fvets-09-1009858

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TYPE OriginalResearch PUBLISHED 29September2022 DOI 10.3389/fvets.2022.1009858 OPENACCESS EDITEDBY T.BasRodenburg, UtrechtUniversity,Netherlands REVIEWEDBY RegulaBettschart, UniversityofZurich,Switzerland NancyDeBriyne, FederationofVeterinariansof Europe,Belgium *CORRESPONDENCE MathildeCoutant mathilde.coutant@anis.au.dk SPECIALTYSECTION Thisarticlewassubmittedto AnimalBehaviorandWelfare, asectionofthejournal FrontiersinVeterinaryScience RECEIVED 02August2022 ACCEPTED 02September2022 PUBLISHED 29September2022 CITATION CoutantM,MalmkvistJ,KaiserM, FoldagerLandHerskinMS(2022) Piglets’acuteresponsestolocal anestheticinjectionandsurgical castration:E ectsoftheinjection methodandintervalbetweeninjection andcastration. Front.Vet.Sci.9:1009858.doi:10.3389/fvets.2022.1009858 COPYRIGHT ©2022Coutant,Malmkvist,Kaiser,FoldagerandHerskin.Thisisan open-accessarticledistributedunder thetermsofthe CreativeCommonsAttributionLicense(CCBY) .Theuse,distributionorreproductioninother forumsispermitted,providedthe originalauthor(s)andthecopyright owner(s)arecreditedandthatthe originalpublicationinthisjournalis cited,inaccordancewithaccepted academicpractice.Nouse,distribution orreproductionispermittedwhich doesnotcomplywiththeseterms.Piglets’acuteresponsestolocal anestheticinjectionandsurgical castration:E ectsofthe injectionmethodandinterval betweeninjectionandcastration MathildeCoutant1*,JensMalmkvist1,MarianneKaiser1,LeslieFoldager1,2andMetteS.Herskin11DepartmentofAnimalScience,AarhusUniversity,Tjele,Denmark,2BioinformaticsResearchCentre,AarhusUniversity,Aarhus,Denmark

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ieFoldager1,2andMetteS.Herskin11DepartmentofAnimalScience,AarhusUniversity,Tjele,Denmark,2BioinformaticsResearchCentre,AarhusUniversity,Aarhus,Denmark Althoughappliedinsomecountries,e cacyoflocalanestheticsbasedonprocainetomitigateacuteresponsestopigletcastrationremainsquestioned.Thispaperpresentsresultsfromafactorialstudyexaminingthee ectsoftwomethodsofinjectionofaprocaine-baseddrug(intra-funicular,IF,vs.intra-testicular,IT),andfourintervalsbetweendruginjectionandcastration(2.5,5,10,and30min)onacuteresponsesof3 4dayoldpiglets.Thestudyinvolved597malepiglets,and13treatments:surgicalcastrationwithoutanesthesia(CC),localanesthesiafollowedbycastrationinvolvingallcombinationsofinjectionmethodandinterval,andshamhandlingseparatedbythesamefourintervals(SH).Responsesofpigletstodruginjection,castrationandshamhandlingwereevaluatedbasedonquantificationofintra-proceduralvocalizationsandlegmovements,aswellassalivacortisolconcentrationinsamplestakenbeforeandaftercastration.Nodi erenceswerefoundbetweenIFandthesimplerITinjectionmethod.Intervalsof2.5or30minledtostrongerpigletresponsesthantheotherintervals.Overall,treatmentsinvolvinganesthesialedtosignificantlystrongerresponsesthanshamhandling,duringbothinjectionandcastration.Alltreatments,evenshamhandling,ledtoasignificantincreaseinsalivacortisol,withnodi erencesbetweenanesthesiatreatmentsandcontrols.Basedontheseresults,castration5 10minafterintra-testicularinjectionofprocaineseemstobepreferableascomparedtotheothertreatmentstested.However,pigletsstillshowedmeasurablesignsofpainandstressduringbothinjectionandcastration,whilehandlingalone(includingtheuseofacastrationbench)triggeredanoticeablestressresponse.Inlightofthesefindings,theoverallbenefitoftheprocedureintermsofpigletwelfareremainsarguable. KEYWORDS

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castrationbench)triggeredanoticeablestressresponse.Inlightofthesefindings,theoverallbenefitoftheprocedureintermsofpigletwelfareremainsarguable. KEYWORDS localanesthesia,castration,pig,pain,stress,acuteresponsesFrontiersin VeterinaryScience 01 frontiersin.org

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Coutantetal.10.3389/fvets.2022.1009858 Introduction SurgicalcastrationisaroutinepracticethatinvolvesmillionsofpigsyearlyintheEUandotherglobalregions.Theprocedure, consistingoftheremovalofthemalepiglets’testesviaseveringofthespermaticcords,isprimarilyperformedtoprevent boartaint,perceivedbyhumansasanunpleasantodor,and potentiallypresentinmeatfromentiremales( 1 ).Implementedwithoutpainmitigation,surgicalcastrationleadstosubstantial painandstressinpiglets,asmeasuredforexamplebyahigher prevalenceofhighfrequencyvocalizations( 2 , 3 ),increasedplasmacortisolconcentrations( 4 7 ),andin-penbehavioralalterations( 5 , 8 , 9 )comparedtoshamhandledpiglets.Asaresult,painmitigatingstrategiessuchasadministrationof non-steroidalanti-inflammatorydrugs(NSAIDs)and/orlocal anestheticspriortotheprocedurehavebeendevelopedand implemented.Studiessuggestthatlocalanesthesialimitsthe expressionofhighpitchvocalizations( 2 , 10 , 11 ),reduceslevelsoflegmovementsinterpretedasresistance( 11 14 ),anddecreasestheplasmacortisolresponse( 10 , 15 ).Inaccordancewiththe‘EuropeanDeclarationonalternativestosurgical castrationinpigs’,statingthatfrom2012,allpigletsshould receiveprolongedpainrelief( 16 ),variousEuropeancountrieshaveenforcedtheuseoflocalanestheticspriortopiglet castration,andthepracticeiscommon,butnotsystematic,in theEuropeanpigindustry( 17 ).InDenmark,thefourthlargestproducerofpigsinEurope( 18 ),systemicpainreliefusingNSAIDshasbeenmandatorysince2009,andtheadministrationoflocalanestheticsbecame acode-of-practicein2019,followinganinitiativefromthe Danishpigindustry( 19 ).Farmersandherdsmenareallowedtoadministertheanestheticsthemselvesaftercompletinga trainingledbyaveterinarian,followingtheguidelinesprovided bytheDanishVeterinaryandFoodAdministration[DVFA, ( 19

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eanestheticsthemselvesaftercompletinga trainingledbyaveterinarian,followingtheguidelinesprovided bytheDanishVeterinaryandFoodAdministration[DVFA, ( 19 )].Theseincludeadministrationoftheanestheticusinganintra-funicularinjection(injectionintothespermaticcord, testis,andscrotalskin),alsoreferredtoasa three-step method,anda5to10mintimeintervalbetweeninjectionand castration( 19 ).Theprocedureisperformedusingoneofthetwoanestheticsdrugs(bothprocaine-based)legalizedforpiglet castrationinDenmark.Yet,moststudiesinvestigatingtheefficacyoflocalanesthesiatomitigatepigletpainhavebeencarriedoutinlaboratory- likeconditions,involvingproceduresperformedbyskilled veterinarians[e.g.,( 10 , 12 )].Thereby,theprocedureslikelyconsideredsubtleaspectsoftheinjectiontechniquessuchasthe speedorthepressureappliedonthetestes,whichpotentially mayaffectthepaintriggeredaswellasadministrationefficacy ( 20 , 21 ).Otherstudieshavebeenperformedon-farm,butstillinvolvedatrainedveterinarianfortheadministrationoflocal anestheticandcastration( 22 , 23 ).Thus,intheabsenceofacomparativestudy,itisnotknownwhetherherdsmen,despite theirmandatorytraining,performcomplexproceduressuchasintra-funicularinjectionstoasimilarlevelasexperienced veterinarians.Consequently,thepossibilityfordivergingefficacy oftheprocedurewhenperformedinpractice,ascompared tostudiesledbyveterinarians,meansthatpredictionsforthe actualwelfareimpactofcastrationinvolvinglocalanesthesia,as commonlyperformedcommercially,maybeuncertain.Afield trialsetupwasthereforeimplementedinthepresentstudy.Whiletheefficacyoflocalanesthesiaadministeredpriortopigletcastrationhasbeenmostlyreportedinstudiesusing lidocaine,procaineistheactiveingredientmostfrequently usedasanestheticforpigcastrationintheEU( 17

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opigletcastrationhasbeenmostlyreportedinstudiesusing lidocaine,procaineistheactiveingredientmostfrequently usedasanestheticforpigcastrationintheEU( 17 ).Textbooksreviewinglidocaineandprocainereportdifferencesintermsof onsetofaction,efficacyandpotency( 24 26 ).Inaddition,recentstudiessuggestedunsatisfactorypainmitigationofprocaine administeredalonebeforesurgicalcastrationof3 7dayold piglets( 12 , 22 , 27 ).Irrespectivelyofthedrugused,knowledgeonthewelfareimpactofcastrationwithprioradministrationoflocal anestheticsremainslimited,asonlyfewstudieshavereported piglets’responsetotheinjectionitself.AsreviewedbyKongsted etal.( 28 ),studiesreportingeffectofdifferentmethodsofinjectionarealsoscarce.Inaddition,theeffectofspecific modalitiesoftheanestheticadministrationremainpoorly documented.Forinstance,whilethemethodofinjectionis oftendescribedwithregardstotheanatomicallocation(intra- funicular,intra-testicular),descriptionsoftheorientationofthe needle,pressureappliedonthetestes,ordistributionofthe injectedliquidisoftenlacking( 29 ).Potentialeffectsoftheintervalbetweeninjectionoftheanestheticandcastrationhave beendiscussedwithregardstotheonsetofaction,but,toour knowledge,onlyonestudycomparedtheimpactofintervalon theacuteresponsetocastrationforaspecificdrug( 30 ).Theaimofthepresentstudywastocomparetheeffectoftwomethodsofinjectionofprocaineasalocalanesthetic andfourtimeintervalsbetweeninjectionandcastrationon piglets’responsestoinjectionandcastrationinfieldtrial conditionsresemblingcommercialpractice.Acuteresponses wereevaluatedbasedonvocalizationsandnumberofleg movements,interpretedasresistance,duringinjectionand castration,aswellassalivacortisolconcentrationsbeforeand aftersurgicalcastration. Materialsandmethods

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ments,interpretedasresistance,duringinjectionand castration,aswellassalivacortisolconcentrationsbeforeand aftersurgicalcastration. Materialsandmethods AnimalsTheexperimentwascarriedoutbetweenJulyandOctober2020andwasconductedinaDanishconventionalsowherd withapproximately1,300sowsgivingbirthto(Landrace×Yorkshire)×Duroccrossbredpiglets.Sowswereloose-housedinfarrowingpensmeasuring3.1×2.8m,setasacrateinthefirstweekpost-farrowing,i.e.,duringtheexperiment. Frontiersin VeterinaryScience 02 frontiersin.org

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Coutantetal.10.3389/fvets.2022.1009858 Trialswereconducted2daysperweek,correspondingtotheroutinedaysofcastrationatthefarm.Earlierintheweek,litters reaching3to4daysofageontheweekdayofexperimentation (withday0definedasthedayofbirthofthelastpigletina litter),andcountingatleastsixmales,wereclinicallyassessed, andmalepigletsselected.Allselectedpigletswereclinically healthyandfreeofovertanatomicalmalformations.Piglets weighting<0.9or>2.3kgonthedayofselectionwerenotincludedinthestudy,duetoariskofimproperfitinthe castrationbenchduringtesting.Eachofsixmalepigletsselected withinalitterwasrandomlyassignedtooneof13treatment groups( Table1 )accordingtoarandomizationplanbalancingtreatmentsbetweenlitters,experimentaldays,andexperimental weeks.Onthedayofcastration,thehealthstatusofexperimental pigletsandsowswerere-assessed,andpigletswereexcluded fromtheexperimentiftheinclusioncriteriawerenotfulfilled (severediarrhea,lameness,orsowrectaltemperaturehigher than39◦C).Allpigletswereidentifiedbyanumberwrittenontheirbackusingafood-safemarker.Experimentalpigletswereallowed tobecross-fosteredinthefirstdaysoflife,butcouldnotbe movedfromtheirlitterafterselection.Cross-fosteringofnon- experimentallittermateswaspermitteduptothemorningprior tocastration.Thepigletswereadministeredasuspensionof 45mgtoltrazuriland200mggleptoferron(ForcerisTM,1.5mL,CevaAnimalHealthA/S,Libourne,France)onday1after farrowing.Theexperimentalpigletswerenoteartagged, taildocked,orteethclippedbeforecastration.Inorderto avoidconfoundingoftheresultsontheefficacyofthelocal anesthetic,pigletswereadministeredanNSAID(intramuscular injectionof1.5mgmeloxicam;Melovem,0.3mL,Dopharma, TheNetherlands)asanalgesicaftercompletionofthedata collectionandwithin24haftercastration.

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ramuscular injectionof1.5mgmeloxicam;Melovem,0.3mL,Dopharma, TheNetherlands)asanalgesicaftercompletionofthedata collectionandwithin24haftercastration. StudydesignOnthedayofcastration,experimentalpigletswereweighed.Salivasamplesweretakenapproximately35to40minbefore bringingthepigletstothetestingarea,acalmroomoutside thefarrowingroom.Allexperimentalpigletsplusoneextra littermateselectedatrandomweretransportedtogetherina plasticbox(size:71.5×53.0×39.5cm)layeredwithstraw,andplacedunderneathaheatlamp(averaging20◦C,rangingfrom15to25◦Catrecipients)uponarrivalinthetestingarea.Pigletswereinjected,castratedorshamhandledonebyone,respecting arandomizedtestingorder,andfollowingapredefinedschedule ensuringthattheexperimentalintervalsbetweenprocedures wererespected.Duringallprocedures,pigletswerefixatedwhile lyingontheirback,inacommerciallyavailablecastration bench(UnitronA/S,Kolding,Denmark).Fortheexperimental purpose,thebenchwasmodifiedtoenablelargeramplitudesof frontlegmovements,andmorenaturalopeningofthemouthduringvocalizing.Tofurtherensureaproperfitinthebench, consideringthevariationinpiglets’bodysize,asoftmaterial (5-mmyogamatt;Figurein SupplementaryFigureS1 )couldbeplacedinthebench.In-betweenprocedures,pigletswerereturnedtotheheatedboxwiththeirlittermates.Immediatelyaftercastrationor lastshamhandling,pigletswereindividuallysubjectedto complementarytestingnotreportedinthepresentpaper,and broughtbacktothesowinthefarrowingpen.Onaverage 17minaftercastrationorlastshamhandling,asecondsaliva samplewastakeninthefarrowingunit.Laterintheafternoon, approximately6haftercastration,alastsalivasamplewastaken forcortisoldetermination.Inaccordancewiththeclinicaltrialpermit(describedbelow),pigletswerecloselymonitoredfordrug-relatedside

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astsalivasamplewastaken forcortisoldetermination.Inaccordancewiththeclinicaltrialpermit(describedbelow),pigletswerecloselymonitoredfordrug-relatedside effectsupto72hafteranestheticinjectionandcastration.Twoexperimenterswerepresentinthetestingarea:anexperimenterperformingtheprocedures,andanexperimenter startingandstoppingtherecordings.Thesetwowerenot blindedtotheexperimentaltreatments.Allotherexperimenters, selectingthepiglets,samplingthem,recordingthedataand creatingthedatasetswereblindeduntilthestartofthe statisticalanalysis. TreatmentsAtotalof597pigletswereassignedtooneofthirteentreatments( Table1 ):Surgicalcastrationwithoutlocalanesthesiainvolvingasinglestayinthebench(control-castrated;CC), intra-funicular(IF)injectionof0.5mLoflocalanestheticper testisandsubsequentcastrationafter2.5min(IF02),5min (IF05),10min(IF10)or30min(IF30),intra-testicular(IT) injectionof0.5mLoflocalanestheticpertesticleandsubsequent castrationafter2.5min(IT02),5min(IT05),10min(IT10)or 30min(IT30),shamhandling(SH)withtwostaysinthebench withouttissuedamageinflicted(shamanesthesiaandsham castration),separatedby2.5min(SH02),5min(SH05),10min (SH10)or30min(SH30). ProceduresAllsurgicalandinjectionprocedureswereperformedbythesameexperimenter,anexperiencedfarmstafffromAarhus Universitytrainedinaccordancewithstandardsfromthe DVFA( 19 ).Toachieveauniforminjectiontechniqueamongtheexperimentalpiglets,theexperimenterreceivedadditional trainingledbyaveterinarianandpracticedthedifferent proceduresonapproximately50pigletsbeforeexperimentation. Forallpiglets,theprecisedurationofeachprocedure(injection, castration,shamhandling)wasrecordedtothenearestsecond. Frontiersin VeterinaryScience 03 frontiersin.org

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Coutantetal.10.3389/fvets.2022.1009858TABLE1Descriptionofthe13treatmentgroupsinvolvedinthestudy. IF02IF05IF10IF30IT02IT05IT10IT30CCSH02SH05SH10SH30 IMIFIFIFIFITITITIT SHSHSHSHTI2.5510302.551030 2.551030N50505049504950495050252550 ApartfromCC(controlcastrated),alltreatmentswereacombinationofamethodofinjection(IM):IF,intra-funicularinjection;IT,intra-testicularinjection;SH,shamhandling;andtimeintervalbetweeninjectionandcastration(TI,min):02,2.5min;05,5min;10,10min;30,30min.N,numberofpigletstestedpertreatment.AnestheticdrugTheanestheticusedinthestudywasaprocainehydrochloride2%solution(ProcamidorR Vet.,20mg/mL,RichterPharmaAG,Wels,Austria).Theproductwas administeredusinganautomaticsyringe(PrimaTechR ;0.5mLin0.1mLincrements)witha25Gneedlefortheintra-funicular injection(0.5×16mm,BDMicrolanceTM3,BD,NewJersey,USA).Fortheintra-testicularinjection,inordertogetthe shortestneedlelengthpossible,a26Gneedle(0.45×12mm,StericanR Insulinneedle,BBraunMedicalSA,Barcelona,Spain)wasused,togetherwitha5mmplasticstopper.Needles werechangedbetweeneachpiglet. InjectiontypesPigletswerefixatedindorsalrecumbencypositioninthecastrationbench,andtesteswerefixedcarefullyinthedistal endofthescrotum.Thescrotumareawasnotdisinfected priortoanestheticinjectionnorcastration.Therighttestis wasfixedcaudallybetweenthethumbandindexfingerof theexperimenter,applyingasteadybutlowpressureduring thefixation.Fortheintra-funicularinjection,theneedlewas insertedata45-degreeanglepointingindorsaldirection anda10-degreeanglepointinginlateraldirectionfroma caudocranialview( Figure1 ).Theneedlewasinsertedinitsfulllength(16mm)throughthecenterofthetestisandaiming forthespermaticcord.Theanestheticwasadministeredby continuouslydispensingthedrugwhilewithdrawingtheneedle, andreleasingthetestis,alsoreferredtoas pushandpull

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permaticcord.Theanestheticwasadministeredby continuouslydispensingthedrugwhilewithdrawingtheneedle, andreleasingthetestis,alsoreferredtoas pushandpull technique ( 19 ).Aftereachinjection,adropoftheanestheticwasleftonthesurfaceoftheskinofthescrotum(Cutisscroti).Fortheintra-testicularinjection,theneedlewasinsertedinthe centeroftherighttestis,inadorsaldirectionatanangleof90 degreesfromacaudocranialview( Figure1 ).Acustom-made5mmplasticstopperwasplacedoneachneedletoensurea standardizedneedlelengthof7mm.Theanestheticwasinjected slowlyintothetesticle(overapproximately3s)whilegradually looseningthegriparoundthetesticle.Theprocedureswerethen repeatedforthelefttesticle,andthepigletremovedfromthe castrationbenchandplacedinaheatedareawithlittermates untilcastration. FIGURE1 Thetwomethodsofinjectionofthelocalanesthetic.(A)Theangleofinsertionoftheneedleisshownfortheintra-funicular (IF)and(B)intra-testicular(IT)methods. CastrationAfterfixationinthecastrationbench,adisposablescalpel(Scalpelno.24,carbonsteelsterileblade,Swann- Morton,Sheffield,England)wasusedtoperformanincision (approximately1cm)throughthescrotalskinandspermatic fasciae.Therighttestiswasthengentlypressedbetweenthe indexandthethumboftheexperimenteruntilfullyoutsideof thescrotum.Thetestiswasthencarefullyliftedvertically,and thespermaticcordcutafewmillimetersbelowthetestisusing thescalpel.Theincisionwasrepeatedonthelefttestis.Anew scalpelwasusedforeachpiglet.Avideooftheprocedureis availablein SupplementaryVideoS1 .ShamhandlingPigletswerefixatedinthecastrationbench,aspreviouslydescribed,foradurationofapproximately25s(corresponding totheaveragedurationoftheproceduresoflocalanesthesiaand castrationasassessedinapilotstudy),duringwhichtheydid notexperienceanytissuedamagenorphysicalstimulationofthe groinarea. Frontiersin

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ocalanesthesiaand castrationasassessedinapilotstudy),duringwhichtheydid notexperienceanytissuedamagenorphysicalstimulationofthe groinarea. Frontiersin VeterinaryScience 04 frontiersin.org

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Coutantetal.10.3389/fvets.2022.1009858 EthicalandotherpermitsThestudywasperformedincompliancewiththeEUDirective2010/63/EUforanimalexperiments,theMinistryof Food,AgricultureandFisheries,andTheDanishVeterinary andFoodAdministrationunderact474of15.May2014and executiveorder2028of14.December2020.Theexperiment wasapprovedasaclinicaltrialbytheDanishMedicalAgency (referencenumber2020061784).Allprocedureswereethically evaluatedandapprovedbytheDanishAnimalExperiments Inspectorate(Approvalnumber2019-15-0201-00263). Datacollection VocalizationsThevocalresponsesofthepigletswererecordedduringeachprocedure,usingamicrophone(SennheiserE614,Sennheiser Wennebostel,Germany)fixed30cmaheadofthepiglet’ssnout, attheleveloftheheadofthepiglet.Themicrophonewas connectedtoanamplifier(AudioboxUSBR 96,PreSonus,Louisiana,USA)connectedtoacomputer,fromwhich recordingsweremanuallystartedandstoppeduponpiglet’s placementandremovalfromthecastrationbench.Duration ofeachprocedurewasrecorded.Allvocalfileswereanalyzed inRavenPro1.6bioacousticsanalysissoftware(CornellLab ofOrnithology,Ithaca,NewYork,USA)usingtheband limitedenergydetectorfunction,asdescribedinCoutant etal.( 27 ).Thisfunctionallowedeachintra-proceduralcalltobeautomaticallydetectedbasedonapre-setofparameters (datain SupplementaryTextS1 ),andcharacterizedintermsofnumber,duration,energy,andentropy.Afterrunning theautomaticcalldetection,eachproceduralrecordingwas manuallycheckedtoensurethateverycallwasproperly selected,andtode-selectsurroundednoiseorexperimenter’s voiceswrongfullydetectedasacall.Forallpiglets,vocal characteristicsofeachprocedurewerethendefined( Table2 )andanalyzed.Theseprocedureswereperformedbyoneperson (MC),blindedtotheexperimentaltreatments.Anauditory exampleofapigletvocalizingduringcastrationcanbeconsultedin

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.Theseprocedureswereperformedbyoneperson (MC),blindedtotheexperimentaltreatments.Anauditory exampleofapigletvocalizingduringcastrationcanbeconsultedin SupplementaryVideoS1 .ResistancemovementsFourdistincttypesoffrontlegmovements,interpretedasresistancemovements,wererecordedduringeachprocedure usingacamera(GoProHERO7Black,GoPro,SanMateo, California,USA;60framespersec,FPS)placedonastand 30cmtotherightofthecastrationbench,approximately50cm abovethebench.Thisdistanceallowedafullpictureofthe piglets’frontlegs.Resistancemovementswerequantifiedusinga novelmethoddevelopedinCoutantetal.( 27 ).Videoclipswereobservedatlowspeed(5FPS)todetectfourtypesofmovements: flexion,extension,kick,andblow( Table3 ; Figure2 ).Two TABLE2Descriptionofthevocalparametersanalyzedforeachpigletduringinjectionoflocalanesthetic,castration,orshamhandling,allperformedwhilethepigletwasinthecastrationbench. Parameter(unit)Description CallproportionProportionoftimespentvocalizingduringtheprocedure,calculatedascallduration/procedure duration.Callpersecond(s−1)NumberofcallspersoftheprocedureMeancallduration(s)Averagedurationofacallduringtheprocedure,calculatedassumofcalldurations/numberof calls.Meanenergy(dB)Averageenergy,calculatedasanaverageoftheenergyofeachcallduringtheprocedure.Maxenergy(dB)Maximumvalueofenergyrecordedforallcallsduringtheprocedure.Maxpower(dB)Themaximumpowerrecordedforallcallsduringtheprocedure,relativetothespecificrecording set-up.Aggregatedentropy(kilobits)Aggregateddisorderfortheprocedureobtainedbyanalyzingtheenergydistributionwithineachcall. Higherentropyvaluescorrespondtogreater disorderinthesoundwhereasapuretonewould havezeroentropy( 31 ).Maxentropy(kilobits)Highestvalueofdisorderrecordedforallcallsduringtheprocedure. observers,blindedtotheexperimentaltreatments,weretrained

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ntropy( 31 ).Maxentropy(kilobits)Highestvalueofdisorderrecordedforallcallsduringtheprocedure. observers,blindedtotheexperimentaltreatments,weretrained torecognizeandcountthesebehaviors,andpracticedrecording onapproximately100randomvideoclips,usingtheBehavioral ObservationResearchInteractiveSoftware[BORIS;( 32 )].Eachvideosequencewasthenanalyzed,andtheoccurrenceof eachtypeofbehaviorwascountedforeachfrontleginthe intervalbetweenclosingandopeningofthecastrationbench. Movementsthatweretoosuddentobecategorizeddespite thelowspeedofvideoanalysiswerenotcounted.Reversely, movementsperformedrelativelyslow(duration>1s)werenotconsideredasresistanceandthereforenotrecorded.Inaddition, durationofblockinginthebench,correspondingtoalegbeing mechanicallyunabletomoveduetophysicalblocking,were alsorecorded.Thetwoobserversshowedahighinter-observer reliabilitywithanintraclasscorrelationcoefficient(ICC)close to96%(95%confidenceinterval:90 99%;comparing15video clips).Anexampleoflegresistancemovementsperformedby apigletduringcastrationcanbeconsultedinthevideoin SupplementaryVideoS1 .SalivacortisolconcentrationsForbaseline,onesalivasampleperpigletwascollectedinthehomepenonaverage35(±14;SD)minbeforethefirstprocedure.Forchangesinsalivacortisolinresponsetothe Frontiersin VeterinaryScience 05 frontiersin.org

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Coutantetal.10.3389/fvets.2022.1009858TABLE3Descriptionofthelegresistancemovementsrecorded duringinjectionoflocalanesthetic,castration,orshamhandling,allperformedwhilethepigletwasinthecastrationbench. CategoryDescription FlexionPigletverticallybendshisfrontleg,provokingaflexionoftheelbowofatleast90degrees.ExtensionPigletfullyextendshisfrontlegwhileloweringtheheadinthebench.Maybeaccompaniedbytremblingofthe legand/orbyasubtleliftofthepiglet’sback.KickPigletfrontlegperformsasuddenupwardsmovement,changingfromaflexiontoatenseupwardsposition.BlowPigletsuddenlydrawsbackhisfrontlegforwardsorbackwardsforatleasthalfabenchlength,froma normaluprightpositiontoanextendedposition,with littleornoflexionoftheelbow.LegblockedPiglet’sfrontlegisblockedinthebenchcone,preventingmovement. Eachlegwasscoredseparately. Figure2 showsavisualizationofthelegmovements.procedures,onesampleperpigletwascollectedonaverage17 (±9)minaftercastrationandagainapproximately6haftercastration(5h47min±26min).SalivasampleswereperformedusinganovelmethodrecentlyusedinCoutantetal.( 27 );acottonswab(SalivetteR ,Sarstedt,Aktiengesellschaft&Co.,Numbrecht,Germany)wascutinpieces(approx.2.0×0.5cm),soakedinconcentratedapplejuice(nectarfromconcentrated juice,min60%,RynkebyFoodsA/S,Ringe,Denmark)for1h, anddriedinanelectricovenat60◦Cfor5h.Apilotstudyrevealedanincreaseinsalivaproductionwiththismethod comparedtotheuseofanon-pre-soakedpieceofcotton. Similarresultswereobtainedwithsoakingthecottonpieces incitricacid(freshlemonjuice),butafterthismethod,saliva samplingseemedmoreaversiveforthepiglets,andthemethod wasthereforeabandoned.Duringsampling,thecottonswab wasfixedattheendofastraightpeanclamp,andgently introducedintothepiglet’smouth,whilethepigletwasheld intheexperimenter’sarms.Thecottonwasrotatedgentlyin

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wasfixedattheendofastraightpeanclamp,andgently introducedintothepiglet’smouth,whilethepigletwasheld intheexperimenter’sarms.Thecottonwasrotatedgentlyin thepiglet’smouthfor30to45s,withinsistencearoundthe salivaryglands.Thisprocedurewasperformedbyoneoffour trainedexperimentersblindedtotheexperimentaltreatments. Thesamplewasthenplacedinanexperimentaltube(provided aspartoftheSalivetteR ),labeled,andstoredat−18◦Cuntilcortisolconcentrationdeterminationatthedepartmental laboratory.Samplesweredefrostedandcentrifugedfor6min at1,000×g.Concentrationsofcortisolweredeterminedusingadirectenzymeimmunoassaywithoutextractionand previouslyvalidatedforsaliva(ArborAssays,Cat.K003-H1W, Michigan,USA).Withthismethod,theantiserumcross-reacts withcortisolandsomecortisolmetabolites,andvalueshave tobeinterpretedascortisolimmunoreactivity.Theintra-assay FIGURE2 Visualrepresentationofthefourlegresistancemovements recordedduringinjectionoflocalanesthetic,castration,or shamhandlingwhileinthecastrationbench.F,Flexion;K,Kick;E,Extension;B,Blow.Thevisualizationsonlyshowtheleg movements,seefurtherdescriptionofthecategoriesin Table3 . coefficientofvariationwas3.7and5.6%,respectively,forlow andhighcontrol,andtheinter-assayvariationwas7.2and9.8% forlowandhighcontrol,respectively.Theminimaldetectable concentrationwas45pg/mL.Theprocedureoutlinedbythe manufacturerwasfollowed. StatisticalanalysisThreepigletswereremovedfromtheanalysis:twopigletsduetoexperimentalissuesduringtheprocedures,andone duetocryptorchidism(onlyonetestisdescended)discovered duringinjectionofthelocalanesthetic.Inaddition,for twopiglets,insufficientamountsofsalivarenderedcortisol analysisimpossible,andeightpigletswereexcludedfromthe resistancemovementanalysisduetotechnicalissueswith

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ets,insufficientamountsofsalivarenderedcortisol analysisimpossible,andeightpigletswereexcludedfromthe resistancemovementanalysisduetotechnicalissueswith therecordingofthevideos(fiveforinjectionandthree forcastration).Malfunctioningandtechnicalissuesofthe vocalizationrecordingset-upresultedin168missingfilesfor anesthesiaand182missingfilesforcastration,leavingvalid vocalizationdatafrom376pigletsforanesthesiaand412piglets forcastration.Vocalizationsandresistancemovementswereanalyzedseparatelyforlocalanesthesiaandcastration.Duringthe injectionofthelocalanesthetic,timeintervalhadnotyetany bearing,andthusforthefirstprocedureonlythreetreatments Frontiersin VeterinaryScience 06 frontiersin.org

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Coutantetal.10.3389/fvets.2022.1009858 wererelevant:injectionbyintra-funicularmethod(IF),injection byintra-testicularmethod(IT),andshamhandling(SH).Thevocalizationparameters( Table2 )werelog-transformedifnecessary(toobtainnormality)andanalyzedinalinearmixed effectsmodelwithtreatmentasthemainexplanatoryvariable, weight(range:0.94 2.68kg),age(3or4days),timeofday whenstartingtheprocedure(decimalh,range:8.21 14.66)and durationofprocedure(range:21 117s)ascovariates,andlitter asarandomeffect.Rateofvocalizationsandothervariables wheredurationoftheprocedurewasanintegratedpartof thecalculatedresponse,wereanalyzedbysimilarmodels,but withoutdurationoftheprocedureascovariate.Thecountsofeachtypeofresistancemovementsweresummedperpigletduringeachprocedure( Table3 ).Totalobservationdurationwasdefinedasthesumofobservation timeperleg,subtractingthedurationofleftlegandrightleg occasionallybeingblockedwhileinthecastrationbench.The sumofpiglets’resistancemovementswasanalyzedbyanegative binomialmixedeffectsmodelincludingtreatmentasmain explanatoryvariable,weight,ageandtimeofdayascovariates, logarithmoftotalobservationduration(range:1 211s)asoffset, andlitterasarandomeffect.Changesinsalivacortisolinresponsetotheprocedureswerelogtransformedandanalyzedinamixedmodelwithtreatment, samplingpoint(early-late)andtheirinteractionasfixedeffects ofmaininterest,andweight,age,timeofsampling(decimalh, range:7.82 20.50),andbaselinecortisolconcentration(range: 2,001 48,570pg/mL)ascovariates,withsamplepointasa repeatedmeasure,andlitterasrandomeffect.Foralloutcomes,initialmodelswerereducedbystepwiseremovaloffixedeffectsatP>0.10,startingwiththeinteractionofhighestorder,however,neverremovingthemaineffect forthevariablesofkeyinterest.Inlinearmixedeffects

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emovaloffixedeffectsatP>0.10,startingwiththeinteractionofhighestorder,however,neverremovingthemaineffect forthevariablesofkeyinterest.Inlinearmixedeffects models,Satterthwaite’sapproximationofdenominatordegrees offreedomwasused.Deviationsfromassumptionofnormality andvariancehomogeneityweremonitoredvisuallybyplotting residualsateachstep.Covariateswithsignificanteffectswere maintainedinthefinalmodels,buteffectsnotreported,atthe exceptionofweight.Incasethefinalmodelshowedsignificanteffectofthetreatments(P≤0.05),pairwisecomparisonsbetweentreatmentswereperformedwithp-valuesadjustedformultiplecomparisonsusingtheTukey-Kramermethod(markedbyPadj).Foranalysesofcastration,however,Tukey-Kramerwasnotappliedasthe 13treatmentgroupswouldresultin78pairwisecomparisons, manyofwhichwerenotrelevantfortheaimsofthe presentstudy.Instead,intervalswerecomparedwithininjection methods,andmethodswerecomparedwithinintervals,using unadjustedP-valuesreportedasP.AllcalculationswereperformedusingSAS9.4(SASInstituteInc.,Cary,North Carolina,USA).Alldatausedforstatisticalanalysiscan beconsultedinthedatasetin SupplementaryDatasetS1 .Descriptivemeasuresarepresentedasaverage±standarderror. Results VocalizationsVocalresponsesofpigletsduringadministrationofthelocalanestheticwereaffectedbythetreatments.Asignificant differencewasobservedin6outof8indicators( Table4 ),i.e.,forcallproportion,callpersecond,meanenergy,maxenergy, maxpower,andaggregatedentropy.Acrossallindicators,no differenceswerefoundbetweenthetwomethodsofinjection ofthelocalanesthetic,andbothtypesofproceduresresulted inhighervaluesofvocalparameterscomparedtosham handled(i.e.,notinjected,notcastrated)piglets.Therewas noeffectoftreatmentsintermsofmeancalldurationand

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d inhighervaluesofvocalparameterscomparedtosham handled(i.e.,notinjected,notcastrated)piglets.Therewas noeffectoftreatmentsintermsofmeancalldurationand maxentropy.Atcastration,allvocalizationindicatorsshowedsignificanttreatmentdifferences( Table5 ),exceptformaxentropy.Generally,IFdidnotdiffersignificantlyfromIT,except inmeancallduration,whereIFresultedinlongercalls thanITwhenpigswerecastrated2.5minafteranesthetic injection,whileITresultedinlongercallsthanIFwhenpigs werecastratedafter30min.Pigletscastrated2.5minafter IFinjectionshowedmeancalldurations,maximumenergy andmaximumpowerlevelsnotdifferingsignificantlyfrom CC.Additionally,nodifferenceswerefoundbetweenvocal responsesofpigletscastrated30minafterITinjectionsand theonesofCCintermsofcallproportion,meancall durationandmaxenergy.Infiveoutofeightindicators (i.e.,callproportion,meancallduration,meanenergy,max energy,andmaxpower),nodifferenceswerefoundbetween vocalresponsesofITandIFvs.SH,whenpigletswere castratedafter5and10min.Vocalresponsesofpigletsin thetreatmentsIFandITwere,though,greaterthanSHin thecaseofcastrationafter2.5and30min.ITandIFvocal responsesweregreaterthanSH,regardlessoftheinterval betweenlocalanesthesiaandcastration,anddidnotdiffer significantlyfromthoseofCC,intermsofcallpersecond andaggregatedentropy.Pigletweightwasassociatedwith meanenergy(F1,289=7.1,P=0.008),maxenergy(F1,281=4.6,P=0.033),andmaxpowerparameters(F1,277=7.5,P=0.007),withgreatervaluesobservedinheavierpigs.Overall,piglets’vocalresponsestocastrationdidnotdiffer betweenintra-testicularandintra-funicularinjections.Stronger responses,oftencomparabletothoseofpigletscastratedwithout anesthesia,wereshownbypigletscastrated2.5and30min afteranestheticinjection,whilemostvocalparametersdidnot

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encomparabletothoseofpigletscastratedwithout anesthesia,wereshownbypigletscastrated2.5and30min afteranestheticinjection,whilemostvocalparametersdidnot differfromshamhandledanimalswhenpigletswerecastrated 5and10minafterlocalanesthesia.Fortwoindicators,i.e., callrateandaggregatedentropy,thevocalparametersof pigletscastratedafterinjectionoflocalanestheticdidnotdiffer significantlyfromthoseofpigletscastratedwithoutanesthesia, regardlessofthetimeintervalbetweenlocalanesthesia andcastration. Frontiersin VeterinaryScience 07 frontiersin.org

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Coutantetal.10.3389/fvets.2022.1009858TABLE4Averages(±SE)ofvocalparametersrecordedduringinjectionofthelocalanesthetic. IFITSHF-testP Callproportion0.63±0.01a0.64±0.01a0.47±0.02bF2,314=29.4<0.001Callpersecond(s−1)0.90±0.02a0.88±0.02a0.66±0.03bF2,314=39.3<0.001Meancallduration(s)0.75±0.030.76±0.020.72±0.03F2,311=1.60.201Meanenergy(dB)84.80±9.17a85.13±9.12a46.41±10.10bF2,310=5.60.004Maxenergy(dB)319.98±8.54a318.51±7.94a288.21±10.46bF2,311=3.20.041Maxpower(dB)−14.24±0.74a−14.40±0.67a−17.06±0.89bF2,311=4.30.015Aggentropy(kilobits)130.54±3.27a122.08±2.85a108.82±4.40bF2,330=33.8<0.001Maxentropy(kilobits)5.94±0.015.91±0.015.93±0.02F2,312=1.10.340 a,bDifferentletterswithinarowindicatesignificantdifferencesbetweentreatments,Padj≤0.05;IF,intra-funicularinjection(n=138);IT,intra-testicularinjection(n=135);SH,shamanesthesia(n=103).Fn,ddenotesanF-testonn,numeratorandd,denominatordegreesoffreedomfortheeffectoftreatment.ResistancemovementsPiglets’legmovementsduringinjectionofthelocalanestheticdifferedsignificantlyamongtreatments,withgreater levelsofmovementsobservedinIFandITthaninSH.Theleg movementsobservedduringintra-testicularandintra-funicular injectionsdidnotdiffersignificantly,althoughpigletsinjected bytheintra-funicularmethoddisplayedanaverageof17.6% morelegmovementsthanpigletsinjectedbytheintra-testicular method.Atcastration,thenumberofresistancemovements differedsignificantlyamongtreatments( Table6 ).Regardlessofthetimeintervalbetweeninjectionandcastration,IFdidnot leadtoasignificantlydifferentresponsefromIT,andboth methodsresultedinmoreresistancemovementsthanSH.Piglets castratedatvariousintervalsafterIFinjectiondidnotdiffer significantlyintheirresponse,butpigletscastratedafter30min

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moreresistancemovementsthanSH.Piglets castratedatvariousintervalsafterIFinjectiondidnotdiffer significantlyintheirresponse,butpigletscastratedafter30min showedaresponsethatdidnotdiffersignificantlyfromthe oneofCC.Similarly,pigletscastratedafterITinjectiondidnot differsignificantlyintheirresponse,regardlessoftheinterval.In addition,levelsofresistancemovementsofpigletscastratedafter 2.5or30mindidnotdiffersignificantlyfromtheonesofCC. AllSHpigletsrespondedsignificantlylessthancastratedpiglets, regardlessofintervalbetweenstaysinthecastrationbench. Overall,thelevelofresistancemovementsduringcastration didnotdifferbetweeninjectionmethodsnorintervalbetween procedures.Responsesofpigletscastrated2.5or30minafter injectionofanestheticledtoresponsesthatdidnotdiffer fromthoseofpigletscastratedwithoutanesthesia.Inaddition, anesthetisedpiglets,regardlessofthemethod,showedmore resistancemovementsthanshamhandledpiglets. SalivacortisolconcentrationsAsexpected,baselinecortisolconcentrationsfromsamplesobtained35 40minbeforetheproceduresdidnot differsignificantlyamongtreatments(approx.9,700pg/mL,F12,503=0.8,P=0.673;Tablein SupplementaryTableS1 ).Aweighteffectwasobserved(F1,485=12.2,P<0.001),withhighervaluesofbaselinecortisolinlighterpiglets.Theinteractionbetweentreatmentsandtimingofsamplingwasnotsignificant(F12,1044=1.56,P=0.097).Afterremovingtheinteractionfromthemodel,themaineffectoftreatmentwas alsonotsignificant,althoughatendencywasobserved(F12,1112=1.67,P=0.067).However,theaveragecortisolconcentrationsfollowingtheproceduresweresignificantlyaffectedbythe

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nificant,althoughatendencywasobserved(F12,1112=1.67,P=0.067).However,theaveragecortisolconcentrationsfollowingtheproceduresweresignificantlyaffectedbythe samplepoint(F1,1056=708.3,P<0.001),withhigherconcentrationsobserved17minaftercastration(17,378±8,216pg/mL)than6haftercastration(9,479±5,672pg/mL).Inthismodel,aneffectofweightwasalsoobserved(F1,456=6.2,P=0.013),withhighercortisolconcentrationinlighterpiglets.Consideringthesignificantdifferencebetweencortisolconcentrationsmeasuredat17minand6hpost-procedure, twoseparateanalyseswereperformed.At17min,treatment groupsdifferedincortisolconcentration( Table7 ).AcrossIFandIT,nodifferencewasfoundamongintervalsbetween injectionandcastration,butbothinjectionmethodsledto, ortendedtoleadto,agreaterresponsethanshownbysham pigletsat10and30min.WithintheIFtreatments,piglets showedagreatercortisolresponsewhencastrated30minafter administrationofthelocalanestheticcomparedto2.5and 5min.PigletsanesthetisedbytheITmethoddidnotdiffer significantly,irrespectiveofthetimeintervalbetweeninjection ofthelocalanestheticandcastration.Regardlessofthemethod ofinjectionofthelocalanesthetic,pigletscastratedafter10 and30minshowedagreatercortisolresponsethanCC.Within SH,nodifferenceswereobservedforintervals2.5,5,and 30min,butpigletsoffereda10minintervalbetweenstays inthecastrationbenchhadalowercortisolconcentration comparedtointervalsof2.5minand5min.Thelatterintervals werealsosignificantlydifferentfromCC.Cortisolresponses decreasedwithpigletweight(F1,356=9.49,P=0.002).Overall,cortisolresponses17minaftercastrationdidnotdifferbetween injectionmethods,butgreaterresponseswereobservedin Frontiersin VeterinaryScience 08 frontiersin.org

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Coutantetal.10.3389/fvets.2022.1009858TABLE5Averages(±SE)ofvocalparametersrecordedduringcastration. IFITSHF-testP

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Coutantetal.10.3389/fvets.2022.1009858TABLE5Averages(±SE)ofvocalparametersrecordedduringcastration. IFITSHF-testP Callproportion2.5min0.61±0.03b,10.59±0.03b,10.43±0.04b,2F12,370=6.8<0.0015min0.59±0.03b0.60±0.03b0.51±0.07b10min0.55±0.03b,10.58±0.02b,1/20.44±0.05b,230min0.60±0.03b,10.66±0.03ab,10.43±0.04b,2CC0.72±0.02a0.72±0.02a0.72±0.02aCallpersecond2.5min0.87±0.0510.87±0.0510.68±0.05b,2F12,363=6.7<0.0015min0.91±0.0410.89±0.0510.63±0.07b,210min0.95±0.0410.94±0.0310.69±0.07b,230min0.94±0.0410.87±0.0410.64±0.05b,2CC0.92±0.040.92±0.040.92±0.04aMeancallduration(s)2.5min0.75±0.05ab,10.70±0.05abc,20.63±0.04b,2F12,364=2.40.0065min0.70±0.05bc0.70±0.05b0.81±0.11b10min0.60±0.04c0.62±0.03c0.67±0.08b30min0.68±0.04bc,20.81±0.05ab,10.68±0.05b,2CC0.82±0.04a0.82±0.04a0.82±0.04aMeanenergy(dB)2.5min34.58±15.75b,152.37±16.6b,10.15±15.52b,2F12,358=4.8<0.0015min25.88±16.43b29.59±18.05b42.88±29.40b10min19.28±19.23b12.93±11.93b−2.34±21.16b30min50.27±18.81b,159.10±18.68b,16.74±15.40b,2CC131.20±16.32a131.20±16.32a131.20±16.32aMaxenergy(dB)2.5min299.20±16.71ab,1302.53±15.36b,1244.89±18.90b,2F12,359=3.5<0.0015min273.55±18.78bc285.58±19.65b271.39±24.91b10min265.70±20.45c268.42±17.46b250.20±26.68b30min292.07±18.28bc,1302.51±14.16ab,1243.05±18.59b,2CC349.24±11.32a349.24±11.32a349.24±11.32aMaxpower(dB)2.5min−15.22±1.52ab,1−15.20±1.34b,1/2−20.20±1.79b,2F12,359=3.20.0025min−18.42±1.73c−17.44±1.73b−18.65±2.02b10min−17.87±1.64bc−19.36±1.59b−20.52±2.20b30min−15.92±1.54bc,1−15.92±1.15b,1−20.39±1.54b,2CC−11.83±1.04a−11.83±1.04a−11.83±1.04aAgg.entropy(kilobits)2.5min136.12±7.231136.52±10.251110.08±9.03b,2F12,363=6.30.0015min144.72±12.551138.54±7.391100.73±11.52b,210min149.04±7.251141.56±6.841103.88±9.52b,230min149.11±7.481133.58±5.921108.61±9.11b,2CC148.54±6.23148.54±6.23148.54±6.23aMaxentropy(kilobits)2.5min5.97±0.035.96±0.035.93±0.02F12,361=1.70.0675min6.00±0.036.00±0.035.97±0.0310min5.99±0.045.99±0.045.98±0.0430min5.96±0.035.98±0.036.00±0.02CC5.89±0.035.89±0.035.89±0.03

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35.93±0.02F12,361=1.70.0675min6.00±0.036.00±0.035.97±0.0310min5.99±0.045.99±0.045.98±0.0430min5.96±0.035.98±0.036.00±0.02CC5.89±0.035.89±0.035.89±0.03 1,2Differentnumberswithinarowindicatesignificantdifferencesbetweeninjectionmethodswithinintervalsforthegivenparameter.a−cDifferentletterswithinacolumnindicatesignificantdifferencesbetweenintervalwithininjectionmethodfortthegivenparameter;IF,intra-funicularinjectionandcastrationafter2.5min(n=35),5min(n=33),10min(n=33)or30min(n=36);IT,intra-testicularinjectionafter2.5min(n=30),5min(n=35),10min(n=36)or30min(n=35);SH,shamhandlingatanintervalof2.5min(n=35),5min(n=16),10min(n=19)or30min(n=35);CC,castrationwithoutpainmitigation(control-castrated,n=35).Fn,ddenotesanF-testonn,numeratorandd,denominatordegreesoffreedomfortheeffectoftreatment. Frontiersin VeterinaryScience 09 frontiersin.org

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Coutantetal.10.3389/fvets.2022.1009858TABLE6Averages(±SE)oflegresistancemovementsrecordedduringcastration. IFITSHχ2-testP Anaest.28.44±1.26a23.85±1.02a14.32±1.08bX2 2=172.4<0.001Cast.2.5min24.62±2.13b,130.26±2.71ab,113.96±1.77b,2<0.0015min24.23±2.58b,126.49±2.63b,111.08±2.58b,210min30.22±3.85b,126.96±2.65b,116.08±3.34b,2X2 2=132.430min29.51±2.41ab,130.96±3.00ab,115.69±2.01b,2CC41.06±2.72a41.06±2.72a41.06±2.72a Atanesthesia(Anaest.),a,bdifferentletterswithinarowindicatesignificantdifferencesbetweentreatments,Padj≤0.05;IF,intra-funicularinjection(n=195);IT,intra-testicularinjection(n=196);SH,shamanesthesia(n=148).Atcastration(Cast.),1.2differentnumberswithinarowindicatesignificantdifferencesbetweenmethodswithinintervals,P≤0.05.a−cDifferentletterswithinacolumnindicatesignificantdifferencesbetweenintervalswithinmethods,P≤0.05.IF,intra-funicularinjectionandcastrationafter2.5min(n=50),5min(n=48),10min(n=50)or30min(n=49);IT,intra-testicularinjectionafter2.5min(n=50),5min(n=49),10min(n=49)or30min(n=49);SH,shamhandlingatanintervalof2.5min(n=50),5min(n=25),10min(n=25)or30min(n=48);CC,castrationwithoutpainmitigation(control-castrated,n=49).χ2ndenotesachi-squaredtestonndegreesoffreedomfortheeffectoftreatment. pigletscastrated10or30minafterinjection,comparedto pigletscastratedafter2.5or5min,andtopigletscastrated withoutanesthesia.At6hpost-procedure,piglets’cortisolresponsesdidnotdiffersignificantlyamongtreatments(F12,523=0.7,P=0.721,Tablein SupplementaryTableS1 ).Atthispointintime,cortisolconcentrationswerenotassociatedwithpigletweight (F1,301=0.2,P=0.681). Discussion Weaimedinthisstudytoassesspiglets’acuteresponsestotwomethodsofinjectionofalocalanesthetic,andsurgical castrationfollowingfourdifferenttimeintervalsafterthe

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medinthisstudytoassesspiglets’acuteresponsestotwomethodsofinjectionofalocalanesthetic,andsurgical castrationfollowingfourdifferenttimeintervalsafterthe injection.Acuteresponseswereevaluatedbasedonpiglet vocalizations,legmovementsinterpretedasresistance, andsalivacortisolconcentrationsinsamplesobtainedat twotimepointsaftercastration(atapproximately17min andapproximately6h).Resultsshowednosignificant differencebetweenthetwoinjectionmethods.Greateracute responseswereobservedduringcastrationperformed2.5 and30mincomparedto5or10minaftertheinjections. Salivacortisolconcentrationsinsamplesobtainedfrom pigletscastratedafterinjectionwiththelocalanesthetic didnotdiffersignificantlyfromthoseofpigletscastrated withoutanyanesthesiaorshamhandled.Below,thesefindings arediscussedintermsofmethodologyandinrelationto animalwelfare.Ourresultsshowedthat,overall,administrationoftheprocaine-basedlocalanestheticreducedtheacuteresponses ofpigletstocastration,asmeasuredbythenumberof forelegmovementsinterpretedasresistanceandvocalization characteristics(includingnumber,duration,andintensityof calls),ascomparedtopigletscastratedwithoutanesthesia. Theseresultsarebasedonquantitativerecordingofresistance movementsandautomaticdetectionandcharacterizationofvocalparametersdevelopedinthestudy,andareinlinewith previousresults( 11 , 14 , 23 , 27 ).Thus,thesemethodsseemtobeabletodetectsubtledifferencesinpiglets’acuteresponsesto early-lifeinterventions.Saliva-samplingof3 4dayoldpigletsisdifficultastheyproducerelativelylowamountsofsaliva,anddisplayless spontaneouschewingoncottonswabsduringsamplingthan olderpigletsbeingexperiencedwithsolidfeedintake.The presenttechnique,developedinCoutantetal.( 27 ),showedsuccessfulasitallowedgatheringofenoughsalivatoperform

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letsbeingexperiencedwithsolidfeedintake.The presenttechnique,developedinCoutantetal.( 27 ),showedsuccessfulasitallowedgatheringofenoughsalivatoperform theassays.Inaddition,samplestakenonaverage17minafter castrationshowedsignificantlyhighercortisolconcentrations thanbaselinesamplestakenbefore,andpost-proceduralsamples takenhoursaftertheintervention,indicatingthatthesesamples didrecordarobustresponsetotheprocedure.Thepresentfindingsonsalivacortisolare,however,notinlinewithpreviousstudiesshowingaloweredcortisolresponse tocastrationinpigletsadministeredalocalanestheticprior tocastrationcomparedtopigletscastratedwithoutanesthesia ( 10 , 15 , 33 ).Importantly,previousstudiesreportingtheseresultsanalyzedplasmacortisol,andusedlidocaineastheanesthetic, potentiallyincombinationwithananalgesic.Tothebestof ourknowledge,onlyoneotherstudyreportedsalivacortisol responsestocastrationfollowinginjectionofprocaine( 27 ).Thisstudy,usingasimilardesigntothepresentone,showedno differenceincortisolresponsebetweenpigletscastratedwithand withoutanesthesia.Astudyonthedevelopmentofthecircadian patternofsalivacortisolsecretioninneonatalpigletsreported arelativelyhighvariationincortisolconcentrationsfromsaliva sampledinpigletsupto3daysofage,withastablecircadian patternonlyobservedfrom10daysofageinmales( 34 ).Witharelativelylargeindividualvariationincortisolresponse,even withinatreatment,itcannotbeexcludedthatthevariationin salivacortisolconcentrationsinpigletsasyoungas3to4days ofagemayhavereducedthepossibilitytodetectdifferences inacuteresponses,despiteourrelativelylargesamplesize (calculatedbasedonpreviousplasmacortisolresults).Further Frontiersin VeterinaryScience 10 frontiersin.org

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Coutantetal.10.3389/fvets.2022.1009858TABLE7Averages(±SE)ofsalivacortisolconcentrations(pg/mL)sampledat17minpost-castration. IFITSHF-testP 2.5min16,958±1,100bc17,518±1,277ab18,589±1,255aF12,518=2.50.0035min16,367±1,196bc16,291±816ab18,742±1,750ab10min19,132±1,289ab,119,605±1,482a,113,687±1,289c,230min19,713±1,325a,117,207±936a,1/215,842±1,053ab,2CC15,057±858c15,057±858b15,057±858b 1,2Differentnumberswithinarowindicatesignificantdifferencesbetweenmethodswithinintervals.a,bDifferentletterswithinacolumnindicatesignificantdifferencesbetweenintervalswithinmethods,P≤0.05.IF,intra-funicularinjectionandcastrationafter2.5min(n=50),5min(n=48),10min(n=50)or30min(n=49);IT,intra-testicularinjectionafter2.5min(n=50),5min(n=48),10min(n=49)or30min(n=49);SH,shamhandlingatanintervalof2.5min(n=50),5min(n=25),10min(n=25)or30min(n=49);CC,castrationwithoutpainmitigation(control-castrated,n=49).Fn,ddenotesanF-testonnnumeratorandddenominatordegreesoffreedomfortheeffectoftreatment.studiesinvestigatingtheplasmacortisolresponseofpiglets subjectedtotreatmentssimilartoourstudycouldthereforebe relevant,althoughthegeneralimpactofthesamplingmethod shouldbecarefullyevaluated.Wesuggestthatthiswayof salivasamplingisgenerallypreferableasrelativelynon-invasive comparedtovenepuncture.Inrelationtotheinterpretationofthecortisolresults,concernhasbeenraisedforyearsregardingtheusefulnessof cortisolandotherphysiologicalindicatorstoinformaboutthe affectivecomponentofpain,astheseindicatorsmaybemore relatedtothelevelofarousalinducedbytheprocedure( 35 , 36 ).Ourresultsmaythereforealsoreflectpiglets’stressresponse followingthecombinedproceduresratherthantheabilityof theanesthetictorelieveacutepainonly.Ifalongerduration oftheprocedure(includinginjectionofanesthetic,castration,

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mbinedproceduresratherthantheabilityof theanesthetictorelieveacutepainonly.Ifalongerduration oftheprocedure(includinginjectionofanesthetic,castration, andthetimeintervalin-between)iscorrelatedwithhigher stressresponses,thissuggestioncouldexplainwhy,inourstudy, highercortisolresponseswereobservedinpigletscastrated10or 30minafteradministrationofthelocalanesthetic,comparedto 2.5or5minafter,andcomparedtopigletsnon-anesthetized.It is,however,surprisingthatasimilarpatternwasnotobservedin shamhandledpiglets,wherea10minintervalbetweenstaysin thecastrationbenchledtoalowercortisolresponsethan2.5or 5minintervals.Itispossiblethatalongerintervalbetweenstays inthebenchresultedinadifferentshapeofthecurveofcortisol response,thepeakofwhichisnotknown.Wealsohypothesize thatthelackofsensitivityofcortisolmayhaveresultedin ceilingeffectsfollowinghandlingalone,asalreadysuggestedby previousauthors( 4 , 11 ),especiallyinneonatalpiglets,whoseHPA-axismaybehighlyresponsive( 33 ).Thissuggestionissupported,inourstudy,bycomparablecortisollevelsrecorded inshamhandledandcastratedpiglets.Asaconsequenceofthese knownconcerns,thepresentstudyexaminedacuteresponsesto injectionofanestheticandcastrationacrossseveralindicators includingcortisol,vocalizationsandresistancemovements, therebytakingamulti-modalapproachassuggestedbySheiland Polkinghorne,andBaysingeretal.( 36 , 37 ).Tothebestofourknowledge,onlyoneotherstudyhasinvestigatedtheefficacyofalocalanestheticaspain mitigatingwhenadministeredinpracticebyfarmers.Theauthorsconcludedthatherdsmenwereabletoeffectivelyinject alocalanestheticintra-testicularly,resultingincomparable efficacyofanesthesiaasreportedinotherstudiesinvolving trainedveterinarians( 13 ).Yet,thisassessmentwasconcludedwithnodataorconsiderationofthepiglets’responsetothe

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esthesiaasreportedinotherstudiesinvolving trainedveterinarians( 13 ).Yet,thisassessmentwasconcludedwithnodataorconsiderationofthepiglets’responsetothe injectionofthelocalanestheticitself.Ourstudywasnot designedtoassessherdsmen’sabilitytoadministerthelocal anestheticandsubsequentlyperformcastration,butratherto studytheacuteresponseofthepiglets,withapproximationto on-farmpractice.Afewadaptationstothecastrationroutine ofthecommercialfarmhadtobeimplementedforthesake ofdatarecordingandstandardizationthough.Pigletswere broughtoutsideofthefarrowingroomtobeinjectedwith thelocalanestheticandcastratedwhileinacastrationbench, whereasmanyfarmersperformcastrationinthefarrowing room,placingthepigletsupsidedownorontotheherdsman’s lapduringbothprocedures.Althoughtheimpactofcastration performedwhilethepigletisheldupsidedownhasrecently beenreported( 38 ),comparisonofthepotentialimplicationsofdifferentrestrainingtechniquesforpiglets’responsestoinjection oflocalanestheticshasnotyetbeeninvestigated.Wecannot excludethatfixationinthecastrationbenchledtoadifferent stressresponsecomparedtohandlingonthelap,however,we wouldexpectthattheplacementofpigletsinacalm,heatedarea withlittermatesbetweeninjectionandcastrationcontributed tolimitingthestressresponsecomparedtothetypicalon- farmpracticeofplacementinacartwheelinthefarrowing room.Similarly,applicationoftheproceduresinacalm environmentmaylimittheemotionalsocialcontagionofstress responses(amonglittermates,andwiththesow)potentially happeningwhenseverallittersareprocessedsimultaneously inthefarrowingroom( 39 ).Yet,furtherstudiesarerequiredtoinvestigatethesesuggestions.Throughoutthestudy,the sametrainedherdsman,hiredforthisspecificrole,performed theproceduresonallexperimentalpiglets.Thiswaschosen

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igatethesesuggestions.Throughoutthestudy,the sametrainedherdsman,hiredforthisspecificrole,performed theproceduresonallexperimentalpiglets.Thiswaschosen tostrengthenaccuracy,lowervariationandthusincreasethe powertodetectdifferenceswhencomparingexperimental treatments.Whethertheresultscantranslatedirectlyintoafarm settingishowevernotknown.Furtherstudiescouldinvestigate theefficacyoftheadministrationofalocalanestheticwhen Frontiersin VeterinaryScience 11 frontiersin.org

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Coutantetal.10.3389/fvets.2022.1009858 performedbyfarmemployees,aspartoftheirdailyroutinesin thefarrowingroom.Ourstudydidnotshowasignificantdifferenceinacuteresponsesofpigletsexposedtotheintra-testicularvs.intra- funicularinjectionmethods.Thisresultisinlinewithaprevious studyperformedinpigletsundergeneralanesthesia,andalso comparingthetwomethods( 40 ).Itcanberemarked,though,thatthestudybyHaga&Ranheim( 40 )includedalidocaine-baseddrug,andinvolvedpigletsof22daysofage,thus significantlyolderthaninthepresentstudy.Similarly,arecent studyfoundnodifferenceinvocalizationandlegmovements inpigletsadministered0.5mLofprocainebyanintra-funicular injection,vs.0.3mLofthedrugadministeredbyintra-testicular injection( 23 ).Thelatterstudyinvolvedtheadministrationofdifferentvolumesofanestheticforthetwoinjectionmethods, evaluatingthecombinedeffectsoftheintra-testicularpressure resultingfromthevolumeofliquidinjected( 20 ),theanestheticefficacyobtainedfromthedoseofanestheticadministered,and thepainresponsefromtheareaofinjection( 21 ).Althoughthepresentresultsdidnotreachstatisticalsignificance,our datashowedcloseto20%moreresistancemovementsduring injectionoftheanestheticinpigletssubjectedtoanintra- funicularinjectionvs.intra-testicularinjection.Theintra- funicularinjectionisquitecomplex,requiringadeeper,more precise,injectionintothetesticleandtowardthespermatic cord.Precisionoftheinjectionisconsideredimportantfor theefficacyoftheanesthetic( 41 ),whilethespeedofthedruginjectionandtheintra-testicularpressuremayaffectpainfrom theinjection( 21 ).Ifoneoftheinjectionmethodsistobeapplied,wethereforeseeanadvantageoftheuseoftheintra-testicular injection,asitappearstobeeasierandfastertoperform thanthecurrentlyusedintra-funicularinjectiontype( 23

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foreseeanadvantageoftheuseoftheintra-testicular injection,asitappearstobeeasierandfastertoperform thanthecurrentlyusedintra-funicularinjectiontype( 23 ),withcomparableefficacyatcastration.Yet,penetrationofthetesticle bytheneedle,andincreasedintra-testicularpressureresulting fromtheintra-testicularinjectionareconsideredpainful( 41 ).Inourstudy,injectionofthelocalanesthetic,regardlessofthe methodofinjection,ledtoarateofresistancemovementsclose todoubledcomparedtohandlingwithoutinjection.Inaddition, thedurationoftheprocedurewasaffectedbypigletweight (resultsnotshown),withlongerproceduresobservedinlighter piglets,potentiallybecausefixationofthetestesandinsertion oftheneedleatacorrectanglemayhavebeenmoredifficult insmallpiglets.Weightandageeffectswerealsoobservedin thevocalresponsesofthepigletstotheadministrationofthe anestheticandtocastration,andinthesalivacortisolresponses aswell.Theseeffectsmayberelatedtoadifferentfitinthe castrationbenchforsmallerpiglets,and/ortoweight-related characteristics,suchasasmallerthoraciccage,potentially affectingtheperformanceofvocalizations.Itis,however,also possiblethatpiglets’experienceofanesthesiaandcastration varybasedontheirweight,asthevolumeofanestheticinjected wasfixed,andthedoseofanestheticadministeredperkg thereforedecreasedwithanincreaseinweight.Thispossibilityis,however,poorlysupportedbyourdataasweighteffectswere inconsistentandnotrecurrentacrossindicators.Yet,future studiescouldfocusontheeffectsofanestheticinjectionand subsequentcastrationappliedinpigletsacrossawiderweight range,e.g.,acrossthelegalagerangeof2to7daysforsurgical castrationafterlocalanesthesia.Theeffectofintervalbetweeninjectionoflocalanestheticandcastrationispoorlyinvestigatedintheliterature,andseem toonlyhavebeenincludedinonestudybyCourboulayetal. (

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fintervalbetweeninjectionoflocalanestheticandcastrationispoorlyinvestigatedintheliterature,andseem toonlyhavebeenincludedinonestudybyCourboulayetal. ( 30 ).Thestudycomparedpiglets’responsetocastration15or30minfollowingadministrationofprocaine,andfoundreduced plasmacortisolresponseinpigletscastrated30mincompared to15minafterinjection( 30 ).Theinterpretationofthisresultintermsoftheefficacyoftheanestheticcanbequestioned, though,ascortisolresponsesmaynotbeaccurateindicators ofnociceptivepain,asalreadydiscussed.Theauthorsofthe studymentionedthatpreviousstudieshavesuggestedanonset of20minforprocaine,andthatmostworkevaluatingthe efficacyofprocaineuseda10to30minintervalbetweendrug administrationandsurgery( 30 ).Interestingly,guidelinesgiventoDanishfarmers,andbasedonrecommendationsfromthe manufacturersofProcamidorR Vet.,recommendanintervalof5to10min.Thistimingisinlinewithourresults,showingthe bestefficacyafter5to10mincomparedtotheotherintervals examined.Thisgapbetweentheintervalcommonlyusedin theliteratureandtheintervalrecommendedinpracticecould beexplainedbypotentialdiscrepanciesbetweenpre-existing knowledgeonthelowdiffusingabilityandonsetofactionof procaine( 26 ),andtheformulaandcharacteristicsofspecificprocaine-basedproductsavailablecommercially.Considering therelativelylowrateofdiffusionofprocaine( 24 ),itcannotbeexcludedthatanintervalinferiorto5mindoesnotresult insufficientneuronalblocking.Inaddition,astudyusing lidocaineinjectedintra-testicularlyshoweda90%reductionin concentrationoftheproductinthetestesbetween3and40min post-injection,withaverylowconcentrationinthespermatic cordat40min,suggestingthatarelativelylongintervalbetween injectionandcastrationwouldreducetheanestheticefficacy ( 41

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averylowconcentrationinthespermatic cordat40min,suggestingthatarelativelylongintervalbetween injectionandcastrationwouldreducetheanestheticefficacy ( 41 ).Evenwithapotentiallowerrateofdiffusion,asimilareffectmaybeseenforprocaineasof30minpost-injection.Aninterval of5to10minbetweentheadministrationofprocaineand surgicalcastrationthereforeseemspreferableascomparedto2.5 or30min.Inpractice,thisintervalalsopresentstheadvantageof reducingtheoveralldurationofhandling,ascomparedtothe20 or30minusedtheliterature,limitingthetimespentawayfrom thesowandhomepen,andresultingstressandheatloss.Overall,ourresultsshowedthatinjectionofprocaineasalocalanestheticallowssomemitigationofpiglets’acute responsestocastration,butleadstosignificantresponsesduring theinjectionoftheanesthetic.Pigletsinthepresentstudydid notreceiveanyanalgesic[asismandatoryinDenmark( 19 )]untilafterdatacollectionwasover,asouraimwastoassess theefficacyofthelocalanesthetic,andbecausethecomparison Frontiersin VeterinaryScience 12 frontiersin.org

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Coutantetal.10.3389/fvets.2022.1009858 ofthedifferentintervalswouldhavebeenconfoundedbythe useofananalgesicgivenataspecifictimebeforecastration.In practice,however,analgesicsareadministeredonlyfewminutes beforecastrationoratthesametime,andarethereforelikely onlyefficientinmitigatingpost-castrationpainandnottheacute responsetocastration,asexaminedinthecurrentstudy( 42 ).Inthefuture,theuseoflocalanestheticspriortopigletcastrationinEuropemaybefurtherimprovedbytheapproval ofotherlocalanestheticsthanprocaine,suchaslidocaine, whichmayhaveanincreasedefficacyatcastrationcomparedto procaine( 23 ).Yet,inbothcases,theprocedurerequiresneedleinjectionandextrahandling,which,inourstudy,resultedin clearstressresponses,eveninpigletsnotexposedtoanytissue damagebutkeptinacastrationbench.Inconclusion,whileourstudyprovidedfurtherinsightsintotheadministrationofaprocaine-basedlocalanesthetic priortopigletcastration,andhasprovidedresultsallowinga refinementoftheprocedure,wecannotoverseethatcastration withlocalanesthesiaremainsawelfareconcern.Thisconcern hasbeenraisedpreviously[e.g.,( 11 , 12 , 27 )],andisinlinewiththe‘EuropeanDeclarationonalternativestosurgical castrationinpigs’,adocumentproducedafterconsultation ofvariousstakeholders,judgingcastrationwithanesthesiaas anunsuitablealternative( 16 ).Thus,furtherresearchintothepracticalapplicationofalternativestosurgicalcastrationor alternativestotheuseoflocalanesthesiaisstillrecommended. Dataavailabilitystatement Theoriginalcontributionspresentedinthestudyareincludedinthearticle/ Supplementarymaterial ,furtherinquiriescanbedirectedtothecorrespondingauthor. Ethicsstatement TheanimalstudywasreviewedandapprovedbytheDanishAnimalExperimentsInspectorate.Writteninformedconsent wasobtainedfromtheownersfortheparticipationoftheir

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TheanimalstudywasreviewedandapprovedbytheDanishAnimalExperimentsInspectorate.Writteninformedconsent wasobtainedfromtheownersfortheparticipationoftheir animalsinthisstudy. Authorcontributions ThestudywasdesignedbytheprojectgroupinvolvingMH,JM,MC,andMKwithadvicefromLF.MKdeveloped theanesthesiainjectionsandtrainedtheherdsman.MCwasin chargeofthedatacollection,dataprocessing,anddataanalysis withsupervisionfromJM,LF,andMH.LFperformedthe samplesizecalculations.MCdraftedthemanuscript.Allauthors contributedtothearticleandapprovedthesubmittedversion. Funding ThisstudywascommissionedandfundedbytheDanishVeterinaryandFoodAdministration(DVFA)aspartofthe agreementwithAarhusUniversityonresearch-basedpolicy support,aswellasbyAarhusUniversity.Thefundershadnorole instudydesign,datacollectionandanalysis,decisiontopublish, orpreparationofthemanuscript. Acknowledgments TheauthorsthanktheDanishpigfarmerNielsAageArveandhisstaffforprovidingpigletsandfacilitiesforthestudy andfortheirflexibility.TechniciansfromAarhusUniversity involvedintheprojectareacknowledgedfortheirhardworkand dedication.WealsothankKarolineV.Dohrmannforherhelp inthedataanalysis,HanneKongsted,AarhusUniversity,forher qualifiedadviceinthedesignofthestudy,andTorbenLarsen, AarhusUniversity,forhelpingdevelopingthesalivasampling methodandconductingthecortisolassays. Conflictofinterest Theauthorsdeclarethattheresearchwasconductedintheabsenceofanycommercialorfinancialrelationships thatcouldbeconstruedasapotentialconflict ofinterest.ThereviewerNDdeclaredapastcollaborationwithoneoftheauthorsMHtothehandlingEditor. Publisher’snote Allclaimsexpressedinthisarticlearesolelythoseoftheauthorsanddonotnecessarilyrepresentthoseoftheiraffiliated organizations,orthoseofthepublisher,theeditorsandthe

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sexpressedinthisarticlearesolelythoseoftheauthorsanddonotnecessarilyrepresentthoseoftheiraffiliated organizations,orthoseofthepublisher,theeditorsandthe reviewers.Anyproductthatmaybeevaluatedinthisarticle,or claimthatmaybemadebyitsmanufacturer,isnotguaranteed orendorsedbythepublisher. Supplementarymaterial TheSupplementaryMaterialforthisarticlecanbefoundonlineat: https://www.frontiersin.org/articles/10.3389/fvets.2022.1009858/full#supplementary-material SUPPLEMENTARYVIDEOS1 Videooftheintra-testicularinjectionprocedure. SUPPLEMENTARYDATASETS1 Datasetusedforstatisticalanalysis.Frontiersin VeterinaryScience 13 frontiersin.org

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Coutantetal.10.3389/fvets.2022.1009858SUPPLEMENTARYFIGURES1 Theadaptedcastrationbenchusedduringinjection,castrationandshamhandling.Pictureofthebench(1a),withmicrophoneforvocalisationrecording(1b),cameraforrecordingoftheresistancemovements(1c),andsoftlayermattensuringthefittingofeachpigletduringtheprocedures(2).SUPPLEMENTARYTEXTS1 DescriptionoftheparametersusedforcalibrationofthebandlimitedenergyautomaticdetectionofcallsintheRavenProsoftware. SUPPLEMENTARYTABLES1 Salivacortisolconcentrationsrecordedforeachsamplingtime. References

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dlimitedenergyautomaticdetectionofcallsintheRavenProsoftware. SUPPLEMENTARYTABLES1 Salivacortisolconcentrationsrecordedforeachsamplingtime. References 1.BonneauM,LeDenmatM,VaudeletJC,VelosoNunesJR,MortensenAB,MortensenHP.Contributionsoffatandrostenoneandskatoletoboartaint:1.sensoryattributesoffatandporkmeat.LivestProdSci.(1992)32:63 80.doi:10.1016/S0301-6226(12)80012-12.MarxG,HornT,ThielebeinJ,KnubelB,VonBorellE.Analysisofpain-relatedvocalizationinyoungpigs.JSoundVib.(2003)266:687 98.doi:10.1016/S0022-460X(03)00594-73.TaylorAA,WearyDM.Vocalresponsesofpigletstocastration:identifyingproceduralsourcesofpain.ApplAnimBehavSci.(2000)70:17 26.doi:10.1016/S0168-1591(00)00143-X4.CarrollJA,BergEL,StrauchTA,RobertsMP,KatteshHG.Hormonalprofiles,behavioralresponses,andshort-termgrowthperformanceaftercastrationofpigsatthree,six,nine,ortwelvedaysofage.JAnimSci.(2006)84:1271 8.doi:10.2527/2006.8451271x5.LlamasMoyaS,BoyleLA,LynchPB,ArkinsS.Effectofsurgicalcastrationonthebehavioralandacutephaseresponsesof5-day-oldpiglets.ApplAnimBehavSci.(2008)111:133 45.doi:10.1016/j.applanim.2007.05.0196.LonardiC,ScolloA,NormandoS,BrscicM,GottardoF.Cannovelmethodsbeusefulforpainassessmentofcastratedpiglets?Animal.(2015)9:871 7.7.Marchant-FordeJN,LayDC,McMunnKA,ChengHW,PajorEA,Marchant-FordeRM.Postnatalpiglethusbandrypracticesandwellbeing:theeffectsofalternativetechniquesdeliveredseparately.JAnimSci.(2009)87:1479 92.doi:10.2527/jas.2008-10808.HayM,VulinA,GéninS,SalesP,PrunierA.Assessmentofpaininducedbycastrationinpiglets:behavioralandphysiologicalresponsesoverthesubsequent5days.ApplAnimBehavSci.(2003)82:201 18.doi:10.1016/S0168-1591(03)00059-59.SutherlandMA,DavisBL,BrooksTA,CoetzeeJF.Thephysiologicalandbehavioralresponseofpigscastratedwithandwithoutanesthesiaoranalgesia.JAnimSci.(2012)90:2211 21.doi:10.2527/jas.2011-426010.Kluivers-PoodtM,HouxBB,RobbenSRM,KoopG,LambooijE,HellebrekersLJ.EffectsofalocalanestheticandNSAIDincastrationofpiglets,ontheacutepainresponses,growthandmortality.Animal.(2012)6:1469 75.doi:10.1017/S175173111200054711.LeidigMS,HertrampfB,FailingK,SchumannA,ReinerG.Painanddiscomfortinmalepigletsduringsurgicalcastrationwithandwithoutlocalanesthesiaasdeterminedbyvocalizationanddefensebehavior.ApplAnimBehavSci.(2009)116:174 8.doi:10.1016/j.applanim.2008.10.00412.AbendschönN,SenfS,DeffnerP,MillerR,GrottA,WernerJ,etal.Localanesthesiainpigletsundergoingcastration Acomparativestudytoinvestigatetheanalgesiceffectsoffourlocalanestheticsbasedondefensivebehaviorandsideeffects.Animals.(2020)10:1752.doi:10.3390/ani1010175213.HanssonM,LundeheimN,NymanG,JohanssonG.Effectoflocalanesthesiaand/oranalgesiaonpainresponsesinducedbypigletcastration.ActaVetScand.(2011)53:1 9.doi:10.1186/1751-0147-53-3414.SallerAM,WernerJ,ReiserJ,SenfS,DeffnerP,AbendschönN,etal.Localanesthesiainpigletsundergoingcastration-Acomparativestudytoinvestigatetheanalgesiceffectsoffourlocalanestheticsonthebasisofacutephysiologicalresponsesandlimbmovements.PLoSONE.(2020)15:e0236742.doi:10.1371/journal.pone.023674215.BonastreC,MitjanaO,TejedorMT,CalaviaM,YusteAG,ÚbedaJL,etal.Acutephysiologicalresponsestocastration-relatedpaininpiglets:theeffectoftwolocalanestheticswithorwithoutmeloxicam.Animal.(2016)10:1474 81.doi:10.1017/S175173111600058616.EuropeanComission.EuropeanDeclarationonAlternativestoSurgicalCastrationofPigs2020[cited2021Nov25].Availableonlineat:

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1.doi:10.1017/S175173111600058616.EuropeanComission.EuropeanDeclarationonAlternativestoSurgicalCastrationofPigs2020[cited2021Nov25].Availableonlineat: https://ec.europa.eu/food/system/files/2016-10/aw_prac_farm_pigs_cast-alt_declaration_en.pdf (accessedMay15,2022).17.DeBriyneN,BergC,BlahaT,TempleD.Pigcastration:willtheEUmanagetobanpigcastrationby2018?PorcHealManag.(2016)2:1 11.doi:10.1186/s40813-016-0046-x18.NYTfraDanmarksStatistik.UnchangedPigPopulationOf12.7MillionPigs.(2020).Availableonlineat: https://www.dst.dk/Site/Dst/Udgivelser/nyt/GetPdf.aspx?cid=30377 (accessedJune10,2022).19.DanishAgricultureandFoodCouncil.Retningslinjerforkurserilokalbedøvelseafpattegriseforudforkastration.[Guidelinesforcoursesinlocalanesthesiaofpigletspriortocastration].(2019).p.5.Availableonlineat: https://www.foedevarestyrelsen.dk/SiteCollectionDocuments/Dyrevelfaerd%20og%20veterinaermedicin/Veterin%C3%A6rmedicin/L%C3%A6gemidler%20til%20dyr/Retningslinjer%20for%20kurser%20i%20lokalbed%C3%B8velse%20af%20pattegrise%20forud%20for%20kastration.pdf

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rin%C3%A6rmedicin/L%C3%A6gemidler%20til%20dyr/Retningslinjer%20for%20kurser%20i%20lokalbed%C3%B8velse%20af%20pattegrise%20forud%20for%20kastration.pdf (accessedJuly10,2022).20.AllmendingerA,MuellerR,SchwarbE,ChipperfieldM,HuwylerJ,MahlerHC,etal.Measuringtissueback-pressure-Invivoinjectionforcesduringsubcutaneousinjection.PharmRes.(2015)32:2229 40.doi:10.1007/s11095-014-1611-021.ZijlstraE,JahnkeJ,FischerA,KapitzaC,ForstT.Impactofinjectionspeed,volume,andsiteonpainsensation.JDiabetesSciTechnol.(2018)12:163 8.doi:10.1177/193229681773512122.ScolloA,ContieroB,BenedictisGMDe,GalliC,BenattiD,GottardoF,etal.Analgesiaand/oranesthesiaduringpigletcastration partI:efficacyoffarmprotocolsinpainmanagement.ItalJAnimSci.(2021)20:143 52.doi:10.1080/1828051X.2021.187370723.SkadeL,KristensenCS,NielsenMBF,DinessLH.Effectoftwomethodsandtwoanestheticsforlocalanesthesiaofpigletsduringcastration.ActaVetScand.(2021)63:1 9.doi:10.1186/s13028-020-00566-824.WebbAI,PabloLS.Localanesthetics.In:JER,PapichM,editors.VeterinaryPharmacologyAndTherapeutics,9thedition.Iowa:IowaStateUniversityPressUSA,BlackwellPublishing(2009).p.381 97.25.GarciaMD.Localanesthetics.In:GrimmKA,LamontLA,TranquilliWJ,GreeneSA,RobertsonS.,editors.VeterinaryAnesthesiaandAnalgesia:TheFifthEditionofLumbandJones.Hoboken,NJ:JohnWiley(2015).p.332 54.26.PughDM.Localanesthesiaandvoluntarymusclerelaxation.VetApplPharmacolTher.(1991)81 96.27.CoutantM,MalmkvistJ,KaiserM,FoldagerL,HerskinMS.Piglets’acuteresponsestoprocaine-basedlocalanestheticinjectionandsurgicalcastration:effectsoftwovolumesofanesthetic.FrontPainRes.(2022)3.doi:10.3389/fpain.2022.94313828.KongstedH,NielsenC,HerskinM.Hvordanlokalbedøvermanpatteg-riseførkastration? gennemgangafsvenskeogdanskeerfaringer[Howtolocallyanesthetizepigletsbeforecastration?-ReviewofSwedishandDanishexperiences].DCARep.(2018)134.Availableonlineat:

chunk 47 · 226 tokens

ngafsvenskeogdanskeerfaringer[Howtolocallyanesthetizepigletsbeforecastration?-ReviewofSwedishandDanishexperiences].DCARep.(2018)134.Availableonlineat: https://dcapub.au.dk/djfpublikation/index.asp?action=show&id=1276 (accessedJuly10,2022).29.GutzwillerA.KastrationvonFerkelnunterLokalanästhesie[Castrationofpigletsunderlocalanesthesia],(inGerman).AgrarForsch.(2003)10:10 3.30.CourboulayV,HemonicA,PrunierA,InstitutI,RheuL.Evaluationdesdifférentesméthodesdepriseenchargedeladouleurlorsdelacastration[Evaluationofdifferentmethodsofpainmanagementduringcastration].JournRechPorcFr.(2018)50:305 10.31.CharifR,StrickmanL,WaackA.RavenPro1.4User’sManual.Ithaca,NY:CornellLabofOrnithology(2010).32.FriardO,GambaM,BORIS,afree,versatileopen-sourceevent-loggingsoftwareforvideo/audiocodingandliveobservations.MethodsEcolEvol.(2016)7:1325 30.doi:10.1111/2041-210X.12584 Frontiersin VeterinaryScience 14 frontiersin.org

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Coutantetal.10.3389/fvets.2022.1009858 33.PrunierA,MounierAM,HayM.Effectsofcastration,toothresection,ortaildockingonplasmametabolitesandstresshormonesinyoungpigs1.JAnimSci.(2005)83:216 22.doi:10.2527/2005.831216x34.GallagherNL,GilesLR,WynnPC.Thedevelopmentofacircadianpatternofsalivarycortisolsecretionintheneonatalpiglet.BiolNeonate.(2002)81:113 8.doi:10.1159/00004719535.EdeT,LecorpsB,KeyserlingkMAGVon,WearyDM.Symposiumreview:scientificassessmentofaffectivestatesindairycattle.JDairySci.(2019)102.doi:10.3168/jds.2019-1632536.SheilM,PolkinghorneA.Optimalmethodsofdocumentinganalgesicefficacyinneonatalpigletsundergoingcastration.Animals.(2020)10:1 37.doi:10.3390/ani1009145037.BaysingerA,WebbSR,BrownJ,CoetzeeJF,CrawfordS,DedeckerA,etal.Proposedmultidimensionalpainoutcomemethodologytodemonstrateanalgesicdrugefficacyandfacilitatefuturedrugapprovalforpigletcastration.AnimHealthResRev.(2021)22:1 14.doi:10.1017/S146625232100014138.LouM,VenturaB,DeenJ,LiY.Surgicalcastrationchangesstrugglebehaviorandvocalizationsinmalepiglets.JApplAnimWelfSci.(2021)25:1 8.39.ReimertI,FongS,RodenburgTB,BolhuisJE.Emotionalstatesandemotionalcontagioninpigsafterexposuretoapositiveandnegativetreatment.ApplAnimBehavSci.(2017)193:37 42.doi:10.1016/j.applanim.2017.03.00940.HagaA,RanheimB.Castrationofpiglets:theanalgesiceffectsofintratesticularandintrafunicularlidocaineinjection.VetAnaesthAnalg.(2005)32:1 9.doi:10.1111/j.1467-2995.2004.00225.x41.RanheimB,HagaHA,IngebrigtsenK.Distributionofradioactivelidocaineinjectedintothetestesinpiglets.JVetPharmacolTher.(2005)28:481 3.doi:10.1111/j.1365-2885.2005.00675.x42.SchoosA,DevreeseM,MaesDGD.Useofnon-steroidalanti-inflammatorydrugsinporcinehealthmanagement.VetRec.(2019)185:172.doi:10.1136/vr.105170 Frontiersin VeterinaryScience 15 frontiersin.org