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Citation:Miller,R.;Grott,A.;Patzkéwitsch,D.;Döring,D.;Abendschön,N.;Deffner,P.;Reiser,J.;Ritzmann,M.;Saller,A.M.;Schmidt,P.;etal.BehaviorofPigletsinanObservationArenabeforeandafterSurgicalCastrationwithLocalAnesthesia.Animals2023,13,529.https://doi.org/10.3390/ani13030529AcademicEditor:EmanuelaDallaCostaReceived:10January2023Revised:25January2023Accepted:30January2023Published:2February2023 Copyright:©2023bytheauthors.LicenseeMDPI,Basel,Switzerland.ThisarticleisanopenaccessarticledistributedunderthetermsandconditionsoftheCreativeCommonsAttribution(CCBY)license(https://creativecommons.org/licenses/by/4.0/). ArticleBehaviorofPigletsinanObservationArenabeforeandafterSurgicalCastrationwithLocalAnesthesiaReginaMiller1,*,AndreaGrott1,DorianPatzkéwitsch1,DorotheaDöring1,NoraAbendschön2,PaulineDeffner2 ,JudithReiser3,MathiasRitzmann2,AnnaM.Saller3 ,PaulSchmidt4,SteffanieSenf2 ,JuliaWerner3 ,ChristineBaumgartner3,SusanneZöls2,MichaelErhard1 andShanaBergmann1,*

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iser3,MathiasRitzmann2,AnnaM.Saller3 ,PaulSchmidt4,SteffanieSenf2 ,JuliaWerner3 ,ChristineBaumgartner3,SusanneZöls2,MichaelErhard1 andShanaBergmann1,* 1DepartmentofVeterinaryScience,ChairofAnimalWelfare,Ethology,AnimalHygieneandAnimalHusbandry,FacultyofVeterinaryMedicine,LudwigMaximilianUniversityofMunich,LMUMunich,80539Munich,Germany2ClinicforSwine,LudwigMaximilianUniversityofMunich,85764Oberschleißheim,Germany3CenterforPreclinicalResearch,TechnicalUniversityofMunich,81675Munich,Germany4StatisticalConsultingforScienceandResearch,GroßeSeestr.8,13086Berlin,Germany*Correspondence:r.miller@tierhyg.vetmed.uni-muenchen.de(R.M.);s.bergmann@lmu.de(S.B.)SimpleSummary:Surgicalcastrationofpigletsisgenerallyrecognizedasapainfulprocedure.Thus,foranimalwelfarereasons,theGermanAnimalWelfareActstipulatestheuseofeffectiveanesthesiaduringcastration.However,whetherlocalanesthesiaprovidesadequateanalgesiahasbeenanongoingdebateinGermany.Inthepresentstudy,wecomparedthebehaviorof178pigletsallocatedtovarioustestgroupsinanobservationarenabeforeanyoftheappliedprocedures,afteradministrationofthelocalanesthetic,and0,2and24haftersurgicalcastration.Thelocalanestheticandtheinjectiontechniqueswereevaluatedandoptimizedinthreesequentialstudyparts.Overall,theresultsrevealedthatwhenlocalanesthesiawasused,pigletslessfrequentlyshowedpain-associatedbehaviors—suchaschangesintailposition—thanpigletsofthecontrolgroupthathadbeencastratedwithoutlocalanesthesia.Non-castratedpigletsshowedthefewestpain-associatedbehaviorsintheobservationarena.Inseveraltestgroups,thepigletsshowedchangesintailposition,tailwagging,orhunched-backpostureonthedayfollowingtheprocedure.Thesebehaviorsdifferedsignificantlyfromthoseshownbeforetheprocedure.Theadministrationoflocalanestheticsinthepresentstudyconsiderablyreducedcastration-relatedpain.However,becauselocalanesthesiahasalimiteddurationofeffect,adverseeffectsduetocastration-relatedpainwerestillobservableonedayaftercastration.Abstract:Surgicalcastrationofpigletsisgenerallyrecognizedasapainfulprocedure,butthereiscurrentlynogoldstandardfortheassessmentofpainbehaviorinpiglets.However,painassessmentisessentialforevaluatingtheeffectivenessoflocalanesthetics.Inthisstudy,weinvestigatedtheefficacyoffourlocalanestheticsintermsofpainreliefduringandaftersurgicalcastrationinthreesequentialstudyparts.Todoso,wefilmed178pigletsbeforetheappliedprocedures,afterinjectionofthelocalanesthetic,andupto24haftercastration(fiveobservationtimesintotal)inanobservationarenaandcomparedtheirbehaviorbeforeandaftercastrationandbetweentreatmentsandcontrolgroups.Theresultsshowedsignificantdifferencesinthebehaviorofthepigletsbeforeandaftercastrationandbetweenthesham-castratedcontrolgroupandthecontrolgroupcastratedwithoutanesthesia.Thedifferentlocalanesthesiatreatmentgroupsshoweddivergingdifferencestothecontrolgroups.Themostfrequentlyshownpain-associatedbehaviorsofthepigletswerechangesintailpositionandhunchedbackposture.Weobservedareductionbutnocompleteeliminationoftheexpressedpain-associatedbehaviorsafterlocalanesthesia.Severalbehavioralchanges—suchaschangesintailposition,hunchedbackpostureortailwagging—persisteduntilthedayaftercastration.Owingtothelimiteddurationoftheeffectsofthelocalanesthetics,localanesthesiadidnotinfluencelong-termpain.Keywords:animalwelfare;painrelief;procaine;lidocaine;mepivacaine;bupivacaine

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heeffectsofthelocalanesthetics,localanesthesiadidnotinfluencelong-termpain.Keywords:animalwelfare;painrelief;procaine;lidocaine;mepivacaine;bupivacaine Animals2023,13,529.https://doi.org/10.3390/ani13030529https://www.mdpi.com/journal/animals

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Animals2023,13,529 2of13

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Animals2023,13,529 2of13 1.IntroductionTheInternationalAssociationfortheStudyofPaindefinespainasan“unpleasantsensoryandemotionalexperienceassociatedwithactualorpotentialtissuedamageordescribedintermsofsuchdamage”[1].Thisdefinitionisthesameforallanimalspeciesandforhumans,althoughtheintensityandexpressionofpaincandifferbetweenspeciesandbetweenindividualsofthesamespecies,thusmakinganobjectiveassessmentdifficult.Regardingpigs,evenexpertshavedifficultiesinreliablyrecognizingpain[2].However,properassessmentandeliminationofpainaremandatorytocomplywiththerequirementsoftheGermanAnimalWelfareActandtoensurethatthewellbeingoftheanimalsisnotnegativelyaffected.AccordingtoArticle1Section1,Clause2,noonemayinflictunjustifiedpain,sufferingorinjuryonanimals[3].Therefore,asof1January2021,castrationforthepurposeofpreventingboartaintisonlypermittedwitheffectiveanesthesiainGermany.Thisregulationappliestoabout20millionmalepigletsannually[4].Becausealternatives—suchasboarfatteningorimmunocastration—donotyetprevailoversurgicalcastrationinGermany[5],evidence-basedsuitablemethodsofanesthesiathatreliablyreducecastration-inducedpainmustbeapplied.Currently,castrationwithlocalanesthesiaisnotallowedinGermanybecauseitseffectivenessineliminatingpainisstillamatterofdebate.Whereashumansaremostlyabletoevaluateandverballycommunicatetheintensityandqualityofpain,veterinarymedicinemustrelyonnonverbalpainparameters[6].Op-tionsforpainassessmentinanimalsincludethemeasurementofphysiologicalparameterssuchasheartrate,bloodpressureorvariousbloodparameters(e.g.,catecholaminesorcortisol).Becausethemeasurementoftheseparametersusuallyrequireshandlingoftheanimals,theresultsmaybeconfoundedbystress,fearorexternalfactors[7].Especiallyforcastration-inducedpain,butalsoforotherpain-inducingsituations,behaviorobservationswereshowntoprovidereliableassessmentparameters[7].Observationcamerasordirectobservationscandisplaythebehavioroftheanimalswithoutdisturbancebyhandling.Behavioralreactionstopainincludespecificpain-associatedbehaviors—suchashunchedbackposture,tailwaggingandrumpscratching—andunspecificchangesinphysiologi-calbehaviorssuchassucklingorsleeping[8].However,observationofanimalbehavioroveralongperiodisverylaborintensiveandrequirestheexperienceandtrainingoftheobserver[7].Morerecentbehavior-basedassessmentsystemssuchasgrimacescales,whichallowclassifyingpainbasedonanimalmimics,orstandardizedbehaviortestscanfacilitateandstandardizepainassessmentbecausetheyassessonlyspecificbehaviorelementsandrequirenocomprehensiveobservation.Regardingpigs,theuseofthegrimacescaleisstillchallenging,sobehaviorobservationseemstobeamorereliablemethod[7].Thereiscurrentlynogoldstandardfortheassessmentofpainbehaviorinpiglets[7].Therefore,toassesstheefficacyoflocalanesthesiaineliminatingpainduringandaftersurgicalcastration,weanalyzedthepainbehaviorofdifferentlytreatedpigletsundereightdaysofageinanobservationarena.Specifically,wecomparedtheacutepainbehaviorofpigletswhereanesthesiahadbeenadministeredandcastratedpigletswiththebehaviorofnon-castratedpigletsorpigletscastratedwithoutanesthetictreatment.2.MaterialsandMethodsThepresentstudyispartofthejointproject“Effectivenessoflocalanesthesiaineliminatingpainduringpigletcastration,”acollaborationbetweentheCenterforPreclinicalResearchoftheTechnicalUniversityofMunichandtheClinicforSwineoftheLudwigMaximilianUniversityofMunich.Thestudyconsistedofthreesequentialexperimentalparts(part1,part2andpart3)designedtofindthemosteffectivemethodandthemostsuitedagentforanesthesiaduringcastration.ItcompliedwithalllegalrequirementsregardingtheplanningandconductingofanimalresearchaccordingtotheDirective63/2010/EUoftheEuropeanParliamentandoftheCouncilandaccordingtotheGermanAnimalWelfareAct(2019).ThestudywasapprovedbythedistrictgovernmentofUpperBavaria(referencenumberROB-55.2-2532.Vet_02-19-11).

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Animals2023,13,529 3of13

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Animals2023,13,529 3of13 2.1.AnimalsandHousingIntotal,178malehybridfatteningpiglets(crossbreedPiétrain GermanLandraceandGermanLargeWhite)wereincludedinthestudy.Thepigletswerebetweenthreeandsevendaysold(mean SD:part1:5.1 1.1days;part2:6.6 0.7days,part3:6.7 1.5days)andhadaminimumweightof1.4kg.Theaverageweight(mean SD)was2.2 0.5kginpart1,2.2 0.6kginpart2and2.3 0.6kginpart3.Onlyanimalswithcompletelydescendedtesticlesandsoundgeneralconditionswereincludedinthestudyandrandomlyallocatedtothedifferentstudygroups.AllanimalswerehousedonafarminBavaria.Thisbreedingandfatteningfarmhousedeightysows,whichfarrowedinathree-weekrhythm.Sowswerefedamixedration,includingaminimumof200gofrawfiberperday,thatwasproducedandprocessedatthefarm.Thefarrowingpenswere3.8m2insize(length:1.65m,width:2.30m)andhadpartiallyslattedconcretefloorsthatwerecoveredwithrubbermatsinthelyingareaofthesow.Thepigletcreephadasolidconcretefloorbeddedwithsawdustandstraw.Amovablecoverandaninfraredheatlamp(heatlampwithprotectivecase,250W,ARTAS,Armstadt,Germany)wereinstalledabovethepigletcreep.Toavoidpainandthusapossibleinfluenceofadditionalzootechnicalproceduresontheassessedparameters,thepigletswereear-taggedaftercompletingthetrialsandbeforeweaningfromthemothersow,andtheirtailswerenotdocked.Onthedaybeforecastration,allpigletsreceivedanintramuscularironinjection(1mLUrsoferran®,200mgactiveingredientpermilliliterofinjectionsolutionforswine,SerumwerkBernburgAG,Bernburg,Germany).Toallowthecollectionofbehaviordatawithoutinfluenceoftheobservingperson,thepigletswereplacedinanobservationarena(modifiedafterDiGiminianietal.[9]);Figure1atfiveobservationtimepoints(seetimelineinFigure2;foramaximumdurationoftwominutesperobservation).Thisarenaconsistedofaroundblackplastictubwithadiameterof60cmatthebottomand70cmattherim,withaheightof40cm.Thetubhadfourevenlyspreadsideopeningsof4cmdiameterperopening,throughwhichthelensesoffourdigitalcameras(GoProHero7BlackActionCam4K,GoProInc.,SanMateo,CA,USA)wereinserted.Thebottomofthearenawascoveredwithafittedroundhempmat(hempfarrowingmat,5mmthick,GFSTopAnimalShop,Ascheberg,Germany)toensureslip-proofstandingofthepigletsandtoabsorbexcrement.Thehempmatwasreplacedasneeded,e.g.,whensoiled.Tokeepthepigletwarmandprovideadequatelighting,aheatlamp(heatlamp250W,ARTAS,Armstadt,Germany)wasplaced70cmabovethebottomoftheobservationarena.Thearenaandthemetalframeweremountedonawoodenplatform(1 1m)sothatthewholeconstructionwaseasytoliftandmove.Duringtherecordings,theobservationarenawasplacedintheaislenexttothefarrowingpenssothatthepigletscouldalwayshearandsmelltheirmothersowandlittersiblings.

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uringtherecordings,theobservationarenawasplacedintheaislenexttothefarrowingpenssothatthepigletscouldalwayshearandsmelltheirmothersowandlittersiblings. Figure1.Photographoftheobservationarenawithastandingpiglet,modifiedafterDiGiminianietal.[9].Photocredit:S.Bergmann.

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Animals2023,13,529 4of13 Figure2.Experimentalprocedurefromthedaybeforecastrationuntilthedayaftercastrationshowingallfiveobservationtimepointsintheobservationarena.LA=Localanesthetic;OA=videorecordingintheobservationarena.2.2.ExperimentalDesignThestudywasdividedintothreepartsthatwereestablishedstepbysteptooptimizetheappliedlocalanesthesia.Inpart1,4localanestheticsweretestedon71pigletsfrom16litters.Inpart2,2injectiontechniqueswereappliedwith1selectedlocalanestheticon47pigletsfrom10litters.Inpart3,themosteffectivelocalanestheticsandinjectiontechniquesidentifiedinthefirsttwopartswereused[10–12].Inaddition,localanesthesiainpart3wascombinedwithsystemicanalgesia.Part3wasconductedon60pigletsfrom11litters.Inadditiontothetreatmentgroups,eachstudypartincludedtwocontrolgroups:a“handling”groupinwhichinjectionandcastrationwereonlysimulated(shamgroup)andasodiumchloride(NaCl)groupinwhichthepigletswereinjectedwithNaClsolutioninsteadoflocalanestheticandthuswerecastratedwithoutanesthesia.Table1givesanoverviewofthestudygroupsinvestigatedinstudyparts1–3.Table1.Studygroupsadministereddrugsandinjectiontechniquesinstudyparts1–3. StudyPartStudyGroupDrugProductInformationandBrandNameDosageInjectionTechniqueNumberofPiglets 1ProcaTwo-StepPart1Procainehydrochloride+adrenalinePronestesic40mg/mLinjectionsolutionforhorses,cattle,swineandsheep;FATROS.p.A,OzzanoEmilia(Bologna),Italy0.5mLi.t.0.5mLs.c.Two-steptechnique12 LidoTwo-StepPart1Lidocainehydrochloride+adrenalineXylocitin®2%withepinephrine0.001%;mibeGmbHArzneimittel,Brehna,Germany0.5mLi.t.0.5mLs.c.Two-steptechnique12 BupiTwo-StepPart1Bupivacainehydrochloride+adrenalineBupivacaine0.5%,JENAPHARM®;mibeGmbHArzneimittel,Brehna,Germany0.5mLi.t.0.5mLs.c.Two-steptechnique12

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upiTwo-StepPart1Bupivacainehydrochloride+adrenalineBupivacaine0.5%,JENAPHARM®;mibeGmbHArzneimittel,Brehna,Germany0.5mLi.t.0.5mLs.c.Two-steptechnique12 MepiTwo-StepPart1MepivacainehydrochlorideMepidor®20mg/mLinjectionsolutionforhorses;WirtschaftsgenossenschaftdeutscherTierärzteeG,Garbsen,Germany0.5mLi.t.0.5mLs.c.Two-steptechnique12 ShamPart1n/an/an/aShaminjection11 NaClTwo-StepPart1SodiumchlorideNaCl0.9%intravenousinfusion;B.BraunMelsungenAG,Melsungen,Germany0.5mLi.t.0.5mLs.c.Two-steptechnique12

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Animals2023,13,529 5of13 Table1.Cont. StudyPartStudyGroupDrugProductInformationandBrandNameDosageInjectionTechniqueNumberofPiglets 2LidoOne-Step(F)Part2Lidocainehydrochloride+adrenalineXylocitin®2%withepinephrine0.001%;mibeGmbHArzneimittel,Brehna,Germany0.6mLOne-step,fenestratedneedle11 LidoTwo-Step(H)Part2Lidocainehydrochloride+adrenalineXylocitin®2%withepinephrine0.001%;mibeGmbHArzneimittel,Brehna,Germany0.4mLi.t.0.2mLs.c.Two-stepafterHanssonetal.201112 ShamPart2n/an/an/aShaminjection12 NaClOne-Step(F)Part2SodiumchlorideNaCl0.9%intravenousinfusion;B.BraunMelsungenAG,Melsungen,Germany0.6mLOne-step,fenestratedneedle12 3LidoOne-Step(F)Part3Lidocainehydrochloride+adrenaline*Xylocitin®2%withepinephrine0.001%;mibeGmbHArzneimittel,Brehna,Germany0.6mLOne-step,fenestratedneedle12 LidoTwo-Step(H)Part3Lidocainehydrochloride+adrenaline*Xylocitin®2%withepinephrine0.001%;mibeGmbHArzneimittel,Brehna,Germany0.4mLi.t.0.2mLs.c.Two-stepafterHanssonetal.201112 MepiOne-Step(F)Part3Mepivacainehydrochloride*Mepidor®20mg/mLinjectionsolutionforhorses;WDTeG,Garbsen,Germany0.7mLOne-step,fenestratedneedle12 ShamPart3n/a*n/an/aShaminjection12 NaClOne-Step(F)Part3Sodiumchloride*NaCl0.9%intravenousinfusion;B.BraunMelsungenAG,Melsungen,Germany0.6mLOne-step,fenestratedneedle12

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/aShaminjection12 NaClOne-Step(F)Part3Sodiumchloride*NaCl0.9%intravenousinfusion;B.BraunMelsungenAG,Melsungen,Germany0.6mLOne-step,fenestratedneedle12 Abbreviations:n/a=notapplicable;i.t.=intratesticular;s.c.=subcutaneous;*=pigletsweregivensystemicanalgesiapriortotheinjectionoflocalanesthetic(0.4g/kgmeloxicam(Metacam®5mg/mL),injectionsolutionforcattleandswine,BoehringerIngelheimPharmaGmbH&Co.KG,IngelheimamRhein,Germany)and50mg/kgmetamizole(MetamizolWDT500mg/mL,injectionsolutionforhorses,cattle,swineanddogs,WirtschaftsgenossenschaftdeutscherTierärzteeG,Garbsen,Germany).2.3.CastrationOnthedaybeforecastration,thepigletswereweighed,andtheirhealthstatuswasexamined.Toensureindividualidentificationofthepiglets,theyweremarkedwithdistinctsymbolsonthebackbyusingapaintbrushandblackmarkerspray(MSmarkerspray,black,MSSchippers,Kerken,Germany).Beforecastration,thepigletswereweighedagainonthedayofcastrationwithadigitalscale(MSSchippers,Kerken,Germany).Afterwards,thepigletswereindividuallyfilmedintheobservationarenaforamaximumdurationoftwominutestoestablishabaselineofthebehavior.Thisbaselinecouldthenbecomparedtotherecordingsaftertheprocedure.Fortheinjection,thepigletswerefixedinacastrationstand(MSSchippers,Kerken,Germany)andinjectedintobothtesticleswitheitheralocalanestheticorNaClsolutionbymeansofaself-fillinginjector(HSWECO-MATIC®,Henke-Sass,WolfGmbH,Tuttlingen,Germany)fittedwitha25-gaugeneedle(0.5 16mm,B.BraunTravaCareGmbH,Hallbergmoos,Germany).Inpart1ofthestudy,theinjectionwascarriedoutintwosteps(two-stepinjection),duringwhichtheleftandrighttesticleswerefirstinjectedwith0.5mLinjectionsolutionadministeredintratesticularlyandthenwith0.5mLadministeredsubcutaneously

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Animals2023,13,529 6of13

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Animals2023,13,529 6of13 intothescrotum.Calculatedforthemeanweightof2.2kg,pigletsinpart1ofthestudyreceived36.4mg/kgProcaine(Pronestesic40mg/mLinjectionsolutionforhorses,cattle,swineandsheep;FATROS.p.A,OzzanoEmilia(Bologna),Italy),18.2mg/kgLidocaine(Xylocitin®2%withepinephrine0.001%;mibeGmbHArzneimittel,Brehna,Germany),4.5mg/kgBupivacaine(Bupivacaine0.5%,JENAPHARM®;mibeGmbHArzneimittel,Brehna,Germany)and18.2mg/kgMepivacaine(Mepidor®20mg/mLinjectionsolutionforhorses;WirtschaftsgenossenschaftdeutscherTierärzteeG,Garbsen,Germany).Basedonadosageadjustment,lesslocalanestheticwasinjectedinpart2ofthestudy(calculatedforthemeanweightof2.2kg:10.9mg/kgLidocaine).Inpart3ofthestudypigletsreceived10.4mg/kgLidocaine(calculatedforthemeanweightof2.3kg)and12.2mg/kgMepivacaine.Thetwo-stepinjectiontechniqueaccordingtoHanssonetal.[13](two-step[H]injection)usedinparts2and3startedwithanintratesticularinjectionof0.4mLpertesticle,andtheremaining0.2mLwasdeliveredduringwithdrawaloftheneedleinsteadofasubcutaneousinjectionintothescrotum.Thepigletsoftheshamgroup(handlingonly)werealsofixedinthecastrationstand,andaninjectionwassimulatedbypressureexertedwiththeneedlecapwhilethetesticleswerefixedwiththefingers.Theone-stepfenestratedinjectiontechnique(one-step[F]injection)usedforcomparisoninparts2and3wascarriedoutwitha25-gaugeneedle(0.5 10mm)thathadfourlateralopeningsinadditiontothedistalopening,twoofthemproximallyandtwodistally[12].Thus,duringinjection,thelocalanestheticwassimultaneouslyadministereddistallyintothetesticleandproximallyintothesubcutisofthescrotum.Afterwards,thepigletswerefilmedintheobservationarenaformaximallytwominutesandthenplacedbackintothefarrowingpenwiththeirmothersow.Toensurefullactivityoftheadministeredlocalanesthetics,thepigletswerecastratedinthecastrationstandafterawaitingperiodof20min.Aftercleaningthescrotalareawithantisepticsolution(Octenisept®,Schuelke&MayrGmbH,Norderstedt,Germany),thepigletswerecastratedwithasterilesurgicalblade(scalpelbladescarbonsteel,sterile21,HeinzHerenzMedizinalbedarfGmbH,Hamburg,Germany;scalpelhandleno.4,AESCULAPAG&CO.KG,Tuttlingen,Germany).First,theskinandthetunicavaginaliswereincisedononeside.Then,thetesticlewasexposedandremovedafterseveringtheductusspermaticus.Thisprocedurewasrepeatedontheotherside.Thepigletsoftheshamgroupwerefixedinthecastrationstand,andthecastrationwassimulatedbymanualfixationofthetesticlesandpressureexertedwiththedullendofthescalpel.Castrationandinjectionwerestandardizedandalwaysconductedbythesamethreetrainedandalternatingveterinarians.Immediatelyaftercastration,thepigletswereplacedintheobservationarenaforamaximumoftwominutes.Twohoursaftercastration,allpigletswereagainobservedintheobservationarenaformaximallytwominutes.Onthedayaftercastration(approximately24haftercastration),theweightandgeneralconditionofthepigletswerere-assessed,andallpigletswereoncemorefilmedintheobservationarenaformaximallytwominutes.Eachpigletthenreceived0.4mg/kgofananalgesic(Metacam®5mg/mL,injectionsolutionforcattleandswine,BoehringerIngelheimPharmaGmbH&Co.KG,IngelheimamRhein,Germany),whoseadministrationhadbeendelayeduntilthistimetoexcludetheinfluenceofmedicationonthedatacollectedintheobservationarena.Anexceptionwasmadeforthepigletsofstudypart3;immediatelybeforeinjectionorsimulatedinjection,thesepigletsreceivedsystemic,intramuscularlyadministeredanalgesics:0.4g/kgmeloxicam(Metacam®5mg/mL,injectionsolutionforcattleandswine,BoehringerIngelheimPharmaGmbH&Co.KG,IngelheimamRhein,Germany)and50mg/kgmetamizole(MetamizolWDT500mg/mL,injectionsolutionforhorses,cattle,swineanddogs,WirtschaftsgenossenschaftdeutscherTierärzteeG,Garbsen,Germany).2.4.BehaviorintheObservationArenaEachpigletwasplacedintheobservationarenaforamaximumdurationoftwominutesateachofthefollowingfivetimepoints:(i)baseline,i.e.,onthedayofcastration

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Animals2023,13,529 7of13 beforeallappliedprocedures;(ii)afterinjectionorshaminjection;(iii)aftercastrationorshamcastration;(iv)twohoursaftercastrationorshamcastration;(v)andtwenty-fourhoursaftercastrationorshamcastration.Accordingtopredeterminedterminationcriteria,pigletswereremovedfromtheobservationarenaiftheyshowedsignsofstressorfearduringdirectobservation.Thesecriteriaincludedstressreactionssuchasfreezing,high-frequencyvocalization,flightattempts,orfrequentpivoting(seesupplementaryscoresheetTableS1).Therecordedbehaviorwasanalyzedbyfocalanimalsampling,accordingtoMartinandBateson[14]forupto60s.Fortheanalysis,anethogrammodifiedafterHayetal.[8]wasused(Table2).Allobserverswereblindedregardingthestudygroup.Table2.Ethogramforpain-associatedbehaviorsintheobservationarena,modifiedafterHayetal.[8]. FunctionalCircleBehaviorDefinition PainbehaviorTailpositionTailheldstraightandnotcurled,eitherextendedfromtherumporhangingdownTailwaggingRepeatedtailmovementsupanddownorsidetosideHunchedbackpostureKyphosis,frontlegsextendedHunchedbackposturewithpressingagainstthearenawallKyphosis,frontlegsextended,rumppressedagainstthewalloftheobservationarenaSittingBodyweightsupportedbyhindquartersandfrontlegsExcretionbehaviorHunchedbackpostureduringexcretionKyphosisduringexcretionoffeces

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heobservationarenaSittingBodyweightsupportedbyhindquartersandfrontlegsExcretionbehaviorHunchedbackpostureduringexcretionKyphosisduringexcretionoffeces 2.5.StatisticalAnalysisForaninitialdescriptionofthedata,relativefrequencieswerecomputedforallbehaviorvariablesalongstudyparts,studygroupsandtimepoints.Forfurtheranalysis,generalizedlinearmixedmodelsforbinaryresponsevariableswithlogitlinkfunctionwereusedforeachpartindependentlytoestimaterisksforthedifferentbehaviorsalongstudygroupsandtimepoints.Allmodelsincludedstudygroupandtimepointasfixedeffects,theinteractionofthesevariables,andtheweightoftheanimals.Subject-specificvariationwasaccountedforbyrandomeffectsfortheintercept.Comparisonsofestimatedrisksbetweenstudygroupsatgiventimepointswereperformedbyriskratios.Resultsarepresentedasestimatedrisks,riskratios,thecorresponding95%uncertaintyintervalsandp-values.Foranalysis,theRlanguageforstatisticalcomputing[15]wasused.Inter-observerreliabilitywascalculatedwithCohen’skappafromtheagreementbetweentwoobservers.3.ResultsThemostfrequentlyshownpain-associatedbehaviorswerechangesintailposition,hunchedbackposture,andhunchedbackposturewithpressing(againstthearenawall),confirmingpreviouslypublishedfindings[8,16,17].Inallstudyparts,wefoundatleastonesignificantincreaseintheseparametersbetweenthebaselinebehaviorandthebehavioratsubsequenttimepoints.Wealsofounddifferencesbetweenthesubsequenttimepointsinthatthefrequencyofthebehaviorreachedapeakatcastration(changesintailposition,tailwagging)ortwohoursaftercastration(hunchedbackposture,hunchedbackposturewithpressingagainstthearenawall)anddecreasedafterwards.Intheshamgroup,theparameterswereoftenonlyincreasedatsingletimepoints.Theintraclasscorrelationcoefficient(Cohen’skappa)wasK=0.71forchangesintailposition,K=0.78fortailwagging,K=0.78forhunchedbackposture,andK=0.77forhunchedbackposturewithpressingagainstthearenawall.

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Animals2023,13,529 8of13 3.1.StudyPart1:EvaluationofFourLocalAnestheticsinComparisonwithTwoControlGroups(Sham;InjectionwithNaCl,i.e.,CastrationwithoutAnesthesia)ThepigletsofgroupNaClTwo-StepPart1(controlgroup:castrationwithoutanes-thesia)showedasignificantincreaseinthepainparameterchangesinthetailpositionimmediatelyaftercastrationincomparisonwithallothergroups(p=0.044).Twohoursaftercastration,pigletsofgroupBupiTwo-StepPart1showedchangesintailpositionmorefrequentlythanpigletsofgroupLidoTwo-StepPart1(Figure3).

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p=0.044).Twohoursaftercastration,pigletsofgroupBupiTwo-StepPart1showedchangesintailpositionmorefrequentlythanpigletsofgroupLidoTwo-StepPart1(Figure3). Figure3.Studypart1:Effectofthestudygroupontherelativeriskforthepercentageoccurrence(medianwithminimumandmaximum)oftheparameterschangesintailposition,tailwagging,hunchedbackpostureandhunchedbackposturewithpressingagainstthearenawall.Thedifferentstudygroupswerecomparedaccordingtothefiveassessedtimepointsbaseline(beforeanyproce-dure),afterinjection,aftercastration,twohoursaftercastrationandonedayaftercastration.ThestudygroupswereShamPart1(n=11),NaClTwo-StepPart1(n=12),MepiTwo-StepPart1(n=12),BupiTwo-StepPart1(n=12),LidoTwo-StepPart1(n=12)andProcaTwo-StepPart1(n=12).TailwaggingafterinjectionoccurredmoreingroupsLidoTwo-StepPart1,MepiTwo-StepPart1andNaClTwo-StepPart1andlessingroupsShamPart1(controlgroup:handlingonly)andProcaTwo-StepPart1(p 0.014).Onthedayaftercastration,thisbehaviorwasshownmorefrequentlyingroupBupiTwo-StepPart1thaninallothergroups(p 0.016),especiallyincomparisonwithgroupShamPart1(p<0.001)(Figure3).Theparametersofhunchedbackpostureandhunchedbackposturewithpressingagainstthearenawallshowedalmostnosignificantdifferencesinthispartofthestudy(Figure3).WefoundanincreaseinhunchedbackpostureinpigletsofgroupShamPart1twohoursaftercastration,withasignificantdifferencetogroupMepiTwo-StepPart1(p=0.046)(Figure3).Inthispartofthestudy,wefoundthesignificanteffectsofbodyweightonthebehaviorparameteroftailwagging.Heavypigletsshowedthisbehaviormorefrequentlythanlighterones(p=0.028).

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Animals2023,13,529 9of13 3.2.StudyPart2:EvaluationofTwoInjectionTechniquesforLidocaineinComparisonwithTwoControlGroups(Sham;InjectionwithNaCl,i.e.,CastrationwithoutAnesthesia)Instudypart2,changesintailpositionoccurredespeciallyofteningroupsNaClOne-Step(F)Part2andLidoTwo-Step(H)Part2(Figure4).Furthermore,twohoursaftercastrationthepigletsofgroupNaClOne-Step(F)Part2showedahunchedbackposturemoreoftenthanthepigletsofgroupShamPart2(p=0.030)(Figure4).Wefoundnosignificantinfluenceofbodyweightonthefrequencyoftheassessedpainparametersinthispartofthestudy(p>0.50).

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igletsofgroupShamPart2(p=0.030)(Figure4).Wefoundnosignificantinfluenceofbodyweightonthefrequencyoftheassessedpainparametersinthispartofthestudy(p>0.50). Figure4.Studypart2:Effectofthestudygroupontherelativeriskforthepercentageoccurrence(medianwithminimumandmaximum)oftheparameterchangesintailpositionandhunchedbackposture.Thedifferentstudygroupswerecomparedaccordingtothefiveassessedtimepointsbaseline(beforeanyprocedure),afterinjection,aftercastration,twohoursaftercastrationandonedayaftercastration.ThestudygroupswereShamPart2(n=12),NaClOne-Step(F)Part2(n=12),LidoTwo-Step(H)Part2(n=12)andLidoOne-Step(F)Part2(n=11).3.3.StudyPart3:EvaluationofTwoLocalAnestheticsCombinedwithSystemicAnalgesiainComparisonwithTwoControlGroups(Sham;InjectionwithNaCl,i.e.,CastrationwithoutAnesthesia)Asobservedintheprecedingstudypart,immediatelyaftercastrationthepigletsofgroupShamPart3showedchangesintailpositionlessoftenthanthepigletsoftheothergroups,especiallyincomparisonwithgroupLidoTwo-Step(H)Part3(p=0.012).Inaddition,twohoursaftercastration,thepigletsofallothergroupsshowedthisbehaviormoreoftenthanthepigletsofgroupShamPart3(p<0.001)(Figure5).Asfoundinpart2,twohoursaftercastration,thefrequencyofhunchedbackposturewasloweringroupShamPart3thaningroupNaClOne-Step(F)Part3(p=0.036)(Figure5).Inthispartofthestudy,wefoundaninfluenceofbodyweightononepainparameter.Thepossibilityfortheoccurrenceofahunchedbackposturewithpressingagainstthearenawallincreasedwithincreasingbodyweight(p=0.008).

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Animals2023,13,529 10of13 Figure5.Studypart3:Effectofthestudygroupontherelativeriskforthepercentageoccurrence(medianwithminimumandmaximum)oftheparameterchangesintailpositionandhunchedbackposture.Thedifferentstudygroupswerecomparedaccordingtothefiveassessedtimepointsbaseline(beforeanyprocedure),afterinjection,aftercastration,twohoursaftercastrationandonedayaftercastration.ThestudygroupswereShamPart3(n=12),NaClOne-Step(F)Part3(n=12),MepiOne-Step(F)Part3(n=12),LidoTwo-Step(H)Part3(n=12)andLidoOne-Step(F)Part3(n=12).4.DiscussionThefirsttwopublicationsoftheprojectteampresentedresultsof(i)physiologicalparametersassessedinpigletscastratedunderlowdosesofisofluraneandlocalanesthesiaaccordingtoaminimalanesthesiamodel[10]and(ii)defensivemovements,vocalizationandthebehaviorinahandlingchuteassessedinconsciouspigletsunderlocalanesthesiaduringoraftercastration[11].Theresultsshowedthatcastrationwithoutanesthesiaispainful,confirmingpreviousfindings[18].However,findingsontheeffectivenessoflocalanesthesiaduringcastrationhavebeendiverging.Inthepresentstudy,theoccurrenceofspecificacutebehavioralsignsofpaincouldbereducedbytheadministrationoflocalanesthetics.Thisresultagreeswiththoseofotherpublications[13,19].Becausecastration-relatedpaininducesbehaviorchangesthatcanpersistforuptofivedays[8,20],weassessedthepainbehavior2and24haftercastrationbasedonvideorecordingsmadeinanobservationarena.Changesintailpositionwerepreviouslydescribedtobeassociatedwithvariousemotionsinpigsandpiglets[21–23].Theassociationbetweenspecifictailpositionsandpaincouldbedemonstratedmultipletimes[21,24,25].Inourstudy,thisbehavioroccurredmostoftenimmediatelyaftercastration.Moreover,incomparisonwiththebehaviorbeforetheprocedure,changesintailpositionwerestillmarkedlyincreased2or24haftercastration.Furthermore,achangeintailpositionwasshownsignificantlymoreoftenbycastratedthanbysham-castratedpiglets,suggestingapain-inducedexpressionofthisbehavior.Severalstudiesfocusedmoreonthemovementthanonthepositionofthetail[8,16,26,27]andmostlyfoundanincreaseintailmovementsaftercastration.Inthepresentstudy,weassessedtailmovementsandtailpositionseparatelyandfoundanin-creaseintailmovements(tailwagging)ratheronthedayaftercastrationorafterinjectionofthelocalanesthetic.Thisparametercouldbeassociatedwithparesthesia—suchasitching(duetoincreasedbleeding)ornumbness(afteradministrationofthelocalanesthetic).Otherstudiesalsoreportedincreasedtailwaggingaftercastrationwithlocalanesthesia,whereaspigletscastratedwithoutanesthesiashowedlesstailwagging[27].TheconsiderablyhighoccurrenceoftailmovementsinthepigletsofgroupBupiTwo-StepPart1onthedayaftercastrationcouldbecausedbythelong-lastingeffectofbupivacaine,whichinduces

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Animals2023,13,529 11of13 numbness[28].Thus,wecouldnotdemonstratethepain-specificityofthetail-waggingparameter.Thehunched-backpostureparameterhasbeenclassifiedasapainparameterforvariousanimalspecies,e.g.,forcattle[29].Thisbehaviorwaspreviouslyobservedinpigletsaftercastrationandpersisteduptoseveralhoursaftertheprocedure[17,30].Inourstudy,wefoundashort-termincreaseinthisparameteralsointhesham-castratedpiglets—especiallyinstudypart1andconsiderablylessafterpreemptivesystemicpainmedicationinstudypart3.Weassumethatfixationofthetesticlesduringhandlingcanalreadyinducepainstimulithatcauseapiglettochangeitsbodypositionandhunchitsback.Wealsofoundanincreaseinthehunched-backpostureparameterbypressingagainstthearenawallinthesham-castratedpigletsatthetimeofcastration.Toensureanentirelypain-freecontrolgroup,futureassessmentsintheobservationarenashouldincludeacontrolgroupwithouthandling.Ingeneral,wefounddifferencesbetweencastratedandsham-castratedpigletsforonlyafewoftheassessedparameters.Theseresultsareinlinewithotherstudiescomparingthebehaviorofcastratedandsham-castratedpigletsbeforeandaftercastration[7].Differencesbetweenthedifferentlocalanestheticsorinjectiontechniqueswereevenlesspronounced.Itispossiblethatalargernumberofanimalsmightrevealclearerdifferences.However,intheNaClgroupandinthegroupstreatedwithlocalanesthetics,wenoticedasustainedincreaseinthebehavioralpainparameters,whichpersisteduntilthedayaftercastration,evenwiththepreemptiveadministrationofmeloxicamandmetamizole.Variousstudieshaveconfirmedthatthepaininducedbycastrationseemstopersistforseveraldaysandthatswellingsandrednesscanremainvisibleandpalpableforseveraldays[8,11,27].Therefore,perioperativepainmanagementshouldgobeyondtreatingacutecastration-inducedpain.Severalreviewarticlesemphasizedtherelevanceofobservingspecificpainbehavior[7,31].However,continuousobservationisassociatedwithhightechnologicaleffortandlaborinput.Someresearchers,therefore,usedshortobservationtimepointstoconductbehaviorobservationsonpiglets[9].Observationinshorttimeintervals(focalsampling)issuitedfortheassessmentofhighlyfrequentspecificbehaviors,whereasrarelyshownbehaviorparametersareeasytomisswiththismethod.Furthermore,theuseofanarenaliketheoneinourstudyonlyallowstheevaluationofindividualbehavior.Socialbehaviors—suchasplaybehavior—cannotbeassessed.5.ConclusionsToevaluateeffectiveanesthesiainpigletsundergoingcastration,moststudiesfocusontheacutepainatthetimeoronthedayofcastration.However,castration-inducedpainpersistsoveralongerperiod.Thus,foranimalwelfarereasons,theassociatedlong-lastingpainshouldbeincludedintheevaluationofacastrationmethod.Ourresultssuggestthattheadministrationoflocalanestheticsseemstoreducepainduringcastrationbutcannoteliminatetheassociatedlonger-lastingpain,owingtothelimiteddurationofactionofthelocalanesthetics.Therecommendationofaspecificlocalanestheticorinjectiontechniqueisnotpossiblebymeansofthepresentedbehavioranalysisintheobservationarena.However,itwasnoticeablethatsomeofthelocalanesthetics—suchaslidocaineandmepivacaine—ledtostrongerorlonger-lastingdecreasesinthepain-associatedbehaviorparameters.The“changesintailposition,”“hunchedbackposture”and“hunchedbackposturewithpressingagainstthearenawall”parametersestablishedinthisstudyseemtoallowavalidassessmentofpaininpigletsandgeneratedreproducibleresultsfacilitatingdifferentiationbetweenthetwocontrolgroups(NaCl(castrationwithoutanesthesia)andSham(nocastration,onlyhandling)).Becausepain-inducedbehavioralchangesinpigletsareindividualandtransient,furtherstudiesareneededtoevaluatehowfarpainindicatorsdifferaccordingtotheageandraceoftheassessedpiglets.Inthelongterm,astandardizedassessmentprotocolforpaindetectioninpigletswouldbeusefultoallowbettercomparabilitybetweenstudiesandbetterevaluationofthesuitabilityofalternativemethodsofanesthetization.

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Animals2023,13,529 12of13 SupplementaryMaterials:Thefollowingsupportinginformationcanbedownloadedat:https://www.mdpi.com/article/10.3390/ani13030529/s1,TableS1:Terminationcriteriaduringobservationofpigletsintheobservationarena.AuthorContributions:Conceptualization,S.B.,D.P.,R.M.,S.Z.,S.S.andC.B.;methodology,S.B.,D.P.,R.M.,A.G.,D.D.,N.A.,S.S.,P.D.,J.W.,A.M.S.andJ.R.;validation,R.M.,A.G.andS.B.;formalanalysis,P.S.,R.M.andS.B.;investigation,R.M.,A.G.,S.B.,N.A.,P.D.,S.S.,J.W.,J.R.andA.M.S.;resources,M.E.,M.R.andC.B.;datacuration,R.M.,A.G.andS.B.;writing—originaldraftpreparation,R.M.;writing—reviewandediting,S.B.,D.P.,D.D.andA.G.;visualization,R.M.,S.B.andP.S.;supervision,S.B.,D.P.,M.E.,S.Z.andC.B.;projectadministration,C.B.andS.Z.;fundingacquisition,M.R.,S.Z.,M.E.andC.B.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.Funding:ThisresearchwasfundedbytheGermanFederalMinistryofFoodandAgriculture(BMEL,https://www.bmel.de,accessedon25May2022)basedonadecisionoftheParliamentoftheFederalRepublicofGermany,grantedbytheFederalOfficeforAgricultureandFood(BLE,https://www.ble.de,accessedon25May2022grantnumbers:2817HS016,2817HS013).InstitutionalReviewBoardStatement:TheanimalstudyprotocolwasapprovedbytheEthicalCom-mitteeforAnimalExperimentsoftheGovernmentofUpperBavaria,Munich,Germany(referencenumberROB-55.2-2532.Vet_02-19-11).InformedConsentStatement:Notapplicable.DataAvailabilityStatement:Notapplicable.Acknowledgments:Theauthorsareverygratefulforthesupportofallparticipants.Aspecialthankyoutoallourprojectpartnerswhomadethepublicationofthispaperpossible.Invaluablewasthehelpfromthestaffatourdepartmentinsettingup,maintaining,andcleaningthecameraandarenaequipment.ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.Thefundershadnoroleinthedesignofthestudy;inthecollection,analyses,orinterpretationofdata;inthewritingofthemanuscript;orinthedecisiontopublishtheresults.References1.Raja,S.N.;Carr,D.B.;Cohen,M.;Finnerup,N.B.;Flor,H.;Gibson,S.;Keefe,F.J.;Mogil,J.S.;Ringkamp,M.;Sluka,K.A.;etal.TherevisedInternationalAssociationfortheStudyofPaindefinitionofpain:Concepts,challenges,andcompromises.Pain2020,161,1976–1982.[CrossRef]2.Ison,S.;Rutherford,K.Attitudesoffarmersandveterinarianstowardspainandtheuseofpainreliefinpigs.Vet.J.2014,202,622–627.[CrossRef][PubMed]3.TierSchG.Tierschutzgesetz,NeugefasstDurchBek.v.18.5.2006I1206,1313;ZuletztGeändertDurchArt.280Vv.19.6.2020I1328.2020.Availableonline:https://www.gesetze-im-internet.de/tierschg/BJNR012770972.html(accessedon16May2022).4.BLE.SchweinehaltunginDeutschland.2022.Availableonline:https://www.praxis-agrar.de/tier/schweine/schweinehaltung-in-deutschland(accessedon16May2022).5.Backus,G.;Higuera,M.;Juul,N.;Nalon,E.;DeBriyne,N.SecondProgressReport2015–2017ontheEuropeanDeclarationonAlternativestoSurgicalCastrationofPigs.2018.Availableonline:https://www.boarsontheway.com/wp-content/uploads/2018/08/Second-progress-report-2015-2017-final-1.pdf(accessedon16May2022).6.Ison,S.H.;Clutton,R.E.;DiGiminiani,P.;Rutherford,K.M.D.AReviewofPainAssessmentinPigs.Front.Vet.Sci.2016,3,108.[CrossRef][PubMed]7.Sheil,M.;Polkinghorne,A.OptimalMethodsofDocumentingAnalgesicEfficacyinNeonatalPigletsUndergoingCastration.Animals2020,10,1450.[CrossRef]8.Hay,M.;Vulin,A.;Génin,S.;Sales,P.;Prunier,A.Assessmentofpaininducedbycastrationinpiglets:Behavioralandphysiologicalresponsesoverthesubsequent5days.Appl.Anim.Behav.Sci.2003,82,201–218.[CrossRef]9.DiGiminiani,P.;Brierley,V.L.;Scollo,A.;Gottardo,F.;Malcolm,E.M.;Edwards,S.A.;Leach,M.C.TheAssessmentofFacialExpressionsinPigletsUndergoingTailDockingandCastration:TowardtheDevelopmentofthePigletGrimaceScale.Front.Vet.Sci.2016,3,100.[CrossRef]10.Saller,A.M.;Werner,J.;Reiser,J.;Senf,S.;Deffner,P.;Abendschon,N.;Weiss,C.;Fischer,J.;Schorwerth,A.;Miller,R.;etal.Localanesthesiainpigletsundergoingcastration-Acomparativestudytoinvestigatetheanalgesiceffectsoffourlocalanestheticsonthebasisofacutephysiologicalresponsesandlimbmovements.PLoSONE2020,15,e0236742.[CrossRef][PubMed]11.Abendschön,N.;Senf,S.;Deffner,P.;Miller,R.;Grott,A.;Werner,J.;Saller,A.M.;Reiser,J.;Weiss,C.;Zablotski,Y.;etal.LocalAnesthesiainPigletsUndergoingCastration-AComparativeStudytoInvestigatetheAnalgesicEffectsofFourLocalAnestheticsBasedonDefensiveBehaviorandSideEffects.Animals2020,10,1752.[CrossRef]

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Animals2023,13,529 13of13 12.Werner,J.;Saller,A.M.;Reiser,J.;Senf,S.;Deffner,P.;Abendschon,N.;Fischer,J.;Grott,A.;Miller,R.;Zablotski,Y.;etal.EvaluationofTwoInjectionTechniquesinCombinationwiththeLocalAnestheticsLidocaineandMepivacaineforPigletsUndergoingSurgicalCastration.Animals2022,12,1028.[CrossRef]13.Hansson,M.;Lundeheim,N.;Nyman,G.;Johansson,G.Effectoflocalanaesthesiaand/oranalgesiaonpainresponsesinducedbypigletcastration.ActaVet.Scand.2011,53,1–9.[CrossRef]14.Bateson,M.;Martin,P.MeasuringBehaviour:AnIntroductoryGuide;CambridgeUniversityPress:Cambridge,UK,2021.15.RCoreTeam.R:ALanguageandEnvironmentforStatisticalComputing;RFoundationforStatisticalComputing:Vienna,Austria,2015.Availableonline:https://www.r-project.org/(accessedon25May2021).16.Viscardi,A.V.;Hunniford,M.;Lawlis,P.;Leach,M.;Turner,P.V.DevelopmentofaPigletGrimaceScaletoEvaluatePigletPainUsingFacialExpressionsFollowingCastrationandTailDocking:APilotStudy.Front.Vet.Sci.2017,4,51.[CrossRef][PubMed]17.Luna,S.P.L.;deAraujo,A.L.;daNobregaNeto,P.I.;Brondani,J.T.;deOliveira,F.A.;Azeredo,L.;Telles,F.G.;Trindade,P.H.E.ValidationoftheUNESP-Botucatupigcompositeacutepainscale(UPAPS).PLoSONE2020,15,e0233552.[CrossRef][PubMed]18.Prunier,A.;Bonneau,M.;Borell,E.H.;Cinotti,S.;Gunn,M.;Fredriksen,B.;Giersing,M.;Morton,D.;Tuyttens,F.;Velarde,A.Areviewofthewelfareconsequencesofsurgicalcastrationinpigletsandtheevaluationofnon-surgicalmethods.Anim.Welf.2006,15,277–289.[CrossRef]19.Skade,L.;Kristensen,C.S.;Nielsen,M.B.F.;Diness,L.H.Effectoftwomethodsandtwoanaestheticsforlocalanaesthesiaofpigletsduringcastration.ActaVet.Scand.2021,63,1.[CrossRef]20.LlamasMoya,S.;Boyle,L.A.;Lynch,P.B.;Arkins,S.Effectofsurgicalcastrationonthebehaviouralandacutephaseresponsesof5-day-oldpiglets.Appl.Anim.Behav.Sci.2008,111,133–145.[CrossRef]21.D’Eath,R.B.;Jack,M.;Futro,A.;Talbot,D.;Zhu,Q.;Barclay,D.;Baxter,E.M.Automaticearlywarningoftailbitinginpigs:3Dcamerascandetectloweredtailposturebeforeanoutbreak.PLoSONE2018,13,e0194524.[CrossRef][PubMed]22.Li,X.;Zhao,J.N.;Zhao,P.;Zhang,X.;Bi,Y.J.;Li,J.H.;Liu,H.G.;Wang,C.;Bao,J.Behaviouralresponsesofpigletstodifferenttypesofmusic.Animal2019,13,2319–2326.[CrossRef][PubMed]23.MarcetRius,M.;Pageat,P.;Bienboire-Frosini,C.;Teruel,E.;Monneret,P.;Leclercq,J.;Lafont-Lecuelle,C.;Cozzi,A.Tailandearmovementsaspossibleindicatorsofemotionsinpigs.Appl.Anim.Behav.Sci.2018,205,14–18.[CrossRef]24.Tallet,C.;Rakotomahandry,M.;Herlemont,S.;Prunier,A.EvidenceofPain,Stress,andFearofHumansDuringTailDockingandtheNextFourWeeksinPiglets(Susscrofadomesticus).Front.Vet.Sci.2019,6,462.[CrossRef]25.Langhoff,R.R.UntersuchungenüberdenEinsatzvonSchmerzmittelnzurReduktionKastrationsbedingterSchmerzenbeimSaugferkel.Ph.D.Thesis,LMUMünchen,Munich,Germany,2008.26.Yun,J.;Ollila,A.;Valros,A.;Larenza-Menzies,P.;Heinonen,M.;Oliviero,C.;Peltoniemi,O.Behaviouralalterationsinpigletsaftersurgicalcastration:Effectsofanalgesiaandanaesthesia.Res.Vet.Sci.2019,125,36–42.[CrossRef]27.Kluivers-Poodt,M.;Zonderland,J.J.;Verbraak,J.;Lambooij,E.;Hellebrekers,L.J.Painbehaviouraftercastrationofpiglets;effectofpainreliefwithlidocaineand/ormeloxicam.Animal2013,7,1158–1162.[CrossRef][PubMed]28.Garcia,E.R.Localanesthetics.Vet.Anesth.Analg.2015,7,332–354.29.Sprecher,D.;Hostetler,D.;Kaneene,J.Alamenessscoringsystemthatusespostureandgaittopredictdairycattlereproductiveperformance.Theriogenology1997,47,1179–1187.[CrossRef][PubMed]30.Lonardi,C.;Scollo,A.;Normando,S.;Brscic,M.;Gottardo,F.Cannovelmethodsbeusefulforpainassessmentofcastratedpiglets?Animal2015,9,871–877.[CrossRef]31.Baysinger,A.;Webb,S.R.;Brown,J.;Coetzee,J.F.;Crawford,S.;DeDecker,A.;Karriker,L.A.;Pairis-Garcia,M.;Sutherland,M.A.;Viscardi,A.V.Proposedmultidimensionalpainoutcomemethodologytodemonstrateanalgesicdrugefficacyandfacilitatefuturedrugapprovalforpigletcastration.Anim.HealthRes.Rev.2021,22,163–176.[CrossRef][PubMed]Disclaimer/Publisher’sNote:Thestatements,opinionsanddatacontainedinallpublicationsaresolelythoseoftheindividualauthor(s)andcontributor(s)andnotofMDPIand/ortheeditor(s).MDPIand/ortheeditor(s)disclaimresponsibilityforanyinjurytopeopleorpropertyresultingfromanyideas,methods,instructionsorproductsreferredtointhecontent.