelines.Althoughuniformstandardscanserveasaguide,eachindividualcaseisuniqueandwillbejudgedonitsownmerits.
B) AUTHORITY TheIACUCischargedwithandisresponsibleforensuringcompliancewithfederalregulationsunderCampusAdministrativePolicy100-17,ResearchandInstructionUsingAnimalSubjects.Inordertofulfilltheseobligations,theIACUCmustexerciseauthoritygrantedbythePublicHealthService(PHS)PolicyonHumaneCareandUseofLaboratoryAnimalsandtheAnimalWelfareActandRegulations(AWAR)toenforcepoliciesandregulations.UnderthePHSPolicyonHumaneCareandUseofLaboratoryAnimals,theIACUCmustpromptlyreport(viatheInstitutionalOfficial)totheNationalInstitutesofHealth(NIH)OfficeofLaboratoryAnimalWelfare(OLAW)thefollowing:• AnyseriousorcontinuingnoncompliancewiththePHSPolicy;• AnyseriousdeviationfromtheprovisionsoftheGuidefortheCareandUseofLaboratoryAnimals(theGuide);and• AnysuspensionofanactivitybytheIACUC.
TheAWARalsorequiresthatiftheIACUCsuspendsanactivityinvolvinganimals,theInstitutionalOfficial,inconsultationwiththeIACUC,willreviewthereasonsforsuspension,takeappropriatecorrectiveaction,andreportthatactionwithafullexplanationtotheUnitedStatesDepartmentofAgriculture(USDA)andanyFederalagencyfundingthatactivity.
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C) POLICY
i) Determination of Non-Compliance Todeterminewhetheranissueshouldbeclassifiedasnon-compliance,theIACUCwillinitiateaninvestigationinvolvingthePrincipalInvestigator(PI).Iftheactivityinquestionisdeterminedtobenon-compliant,itwillbecategorizedbasedonitsseverity(asdescribedbelow),aComplianceReportwillbepresentedtotheIACUC,andnecessarycorrectiveactionswillbetaken.Minorissuesinvolvinganimalsarenotalwaysinitiallydeemedasnon-compliance.IfsuchissuesareobservedbytheIACUCstaff,thentheinvestigatororhisorherstaffwillbenotifiedandgivenanopportunitytoaddressittoavoidafindingofnon-compliance.Self-reportingisencouraged.Self-reportinggivestheIACUCnoticeandopportunitytoinvestigateandthePIachancetoremedytheissuebeforeitbecomesnon-complianceorallowsthePItoinformtheIACUCofself-correctivemeasurestakentopreventrecurrence.MonitoringandclarificationofinstitutionalpoliciesandguidelinesregardingthecareanduseofanimalsisroutinelyprovidedbytheIACUCtoassisttheinvestigatorsandtomaintainoverallinstitutionalcompliance.
ii) Classification of Non-Compliance TheIACUCclassifiesnon-complianceincidentsintooneoftwocategories:SignificantandMinor.Bothcategoriesaredefinedbelowwithexamplesofnon-compliances,aswellasanoutlineforcorrectiveactionsthathasbeendelineatedbytheIACUC.TheexamplesareprovidedasageneralguidelinetohelpPIsandresearchstaffinunderstandingthisprocess.Assituationsvaryconsiderably,determinationsaremadeonacase-by-casebasisbasedonthetotalityofthecircumstances,includingself-reporting,voluntarycorrectiveactions,andanyotherrelevantconsiderations.a)SignificantNon-ComplianceSignificantnon-complianceindicatesaseriousbreachoflaws,regulations,oruniversitypolicy,whichcompromisesthefunctionoftheIACUCorputsresearchersoranimalsatriskofundueharm.ExamplesofSignificantnon-complianceinclude,butarenotlimitedto:
• AcquiringanimalsforresearchorperformingunapprovedprocedureswithouttheIACUCapproval;
• Performingaprocedureinsuchamannerthatanimalsendurepainorsufferingthatisnotaddressedbytheapprovedprotocol;
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• Performingaprocedurewithimpropertechniqueorsafeguardswhichputseitherthestafforanimalsatrisk;
• Failuretoadheretoproperaseptictechniqueforsurvivalsurgery;• Repeatedorwillfulincidentsofminornon-compliance.• Failuretoprovideadequateanesthesiaoranalgesiaaccordingtoprotocol
iv) Consequences and Resolution of Significant Non-Compliance Incidents Ifanoccurrenceofnon-compliancedirectlyresultsinasignificantnegativeimpacttoanimalwelfare,theAttendingVeterinarianortheIACUCChairortheirdesignate(s)havetheauthoritytoimmediatelystopallproceduresnecessarytoprotectthehealthandwelfareoftheanimals.ThePIwillbecontactedassoonaspossibleandthematterwillbereferredtotheIACUCandInstitutionalOfficialforfurtherinvestigation.Withinthecategoryofsignificantnon-compliance,incidentswillvaryintheirdegreeofseriousness.AnIACUCsubcommitteemayrecommendacorrectiveaction,andamajorityvotebyaquorumofIACUCmemberswilldecideonacorrectiveactionandmayvoteonprotocolsuspension,revocationofresearchprivileges,orothersanctions.IftheIACUCvotesforprotocolsuspension,allproceduresandorderingprivilegesencompassedbythatprotocolmustceaseduringtheperiodofsuspension.Ifaprotocolissuspended,thePIandhis/herdepartmentchairwillbecontactedassoonaspossibleandaletterwillbesenttothePIfromtheIACUCthatrequiresaresponseregardingcorrectiveactionandfuturepreventivemeasures.ThePIalsomaybeaskedtomeetwiththeIACUC,theIACUCChairand/ortheInstitutionalOfficialasaconditionofreinstatement.FollowinganinvestigationthatincludesafulldiscussionofthesituationwiththePI;theIACUCwillmeettodeterminethecorrectiveactionsthatwillberequiredofthePI.AmajorityvoteoftheIACUCwithaquorumpresent,inconsultationwiththeInstitutionalOfficial,mayliftasuspensiononlyafterithasbeendeterminedthattheprotocol’sactivitiescanbeaccomplishedinfullcompliancewiththerelevantrulesandregulationsandthatadequatemeasureshavebeentakentopreventrecurrenceofthenon-compliantactivity.
v) Federal Reporting of Non-Compliance Intheeventofanincidentofsignificantnon-compliance,duringtheinitialinvestigationtheIACUCwilldeterminetheextentofthenon-compliance.Incidentsofsignificantnon-compliancethataredeemedseriousorcontinuingorrepresentaseriousdeviationfromtheprovisionsoftheGuide,includingsuspensions,willrequirereportingtotheOLAWthroughthetheDeanoftheSchoolofScienceortheirdesigneeandmaybereportedtothefederalfundingagencysupportingtheactivity.
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b)MinorNon-ComplianceMinornon-compliancetypicallyarisesininstanceswherepolicyhasbeenviolatedbuttheriskofharmtoresearchersoranimalsisminimalandtheIACUCauthorityorfunctionhasnotbeencompromised.Minornon-compliancecanoftenbecorrectedattheinstitutionallevel.Examplesofminornon-complianceinclude,butarenotlimitedto:
• NotinformingtheIACUCoftheadditionofpersonnel;• Notmaintainingsurgicalandpost-operativecarerecordsperIACUCpolicyand/or
protocolrequirements;• Failuretorespondtoa“HealthCheck”cardandaddresstheproblemorfailureto
monitortheanimalsadequatelyfollowinginvasiveprocedures;• UseofanunapprovedprocedurearearesultinginfailureoftheIACUCtoinspectthis
areaasrequiredbylaworpolicy;• Failuretoobserveapprovedtimeframeforanimalstobeinoff-siteareas(i.e.
laboratories);• Personnelnotattendingtrainingwithintherequiredtimeframeornotmaintaining
updatedoccupationalhealthforms;• Personnelaccessingfacilitieswithoutauthorization;• Inadequatecontrolledsubstancelogsorcontrolledsubstancestorage;• Unapprovedtransferofanimalsfromoneprotocoltoanother;• Improperorunapprovedanimaltransportation;• NotfollowingsafetyprocedureswhenworkinginBSL-2orlowersuchasnot
wearingappropriatepersonalprotectiveequipment;• Housinganimalsinalabwithoutapprovaloroverthetimelimitapprovedinthe
protocol;• Inadequatehousingconditions,e.g.overcrowdedcages,weaningdelays,orfailureto
separateaggressiveanimalsasrequired;• Minorprotocoldeviation,whichdoesnotsignificantlycompromiseanimalwelfare.• Useoforfailuretodisposeofexpireddrugs;• UseofexpiredmedicalmaterialswithoutIACUCapproval.
ConsequencesandResolutionofMinorofNon-ComplianceIncidentsResolutionofminorissuesmaybeachievedthroughcommunicationbetweentheLARCpersonnel,theIACUCstaff,andtheindividuallabpersonnelwithoutnecessitatingIACUCintervention.Ifanissueisnotresolvedorisdeemedmoreserious,itwillbereportedtotheIACUC.The
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followingnotificationprocessmaybeusedtoobtaincomplianceorescalatethenon-compliance.Firstnotification:ThePIwillberequiredtoprovideawrittenresponseregardinghowtheincidentoccurred,howitwascorrectedandhowitwillbepreventedinthefuture.ThisresponsewillbereviewedbytheIACUC,andtheIACUCmayalsorequireadditionalstepsincludingretrainingofinvestigativestaffmember(s).Secondnotification:PossiblerevocationofanimalorderingorfacilityaccessprivilegesdependinguponthecircumstancesandtheresponseofthePI.ThePImayberequiredtoappearbeforetheIACUCorasubcommitteeoftheIACUC.Thirdnotification:TheNon-compliancemaybereclassifiedassignificant(seeabove).Ifreclassified,itmayresultinanypotentialconsequencesofsignificantnon-compliancelistedabove.ThismayincludesanctionsuptoandincludingasuspensionofactivitiesfollowingamajorityvoteoftheIACUCwithaquorumpresentand/orrevocationofaresearchersprivilegesandanyanimalactivitytherein.ThePImayalsoberequiredtomeetwiththeIACUCChairand/ortheInstitutionalOfficial.Athirdnotificationmayrequirereportingtotheappropriatefederalauthorityandanysuspensionofanimalactivitymustbereported(seeabove).
D) References 1PublicHealthService(PHS).1986.PublicHealthServicePolicyonHumaneCareandUseofLaboratoryAnimals.Washington,D.C.:U.S.DepartmentofHealthandHumanServices.(Availablefrom:OfficeforProtectionfromResearchRisks,Building31,Room4B09,NationalInstitutesofHealth,Bethesda,MD20892).2AnimalWelfareActof1966(P.L.89-544)inclusiveofamendments;1970(P.L.91-579;1976(P.L.94-279);1985(P.L.99-198).3AnimalWelfareActRegulations,(9C.F.R.2.31)InstitutionalAnimalCareandUseCommittee.4OfficeforLaboratoryAnimalWelfare.RequirementsforpromptreportingofproblemstoOLAW.OPRRReports,January12,1994.5AcademicPersonnelManual,APM–015,TheFacultyCodeofConduct6TheGuidefortheCareandUseofLaboratoryAnimals,EighthEdition,NationalAcademicPress,2011.
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VII)IACUCGUIDELINES
A) ZEBRAFISH INCLUSION IN IACUC PROTOCOLS PublicHealthServicepolicyrequiresthatalllivevertebrateanimalsbeincludedintheIACUCapprovedprotocol.TheNIHOfficeofLaboratoryAnimalWelfare(OLAW)considersfishspeciestobe“livevertebrateanimals”at“hatching.”Althoughthisisanimprecisestageinzebrafish,OLAWconsiderszebrafishhatchingtooccurat72hours/3dayspostfertilization(dpf).
1. Zebrafish0-3dayspostfertilization(dpf)arenotconsideredlivevertebrateanimalsanddonotneedtobeincludedinSectionCofyourIACUCprotocol.Descriptionoftheirusemaynonethelessbenecessaryforacompletedescriptionofanimalactivities(SectionG:Procedures)andaspartofthejustificationfornumbersofadultbreedingzebrafish(SectionC:Animals).
2. Zebrafish>3dpf(i.e.72hoursorolder)areconsideredlivevertebrateanimalsandmustbeincludedwiththeirnumbersjustifiedintheIACUCprotocol.
SPECIALCONSIDERATIONSFORPAINCATEGORIZATIONOFZEBRAFISH
1. Zebrafishfrom3-8dpfareallconsideredCategoryCanimals,asthesestageshavenotbeenshowntoperceivepainordistress.
2. Zebrafish>8dpfarecategorizedbasedonthespecificproceduresdescribedintheprotocol.
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B) ANESTHESIA GUIDELINES- RODENTS
1. Acclimationperiodandhealthobservation:Animalsshouldbeacclimatedforatleasttwodaysbeforemajorsurvivalsurgeryorgeneralanesthesiaforsurvivalprocedures.Apre-anesthetichealthobservationshouldbeperformedpriortotheprocedure.Thisinvolvesreviewingtheanimal’sgeneralappearance,activity,respiration,andbodyweightorbodyconditionscore.
2. Fasting:Rodentsaregenerallynotfastedbeforeanesthesia.Waterisnotwithheld.
3. Provideheat:Rodentscanquicklybecomehypothermicunderanesthesiaandduringrecoveryfromanesthesia.Preferredheatsourcesduringandafterproceduresincludecirculatingwarmwaterblankets,chemicalheatpacks,andinsulatingmethods(e.g.Saranwrap).Allrequirecarefulmonitoring,asrodentscaneasilyoverheat.Alwaysplaceaninsulatinglayersuchasatowelbetweentheanimalandtheheatsource.Dependinguponthespeciesandprocedure,monitoringofbodytemperaturemaybeindicated.
Useofheatlampsisdiscouragedastheymaycauseanimalstooverheat.Ifaheatlampmustbeused,placethelampfarenoughawayfromtheanimalstopreventhyperthermia,andprovidealargeenoughrecoveryspacethattheanimalcanmoveifitbecomestoowarm.Maintainingathermometerattheleveloftheanimaltomonitorambientsurfacetemperaturecanaidinhyperthermiaprevention.
4. AdministeringtheAnesthetic:Rodentsmaybeanesthetizedwithinjectableorinhalantagents,orwithacombinationofthetwo.Anadequate,evendepthofanesthesiashouldbemaintainedthroughouttheprocedure.Forinhalants,thiscanbeaccomplishedbyadjustingthevaporizerasnecessary.Forinjectables,supplementaldosesoftheagentcanbeadministeredasneeded.AnestheticdosesmustfollowtheapprovedIACUCprotocol.
5. Applyeyeointment:Foranesthesia/sedationlastinglongerthanfiveminutes,eyeointmentisrequiredtopreventcornealdamageduetolossofblinkreflex.
6. MonitoringAnesthesia:Beforesurgeryisstarted,ensurethattheanimalisadequatelyanesthetizedbytestingthepedalwithdrawalreflex(footpadpinchonbothhindfeet).Ifthefootpadpinchcausesaresponse,supplyadditionalanesthesiaandre-testbeforestartingtheprocedure.Anestheticdepthmustberecheckedregularlyforthedurationoftheprocedure.Monitortheanimal’sresponsivenesstopainfulstimuli,characterofrespiration,andcoloroftheears,tail,gums,orfootpads,andadjustanestheticdepthwheneverneeded.
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7. Stayintheroomwithanesthetizedanimalswhiletheyareontheproceduretable.Donotimmediatelyreturnrecoveringanimalstoacagecontainingun-anesthetizedanimals.Animalsshouldbeplacedinaseparaterecoverycagewithhalfthecageonaheatsource,andobservedevery10-15minutesuntilambulatory.Whentheanimalisabletowalknormally,itcanbereturnedtostandardhousing.
8. Post-proceduremonitoring:Monitoranimalsforsignsofdistressordiscomfortduringandafterrecovery.Recordanycomplications,andcontactthelocalveterinarianifproblemsrecur.AdministeranalgesicspertheapprovedIACUCprotocol.Animalsexperiencingpost-proceduralcomplicationsthatcannotbealleviatedshouldbeeuthanizedusingapprovedguidelines.
9. Fluidadministration:Administrationofwarmed(i.e.bodytemperature)fluidscanhelpanimalsmaintainbloodpressureandspeeduprecoveryfromanesthesiaorsurgery.FluidsmaybegivenIP,intravenously,orsubcutaneously.
10. Recordkeepingisrequiredforanesthesia/sedationprocedureslastinglongerthan15minutesfromtheadministrationoftheanesthetic/sedativeagent(s),andforanysurgicalprocedure.Minimumrequireddocumentationincludes:proceduredate,individualanimalorcageID,procedureperformedandalldrugsadministered.Surgicaloranestheticproblemsmustbedocumented.Refertotheprotocolforotherrecordkeepingrequirements.Grouprecordsmaybeutilizedfordocumentationpurposeswhenallanimalsinacageundergothesameprocedure.
Resources:Incaseofemergency,theIACUCveterinariancanbereachedatXXX-XXX-XXXX.
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C) SURGERY GUIDELINES – RODENTS Scope:Theseguidelinesapplytoallsurgicalproceduresperformedonrodents.Section1-SurvivalSurgeryinRodentsSection2-Non-Survival(Terminal)SurgeryinRodentsSection1-SURVIVALSURGERYAnysurgeryconductedonanimalsthatareexpectedtorecoverfromanesthesiaisconsideredsurvivalsurgery.General:RefertotheIACUCAnesthesiaguidelinesformoredetailsaboutrodentanesthesia.Survivalsurgeryonrodentsshouldbeperformedusingsterileinstrumentsandsutures,cleanorsterilesurgicalgloves,andasepticprocedurestoreducemicrobialcontaminationofexposedtissuestothelowestpracticallevel.PROCEDURESPre-Operative:
1. Surgeryshouldbeconductedinanuncluttered,disinfectedareathatpromotesasepsisduringsurgery.Sanitizethecounter/labbenchwithbleachsolution,chlorhexidine,orClidoxbeforesurgery.Theuseofalcoholisdiscouragedduetolongcontacttimerequiredtotakeeffect(15minutes).Usesteriledrapes,cleanabsorbablepadsortowels,andreplacethesematerialsaftereachsurgerysession.
Allinstrumentsandimplantsmustbesterilizedpriortousewithsteamautoclave,glassbeadsterilizerorethyleneoxidegassterilization.Labelpackagedinstrumentsandimplantswithdateofsterilization.
2. Assoonastheanimalisanesthetized,shave/removefurfromthesurgicalsite(anatomicsite).Performthisprocedureinalocationseparatefromwherethesurgeryistobeconducted(i.e.aseparatelabbench).
3. Administeranypre-operativeanalgesicspertheIACUC-approvedprotocol.
4. Applyophthalmicointment(eyelube)totheanimal’seyestopreventcornealdamage.
5. Disinfectthesurgicalsite(anatomicsite)withdilutechlorhexidineorbetadinescrub.Threealternatingscrubseachofdisinfectantandalcohol(orsterilesaline)arerecommended.Alcoholbyitselfisnotanappropriateskindisinfectantforsurgery.
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6. Surgeonsshoulddonacleanlabcoatorscrubtopandamask.Asurgicalbonnetis
alsorecommended.Washanddryhandsand/orapplyfoamorgeldisinfectantbeforeasepticallydonningsurgicalgloves.Placeasteriledrapeoverthenon-sterilepartoftheanimaltopreventcontaminationofinstrumentsandsuturefromexposedfur.
Noteongloves:Sterileglovesarerequiredifyouwillbetouchingtheanimalorthetipsofyoursterileinstrumentswithyourhands.Ifyouwillonlybetouchingthehandlesofyourinstruments,thenclean,non-sterileglovesareacceptable.
Operative:
1. Providetheanimalwithaheatsourceforthedurationoftheprocedure.Circulatingwarmwaterblanketsandheatingpadsarepreferredoverheatlamps.Alwayshaveatowelordrapebetweentheheatsourceandtheanimal.
2. Testthedepthofanesthesiabyperformingatoepinchonbothrearfeet.Iftoepinch
elicitsaresponse,adjustyouranesthesiaaccordingly,andre-testbeforemakingyourincision.
3. Monitortheanimal'svitalsigns,suchasrespiratorypattern,skin/mucousmembranecolor,anddepthofanesthesia.Iftheanimalrespondstostimuliduringsurgery,stoptheprocedureuntiladditionalanestheticisadministeredandtoepinchelicitsnoresponse.
4. Handlesterileinstrumentsasepticallytominimizecontamination.Abeadsterilizermaybeusedmid-procedureifaninstrumentbecomescontaminated.Makesureinstrumentsarecooltothetouchpriortousingontheanimal.Hotinstrumentsareextremelydamagingtotissue.
5. Instrumentsandglovesmaybeusedforaseriesofsimilarsurgeriesprovidedtheyarestillcleanandarere-sterilizedbetweenanimals.Beadsterilizerscanbeusedtore-sterilizeinstruments.Glovesshouldbechangedifsoiled;ifnotsoiled,spraywithdisinfectant.Anewsetofautoclavedinstrumentsshouldbeusedforeverycage.Keepsterileinstrumentsonasterilefieldwhennotinuse.
6. Closeincisionusingappropriatetechniquesandmaterials.Ifthoraxorabdomenhasbeenopened,atwo-layerclosureshouldbeperformed.
Post-Operative:
1. RecovertheanimalaccordingtotheIACUCanesthesiaguidelines.
2. Monitoranimalpost-operativelyforsignsofdistressordiscomfort:abnormalpostureormovement,lackofappetite,increasedattentiontosurgicalsite.ContactLARCiftherearerecurringproblemswithrecovery.
3. Administerpost-operativeanalgesicspertheIACUC-approvedprotocol.
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4. Cleananddryallsurgicalinstrumentsandre-assemblethemtobesterilizedagain.
Disposeofallsoileddrapes,pads,towels,etc.Placeallsharpsinsharpsdisposalcontainer.
5. Minimumrequireddocumentation:animal/cageID,date,anestheticandanalgesicagentsanddoses,surgicalprocedureperformed,andanysurgicaloranestheticcomplications.Iftheprotocoldescribesotherparametersthatwillberecorded,thesemustbedocumentedinadditiontotheaboverecords.
6. Post-opanimalsshouldbemonitoredatleastoncedailyfor3daysforanysignsofdiscomfort,decreasedappetite,ordelayedwoundhealing.Documentanyproblemsandconsultwiththeveterinarianifcomplicationsrecur.
7. Removewoundclipsorskinsutures10-14dayspostoperatively.AnyexceptionsmustbedescribedintheIACUC-approvedprotocol.
Incaseofemergency,contacttheveterinarianatXXX-XXXX.Section2-NON-SURVIVAL(TERMINAL)SURGERYAnysurgeryconductedonanimalsthatarenotallowedtoregainconsciousnessisconsiderednon-survivalsurgery.Thisincludesterminalvascularperfusion.
• Non-survivalsurgeriesrequireneitheraseptictechniquenordedicatedfacilities,providedthatanimalsarenotanesthetizedlongenoughtodevelopinfections.
• Non-survivalsurgeriesnotperformedasepticallyorinadedicatedfacilitymustatleastbeperformedinacleanarea,freeofclutter.Personnelpresentintheareamustobservereasonablecleanlinesspracticesforboththemselvesandtheanimals.
• Noexpireddrugsorfluidsareallowed.Pharmaceutical-gradeagents(USP)mustbeusedunlessanexemptionisapprovedbyIACUC.
• TheIACUCmustapprovemonitoringparametersforthistypeofsurgery.Theprotocolmustalsodescribethelengthoftheprocedureandstepstakentominimizethepossibilityofinfection.
• ThemethodofeuthanasiashouldbeconsistentwiththeAVMAGuidelinesonEuthanasiaandmustbelistedintheapprovedIACUCprotocol.
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D) MAINTAINING ANIMAL STUDY AREAS (RODENTS)
Forallanimalprocedureareas:
• Theanimalusespaceshouldbeclearedofunnecessaryequipmentandclutterpriortobringinganimalstothelab.Allsurfacesintheanimalusespaceshouldbeeasilysanitizable.Materialsmadeofwood,cardboard,orothermaterialsthatcannotbesanitizedshouldberemovedfromthespace.
• Thefumehoodorbenchtopshouldbecleanedpriortousewithanappropriatedisinfectant(i.e.chlorhexidine,bleachsolution).AppropriatePPEshouldbeputonbeforeremovinganimalsfromcages.
• Ifliveanimalswillbekeptinafumehoodforanylengthoftime,allchemicals
shouldberemovedfromthehood.
• Maintainseparateareasforcagestorage,furclipping,surgery,andnon-surgicalprocedures,aseachactivitycreatesdifferentlevelsofcontamination.
• Suppliesorequipmentthatcomeintocontactwithanimalsmustbe
cleaned/disinfectedbeforeusewithbleach,alcohol,orotherappropriatedisinfectant.
• Dirtycagesaretobecoveredatalltimesandstoredinalocationthatdoesnot
interferewithlaboratoryactivities.Alldirtycagesshouldberemovedfromprocedureareasassoonaspossibletominimizeallergenexposure.Cagesmustberemovedfromlaboratoryspacesbytheendoftheday,andshouldnotbestoredinthelaboratoryovernight.
• Ifanimalusegeneratesdirtybedding,feces,blood,orothercontaminants,these
mustbecleanedupimmediately.
• Theanimaluseareamustbedisinfectedimmediatelyafteranimalworkiscompleted.Cleaningthesurfaceinbetweencagesofanimalsishighlyrecommendedtopreventcross-contamination.
• Inventoryofexpireddrugsandsuppliesshouldbecheckedatleastmonthly.Expired
materialsmustbediscardedimmediatelyorlabeled“forinvitrouseonly”andstoredinanon-animalusearea.
Forsurvivalsurgeryareas:
• Asepticsurgeryshouldbeconductedinadedicatedfacilityorspace.Ifanoperating
roomisnotavailable,anareaphysicallyseparatedfromotherlaboratoryactivitiesmaybeacceptable.
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• Surgicallocationsshouldhaveminimaltrafficandcontainsurfacesthatcanbeeasilydisinfected.
• Thesurgicalpreparationoftheanimalshouldbeconductedinanareaseparated
fromwherethesurgerywillbeperformed.Thesametable/benchtopmaybeusedforbothsurgicalprepandsurgery,buttheseareasmustbedistinctfromoneanother.
• Alwaysdisinfectthesurgeryspacepriortouse,andbetweencagesofanimals.Non-
sanitizablematerialssuchaswoodorcardboardshouldnotbepresentinthesurgeryarea.
• Allsurgicalinstrumentsandmaterialsmustbesterilizedbysteamautoclaveor
otherappropriatemeans(refertoIACUCsurvivalsurgeryguidelines)andkeptsterileuntiltheyareused.
• Cleananddisinfectsurgeryareaimmediatelyafteruse.
Foreuthanasiaareas:
• Euthanasiaequipmentmustbecleanedbeforeandafteruse.
• Itispreferabletoeuthanizeanimalsinahood,intheirhomecageifpossible.
• Questionsorcomments:PleasecontacttheChairoftheIACUC
ForSurvivalSurgeriesSurgicalpreparationoftheanimalisperformedinanareaseparatefromwherethesurgerywilltakeplace.
.
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E) EUTHANASIA GUIDELINES FOR RODENTS GeneralGuidelinesEuthanasiamethodsmustbeconsistentwiththeAVMAGuidelinesfortheEuthanasiaofAnimals:2013Edition,andbespecifiedintheapprovedInstitutionalAnimalCareandUse(IACUC)protocol.• Useofananestheticagentforeuthanasiamustbeadministeredatanoverdose,not
ananestheticdose.Toconfirmdeath,anychemicalmethodusedforeuthanasiamustbefollowedbyaphysicalmethodfromwhichtheanimalcannotrecover,suchasdecapitation,exsanguination,cervicaldislocation,bilateralthoracotomy,tissueperfusion,ordissectingofamajororgan.Theanimalmustbecompletelynon-responsivetonoxiousstimuli(hindfootpadpinchoneachfoot)beforeanyphysicalmethodisperformed.Allagentsusedaretobepharmaceuticalgrade.
• ThetechniqueslistedbelowaremethodscommonlyapprovedinSaintMary’s
CollegeofCAIACUCprotocolsfortheeuthanasiaofrodents.OthermethodsoutlinedintheAVMAGuidelinesonEuthanasiaareacceptablewhenapprovedintheIACUCprotocol.
A)ChemicalMethodsCarbonDioxideInhalation/administration:CO2isdeliveredfromapressurizedtankintoanun-crowdedcagetoensurepreciseregulationofgasinflow.Theflowratemustbesettodisplace10-30%ofthechamberorcagevolume/minute,allowingCO2toenterthechamberslowlysothatunconsciousnessandcompletenarcotizationoccurpriortodeath.Prefilledchambersareunacceptable.CO2flowshouldbemaintainedforatleastoneminuteafterrespiratoryarrest;animalsmustbeleftinthechamberforasufficienttimesothatdeathhasoccurredpriortoperformingaphysicalmethod.Wheneuthanizingmice,astandardsizemousecagemaycontainnomorethan2litters.
CageType CageSize CO2FlowRate
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ToensurecompliancewiththeAVMAGuidelines,aprecisionCO2gauge/regulatorwithapressurereducingvalveorflowmetermustbeused.ThetablebelowprovidesthespecificationsfortheCO2flowrateformiceandrateuthanasia.InspectionandverificationofflowratethatisincompliancewiththeAVMAGuidelinesshouldbeconfirmedonanannualbasisbythelab.Whenpossible,euthanizerodentsintheirhomecagetominimizethestressofbeingplacedintoanunfamiliarenclosureandtopreventsocialaggression.Cages/containersusedforeuthanasiamustallowclearvisibility,beasizethatpermitsfullposturetobeexpressed,andbedisinfectedbetweenusestoremovethepotentialdistressthatmaybecausedbyexposuretoremainingpheromone
Mouse Supermouse750(0.263ft3) 2L/minRat Onecage2100(0.970ft3) 8L/min
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InjectableAnestheticOverdose:Intraperitonealinjectionofatleast200mg/kgsodiumpentobarbitalisrecommended;otherinjectableanestheticsmaybeapprovedanddeliveredatanoverdose.Pentobarbitalsolutionscanbeviscousandarebestdilutedtoaconcentrationofnomorethan60mg/ml.Intracardiacinjectionsaresuitableonlyiftheanimalisadequatelyanesthetized.InhalantAnestheticOverdose:Isofluraneinhalationatanoverdosemaybeutilizedasamethodofeuthanasia,eitherbyprecisionvaporizeroropen-dropmethod.Ifopen-dropisofluraneisutilized,itmustbeadequatelyscavengedtopreventpersonnelexposure.Animalsmayneedtobeexposedforprolongedtimeperiodstoensuredeath.Euthanasiawhileunderanesthesia:Whenanimalsarefullyanesthetizedasattheendofanon-survivalsurgery,methodssuchasbilateralthoracotomy,exsanguinationorperfusionareacceptable.TemporaryHoldingCages:Onoccasion,itmaybeusefulforinvestigatorstotemporarilyholdmorethan5micepercage.Forexample:micebeingcollectedforimmediateeuthanasia.Thisisacceptableaslongasthefollowingconditionsaremet:
• Upto10compatiblemicemaybeplacedinatemporaryholdingcageforupto30minuteandholdingcagesareneverleftunattended.
• Iffightingisobserved,micemustbeimmediatelyseparated.• Adultmales≥6weeksoldfromdifferentcagesshouldnotbecombined• Formiceandratpups<7daysold,refertothebelowguidance
B)PhysicalMethodsChemicalmethodsmustbefollowedbydecapitation,exsanguination,cervicaldislocation,bilateralthoracotomy,tissueperfusion,ordissectingofamajororgan,andaftertheanimalhasbeendeterminedtobenon-responsivetonoxiousstimuli.Physicalmethodsofeuthanasiasuchasdecapitationorcervicaldislocationofun-anesthetizedanimalsmaybeapprovedbytheIACUCwithappropriatejustificationintheIACUCprotocol.ThePImustensurethatpersonnelareexperiencedorproperlytrained.RatsandHamsters:Cervicaldislocationmaybeacceptableifperformedonanimalslessthan21daysand/orweighinglessthan200grams.Guineapigs:Cervicaldislocationmaynotbeperformedonguineapigs.FetusesandNeonates• Itisnotnecessarytoremovefetusesforeuthanasiaafterthedamiseuthanizedasthey
areunconsciousinuteroandhypoxiadoesnotevokearesponse.
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• InhaledanestheticsandCO2areacceptablemethodsofeuthanasiaforneonatessolongasadequateexposuretimeisprovided(30-50minutesforCO2exposure)oranadjunctivephysicalmethodisperformedafteraneonateisnonresponsivetonoxiousstimuli.
• Rats,mice,andhamsterslessthan7daysold:Rapiddecapitationmaybe
performedwithsharpscissorsoranadultdecapitator,dependingontissuemass.Itisrecommendedtoremoveallotherliveanimalsfromtheworkspacewhenperformingthisprocedure.
C)MaintenanceandUseofDecapitationEquipment:Equipmentusedforeuthanasiaofun-anesthetizedanimalssuchascommercialguillotines,scissors,orshearsmustbekeptcleanandservicedonaregularbasistoensuresharpnessofblades.Cleananddisinfectaftereachuse.Afinalrinsewith70%ethanolwillpromotedrying.Bladesshouldbesharpenedannuallyunlessnotinuse,ormoreoftenasindicated.Ifreturnedtouse,theblade(s)mustbesharpenedbeforefirstprocedure.Thebladesshouldbecheckedpriortoeachuseforrust,cleanlinessandabilitytomovefreelywithoutresistance.Dullbladesshouldbereplacedorcanbesharpenedbyprofessionalsharpeningservices.SharpeningandmaintenancerecordsaswellassharpeningSOPsneedtobeavailableduringsemi-annualinspectionsoruponIACUCrequest.Labstaffmustbeappropriatelyexperiencedortrained(seePhysicalMethodsabove).Whenusingdecapitationequipment,ensurehandsandfingersareclearofbladepath.Theuseofplasticrestraintcones(i.e.Decapicones®)isrecommendedtorestrainadultanimalsastheyappeartoreducedistressfromhandling,minimizethechanceofinjurytopersonnelandimprovepositioningoftheanimalintheguillotine.Note:Guillotinesshouldbeperiodicallylubricatedwithsilicone.D)DisposalALLanimalcarcassesandtissuesaretreatedasinfectedbiohazardouswasteanddiscardedinbiohazardbags.Thebagsmustbesealedandstoredinclosedwaterproofcontainerswithtight-fittedlidsindesignatedcoldroomsorfreezersuntilremovedbytheanimalwastemanagementcontractors.Donotplacetheredbagsindirtycagesbeingtransportedtothecagewashfacility.E)Training: Onlytrainedindividualsmayperformeuthanasia.TrainingisprovidedinindividualorgroupworkshopsthroughtheIACUCTrainingandCompliance,
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F) GUIDELINES FOR EUTHANASIA OF ZEBRAFISH Whenpossible,euthanasiashouldcompriseatwo-stepprocessconsistingofachemicalmethodfollowedbyaphysicalmethodfromwhichtheanimalcannotrecover.ThemethodsusedmustbespecifiedinyourapprovedIACUCprotocol.AcceptablemethodsofeuthanasiaaredescribedintheAVMAEuthanasiaGuidelinesundertheLaboratoryFinfishsection.Approvedmethodsforadultzebrafish7dayspostfertilization(dpf)andolderinclude:
• Tricaine(MS-222):Immersefishinasolutionoftricainemethanesulfonate(FinquelorTricaine-S).ThesolutionshouldbebufferedwithsodiumbicarbonatetoapHof7.0-7.5.Fishmustremaininthesolutionfor10minutesfollowingcessationofopercular(gill)movement.
• Rapidchilling:Submergefishin2-4⁰Cchilledwater.Fishshouldnotbeindirectcontactwithice.Fishmustremaininthechilledwaterfor10minutesfollowingcessationofopercularmovement.
Zebrafishfry4-7dayspostfertilization:
• Tricaineorrapidchillingmaybeusedasabove,butfryshouldremainsubmergedinsolutionfor20minutesfollowingcessationofopercularmovement
Zebrafishembryos0-3dayspostfertilization:
• Tricaineandrapidchillingareunreliablemethodsofeuthanasiaforembryos<3dpf.Adddilutebleachsolution(1partsodiumhypochlorite6.15%to5partswater)tothewaterfor5minutestoensureembryoniclethality.
Afteroneoftheabovemethodshasbeenperformed,acceptablephysicalmethodsforallstagesofdevelopmentincludemaceration(fornon-transgenicanimals)orplacementoftheanimalcarcassesinthefreezer.References:1)AmericanVeterinaryMedicalAssociation.2013.AVMAguidelinesoneuthanasia,2013update.2)KoerberASandKalishmanJ.2009.PreparingforaSemiannualIACUCInspectionofaSatelliteZebrafish(Daniorerio)Facility.JournaloftheAmericanAssociationforLaboratoryAnimalScience.48:65-75.3)NIH.2009.GuidelinesforUseofZebrafishintheNIHIntramuralResearchProgram.4)WilsonJK,BunteRMandCartyAJ.2009.EvaluationofRapidCoolingandTricaineMethanesulfonate(MS222)asMethodsofEuthanasiainZebrafish(Daniorerio).JournaloftheAmericanAssociationforLaboratoryAnimalScience.48:785-789.
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G) COHOUSING TheGuidefortheCareandUseofLaboratoryAnimals(2011,NationalAcademiesPress,Washington,DC)recommendsphysicalseparationofdifferentanimalspeciesforthefollowingreasons:
• Topreventinterspeciesdiseasetransmission
• Toeliminatethepotentialforanxiety,physiologicandbehavioralchanges
However,theGuidedoesconsidercohousingofspeciesacceptableifthetwospeciesareofsimilarpathogenstatusandbehaviorallycompatible.Theneedtoco-houseanimalsofdifferentspeciescanbetheresultofseveralfactors.Thesecanincludespacelimitations,researchneeds,sentinelrequirementsand/orequipmentavailability.SpeciesApprovedforCohousingintheSameAnimalHousingRoom
1.RatsandMice• Inallsuchcases,theanimalshaveasimilarpathogenstatus.• Animalsarehousedinindividuallyventilatedcages
2.GerbilsandHamsters• Inallsuchcases,theanimalshaveasimilarpathogenstatus.• Theanimalsarehousedinindividuallyventilatedcagescages.
3.GuineaPigsandMice• Inallsuchcases,theanimalshaveasimilarpathogenstatus.• Theanimalsarehousedinindividuallyventilatedcages.
4.Sentinelmiceandotherrodentspecies• Asinglecageofsentinelmiceperrackmaybecohousedinahousingroomforother
rodentspeciessuchasvoles,hamsters,gerbilsandguineapigs.• Sentinelmicearetestedonaquarterlybasisandprovideimportantinformationon
thehealthstatusofthecolony.
5.DifferentSpeciesofMice• Differentspeciesofmicesuchperomyscusorvaryingspeciesofmiceinthegenus
Musmaybecohousedinthesamehousingroom.• Inallsuchcases,theanimalshaveasimilarpathogenstatus.• Thesespeciesarehousedinindividuallyventilatedcagesorstaticmicroisolator
cages.
6.AquaticAnimals• Turtles,frogsandsalamandersmaybehousedinthesamehousingroom.• Separationofeachspeciesoccursatthecage/tanklevel.
Allnetsandanimalhandlingequipmentremainsseparatebetweenspecies.
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H) RAT CAGE DENSITY TheserecommendationsarebasedontheguidelinesprovidedintheGuidefortheCareandUseofLaboratoryAnimals.CageDensityThefollowingtableindicatesthatmaximumnumberofratsthatmaybehousedintheratcages:
*Nomorethan1000goftotalratbodyweightispermissibleinstaticratcagesEnrichmentandSocialHousingRatsmustbeprovidedwithEnvironmentalEnrichment.Provideatleastonepieceofenrichmentineachcage(nestlet,shelter,additionalnestingmaterial,etc.).Ratsaretobesociallyhoused.SinglyhousedratsareacceptableinthefollowingsituationsanddonotneedseparateIACUCapproval:whentheyproveaggressivetowardcagemates,arethelastsurvivingmemberofacageofanimals,orwhenpaired,maximumcageweightisexceeded.OtherreasonsmustbebasedonscientificjustificationapprovedintheIACUCprotocol.BreedingMonogamousBreedingispermitted.Ventilatedrackcagecapacityisonemale,onefemale,andonelitter.Instaticcagesthemalemaynotbehousedinthesamecageasafemaleandherlitter.
• CagesarelabeledwithbirthandP21projectedweaningdates• CagescontainingalitterolderthanP21cannotalsocontainanewbornlitter
Ø Forthewelfareofanewbornlitter,iftwolittersarepresent,thenthenewlitteristransferredtoanewcage.
WeaningLittersaretobeweanedatP21andseparatedintosamesexcagesExceptionstothispolicymustbesubmittedintheIACUCprotocolandapprovedpriortoimplementation.
BodyWeightofEachRat(grams)
StaticCage(143in2)(maximum#ofRats)
VentilatedCage(210in2)(maximum#ofRats)
<100 8 12101to200 6 9201to300 4 7301to400 3 5401to500 2 3>500 1-2* 2
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I)MOUSECAGEDENSITYTheserecommendationsarebasedontheguidelinesprovidedintheGuidefortheCareandUseofLaboratoryAnimals.CageDensityMaximumof5adultmice/cagearepermittedinthestandard75in2mousecagesusedinLARC.EnrichmentandSocialHousingMicemustbeprovidedwithEnvironmentalEnrichment.Provideatleastonepieceofenrichmentineachcage(nestlet,shelter,additionalnestingmaterial,etc).Micearetobesociallyhoused.SinglyhousedmiceareacceptableinthefollowingsituationsanddonotneedseparateIACUCapproval:whentheyproveaggressivetowardcagemates,thelastremainingofacageofanimals,ormiceremovedfrombreedingcages.OtherreasonsmustbebasedonscientificjustificationapprovedintheIACUCprotocol.BreedingMonogamousBreeding(1M:1F)withonelitterispermitted• CagesarelabeledbyLARCwithbirthandP21projectedweaningdates• CagescontainingalitterolderthanP21cannotalsocontainanewbornlitter
(Forthewelfareofanewbornlitter,LARCstaffisauthorizedtoseparatelittersintonewcagesonarechargebasiswhentwogenerationsofpupsarepresent.)WeaningLittersmustbeweanedatP21andseparatedintosamesexcages.FortheoccasionalfindingofsmallsizedpupsthatmaynotbesuccessfullyweanedatP21,thecagemustbelabeledwiththenewexpectedweaningdateandotherlittersmaynotbebornintothesamecage.ExceptionstothispolicymustbesubmittedintheIACUCprotocolandapprovedpriortoimplementation.
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VIII)IACUCSTANDARDPROCEDURES
A) OVARIECTOMY IN RATS AND MICE
1. Theanimalisweighedandanesthetizedfollowingtheguidelinesabove.Surgicalplaneofanesthesiaandperi-operativeanalgesiaarerequired.Multimodalanalgesiaisencouraged.
2. ThesurgeonwillfollowIACUCguidelinesforasepticsurvivalsurgery.3. Ovariesaretypicallyapproachedbytwoseparateflankincisions[Fig1].
Alternatively,experiencedsurgeonsmayuseasinglemidlineskinincisionontheback,iftheycandothiswithoutexcessivetissuemanipulation[Fig2].Skinincisionisapprox.5mminmouseand10mminrat.Animaliskeptinsternalrecumbencyifusingasingleincision,orlaidonhersideifusingtwoincisions.Theskinisseparatedfromtheunderlyingmusclebeforeincisingthemuscle.
Fig2.Midlineincision
Fig1.Flankincision
4.Beforemakingtheincisionthroughthemuscleoverlyingtheovary,thesurgeonconfirmsthelocationoftheovarianfatpad,whichissometimesvisibleunderthemuscle.Ratherthancuttingthemuscle,thetipofsmalldouble-sharpiridectomyscissorsisinsertedjustthroughthemusclelayertoseparatethemusclefibersbyopeningthescissorsinadorsalventraldirection.
5.Theovaryisgentlypulledthroughtheincisionwithabluntforcepsbygraspingthefatpadsurroundingit.Ahemostatorsimilarisplacedattheboundarybetweentheoviduct
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anduterus,aligatureplacedjustbelowthehemostat(nexttotheuterus)andacutismadejustabovethehemostat.Oncetheovaryandoviductareremoved,thehemostatisreleasedandhemostasisisverifiedbeforelettinggotheuterusandallowingittoreturntotheabdomen.Musclelayerisclosed,usuallywithabsorbablesuture.Skinisclosedwithsutureorwoundclips.
6.Forthestandardtwo-incisionapproach,theanimalisturnedover,andthesecondsidetreatedasthefirstwasdone.
7.AnestheticrecoveryfollowstheSaintMary’sCollegeofCAAnesthesiaguidelines.
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B) Vascular Perfusion with Fixatives for Tissue Collection DescriptionoftheProcedure:
• Thisisastandardprocedureforthevascularperfusionofanesthetizedanimalswithfixativesforpreparingtissuesforhistology.Itisanon-survivalsurgery.
• AnimalsshouldbeanesthetizedfollowingtheAnaesthesiaguidelinesabove.
• Theusualrouteofperfusionistranscardial.Perfusionisusuallydonethroughtheaortaorleftventricle.Therightatriumiscutopen.Animalsaretranscardiallyperfusedthroughtheventricularcatheterwithsalinefollowedbyfixatives,oraredirectlyperfusedwithfixatives.Theperfusionsolutiondrainsthroughtheincisionintheatrium.
• Fixativesolutionscanalsobeinfusedthroughotherlargearteriesanddrainedfromanincisioninavein.
• Fixativesareusuallyinfusedundergravityorusingapump.
• Theperfusedanimalisconsideredeuthanized.Tissuesarecollectedforanalysis.
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C) BAR HOLDING FOR GRIP STRENGTH
Descriptionofprocedure:
Inordertoassesslimbstrengthandcoordination,amouseisallowedtohangbyitsforelimbssuspendedfromawireorsmallbar.
ProcedureSteps:
1. Paddingispositionedunderthebarsothattheanimalisnotharmeduponreleasinggriponthebar.
2. Themouseisallowedtohangbyitsforelimbssuspendedfromawireofsmallbar.Miceshouldbeabletoholdontothebarfor30-240seconds.
Agents:
Thisproceduredoesnotrequireanyagents.
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D) FORCED EXERCISE/WALKING WHEEL SYSTEM
Descriptionofprocedure:
Theforcedexercise/walkingwheelsystemisatestthatcanbeusedforphysiologicstudiesaswellasactivitybasedrestorativetherapies.Thewheelsincorporateaswing-hatchsystemforeasyanimalloadingandremoval.ThehandheldLCDinterfacepermitsasingleexercisetimeandnumberofcycles.Theanimalswillbeloadedonthewheelinsequence.Theexerciseprogramconsistsoftrainingratstorunonamotorizedwheelsystemforupto7.0meters/min.7.0meters/min.iswellbelowtheexercisetolerancelevelinrats.
Supplies:
• HarvardInstrumentswithLCDmonitor.
ProcedureSteps:
1.Theinitialtwo-weektrainingperiodinvolvesatrainingperiodinwhichmiceareputintothetrainingwheelandputontheexercisebedandforcedtorunatacertainspeed,foracertainamountoftime.
2.Theinitialexercisespeedwillbe2.5meters/min.foronehour.
3.Thespeedisthenchangedincrementallyupto7.0meters/minforuptoonehour.
Agents:
Thisproceduredoesnotrequireanyagents.
AdverseEffects:
Procedure,AgentorPhenotype
PotentialAdverseEffects
Management
N/A N/A N/A
MonitoringParameters:
MonitoringParameters Frequency PI/LabwillDocumentActivity,Excreta,Grooming Duringexperimental
manipulations(1-2times)Yes,ifabnormal
Appearance,Behavior,Posture
Daily,duringexperiments Yes,ifabnormal
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E) CROSSING ZEBRAFISH
Objectives:Fisharebredinordertogenerateembryosforexperimentsandformaintenanceofthebreedingstock.Transgenicfisharebredinordertogenerateanimalscarryingtransgenesforlabelingorgeneticmanipulationandgeneticcrossesareperformedtoproduceparticulargenotypesandphenotypes.
DescriptionofProcedure:Crossadultfishataratioof1maleto1femalefishinbreedingtanksovernightatadensityof4orlessfishperliterofwater.Thefishwillbemaintainedinacrossingtankconsistingofanupper(breeding)andlower(embryocollection)chamberseparatedbyplasticorwiremesh.
Embryosfromsuccessfulcrosseswillbecollectedthefollowingmorning.Aftercrossing,adultfishwillbereturnedtosystemtanks.Fishwillbeallowedtoremainoffsystemfornolongerthan20hours.
Fishwillbeallowedaminimum2weekrecoveryperiodbetweenproductivecrosses.Fishthathavebeensetupwithoutsuccesswillbealloweda2dayrecoveryperiodbeforetheprocedureisperformedagain.
PotentialAdverseEffects:Therearenoexpectedadverseeffectsassociatedwiththeprocedurehoweverthephenotypeofthecrossshouldbetakenintoaccount.
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F) OOCYTE COLLECTION FROM XENOPUS LAEVIS FROGS Descriptionoftheprocedure:Oocytesmaybeharvesteduptosixtimesperanimal,alternatingovariesforeachprocedure.Theseproceduresmustbeperformedatleastfourweeksapart,withthelastharvestbeingaterminalprocedure.Pre-proceduralpreparationandanesthesia:• Cleanglovesmoistenedwithwatershouldbewornatalltimeswhenhandlingfrogs.All
surgicalproceduresmustberecordedandrecordscheckedtoassurethatthefrogisanappropriatesurgicalcandidate.
• Chooseafemalefrogthatislargeandactive.Transportfrogsinwaterfromtheirhometanktoandfromthelab.Collectthefrogwithanetthatisdedicatedtothetank.
• AnesthetizethefrogwithafreshmixtureofTricainemethanesulfonate(MS222):500mg-2g/literdissolvedindeionizedwater.Thesolutionmustbebuffered.
• SubmergethefrogintheMS222andwaitfortheonsetofanesthesia-itusuallytakesapproximately15-20minutes.
• Asurgicallevelofanesthesiaisconfirmedbygentlypinchingthefleshypartofbothrearfeetwithhemostatsorforcepsandensuringthatthefrogisnon-responsivetopainfulstimuli.
• Placethefrogonacleansurface.• Removecontaminantsontheskinbygentlyswabbingorsprayingdilutesurgical
disinfectionsolution(chlorhexidine,povidone)onlyontheportionoftheskinwheretheincisionwillbemade.
OocyteCollection:• Theuseofsterileinstrumentsisstandardpracticeforsurvivalcollectionsurgeries.
'Cold'sterilizationfluids(suchascidexorzephrin)shouldbeavoided,astheymayintroducepotentiallytoxicchemicalsintothesurgicalsiteorontopermeableamphibianskin.
• Makeasmallincision(1-2cmhorizontalorvertical)ontheabdomenabovethegroinandinbetweenthemidlineandthelateralaspectoftheabdomen.
• Bluntscissorsareusedtodissectthroughthefasciaandmuscletovisualizetheoocytes.Oocytestrandsarethengentlyexternalizedandcut.
• Theincisionisclosedbysuturingboththefasciaandskinlayer-itisrecommendedtoswabtheincisionsitewithlocalanestheticsuchasbupivacainebeforesuturing.Skinshouldbeclosedusinganinterruptedsuturepatterntopreventdehiscence.
• Recoverthefrogintankwaterwithheadelevated(topreventdrowning)andtherestofthebodysubmerged.Recoverytakesupto1hour.MONITORFROGFREQUENTLY(nolessthanonceevery10minutes).
Post-collectionCare:
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• Frogsmaybereturnedtoaseparaterecoverytankinstandardhousingwhenanimalsareabletoswimnormally
• Allpost-surgicalanimalsmustbeappropriatelyidentified.Iftherearemultiplepost-opanimalsinthesametank,theneachanimalmustbeindividuallyidentified(no-absorbablesutureplacedinwebbing,webnotching,etc.).
• Monitorfrogsdailyforaminimumof48hoursforwounddehiscenceorinfectionandthenevery2-3daysfortwoweeks.ContacttheLARCVeterinaryServicesgroupifthereareanyhealthconcerns.
Documentationrequired:• Identificationofmethodusedandlocationontheanimal.• Dateofsurgery,andapplicablecomments(e.g.frogrecoverednormallyfrom
anesthesia).• Dateofsutureremoval.• Euthanasia.Agents:TheprocedurerequiresanestheticMS-222.Analgesic(bupivacaine)isrecommended.AllagentsmustbelistedonSectionIoftheprotocol.Potentialadverseeffectstobeconsidered:Infectionofsurgicalsite,dehiscence