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ArticleLocalAnesthesiainPigletsUndergoingCastration—AComparativeStudytoInvestigatetheAnalgesicE ectsofFourLocalAnestheticsBasedonDefensiveBehaviorandSideE ectsNoraAbendschön1,*,Ste anieSenf1 ,PaulineDe ner1 ,ReginaMiller2,AndreaGrott2,JuliaWerner3 ,AnnaM.Saller3,JudithReiser3,ChristineWeiß1,YuryZablotski1,JohannesFischer3,ShanaBergmann2 ,MichaelH.Erhard2,ChristineBaumgartner3,MathiasRitzmann1andSusanneZöls11ClinicforSwine,CenterforClinicalVeterinaryMedicine,LMUMunich,85764Oberschleißheim,Bavaria,Germany;S.Senf@med.vetmed.uni-muenchen.de(S.S.);P.De ner@med.vetmed.uni-muenchen.de(P.D.);C.Weiss@med.vetmed.uni-muenchen.de(C.W.);Y.Zablotski@med.vetmed.uni-muenchen.de(Y.Z.);Ritzmann@med.vetmed.uni-muenchen.de(M.R.);S.Zoels@med.vetmed.uni-muenchen.de(S.Z.)2ChairofAnimalWelfare,Ethology,AnimalHygieneandHusbandry,LMUMunich,80539Munich,Bavaria,Germany;r.miller@tierhyg.vetmed.uni-muenchen.de(R.M.);a.schoerwerth@tierhyg.vetmed.uni-muenchen.de(A.G.);s.bergmann@tierhyg.vetmed.uni-muenchen.de(S.B.);m.erhard@tierhyg.vetmed.uni-muenchen.de(M.H.E.)3CenterofPreclinicalResearch,TechnicalUniversityofMunich,81675Munich,Bavaria,Germany;julia.werner@tum.de(J.W.);anna.saller@tum.de(A.M.S.);judith.reiser@tum.de(J.R.);fischer.johannes@tum.de(J.F.);christine.baumgartner@tum.de(C.B.)*Correspondence:Nora.Abendschoen@med.vetmed.uni-muenchen.deReceived:27August2020;Accepted:24September2020;Published:26September2020

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artner@tum.de(C.B.)*Correspondence:Nora.Abendschoen@med.vetmed.uni-muenchen.deReceived:27August2020;Accepted:24September2020;Published:26September2020 SimpleSummary:Morethan80millionmalepigletsarecastratedeveryyearwithinthefirstweekoflifemostlywithoutpainreliefintheEU.Castrationisperformedtopreventboartaint,tominimizeaggressiveandsexualbehaviorassociatedwithintactmalesandtogainaconstantqualityofmeat.Itisanimportantanimalwelfareissuetoeliminatepaincausedbycastration.Localanesthesia,meaningthesoleinjectionofalocalanestheticintothetesticlesandinthesurroundingtissueofconsciouspiglets,priortocastrationisacurrentlydiscussedmethodinGermany.Thus,itwastheaimofthepresentstudytoinvestigatethee ectoffourlocalanesthetics(procaine,lidocaine,bupivacaineandmepivacaine)onpainreliefduringsurgicalcastrationinconsciouspiglets.Toassesspain,defensivebehaviorofpigletsundergoinginjectionandcastrationwasevaluated.Inaddition,attentionwaspaidtopossiblesidee ects.Consideringhealing,bleedingandweightgainnonegativeimpactswereobserved,butimpairmentsoflocomotoractivityoccurred.Theresultsindicatethatlidocaineandmepivacaineareabletoachievesignificantpainreliefduringthecastrationprocedure,whereasprocaineandbupivacaineonlyduringtheseveringofthespermaticcord.Abstract:Theaimofthepresentstudywastoinvestigatethee ectoffourlocalanestheticsonpainreliefduringsurgicalcastrationunderstandardizedconditionsinconsciouspiglets.Therefore,71malepiglets(threetosevendays)weredistributedintocontrolgroups(handling,castrationwithoutanesthesiaoranalgesia)andlocalanesthetictrialgroups(procaine,lidocaine,bupivacaine,mepivacaine).Then,20minpriortocastration,animalsofthetreatmentgroups,exceptpigletsinthehandlinggroup,receivedaninjectionofalocalanestheticorsodiumchlorideof0.5mLintratesticularlyand0.5mLsubscrotally.Duringinjectionandcastration,defensivebehaviorwasevaluated.Locomotoractivity,aswellaspostoperativebleeding,woundhealingandaveragedailyweightgainwereassessedtodetectsidee ects.Theinjectioncausedincreaseddefensivemovements,Animals2020,10,1752;doi:10.3390/ani10101752www.mdpi.com/journal/animals

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Animals2020,10,17522of15significantlyinthebupivacainegroup.Lidocaineandmepivacainesignificantlyreduceddefensivemovementsduringcastration,andprocaineandbupivacaineonlyduringseveringofthespermaticcord.Impairmentsoflocomotoractivitywerefoundinpigletsinjectedwithlidocaine,bupivacaineorsodiumchloride.Consideringhealing,bleedingandweightgain,nonegativeimpactswereobserved.Inconclusion,lidocaineandmepivacainewereabletoachievesignificantpainreliefduringthecastrationprocedure,whereasprocaineandbupivacaineonlyduringtheseveringofthespermaticcord.Moreover,theinjectionofbupivacaineseemedtobepainfulitself.Keywords:piglet;castration;localanesthesia;behavior;pain;procaine;lidocaine;bupivacaine;mepivacaine;animalwelfare

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ofbupivacaineseemedtobepainfulitself.Keywords:piglet;castration;localanesthesia;behavior;pain;procaine;lidocaine;bupivacaine;mepivacaine;animalwelfare 1.IntroductionIntheEU,morethan80millionmalepigletsarecastratedeveryyearwithinthefirstweekoflife,mostlywithoutpainrelief[1].Castrationisperformedtopreventboartaint,tominimizeaggressiveandsexualbehaviorassociatedwithintactmalesandtogainaconstantqualityofmeat[2–4].ThemajorityoftheEUmemberstatesarestillcarryingoutsurgicalcastrationwithorwithoutanesthesia.TheexceptionsareIreland,theUnitedKingdom,theNetherlands,PortugalandSpain,where20%orlessofthemalepigsarecastrated[5].AccordingtoEUDirective2008/120/EC,betterpracticesaredemanded[6].InGermany,theAnimalWelfareActprohibitsthesurgicalcastrationofmalepigletswithoutanesthesia,evenundertheageofeightdays,asof1January2021[7].InadditiontoacompletewaiverofsurgicalcastrationbyimplementingthepracticesofboarfatteningorGnRH(gonadotropinreleasinghormone)vaccination,animprovedpracticeofmalepigletcastrationrequiressu cientanesthesiaandanalgesiatoavoidacuteandpostoperativepain.Severalpossibilitiestoensuretheserequirementsarediscussed:injectableanesthesia,inhalationanesthesiaandlocalanesthesia.ThepossibilityofcastrationwithlocalanestheticsiscurrentlynotpermittedinGermany,becausetheevidenceofthee cacyofthismethodisstillcontroversiallydiscussed.Kluivers-Poodtetal.[8],Hanssonetal.[9]andLeidigetal.[10]concludedthattheuseoflocalanestheticsreducespainresponsesduringcastration,whereasPerez-Pedrazaetal.[11]didnotshowanye ect.Bonastreetal.[12]aswellasRauhetal.[13]indicatedthatlocalanestheticsleadtoareductioninpain-relatedresponses,withouteliminatingthepain.Thesepreviousstudiesdi erintermsofusedlocalanesthetics,applicationmethodsandobtainedpainassociatedparametersandarethereforedi culttocompare.Procaineisthelocalanestheticwiththelowestanestheticpotency[14]andtheonlyapprovedlocalanestheticforpigsinGermanyatthemoment.LidocaineiscurrentlyusedforpigletcastrationinNorway,SwedenandItaly[5].Thee ectsofprocaineandlidocaineinpigletsundergoingcastrationhavebeeninvestigatedinvariousstudies[8–13,15]butshowedcontradictoryresults.Furthermore,Bonastreetal.[12]investigatedthee ectofthecombinationoflidocaineandbupivacaine.Bupivacaineisdescribedwiththehighestanestheticpotency[14].Thelocalanestheticmepivacaineiscommonlyusedinhorses.Totheauthors’knowledge,therehasbeennoinvestigationonmepivacaineinconsciouspigletsundergoingcastrationsofar.Toassesspaininpigletsundergoingcastration,di erentmethodsareavailable.Asphysiologicalparametersi.e.,bloodparameters,suchascortisol[12,16–20]adrenalinandadrenocorticotropichormones[16,18,19]andchangesinheartrateandbloodpressure[21]canbeassessed.Furthermore,behavioralvariables,suchasdefensivemovements[10,21,22]andvocalization[8,20,23–27]canbeusedforpainassessment.Additionally,Bilsborrowetal.[28]describedanobjectivetesttoassesspaininpigletswherethenavigationtimethroughahandlingchuteisdetermined.Inafirstalreadypublishedstudypart,thefourlocalanesthetics,procaine,lidocaine,bupivacaineandmepivacaine,wereevaluatedbasedonpain-relatedphysiologicalparametersandlimbmovements

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Animals2020,10,17523of15underaminimalanesthesiamodelusinglowdosesofisoflurane[21].Underlightanesthesia,allfourlocalanestheticswerehighlye ectiveatreducingsignsofnociceptionduringcastration.Theaimofthepresentstudywastoinvestigatethee ectoffourlocalanesthetics(procaine,lidocaine,bupivacaine,mepivacaine)onpainreliefduringsurgicalcastrationunderstandardizedconditionsinconsciouspigletsasasecondpartofacomprehensivestudy.Toevaluatepain,defensivemovementsandvocalizationwereassessed.Todetectsidee ectslocomotoractivity,postoperativebleeding,woundhealing,weightgainandmortalitywereevaluated.2.MaterialsandMethodsDataoffourgroupsofpigletscastratedunderlocalanesthesiawerecollectedandcomparedwithpigletscastratedwithoutlocalanesthesiaandwithonlyhandledpiglets.ThestudywasperformedinaccordancewiththeEUDirective2010/63/EUontheprotectionofanimalsusedforscientificpurposesandtheGermanAnimalWelfareAct(2019).TheresearchprotocolwasapprovedbytheEthicalCommitteeforAnimalExperimentsoftheGovernmentofUpperBavaria,Munich,Germany(referencenumberROB-55.2-2532.Vet_02-19-11).2.1.AnimalsandHousingThepresentrandomized,double-blindedstudywasconductedinanexperimentalfarrow-to-finishfarminBavaria,Germany,with80sowsfarrowinginathree-weekbatch.Thepiglets(Piétrain LargeWhite/Landrace)werehousedinfarrowingunitsonpartiallyslattedfloor,exceptforthenestarea,whichwasmadeupofconcretefloorandprovidedwithshavingsandaninfraredheatlamp.Allpigletsinthestudyreceived1mLofanintramuscularironsupplementation(Ursoferran200mg/mLsolutionforinjectionforpigs,SerumwerkBernburgAG,Bernburg,Germany)thedaybeforecastration.Besides,nootherpainfulprocedures,suchastaildockingoreartagging,werecarriedoutbeforethestartofthestudy.Datawerecollectedfrom71malepigletsbetweenthreetosevendaysoflife(5.2 1.09days,2.2 0.48kg(mean standarddeviation))of16litters.Inclusioncriteriawereagoodgeneralcondition,ahealthysowandaminimumweightof1.4kgonthedayofcastration.Pigletswithanydeviationfromthenormalanatomicalcondition(e.g.,herniascrotalisoringuinalis)wereexcluded.2.2.ExperimentalDesignOnthedaybeforecastration,eachpigletwasweighed(MSweighingplateaumax.100kg,SchippersGmbH,Kerken,Germany),thegeneralconditionsofthepigletswerecheckedandacomputer-generatedsimplerandomizationwasusedtodistributetheanimalstooneofthesixexperimentalgroups(Table1).Additionally,thehandlingchute,accordingtoBilsborrowetal.[28]wastrainedfourtimeswitheachpiglet(twotimeswithoutandtwotimeswithhurdles)(Figure1).

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Animals2020,10,17524of15Table1.Classificationofstudygroups. GroupPreparationActiveAgentInterventionAnimals(n) LA(mg/Animal)Epin.(mg/mL)InjectionCastration PPronestesic40mg/mL1procaine(80)0.02++12LXylocitin®2%2lidocaine(40)0.01++12BBupivacain0.5%3bupivacaine(10)0.005++12MMepidor20mg/mL4mepivacaine(40)-++12NaClNaCl0.9%5--++12H---shaminjectionshamcastration11 P=procaine;L=lidocaine;B=bupivacaine;M=mepivacaine;NaCl=sodiumchloride;H=handling;1Pronestesic40mg/mLsolutionforinjectionforhorses,cattle,pigsandsheepFATROS.p.A,OzzanoEmilia(Bologna),Italy;2Xylocitin®2%withepinephrine,mibeGmbHArzneimittel,Brehna,Germany;3Bupivacain0.5%withepinephrine,JENAPHARM®,mibeGmbHArzneimittel,Brehna,Germany;4Mepidor20mg/mLsolutionforinjectionforhorses,RichterPharmaAG,Wels,Austria;5NaCl0.9%,0.9%SodiumChlorideIntravenousInfusionB.BraunMelsungenAG,Melsungen,Germany;LA:localanestheticagentwithhydrochloride;epin.:epinephrine.

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ustria;5NaCl0.9%,0.9%SodiumChlorideIntravenousInfusionB.BraunMelsungenAG,Melsungen,Germany;LA:localanestheticagentwithhydrochloride;epin.:epinephrine. Figure1.IllustrationofthehandlingchuteaccordingtoBilsborrowetal.[28];dimensions=internaldimensionsinmm.Onthemorningofthecastrationday,pigletspassedthroughthehandlingchutethreeconsecutivetimestodeterminethebaseline.Duringinjectionandcastration,allpiglets,includingthehandlinggroup,werefixedinacastrationcradle(SchippersGmbH,Kerken,Germany).Thepigletsweregivenatleastfiveminutestorestbeforetheinjection.Allpigletsexceptthoseofthehandlinggroup(H,onlyfixation)wereinjectedwithatotalvolumeof2mLwiththerespectivelocalanesthetic(procainehydrochloride(P),lidocainehydrochloride(L),bupivacainehydrochloride(B)andmepivacainehydrochloride(M))or0.9%sodiumchloride(NaCl)accordingtothestudygroup.Then,0.5mLwereinjectedintratesticularlyand0.5mLsubscrotallyforeachtesticle.Fortheinjectionanautomaticself–fillingsystem1mLsyringe(HSWECO–MATIC®,Henke-Sass,WolfGmbH,Tuttlingen,Germany)witha25Gsizedcannula(0.5 16mm,B.BraunTravaCareGmbH,Hallbergmoos,Germany)wasused.Theintratesticularinjectionwasperformedbyfixingthetesticlebetweenthethumbandindexfingercaudally.Directlyfollowingtheintratesticularinjectionthesubscrotalsinglepointinjectionwasperformedbyreleasingthefixedtesticle,retractingthecannulafromitbutleavingthecannulaundertheskinandmakingaskinfoldofthescrotum.Theintratesticularandsubscrotalinjectionwasrepeatedforthesecondtesticle.Thereafter,thepigletswerereturnedintothefarrowingpenforaperiodof20min.Afterwards,allpigletswereremovedagainfromthefarrowingpenand,withtheexceptionofgroupH(onlyfixationandsimulatedinterventions),castrated.Therefore,thescrotalareawas

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Animals2020,10,17525of15cleanedwithanantisepticsolution(octenisept®aqueouswoundandmucousmembraneantiseptic,colorless,Schuelke&MayrGmbH,Norderstedt,Germany).Usingasterilescalpel(scalpelbladescarbonsteel,sterile21,HeinzHerenzMedizinalbedarfGbmH,Hamburg,Germany;scalpelhandleno.4,AESCULAPAG&CO.KG,Tuttlingen,Germany)foreverypiglet,scrotaltissueandtunicavaginaliswereopenedbyaskinincisionparalleltotheraphescrotiandthefirsttesticlewasremovedbyseveringofthespermaticcordwiththescalpel.Skinincisionandseveringofthespermaticcordwererepeatedforthesecondtesticle.Theinjectionandcastrationwereperformedbythreetrainedveterinarians.Aftersurgery,nodisinfectantwasappliedonthescrotalareatoavoidbehavioralchangesbyirritatingsubstances.GroupNaClservedaspositivecontrolforpain-relatedbehaviorinducedbyinjectionandcastrationwithoutpainrelief.Asnegativecontrol,pigletsfromgroupHunderwentsimulatedinjectionandcastrationtomeasurestressrelatedbehavior.Inordertoavoidananalgesice ectonthepain-relatedbehaviorduringandafterinjectionandcastration,meloxicam(0.4mg/kg)(Metacam®5mg/mL,solutionforinjectionforcattleandpigs,BoehringerIngelheimPharmaGmbH&Co.KG,IngelheimamRhein,Germany)wasnotadministereduntilonedayaftercastration.2.3.DataSamplingFordatasampling,evaluationofrecordingsandalsodataanalysisforallpersonsinvolvedwereblindedatanytimewiththeexceptionforgroupH(handling).2.3.1.DefensiveBehaviorToevaluatedefensivemovements,pigletswerefilmedduringinjectionandcastrationprocedure(SamsungGalaxyS6-SamsungElectronicsGmbH,Schwalbach,Germany).AnalyzingofthedefensivemovementsaccordingtoascoremodifiedbyLeidigetal.[10](TableS1)wasalwaysperformedbythesameblindedperson.“Injection”,“skinincision”and“severingofthespermaticcord”wereevaluatedseparatelyaccordingtointensity(0–4)andduration(0–3).Theintensitywasscaledinto0=nomovements,1=movingofonelimb,2=movingoftwolimbs,3=movingofthreelimbs,4=movingofallfourlimbs.Thedurationwasscaledinto0=nomovements,1=onesinglemovement,2=repeatedmovements(2–4),3=continuousmovements(>4).Defensivemovementswereevaluatedfortherightandlefttesticleseparately.Thesinglescoresresultedinamaximumofsevenforeachtesticleandstep.Thescoresofbothtesticlesandeachstepweresummedupforevaluation.Thisresultsinacombinedtotalscorefor“injection”,“skinincision”and“severingofthespermaticcord”from0to14.Onthebasisoftheaudiooftheserecordings,ablindedpersonevaluatedvocalizationwith“Yes”or“No”.Vocalizationwasassessedwith“Yes”,iftherewasanacuteonsetofincreasedvocalizationduringtheinjectionand/orcastrationprocedure.Moreover,twoadditionalblindedpersonsevaluatedtherecordingsofdefensivemovementsindependentlytoensurethereliabilityoftheobserver.2.3.2.HandlingChuteBehaviorToevaluatethenavigationtimeaccordingtoBilsborrowetal.[28],pigletspassedthroughthehandlingchuteafterinjectionandcastration,twohoursaftercastrationandonedayaftercastration.Thehandlingchutehadalengthof1400mm,widthof210mm,heightof400mm,two70mmhurdles(distancebetweenthehurdles:600mm)andanopentoptorecordthedurationandqualityoftherun(PanasonicLUMIXDMC-FZ8,MatsushitaElectricIndustrialCo.,Ltd.,Osaka,Japan)(Figure1).Additionally,astructuredthermalmatwasusedasabaseforthehandlingchutetoavoidslipping.Thenavigationtimewasdetermined,basedontherecordings,startingwhenthepigletsbeganrunningandendingwhenallfourlimbswereoverthelasthurdle.Particularattentionwaspaidtoimpairmentsofthelocomotoractivity(unsteadygait,stumbleuponthehurdles,sitting/lyinginthehandlingchute).2.3.3.PostoperativeAssessmentPigletswereweighedandwoundhealingwasscoredaccordingtoZankl[29](TableS2)ondays1,7,14and21aftercastrationtoassessthepostoperativeperioduptoday21.Castrationwoundswere

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Animals2020,10,17526of15rankedonthebasisofwoundhealing(0=withoutspecificfindings(dry,fullyclosedwound),1=minorfindings(swelling,redness),2=openwound),woundsecretion(0=nosecretion,1=serous/bloodysecretion,2=purulentsecretion)andtextureandsizeofthespermaticcord(0=hardlypalpable,1=upto1cm,softtoroughandelastic,2=strongerthan1cm,softtoroughandelastic,3=strongerthan1cm,roughorfluctuating).Thescorewassummedupforevaluationwithamaximumofseven.PostoperativebleedingwasquantifiedtwohoursaftercastrationusingascoreadaptedbyEnzetal.[30](TableS3).Thescorerangedfrom0(nobleeding)toamaximumof3(severebleeding,perinealareaandhindlimbsbloodstained).Inaddition,mortalitywasrecordeduntilday21postcastration.2.4.StatisticalAnalysisForthestatisticalanalysis,allexperimentalgroupsweretestedforageandweightondayofcastration,defensivemovements,vocalization,navigationtime,woundhealing,postoperativebleeding,andaveragedailyweightgain.ThedistributionofallcontinuousparameterswastestedusingShapiro–Wilknormalitytest.Fornon-normallydistributedparametersdefensivemovements,woundhealingandpostoperativebleedingaKruskal–Wallistestwasperformed.DunntestformultiplecomparisonsbetweenthegroupsP,L,B,M,HandNaClwithBenjamin–HochbergcorrectionsfollowedtheKruskal–Wallistest.Fornormallydistributedparameters(averagedailyweightgain)ANOVAwasusedwithStudent’st-testforpairwisecomparisons.Nonparametricbootstrapwasusedforobtainingconfidencelimitsforthepopulationmeanwithoutassumingnormality.FormostlynormaldistributedparametersageandweightondayofcastrationaMann–Whitney-Utestwasperformed.VocalizationwasanalyzedusingpairwiseFisher’stestswithBenjamin–Hochbergcorrections.Navigationtimewasanalyzedusingmixed-e ectslinearmodelduetothepresenceofrepeatedmeasures,withtime,groupandinteractionbetweenthemasfixede ectsandarandome ectofindividualpiglet.Duetothenon-normalityandheteroscedasticityoftheresiduals,theresponsevariablewaslog-transformed.Statisticalsignificancewasconsideredatp<0.05.ThesestatisticalanalyseswereperformedusingRstatisticalsoftwareversion3.6.1,mainlywith“ggstatsplot”[31],“lme4”[32]and“emmeans”[33]packagesandIBMSPSSStatisticsforWindows,Version26.0(IBMCorp.,Armonk,NY,USA).Forthescoreusedtoevaluatethedefensivemovements,theIntraclassCorrelationCoe cients(ICC)andtheir95%confidentintervalswerecalculatedusingIBMSPSSStatisticsforWindows,Version26.0,basedonmeanrating,absoluteagreementand2-waymixed-e ectsmodel.TheICCvaluesrangedbetween0and1,withvaluescloserto1representingstrongerreliability.3.ResultsDayofcastrationvariedbetweenthirdandseventhdayoflife(dl)ofthepigletsandthemeanageofthegroupsdi eredbetween4.75 0.97dl(Mean standarddeviation(SD))ingroupNaCland5.58 1.40dlingroupMatcastrationday(p 0.05).Meanbodyweightondayofcastrationdi eredsignificantlybetweenpigletsofgroupM(2.53 0.59kg)andgroupsL(2.08 0.44kg)(p<0.05)andnumericallybetweengroupMandB(2.09 0.42kg)(p<0.1)(Table2).Table2.Meanvaluesandstandarddeviationofageandbodyweightondayofcastration.

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8 0.44kg)(p<0.05)andnumericallybetweengroupMandB(2.09 0.42kg)(p<0.1)(Table2).Table2.Meanvaluesandstandarddeviationofageandbodyweightondayofcastration. GroupAgeonDayofCastrationWeightonDayofCastration MeanValueSDMeanValueSD procainen=125.00dl 0.952.23kg 0.44lidocainen=125.58dl 0.902.08kga 0.44bupivacainen=125.17dl 1.272.09kg 0.42mepivacainen=125.58dl 1.342.53kgb 0.59NaCln=124.75dl 0.972.14kg 0.44handlingn=115.09dl 0.942.32kg 0.45 Di eringsuperscriptswithinacolumnindicatesignificant(p<0.05)di erencesbetweengroups;n=animals;dl=dayoflife;SD=standarddeviation.

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Animals2020,10,17527of153.1.DefensiveBehaviorTheIntraclassCorrelationCoe cients(ICCs)fortheevaluationofthedefensivemovementsaccordingtothemodifiedscoreofLeidigetal.[10]were0.97(confidenceinterval(CI):0.95–0.98)forinjection,0.96(CI:0.94–0.98)forskinincisionand0.98(CI:0.97–0.99)forseveringofthespermaticcord.AsshowninFigure2,duringinjection,onlygroupBshowedsignificantlymoredefensivemovementscomparedtogroupsHandL.Amongtheothergroups,nosignificantdi erencecouldbedetectedduringtheinjection.DuringskinincisiongroupsL,MandHshowedsignificantlyfewerdefensivemovementscomparedtogroupNaCl.IngroupsPandB,nosignificantdi erencewasfoundcomparedtogroupNaCl.GroupsNaClandPshowedsignificantlymoredefensivemovementscomparedtogroupHduringskinincision.Nosignificantdi erencewasfoundbetweengroupBandtheothergroupsduringskinincision.ThehighestmeanscoreofdefensivemovementswasfoundingroupNaClduringtheseveringofthespermaticcord.Regardingtheseveringofthespermaticcord,alllocalanestheticgroups(P,L,B,andM)andgroupHshowedsignificantlyfewerdefensivemovementscomparedtogroupNaCl.Apartfromthis,nodi erencesamongthegroupswerefoundduringtheseveringofthespermaticcord. Figure2.Meanvalues,confidenceintervalsandp-valuesofthescoreusedtoevaluatethedefensivemovementsindi erentgroupsduringinjection,skinincisionandseveringofthespermaticcord;outlierswerecalculatedusingTukey´smethodandweretaggedonthepicturewithavalueinsteadofthedatapointtoavoidclutter.

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Animals2020,10,17528of15Duringinjection,nosignificantdi erencewasfoundamongthegroupsregardingvocalization.Threeofthe10pigletsingroupHshowedanacuteonsetofincreasedvocalizationduringshamskinincisionandshamseveringofthespermaticcordincontrastto11of12pigletsingroupNaCl(p<0.05),asshowninTable3.Duringtheseveringofthespermaticcord,onepigletingroupMshowedanacuteonsetofincreasedvocalizationincontrastto11ingroupNaCl(p<0.05).Table3.Numberofpigletswithincreasedvocalization/numberofpigletspergroupduringinjection,skinincisionandseveringspermaticcord. GroupNumberofPigletswithIncreasedVocalization InjectionSkinIncisionSeveringSpermaticCord handling6/103/10a3/10aNaCl10/1211/12b11/12bprocaine11/129/126/12lidocaine9/126/123/12bupivacaine10/129/123/12mepivacaine8/128/121/12apn.s.<0.05<0.05

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d handling6/103/10a3/10aNaCl10/1211/12b11/12bprocaine11/129/126/12lidocaine9/126/123/12bupivacaine10/129/123/12mepivacaine8/128/121/12apn.s.<0.05<0.05 Di eringsuperscriptswithinacolumnindicatesignificant(p<0.05)di erencesbetweengroups;n.s.=notsignificant.3.2.HandlingChuteBehavior3.2.1.NavigationTimeAsshowninTable4,thebaselineofnavigationtimeofthegroupsdi eredbetween5.5 2.3s(S)(mean SD)(NaCl)and7.6 4.1s(L)(p 0.05).Afterinjection,groupMpassedthroughthehandlingchutewithin3.8 1.8s(fastest)andgroupLwithin9.4 7.6s(slowest)(p<0.05).Aftercastration,thefastestnavigationtimewasmeasuredforgroupMwith3.5 1.25sandtheslowestforgroupBwith8.9 11.47s(p<0.05).Twohoursaftercastration,groupMnavigatedthehandlingchutewithin2.4 0.51s,significantlyfasterthangroupsL(5.0 5.41s),B(4.5 2.3s)andP(4.1 1.4s)(p<0.05).Regardingthenavigationtime,nosignificantdi erencebetweencontrolgroupsNaClandHcouldbedetectedatanytime(p 0.05).Table4.MeanvaluesandstandarddeviationofnavigationtimeinsecondsofthepigletsthroughthehandlingchuteaccordingtoBilsborrowetal.[28]indi erentgroupsbeforeinjection,afterinjection,aftercastration,twohoursaftercastrationandonedayaftercastration. GroupInjectionCastration Before1AfterAfter2hAfter1DayAfter Procainen=126.02 3.905.39 3.126.78 2.78a4.12 1.40b3.38 0.74Lidocainen=127.64 4.07x9.50 7.56a6.40 4.404.97 5.41xb4.45 2.75xBupivacainen=126.46 2.795.90 3.76x8.97 11.47x4.51 2.29b4.56 2.33Mepivacainen=125.86 2.263.82 1.79b3.49 1.25b2.41 0.51a3.21 1.18NaCln=125.53 2.26x4.51 1.394.13 1.11x3.70 1.57x2.85 0.56xHandlingn=116.09 1.964.61 1.365.39 3.973.24 0.99xx3.88 2.13pn.s.<0.05<0.05<0.05n.s.

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a3.21 1.18NaCln=125.53 2.26x4.51 1.394.13 1.11x3.70 1.57x2.85 0.56xHandlingn=116.09 1.964.61 1.365.39 3.973.24 0.99xx3.88 2.13pn.s.<0.05<0.05<0.05n.s. Di eringsubscriptswithinacolumnindicatesignificant(p<0.05)di erencesbetweengroups;n=animals;xn=11;xx=n=10;1meanvaluefromthreeconsecutiveruns;h=hours;n.s.=notsignificant.3.2.2.LocomotorActivityAsshowninTable5,afterinjection,castrationandtwohoursaftercastration,animpairmentofthelocomotoractivityoccurredingroupsNaCl,L(twopigletsineachcase)andB(threepiglets).Twopigletsdiedonedayaftercastration.

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Animals2020,10,17529of15Table5.Impairmentsoflocomotoractivityatdi erenttimesofindividualpiglets. GroupPigletWeightinkgInjectionCastration AfterAfter2hAfter1Dayafter NaCl1n=1211.8refusingthehurdles22.9notabletowalkorstanddead L2n=1211.85notabletowalkorstanddead21.8unsteadygait B3n=1211.4tripping,lying,kyph.spinetripping22.35unst.gait,ref.thehurdles32.5unsteadygaitunsteadygait

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n=1211.85notabletowalkorstanddead21.8unsteadygait B3n=1211.4tripping,lying,kyph.spinetripping22.35unst.gait,ref.thehurdles32.5unsteadygaitunsteadygait 1Injectionofsodiumchloride;2injectionoflidocaine;3injectionofbupivacaine;n=animals/group;kyph.=kyphotic;unst.=unsteady;ref.=refusing;h=hours.3.3.PostoperativeAssessment3.3.1.WoundHealingOnedayaftercastration,onepigletingroupBhadaheightenedwoundhealingscoreofthree.Ondays7,14and21aftercastration,thewoundhealingscoreofallpigletswasbelow3,withoutdi eringsignificantlybetweengroupsatanytime.3.3.2.PostoperativeBleedingPigletsofgroupNaClshowedtheslightestpostoperativebleedingscore(1.25 1.42(Mean SD)).Postoperativebleedingscoresofthelocalanestheticgroupswere2.08 0.97(P),1.75 1.42(L),1.67 1.38(B)and2.08 1.38(M).Nosignificantdi erenceswerefoundbetweenthelocalanestheticgroupsorbetweenanyofthelocalanestheticgroupsandgroupNaCl.3.3.3.MortalityRateandAverageDailyWeightGainIntotal,three(4.2%)of71pigletsdiedduringthetrialperiodfromcastrationdayuntilweaning.Twoofthemwerefromthesamelitteranddiedthesamedayaftercastration(groupsNaClandL).Thepigletinjectedwithlidocainewasexaminedhistopathologically.Theautopsyshowedaseverehemorrhageintotheabdomen.Thehistologicalexaminationofthelungsshowedahemorrhagicandnecrotizingpneumonia.Anotherpiglet(groupNaCl)diedbetweendaystwoandsevenaftercastrationduetocrushingbythesow.Noe ectoftreatmentwasfoundonaveragedailyweightgain(ADW).TheADWofthepigletsrangedbetween0.131and0.353kg,exceptforoneoutlier(0.075kg)ingroupNaCl.Betweengroups,ADWdi eredbetween0.195 0.049kg(Mean SD)ingroupLand0.247 0.036kgingroupM.4.DiscussionSeveralstudieshavealreadybeenconductedonlocalanesthesiaasanalternativetopigletcastrationwithoutanesthesiabuttheydi eredinstudydesigns,localanestheticsusedandyieldedheterogeneousresults[8–13].Thissecondpartofacomprehensivestudyexaminedthefourlocalanesthetics,procaine,lidocaine,bupivacaineandmepivacaine,inconsciouspigletsduringcastrationinthefirstweekoflife.Partoneofthecomprehensivestudyshowedthattheintratesticularinjectionwithasubcutaneousdepotofalocalanestheticpriortocastrationreducesnociception-relatedcardiovascularresponsesandlimbmovementsinpigletsunderanesthesia[21].

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Animals2020,10,175210of15SimilartoLeidigetal.[10]andRauhetal.[13],weuseddefensivemovementsasasuitableparametertoevaluatepainresponsesinconsciouspiglets.Inthepresentstudy,theICCvaluesforthescoreusedtoevaluatedefensivemovementsweremorethan0.9for“injection”,“skinincision”and“severingofthespermaticcord”,whichindicatesanexcellentInterraterReliability[34].ThehighestmeanscoreofdefensivemovementsoccurredingroupNaCl(castrationwithoutanesthesia)duringseveringofthespermaticcord.ThisisinlinewiththefindingsofRauhetal.[13]andthusitconfirmsthesuggestionbasedonthevocalizationofTaylorandWeary[35]thatpullingandseveringofthespermaticcordsarethemostpainfulcomponentsofcastration.AlmostthesamescorewasobservedingroupNaClduringskinincision,whichisconsistentwithseveralstudiesthatthewholecastrationprocedurewithoutanesthesiacausesseverepain[8,10,35,36].Priorstudiesshowed,basedondefensivemovements,thattheapplicationoflidocaineintratesticularlyandsubscrotallyreducespainresponsesduringthecastrationprocedure[9,13].Inthepresentstudy,theinjectionofprocaine,lidocaine,bupivacaineormepivacaineledtoasignificantreductionindefensivemovementscomparedtocastrationwithoutanesthesiaduringtheseveringofthespermaticcord,whereasduringskinincisionasignificantreductionwasobservedonlyaftertheinjectionoflidocaineormepivacaine.Anexplanationforthedi erente ectsoflocalanesthesiaonskinincisionandseveringofthespermaticcordcouldbethatwiththesubscrotalandintratesticularapplicateddepotsusedinthepresentstudy,thelocalanestheticsdidnotreadilydi useintotheskin.Ranheimetal.[37]discovered,throughautoradiograms,thatradiolabeledlidocaineinjectedintothetestisandsubcutaneouslyintothescrotumdoesnotreadilydi usethroughthetunicavaginalis.However,theassumptionthatthelocalanestheticsdidnotreadilydi useintotheskindoesnotexplainthedi erentextentofthereductionindefensivemovementsbetweenlocalanestheticsduringskinincision.Theinjectionoflidocaineandmepivacaineledtoasignificantreductionindefensivemovementsduringskinincision,whileprocaineandbupivacaineapplicationdidnot.Anotherpossibleexplanationliesinthephysiochemicalpropertiesoftheusedlocalanesthetics.Localanestheticsareweakbasesandthusexistinequilibriumbetweentheneutral,non-ionized,lipid-solubleandtheionized,water-solubleform.ThepositionofequilibriumcanbedefinedusingpKa.ThepKaofamoleculerepresentsthepHatwhich50%existsinthenon-ionized(lipid-soluble)and50%intheionizedform(water-soluble)[38].Themainaccessoflocalanestheticstothecellisbypenetrationofthelipophilicneutralformthroughthelipidmembrane[14,39].Therefore,alocalanestheticwithalowpKa(mepivacaine:7.72;lidocaine:7.77;bupivacaine:8.1;procaine:8.89)willhaveagreaterproportionofthenon-ionizedlipid-solubleformatphysiologicalpH,resultinginbetterdi usionintothesurroundingtissueandamorerapidonsetofaction.Moreover,therelativeanestheticpotencyisofimportanceaswell.Themaindeterminantoflocalanestheticpotencyisthelipidsolubility[40,41].However,wecouldnotprovethatthelocalanestheticwiththehighestlipidsolubilityandthehighestrelativeanestheticpotency,inthiscasebupivacaine,producedthegreatestreductioninpainrelatedbehavior.Accordingtotheseresults,weassumethatthespermaticcordwasanesthetizedmoree cientlythanthescrotalskin.Forfutureresearch,optimizingtheapplicationmethodisneededtoimprovetheanesthesiaofthescrotalskin.Toevaluatepaincausedbytheinjection,irrespectiveoftheacidityoflocalanestheticsolutions,sodiumchloride(NaCl)wasinjectedintothetesticlesofthepigletscastratedwithoutanesthesia.Inthepresentstudy,pigletsthatreceivedanintratesticularandsubscrotalinjectiontendedtohavehigherscoresofdefensivemovementsandvocalizationthanpigletsthatwerehandledonly(H).ThelackofsignificancebetweenthecontrolgroupsHandNaClcouldresultfromthestressandfearprovokedbythefixationofthepigletinasupinepositionandthefixationofthetesticles,whichmayhaveoverlappedthedefensivebehaviorcausedbytheinjection.Moreover,pigletsinjectedwithbupivacaineshowedsignificantlymoredefensivemovementsthanpigletsinjectedwithlidocaineorpigletsthatwereonlyfixated.However,thisresultiscontradictorytotheassumptionthatthepaininducedbyinjectionoflocalanestheticsisbelievedinparttoberelatedtotheacidity,sinceBupivacain0.5%withepinephrine0.0005%JENAPHARM®(mibeGmbHArzneimittel,Brehna,Germany)andXylocitin®2%(mibeGmbHArzneimittel,Brehna,Germany)withepinephrine0.001%havethesamepH.Theresults

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Animals2020,10,175211of15ofthepresentstudyindicatethattheinjectionofbupivacaineseemedtobepainfulitself.Additionally,thesolefixationofthetesticlesmightinducedefensivemovements,asseeninthehandlinggroup,buttoalesserextentcomparedtoinjectinganysubstances.Wearyetal.[23]validatedvocalmeasuresasareliableindicatorofacutepainduetocastration.Severalstudiesconfirmedthatthevocalizationofpigletscastratedwithoutanesthesiadi ersfrompigletsthatwereshamcastrated[24]orcastratedwithanesthesia[8,25,26].Whiteetal.[25]assessedvocalizationduringthecastrationprocedureofpigletswithfrequencyofhighestenergy(HEF).Wearyetal.[23],Tayloretal.[24]andPuppeetal.[27]usedhighfrequencycalls.Incontrast,Marxetal.[26]classifiedcallsduringcastrationintothreedi erentcalltypes(grunt,squeal,scream)anddemonstratedthatpigletscastratedwithoutanesthesiaproducedalmosttwiceasmanyscreamsaspigletscastratedwithlocalanesthesia.Itisnecessarytomentionthat,intheirstudies,fortherecordingsofthevocalizationseparateroomswereusedtopreventinterferingnoises[23,26,27].Furthermore,collecteddatawereelaboratelyanalyzedwithspecialprograms[23–27].Inthepresentstudy,vocalizationswererecordedinthenurseryandnotinaseparateroom,sotheoccurrenceofinterferingnoisescouldnotbeprevented.Asapracticablemethodtoassessvocalization,despitetheinterferingnoises,anacuteonsetofincreasedvocalization,comparablewiththecalltype“scream”wasevaluatedinthepresentstudy,inorientationtotheclassificationofMarxetal.[26].Sincenocomputerprogramwasusedfortheevaluationofthevocalizationinthepresentstudy,itisdi culttocomparetheresultswithpriorstudies.Nonethelesstheresultsobtainedaresimilartoformerstudies[8,10,23–26,35].Pigletscastratedwithoutanesthesia(NaCl)reactedsignificantlymoreoftenwithanacuteonsetofincreasedvocalizationcomparedtopigletsthatwerehandled(H).IncontrasttoMarxetal.[26]andKluivers-Poodtetal.[8]nosignificantdi erenceoccurredbetweenthecontrolgroupsandpigletscastratedwithlocalanesthesiaduringthecastrationprocedure,withtheexceptionofpigletsinjectedwithmepivacaineduringtheseveringofthespermaticcord.Thiscouldbeduetotheusedmeasurementofvocalizationinthepresentstudyandtheresultinglowersensitivity.Thenavigationtimethroughthehandlingchutewasdeterminedwiththeassumptionthatthepresenceofpainwouldresultinlongernavigationtimes[13,28].AccordingtoBilsborrowetal.[28],pigletscastratedwithoutanesthesiahadasignificantlylongernavigationtimethroughahandlingchute0and15minaftercastrationthanshamcastratedpiglets[28].Davisetal.[42]andRauhetal.[13]validatedtheseresults.However,nosignificantdi erenceregardingnavigationtimewasfoundbetweencontrolgroupscastratedwithoutanesthesiaandshamcastratedinthepresentstudy.Incontrast,pigletsinjectedwithmepivacainehadasignificantlyshorternavigationtimeafterinjection,castrationandtwohoursaftercastration,possiblybecauseofsignificantweightdi erences.Pigletsinjectedwithmepivacaineweresignificantlyheavier.Thesignificantdi erencesregardingtheweightondayofcastrationcouldbecausedbythesmallnumberofpiglets.Sincenosignificantdi erencewasfoundbetweenbothcontrolgroupsinthepresentstudy,thehandlingchutewasnotsuitabletoassesspainobjectivelyfollowingcastrationinthepresentstudy.However,therecordingswerenotonlyusedtomeasurenavigationtime,butalsotogetevidenceofimpairmentsoflocomotoractivity.Theimpairmentsoccurred,asshowninTable5,asanunsteadygait,kyphoticspine,tripping,sittingorlyinginthehandlingchuteorrefusingthehurdles.Twopigletsinjectedwithbupivacaineshowedsuchimpairmentsafterinjection.Thisisinlinewiththeresultsofdefensivemovements,wherepigletsinjectedwithbupivacaineshowedthehighestscoreduringinjection.However,wecouldnotexplaintheseresults.Furthermore,aftercastration,onepigletinjectedwithsodiumchloride,onepigletinjectedwithlidocaineandtwopigletsinjectedwithbupivacaineshowedimpairments.Inaddition,onepigletinjectedwithbupivacaineshowedanimpairmenttwohoursaftercastration.TheimpairmentsofindividualpigletsofgroupsNaCl,LandBaftercastrationcouldbeexplainedbypainitself.Moreover,localanestheticscouldcausesidee ects,suchasimpairmentsoflocomotoractivity.Sinceeverypigletwasinjectedwithastandardizeddosageoflocalanestheticregardlessofitsweight,anoverdoseinlighterpigletsmightbeconceivable.Furthermore,itispossiblethatheavierpigletshadalossofe ectiveness,althoughwecouldnotobserveane ect.However,thepiglets

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Animals2020,10,175212of15concernedwerenotamongthelightest.Furtherstudiesarenecessarytoinvestigatehowdi erentdosages,basedonindividualweights,influencethee ectivenessoflocalanesthesiaandtheoccurrenceofsidee ects.Moreover,aninadvertentintravenousinjectionofthelocalanestheticscouldresultincentralnervoussystemandcardiovasculartoxicity.Lipid-solublelocalanesthetics—i.e.,bupivacainefollowedbylidocaineormepivacaine—aremorepotentandthereforecausemoresystemictoxicitythanlesslipid-solubleagents,suchasprocaine,whichisdescribedwithaminimalpotentialforsystemictoxicity[14].Twopigletsofthesamelitter(groupNaClandgroupL)stoodoutwhentheyweresupposedtocompletethehandlingchutetwohoursaftercastration.Theirgeneralconditionwasseverelydisturbedtwohoursaftercastrationandtheywereunabletowalkorstand.Bothpigletsdiedthesameday.Priorstudiesonlocalanesthesia,whichappliedlidocaine[8,9,12,15]orprocaine[15],didnotshowanye ectonmortalityduetolocalanesthesia.Bonastreetal.[12]alsodidnotshowanye ectonmortalitycausedbythelocalanesthetics,lidocaineandbupivacaine.Priortothepresentstudy,nodatawereavailableonthemortalityofpigletsthatwereinjectedwiththelocalanestheticmepivacainepriortocastration.Inthepresentstudy,thedeceasedlidocaineinjectedpigletwasexaminedhistopathologically.Theautopsyshowedaseverehemorrhageintotheabdomen,probablyassociatedwithanincreasedbleedingpropensity.Furthermore,wecannotexcludethattheseverehemorrhageintotheabdomencouldhavebeencausedbythesurgicalcastrationitself.Thehistologyofthelungsshowedahemorrhagicandnecrotizingpneumonia,whichismostlikelycompatiblewithaninfarction.Thus,inadditiontothebleeding-inducedlossofprocoagulatorysubstances,aniatrogenicintroductionoflocalanestheticintothebloodstreamwiththrombusformationandapronouncedcardiotoxicityshouldalsobeconsidered.SinceonepigletofgroupNaCldiedaswell,andbothpigletswerefromthesamelitter,itismorelikelythatacoagulopathymayhavecausedtheirdeath.Unrestrictedlocomotoractivity,especiallyinsucklingpiglets,isessentialinordertoavoidlossesforexampleduetocrushingbythesow.Moreover,itcanbeasignofsidee ectoftheinjectedbupivacaineorlidocaine.Furtherresearchisneededtoinvestigateifitispossibletoreducethedosagewithoutreducingthee ectoftheanesthesia.AsdescribedbyHofmannetal.[15]andZankletal.[43],localanestheticshavenoimpactonwoundhealingaftercastration.Inthepresentstudy,onlyonepigletinjectedwithbupivacaineshowedaheightenedscoreonedayaftercastration.Thispigletdefecatedduringcastration,whichpossiblyledtoacontaminationofthewoundandconsequentlytoanincreasedwoundscore,demonstratingthatthecastrationshouldbeperformedassterileaspossible.Inthepresentstudypigletswerecastratedonlyusingascalpelandnoseverepostoperativebleedingoccurredinthescrotalarea.Theuseoflocalanestheticsdidnotinfluencebleedingscoretwohoursaftercastration,thoughlocalanestheticscontaindi erentamountsofepinephrineornone(Mepidor20mg/mL).Itwasnoticedthatpigletscastratedwithoutanypainreliefrankedthelowestconsideringpostoperativebleeding.Anexplanationcouldbethat,duetothepaincausedbycastrationwithoutpainrelief,theadrenaline,whichwasreleasedfromadrenalglands,ledtoagreatervasoconstrictionthantheepinephrine,whichiscontainedinthedrugs.Moreover,neithercastrationwithoutanesthesianorcastrationwithlocalanesthesiahadanegativeimpactondailygrowthperformanceuntil21daysaftercastration.ThisisinlinewiththefindingsofKluivers-Poodtetal.[8]andBonastreetal.[12],whodescribedthatneitheranestheticsnormeloxicamhaveane ectonmortalityorgrowthperformanceinpiglets.Incontrast,Tellesetal.[44]demonstratedthattheuseoflocalanestheticpriortocastrationappearstohavepositivee ectsonlong-termweightgainofpigs.Whetherlocalanesthesiahasaninfluenceonlong-termweightgaincouldnotbeevaluatedinthepresentstudy,becausetheweightsoftheanimalswereonlyrecordeduntilthe21stdayaftercastration.Limitationsofthepresentstudyarethedi erentpigletagesandweightsondayofcastration,useofcommerciallyavailablelocalanestheticproductswithdi erentconcentrationsofepinephrineornoepinephrineatall,andthefactthatthedosagewasbasedonvolumeandnotonweight.Furthermore,thesmallsamplesizeshouldbementionedasalimitationofthestudy.

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Animals2020,10,175213of155.ConclusionsItcanbeconcludedthatthelocalanestheticslidocaineandmepivacainewereabletoachievesignificantpainreliefduringskinincisionandduringseveringofthespermaticcordunderstandardizedconditionsinconsciouspiglets.Thesubscrotallyandintratesticularlyapplicatedlocalanestheticsprocaineandbupivacainecouldonlyrelievepainsignificantlyduringseveringofthespermaticcordbutnotduringskinincision.Moreover,duetothesignificantlyhigheroccurrenceofdefensivemovementstheintratesticularandsubscrotalinjectionofbupivacaineseemedtocausepainitself.Nonetheless,impairmentsoflocomotoractivityaftertheapplicationoflidocaine,bupivacaineandsodiumchlorideoccurred.Injectionofthelocalanestheticsdidnotcausenegativeimpactsongeneralcondition,woundhealing,weightgainorbleedingofthepigletsaftercastration.SupplementaryMaterials:Thefollowingareavailableonlineathttp://www.mdpi.com/2076-2615/10/10/1752/s1,TableS1:ScoreusedtoevaluatethedefensivemovementsaccordingtoLeidigetal.[10]duringinjection,skinincisionandseveringofthespermaticcord;TableS2:ScoreusedtoevaluatethecastrationwoundsaccordingtoZankl[29];TableS3:ScoreusedtoevaluatethepostoperativebleedingaccordingtoEnzetal.[30].AuthorContributions:Conceptualization,S.Z.,S.S.,C.W.,C.B.andS.B.;methodology,N.A.,S.S.,P.D.,R.M.,A.G.,J.W.,A.M.S.,J.F.andJ.R.;validation,N.A.,S.Z.,S.S.andP.D.;formalanalysis,Y.Z.andN.A.;investigation,N.A.,S.S.,P.D.,S.Z.,J.W.andA.M.S.;resources,M.R.,C.B.andM.H.E.;datacuration,N.A.,S.S.andS.Z.;writing—originaldraftpreparation,N.A.;writing—reviewandediting,S.Z.,S.S.,P.D.,S.B.,C.B.,A.M.S.,J.R.andR.M.;visualization,N.A.,S.Z.andY.Z.;supervision,S.Z.,S.B.andC.B.;projectadministration,S.Z.andC.B.;fundingacquisition,M.R.,S.Z.,M.H.E.andC.B.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.Funding:ThisresearchwasfundedbytheGermanFederalMinistryofFoodandAgriculture(BMEL,https://www.bmel.de)basedonadecisionoftheParliamentoftheFederalRepublicofGermany,grantedbytheFederalO ceforAgricultureandFood(BLE,https://www.ble.de,grantnumber:2817HS016,2817HS013).Acknowledgments:Theauthorsthankthesta oftheresearchfacilityoftheTechnicalUniversityofMunichfortheirsupportbyconductingtheprojectandahkService&SolutionsGmbHfortheillustrationofthehandlingchute.Inaddition,thefinancialsupportoftheCostactionCA15215IPEMAtocoverthearticleprocessingchargesofthispaperisgreatlyacknowledged.ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.Thefundershadnoroleinthedesignofthestudy;inthecollection,analyses,orinterpretationofdata;inthewritingofthemanuscript,orinthedecisiontopublishtheresults.References1.Fredriksen,B.;Font,I.F.M.;Lundstrom,K.;Migdal,W.;Prunier,A.;Tuyttens,F.A.;Bonneau,M.PracticeoncastrationofpigletsinEurope.Animal2009,3,1480–1487.[CrossRef][PubMed]2.Rydhmer,L.;Zamaratskaia,G.;Andersson,H.;Algers,B.;Guillemet,R.;Lundström,K.Aggressiveandsexualbehaviourofgrowingandfinishingpigsrearedingroups,withoutcastration.ActaAgric.Scand.AAnim.Sci.2006,56,109–119.[CrossRef]3.Barton-Gade,P.A.Meatandfatqualityinboars,castratesandgilts.Livest.Prod.Sci.1987,16,187–196.[CrossRef]4.Bünger,B.;Schrader,L.;Schrade,H.;Zacharias,B.Agonisticbehaviour,skinlesionsandactivitypatternofentiremale,femaleandcastratedmalefinishingpigs.Appl.Anim.Behav.Sci.2015,171,64–68.[CrossRef]5.DeBriyne,N.;Berg,C.;Blaha,T.;Temple,D.Pigcastration:WilltheEUmanagetobanpigcastrationby2018?PorcineHealthManag.2016,2.[CrossRef]6.O cialJournaloftheEuropeanUnion.COUNCILDIRECTIVE2008/120/ECof18December2008LayingdownMinimumStandardsfortheProtectionofPigs.Availableonline:https://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32008L0120&from=EN(accessedon19August2020).7.FederalMinistryofJusticeandConsumerProtection.Tierschutzgesetz.Availableonline:https://www.gesetze-im-internet.de/tierschg/BJNR012770972.html(accessedon19August2020).8.Kluivers-Poodt,M.;Houx,B.B.;Robben,S.R.M.;Koop,G.;Lambooij,E.;Hellebrekers,L.J.E ectsofalocalanaestheticandNSAIDincastrationofpiglets,ontheacutepainresponses,growthandmortality.Animal2012,6,1469–1475.[CrossRef]

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F.G.;Luna,S.P.L.;Teixeira,G.;Berto,D.A.Long-termweightgainandeconomicimpactinpigscastratedunderlocalanaesthesia.Vet.Anim.Sci.2016,1–2,36–39.[CrossRef] ©2020bytheauthors.LicenseeMDPI,Basel,Switzerland.ThisarticleisanopenaccessarticledistributedunderthetermsandconditionsoftheCreativeCommonsAttribution(CCBY)license(http://creativecommons.org/licenses/by/4.0/).