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Citation:Werner,J.;Saller,A.M.;Reiser,J.;Senf,S.;Deffner,P.;Abendschön,N.;Fischer,J.;Grott,A.;Miller,R.;Zablotski,Y.;etal.EvaluationofTwoInjectionTechniquesinCombinationwiththeLocalAnestheticsLidocaineandMepivacaineforPigletsUndergoingSurgicalCastration.Animals2022,12,1028.https://doi.org/10.3390/ani12081028AcademicEditors:PeterWhiteandDominiqueVanderSaagReceived:21March2022Accepted:12April2022Published:14April2022Publisher’sNote:MDPIstaysneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalafﬁl-iations. Copyright:©2022bytheauthors.LicenseeMDPI,Basel,Switzerland.ThisarticleisanopenaccessarticledistributedunderthetermsandconditionsoftheCreativeCommonsAttribution(CCBY)license(https://creativecommons.org/licenses/by/4.0/). ArticleEvaluationofTwoInjectionTechniquesinCombinationwiththeLocalAnestheticsLidocaineandMepivacaineforPigletsUndergoingSurgicalCastrationJuliaWerner1 ,AnnaM.Saller1,JudithReiser1,SteffanieSenf2 ,PaulineDeffner2,NoraAbendschön2,JohannesFischer1,AndreaGrott3,ReginaMiller3,YuryZablotski2 ,KatjaSteiger4 ,ShanaBergmann3,MichaelH.Erhard3,MathiasRitzmann2,SusanneZöls2andChristineBaumgartner1,*1CenterofPreclinicalResearch,TechnicalUniversityofMunich,81675Munich,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.)2ClinicforSwine,CenterforClinicalVeterinaryMedicine,LMUMunich,85764Oberschleissheim,Germany;s.senf@med.vetmed.uni-muenchen.de(S.S.);p.deffner@med.vetmed.uni-muenchen.de(P.D.);nora.abendschoen@gmail.com(N.A.);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.)3ChairofAnimalWelfare,Ethology,AnimalHygieneandHusbandry,LMUMunich,80539Munich,Germany;a.schoerwerth@tierhyg.vetmed.uni-muenchen.de(A.G.);r.miller@tierhyg.vetmed.uni-muenchen.de(R.M.);s.bergmann@tierhyg.vetmed.uni-muenchen.de(S.B.);m.erhard@tierhyg.vetmed.uni-muenchen.de(M.H.E.)4InstituteofPathology,SchoolofMedicine,TechnicalUniversityofMunich,81675Munich,Germany;katja.steiger@tum.de*Correspondence:christine.baumgartner@tum.deSimpleSummary:Paineliminationduringthesurgicalcastrationofmalepigletsisanimportantanimalwelfareissue.Bansoncastrationwithoutpainreliefarependinginmostpork-producingEuropeancountries.SurgicalcastrationofsucklingpigletswithoutanesthesiahasbeenprohibitedinGermanysinceJanuary2021.Boarfattening,gonadotropin-releasinghormone(GnRH)vaccinationandsurgicalcastrationunderisoﬂuraneorinjectionanesthesiaarepermittedaslegalalternatives.Thesealternativeshaveadvantagesanddisadvantages,andafeasibleandsimplemethodisstillneeded.Thelegalbasisforanotheralternative,i.e.,theapplicationofalocalanestheticbythefarmerpriortocastration,hasbeencreatedinGermany,butevidenceofitsefﬁcacyremainscontroversial.Thepresentstudydevelopedafeasibleandeffectivemethodforlocalanesthesiainpigletcastration.Twodifferentinjectiontechniquesincombinationwiththelocalanestheticslidocaineandmepivacainewereinvestigatedusingnociceptiveparameters,injectionpressureandtissuedistribution.Bothinjectiontechniquessigniﬁcantlyreducedthenociceptiveparametersregardlessofthelocalanestheticusedandachievedsimilardistributionpatternsandinjectionpressures.Onemethodperformedslightlybetterintheexperimentalsetup,basedoninjectiondurationandhandling.Abstract:Thepresentstudyevaluatedtheeffectsoftwoinjectiontechniquesincombinationwithlidocaineormepivacaineforpigletsundergoingcastration.Toimproveeaseofuse,acannulawithsideholes(one-stepfenestrated(F))wasinventedtosimultaneouslydeliveralocalanestheticintothetestisandscrotumandwascomparedtoatwo-stepinjectiontechnique.Thedistributionofalidocaine/contrastagentmixtureusingthetwomethodswasexaminedusingcomputedtomography.Pigletswererandomlydividedintotreatmentgroups:handling,castrationwithoutpainreliefandcastrationafterlidocaineormepivacaineinjectionusingtheone-stepFortwo-stepmethod.Acutephysiologicalresponsestonoxiousstimuliwereevaluatedbymeasuringthemeanarterialbloodpressure(MAP),heartrate(HR)andnocifensivemovements.Fosproteinexpressioninthespinaldorsalcordwassemi-quantitativelyanalyzed.Bothinjectiontechniquesachievedsimilardistributionpatterns.Theone-stepFmethodwasfasterandeasier.InjectionwasnotassociatedwithsigniﬁcantchangesinMAPorHR,butMepi1andNaClelicitedsigniﬁcantlyincreasednocifensivemovements.BothtechniquessigniﬁcantlyreducedMAPandnocifensivemovementswhenthespermaticcords Animals2022,12,1028.https://doi.org/10.3390/ani12081028https://www.mdpi.com/journal/animals Animals2022,12,1028 2of19 werecut,regardlessofthelocalanesthetictype.ComparedtoNaCl,onlythelidocainetreatmentssigniﬁcantlyreducedHRduringskinincision.Lido2signiﬁcantlyreducedFosproteinexpression.Keywords:piglet;castration;localanesthesia;pain;bloodpressure;heartrate;defensivemovements;fosprotein;distribution;injection 1.IntroductionSurgicalcastrationofmalepigletswithoutpainreliefisamajoranimalwelfareconcern.Basedonthecurrentknowledge,surgicalcastrationisconsideredapainfulprocedureregardlessofthepiglet’sage[1,2].Themainreasonsforthecastrationofmalepigletsaretoavoidboartaintincarcasses,toreduceaggressiveandsexualbehaviorsthatarespeciﬁctointactmales,andtoensureconstantmeatquality[3–6].Europeanlegislationallowssurgicalpigletcastrationwithoutanestheticuptoanageof7days[7].Thereisagrowingconsensustocompletelybansurgicalpigletcastrationbyimplementingthepracticeofboarfatteningorimmunocastration.However,surgicalcastrationremainsacommonhusbandryprocedureinEurope,butitishandledverydifferentlyinindividualmemberstates.Bansoncastrationwithoutpainreliefarestillpendinginmostpork-producingEuropeancountries[8].SeveralEuropeancountrieshaverecentlycommittedtoabandoningthepracticeofsurgicalcastrationwithoutanesthesia.SurgicalcastrationdoesnotplayamajorroleinPortugal,Spain,Ireland,theUnitedKingdomandtheNetherlandsbecausetheproductionofboarsiscommon[8,9].Commonanestheticapproachesforsurgicalpigletcastrationareinhalationalanesthe-siawithisoﬂurane(Switzerland,Germany)orCO2/O2(Netherlands)orinjectionanesthesia(Croatia,Bulgaria,Germany,Portugal,Switzerland)[9].Anotheralternativeistheappli-cationofalocalanestheticpriortocastration,whichispracticedinsomeScandinaviancountries[8,10].ThelegalrequirementsfortheuseoflocalanesthesiaforpigletcastrationarestricterinGermanybecausetheynecessitatenotonlypainreliefbuteffectivepaineliminationduringcastration.LocalanesthesiaisnotpermittedforpigletcastrationinGermanyforthefollowingreasons:evidenceofefﬁcacyiscontroversial,andnoapprovedveterinarylocalanestheticproductforpigletcastrationiscurrentlyavailableontheGermanmarket.Severalstudieshaveinvestigatedlocalanesthesiaforpigletcastrationandreporteddivergentresults[10–18].Ameaningfulcomparisonofthedataisdifﬁcultduetodifferentstudyprotocols,suchasthechoiceoflocalanesthetics,injectiontechniquesandoutcomemeasures.Furtherstudiesandapproachesareneededtoconﬁrmeffectivepaineliminationduringcastration.Thepresentstudyisbasedontheresultsoftwopreviousstudies,whichevaluatedtheeffectsoffourlocalanestheticsinisoﬂurane-anesthetizedandawakepigletsundergoingcastration[19,20].Onlylidocaineandmepivacainewerefurtherinvestigatedinthepresentstudybecausetheseagentsshowedthemostpromisingresultsforpainreductionduringcastration.Bothlocalanestheticsachievedsigniﬁcantreductionsinmeanarterialbloodpressure(MAP)duringallcastrationstepsunderlightisoﬂuraneanesthesiaandproducedadecreaseinheartrate(HR)changesduringcastration,exceptforlidocaineafterskinincision[19].Bothlocalanestheticsreduceddefensivebehaviorduringskinincisionsandcuttingofthespermaticcordinconsciouspiglets[20].However,theinjectiontechnique(intratesticularinjectionof0.5mL,retractingcannulaintosubscrotaltissue,makingaskinfoldandinjectionof0.5mLsubscrotally)usedbySalleretal.[19]andAbendschönetal.[20]appearedtobechallenginginawakepiglets.Theinjectiontechniqueinducednocifensivebehaviorinresponsetotheinjectionofalocalanesthetic.Therefore,thepresentstudyinvestigatedamorepracticalandlesspainfulmethodfortheinjectionoflocalanesthesiawhilemaintainingtheefﬁcacydemonstratedinSallerandAbendschön[19,20].Theinjectionvolumewasreducedfrom1mLto0.6mLperside,andtwodifferentinjectionmethodswereevaluated.Onetechniqueuseda Animals2022,12,1028 3of19 two-stepinjectionprocessmodiﬁedbyHanssonetal.[11].Theothertechniqueusedaspeciallydesignedcannulatosimultaneouslyapplyalocalanestheticintratesticularlyandsubcutaneouslyinonlyonestep(theone-stepfenestrated,orF,method).Thepresentstudyevaluatedandcomparedtwodifferentinjectiontechniquesandtwolocalanestheticsforeffectivenessduringpigletcastrationandimprovementofinjectionpainbasedoncardiovascularchanges,nocifensivemovementsandFosexpression.Tissuedistributionbycontrast-enhancedcomputedtomography(CT)andinjectionpressureofbothapplicationmethodswerealsocompared.2.MaterialsandMethods2.1.AnimalsFifty-twoclinicallyhealthyhybridGermanLandrace/GermanLargeWhitexPietrainmalepigletsaged3to7days(4.8 1.3days)wereincludedinthecastrationstudy.TwelvepigletswereusedforsemiquantitativedistributionexperimentsusingCTimaging.SowsandpigletswerehousedintheanimalhusbandryunitoftheClinicforSwine(Oberschleißheim,Bavaria,Germany),accordingtotheGermanOrderfortheKeepingofProductiveAnimalsandtheEUDirective2010/63/EUforanimalexperiments.Forallpiglets,thefollowingadditionalinclusioncriteriawereapplied:abodyweightover1.4kg(mean:2.1kg 0.5kg)andtwotesticleswithoutanydeviationfromthenormalanatomicalcondition.Pigletsreceivedanoralbolusofiron(1mLperpiglet;Ursoferran®150mg/mL,SerumwerkBernburgAG,Bernburg,Germany)duringtheﬁrst10hoflife.Notoothclipping,eartaggingortaildockingwasperformed.ThestudywasperformedincompliancewithEUDirective2010/63/EUforanimalexperimentsandtheGermanAnimalWelfareAct(2020).TheEthicalCommitteeforAnimalExperimentsoftheGovernmentofUpperBavaria,Munich,Germany,approvedtheexperiments(ReferenceNumberROB-55.2-2532.Vet_02-19-11).2.2.DistributionStudyToinvestigatethedistributionofalocalanestheticappliedusingthetwodifferentinjectiontechniques,12isoﬂurane-anesthetizedpigletswererandomlyassignedtothetwoinjectionmethods(n=6).Atotalvolumeof0.6mLcomprisedofamixtureofcontrastagent(Imeron®300M,300mgIod/mL,InjektionslösungIomeprol,BraccoImagingDeutschlandGmbH,Konstanz,Germany)diluted1:10inlidocainehydrochloride2%(Xylocitin®2%,MibeGmbHArzneimittel,Sandersdorf-Brehna,Germany)wasinjectedwitheithertheone-stepFortwo-stepmethodasdescribedbelow.Thesametwopeopleperformedﬁxationandinjection.Pigletsreceivedanintramusculardoseof50mg/kgmetamizole(METAMIZOLWDT,500mg/mL,InjektionslösungfürPferde,Rinder,SchweineundHunde,WDT—WirtschaftsgenossenschaftdeutscherTierärzteeG,Hanover,Germany)30minpriortotheinductionofanesthesia.CTwasperformedusingadual-layerspectralCT(IQonSpectral-CT,PhilipsHealthcare,Best,TheNetherlands)everytwominutesuntil20minafterinjection.ThefollowingevaluationparameterswereusedforallCTscans:120kV,74mAand2-mmthickness.ImageanalysiswasperformedusingastandardDICOMViewer(LARAView,DEKOMMedical,Hamburg,Germany),and3Dreconstructionwasperformedusingsemiautomatedsoftware(IntelliSpacePACS,PhilipsHealthcare,Best,theNetherlands).ThreeindependentandblindedratersperformedsemiquantitativeanalysesusingaCTdistributionscoringsystem(Table1).Directlyafterinjectionandafter20min,CTscanswereevaluatedforthedistributionandaccumulationofthecontrastagentinthetesticulararea.2.3.InjectionPressureandInjectionTimeTheinjectionpressureofthetwomethodsusedintheexperimentswasmeasuredinfourpigletsinjectedusingthetwo-stepmethodandsevenpigletsinjectedusingtheone-stepFmethod.Immediatelyaftereuthanasiainjectionwith0.6mL0.9%sodiumchloride(B.Braun,Melsungen,Germany)wasperformedviatheone-stepFortwo-stepmethod,as Animals2022,12,1028 4of19 described.Beforeinjection,thesystemwasﬂushedwith0.9%sodiumchloride,andallairbubbleswereremovedfromthehoses.Pressurewasmeasuredviaapressuresensor(Xtrans,CodanpvbMedicalGmbH)andrecordedeveryfourseconds(EIM-B,EIM-A,HAEMO-DYNsoftware,HugoSachsElektronik—HarvardApparatusGmbH,March-Hugstetten,Germany).Injectionsintotheairwereperformedtodeterminethebaselinesystempressureduetotheequipment.Themeanmaximumpressuresofbothinjectiontechniquesintothetesticulartissueandairwerecalculated.Todeterminethedurationofinjection,thetimefrominjectionintotheﬁrsttestistowithdrawalofthecannulafromthesecondtestiswasmeasured.Table1.CTdistributionscoringsystem. AnatomicalStructuresDistributionAccumulationScore Scrotalskinn.a.no0n.a.yes1 Testis 0++1+++2+++++++3 Testicularsheath 0++1+++2+++++++3 Spermaticcord 0++1++++2 Maximumscorepertestis9Maximumscoreperpiglet18 n.a.=notassessed, =notpresent,+=moderate,++=intense.2.4.CastrationStudyThiswasarandomized,double-blindexperimentalstudy.Acomputerprogramforsimplerandomizationwasusedtodividethe52pigletsfrom17Lamongthesixexperimentalgroups(n=9):(1)handling:shamhandlingwithoutinjectionandcastration;(2)NaCl:injectionwith0.9%sodiumchlorideusingtheone-stepFmethodandcastrationwithoutpainrelief;(3)Lido1:2%lidocainehydrochlorideusingtheone-stepFmethod;(4)Lido2:2%lidocainehydrochlorideusingthetwo-stepmethod;(5)Mepi1:2%mepivacainehydrochlorideusingtheone-stepFmethod;and(6)Mepi2:2%mepivacainehydrochlorideusingthetwo-stepmethod(Table2).Table2.Classiﬁcationofstudygroups. HandlingNaClLido1Lido2Mepi1Mepi2 npergroup997999InjectionmethodShamOne-stepFOne-stepFTwo-stepOne-stepFTwo-stepInjectionsolution-NaClLidocaine2%Lidocaine2%Mepivacaine2%Mepivacaine2%Injectionvolume-0.6mL0.6mL0.4+0.2mL0.6mL0.4+0.2mLCastrationShamYesYesYesYesYes Shaminjection:Thepigletwasﬁxedforinjection.Thetesticleswereﬁxedandmerelytouchedwithaneedlecap.Shamcastration:Thepigletwasﬁxedforcastration.Thetesticleswereﬁxed,andaskinincisionwassimulatedwiththebackofthescalpelhandle. Animals2022,12,1028 5of19 2.5.AnesthesiaEachpigletwasanesthetizedwiththeindividuallydeterminedminimumalveolarconcentrationofisoﬂurane.Maskinductionwasperformedwith5%isoﬂurane(IsoﬂuraneBaxtervet.,BaxterDeutschlandGmbH,Unterschleißheim,Germany)inoxygen.Themain-tenancedoseofisoﬂuranewasindividuallyevaluatedforeachpigletusinganinterdigitalpinchtomaintainlightanesthesia.Thedepthofanesthesiawasconsideredsufﬁcientwhenanimalsstillshowedapedalwithdrawalreﬂexinresponsetoaninterdigitalpinch,butthestimulusdidnotawakentheanimals.Ifnowithdrawalreactionwasobserved,theisoﬂuraneconcentrationwasreducedby0.2%.Prolongedpaddlingand/ormovementoftheforelimbs,backorheadwerejudgedassignsthatthestateofnarcosiswastoolight,andtheinspiredisoﬂuraneconcentrationwasincreasedby0.2%.Afterathree-minutestabilizationperiod,anotherinterdigitalpinchwasappliedtotheotherhindlimbandtheconcentrationofinspiratoryisoﬂuranewasmaintainedorfurtherincreaseddependingonthemovementintensity.Afteranotherstabilizationperiod,theinjectionwasperformed.Thepigletsbreathedspontaneouslyduringtheentireperiodofnarcosis.Inspiratoryandexpiratoryisoﬂuranelevelswerecontinuouslymonitored(Vamos®plus,DrägerMed-icalDeutschlandGmbH,Unterhaching,Germany).AmoredetaileddescriptionoftheexperimentalsetupandthemethodusedtoadjustthedepthofanesthesiamaybefoundinSalleretal.[19].Aftercastration,anesthesiawasmaintainedforanother90minbeforethepigletswereeuthanizedwithanintravenousoverdoseofpentobarbital(Euthadorm500mg/mLInjektionslösung,CPPharma,Hanover,Germany),andthespinalcordofeachanimalwasthenremovedforFosanalysis.2.6.NociceptiveMeasurementsItwaspresumedthatallpigletsexperiencedsimilarstressfromthebasicexperimentalsetup,suchasinductionofanesthesiaandestablishmentofmeasurements.Fortheapplicationofthemeasurementdevicesandtheﬁnalexperimentalprocedure,pigletswereplacedinasupineposition.Warmwaterbottleswerearrangedaroundthepiglettomaintainbodytemperatureandpreventcooling.Eyeointment(BepanthenAugen-undNasensalbe,BayerVitalGmbH,Leverkusen,Germany)wasapplied,andcottonwoolwasplacedintheexternalauditorycanaltominimizetheimpactofbackgroundnoiseonthemeasurementresults.Alocalanestheticcream(Emla®,AstraZenecaGmbH,Wedel,Germany)wasappliedtotheventralskinofthethroatfor20minbeforeskinincision.Theregionforvascularaccesswasinﬁltratedwithamaximumof0.3mLlidocaine(2%lidocaine,bela-pharmArzneimittelfabrik,Vechta,Germany)subcutaneouslypriortotheskinincision.Withgentlepreparation,avascularaccesssheath(3F,Baltextrusion,Montmorency,France)wasintroducedintotheleftcarotidartery,andinvasivebloodpressurewasmeasuredusingamicrotipcatheter(FISO-LSFiberOpticPressureCatheter,FOP-LS-2FR-10,FISOTechnologiesInc.,Quebec,QC,Canada).Beforeeachmeasurement,thecatheterwascalibratedtoroomair.Systolicbloodpressure,diastolicbloodpressure,MAPandanelectrocardiogram(ECG)wererecordedcontinuously(PLUGSYSmodule,EIM-B,EIM-A,heartratemodule,HAEMODYNsoftware,HugoSachsElektronik—HarvardApparatusGmbH,March-Hugstetten,Germany,FFP-LSandEvolutionSoftware,FISOTechnologiesInc.,Quebec,QC,Canada).BaselinevaluesforMAPandHRweredeterminedoveroneminutepriortoinjectionandcastration.MeanMAPandHRvalueswerecalculated.Withinoneminuteaftereachevent,themaximumchangefrombaselinewasidentiﬁed,andthepercentdeviationfrombaselinewasdetermined.Duringtheentireperiodofanesthesia,oxygensaturation,HR(2500AVET,NoninMedicalInc.,Plymouth,MN,USA),bodytemperature(PLUGSYSThermocoupleAmpliﬁerModule(TCAM),HAEMODYNsoftware,HugoSachsElektronik—HarvardApparatusGmbH,March-Hugstetten,Germany),respiratoryfrequencyandend-tidalCO2weremonitored(Vamos®plus,DrägerMedicalDeutschlandGmbH,Unterhaching,Germany). Animals2022,12,1028 6of19 Nocifensivemovementswereassessedineverypigletduringinjection,skinincisionandcuttingofthespermaticcord.Thepersonwhoimmobilizedthepigletassessedthemovementofthefourlimbsandthetensionofthespinewhileblindedtothetreatments.Forthehandlinggroup,noblindingwaspossiblebecausethedifferenceswereobvious(i.e.,shaminjectionandshamcastration).ThequalityofthenocifensivemovementswasevaluatedusingaspeciﬁcscoreadoptedfromSalleretal.[19].Allfourlimbswereassessedindividuallyforeachtesticleasfollows:score1=onemovement,score2=twotothreemovements,andscore3=morethanthreemovements,longlasting.Theback/spinewasevaluatedseparatelyforeachtesticle:score1=musclecontractionandscore2=movement.Amaximumscoreof28points,with14pointspertesticle,couldbereachedperevent(injection,skinincisionandcuttingofthespermaticcord).2.7.InjectionThefollowinglocalanestheticswereused:2%lidocainehydrochloride(Xylocitin®2%,MibeGmbHArzneimittel,Brehna,Germany)intheLido1andLido2groupsand2%mepivacainehydrochloride(Mepidor®20mg/mLsolutionforinjectioninhorses,RichterPharmaAG,Wels,Austria)intheMepi1andMepi2groups.TheNaClgroupwasinjectedwith0.9%sodiumchloride(B.Braun,Melsungen,Germany)usingtheone-stepFmethod.Theinjectionwasmerelysimulatedinthehandlinggroupbyﬁxingandgentlytouchingthetesteswithaneedlecap.Forbothinjectiontechniques,anautomaticself–filling1mLsyringe(HSWECO–MATIC®,Henke-Sass,WolfGmbH,Tuttlingen,Germany)wasused,andneedleswerechangedbe-tweeneachpiglet.Tostandardizetheprocedureasmuchaspossible,thesametwopersonsalwaysperformedtheinjectionandcastrationthroughoutthestudy.Fortheinjectionandcastrationprocedures,onepersonconstrainedthepigletsinthesupinepositionwithbothpalmsunderthebackwhilekeepingthehindlimbscraniallyusingthethumbs.Thepersonperformingtheinjectionandcastrationpressedthetesticlescaudallyintothescrotalsacandﬁxedthembetweenthethumbandindexﬁnger.Theone-stepFinjectionwasperformedusingaspecialcannulainventedbytheauthors.Theobjectiveindevelopingthisdevicewastocreateacannulaforadministeringalocalanestheticforcastrationofapigletthatiseasytohandleandfacilitatesefﬁcientandproperlocalanesthetizationofthetesticlesandscrotalskin.Thenewlyinvented25Gcannula(0.5 10mm)containedtheusualdistalopeningandtwoproximalandtwodistallateralopeningsforsimultaneousreleaseofthelocalanesthetic.Thespecializedcannulasweremanufacturedindividually.Thelateralholesweredrilledusinglasertechnology.Theneedlewasinsertedcaudallytoitsfulllengthsothatthedistalopeningandthedistallateralopeningwerepositionedinthetesticleandtheproximallateralopeningwasarrangedinthesubcutaneoustissueofthescrotum(Figure1).Avolumeof0.6mLofalocalanesthetic(Lido1,Mepi1)orsodiumchloride(NaCl)wasadministeredperside.Thetwo-stepinjectionwasmodiﬁedbyHanssonetal.[11].Intheﬁrststep,acom-merciallyavailable25Gneedle(0.5 16mm,B.BraunTravaCareGmbH,Hallbergmoos,Germany)wasinsertedcaudallyforapproximatelythree-fourthsofitslengthsothatthetipoftheneedlewaspositionedintratesticularly.Avolumeof0.4mLofthelocalanestheticwascarefullyinjected.Inthesecondstep,theneedlewaswithdrawn,andavolumeofap-proximately0.2mLwasevenlydistributedovertheinjectioncanaltothesurface(Figure1).Intotal,approximately0.6mLwasinjectedpertestis.2.8.CastrationSurgicalcastrationwasperformed20minafterinjectiontoensurethatbothlocalanestheticswerefullyeffective.Twoverticalskinincisionsweremadeparalleltotheraphescrotiusingascalpelandthetesticlesweregentlypressedout.Toevaluatenoxiousstimulationoftheskinincisionindependently,aperiodoftwominuteswasallowedtopassforstabilizationoftheparametersbeforethespermaticcordswereseveredusingan Animals2022,12,1028 7of19 emasculator.Castrationwasmerelysimulatedinthehandlinggroupbyholdingtheintacttesticlesinpositionandgentlytouchingtheskinwiththebluntbackofthescalpelhandle. Figure1.Schematicoverviewoftheone-stepFandtwo-stepmethods.a—Scrotalskin,b—testis,c—Spermaticcord.OnestepFmethod:theneedlewasinsertedcaudallytoitsfulllengthsothatthedistalopeningandthedistallateralopeningwerepositionedinthetesticleandtheproximallateralopeningwasarrangedinthesubcutaneoustissueofthescrotum.Atotalof0.6mLofalocalanestheticwasadministered.Two-stepmethod:Step(1)theneedlewasinsertedforapproximatelythree-fourthsofitslengthandavolumeof0.4mLofthelocalanestheticwasintratesticularlyinjected.Step(2)Theneedlewaswithdrawn,andavolumeof0.2mLwasevenlydistributedovertheinjectioncanal.2.9.FosProteinExpressionNinetyminutesafterthecastrationwasﬁnished,thepigletwaseuthanized,andlum-barandsacralsegmentsofthespinalcordwerecarefullyremovedfromthevertebralcanal.Usingthevertebraeaslandmarks,thespinalcordwasdividedintolumbar(L1,L2,L3)andsacral(S1–S3)spinalcordsegments,ﬁxedin38%(w/w)neutralbufferedformaldehydeforatleast48handembeddedinparafﬁn(LeicaASP300S,Leica,Wetzlar,Germany).Crosssections(2 m)wereproducedstartingfromthecranialcutsurface.Serialsectionswerestainedwithhematoxylinandeosinusingastandardprotocol.ImmunohistochemistrywasperformedtoinvestigateFosproteinexpression(anti-c-Fosantibody,ab209794,Abcam,Cambridge,UK,dilutedinantibodydiluent1:100)usingaLeicaBondRXmsystem.Brieﬂy,afterdeparafﬁnization,epitoperetrieval1wasperformedusingcitratebuffer(pH6)for30min.Forprimaryantibodybindingdetectionandvisualization,aPolymerReﬁneDetec-tionkitwith3,3’-diaminobenzidine(DAB)andnosecondaryantibodywasused.SlidesweredigitizedusingaLeicaAT2scanningsystem.ThespinaldorsalhornwasidentiﬁedasthegraymatterdorsaltoalinedrawnfromthecentralcanaltothelateralborderofthegraymatterusingAperioImagescopeSoftwareversion12.4.0.7081(LeicaBiosystems,Wetzlar,Germany).AGermanboard-certiﬁedpathologistblindedtothesampleidenti-tiesperformedsemiquantitativeanalysisofthestainingintensityofFos-positiveneurons.Fourdifferentsegmentsofeachanimalwereevaluated.Scoreswereassignedasfollows:0=nostaining,1=slightstaininginsingleneurons( 25%),2=slightstaininginsomeneurons( 50%),3=moderatestaininginsomeneurons( 50%),4=strongstaininginsomeneurons( 50%),5=strongstaininginallneurons.2.10.DataandStatisticalAnalysisForthestatisticalanalysis,groupsweretestedforchangesinMAP,HRandFosproteinexpression,andsigniﬁcantdifferencesinage,weightandend-tidalisoﬂuraneconcentrationandnocifensivemovementswereobserved.ThedistributionofallcontinuousparameterswastestedusingtheShapiro–Wilknormalitytest.One-wayANOVAwasperformedfordatawithnormallydistributedresiduals.Ifsigniﬁcant,pairwiseStudent’st-testsorGames–HowelltestswithBenjamini Animals2022,12,1028 8of19 andHochbergp-valueadjustmentformultiplecomparisonswereperformed.TheKruskal–Wallistestwasperformedfornon-normallydistributedresiduals.Ifsigniﬁcant,Dunn’stestofmultiplecomparisonsusingranksumswithBenjaminiandHochbergp-valueadjustmentformultiplecomparisonswasperformed.Forthestatisticalanalysisofthedistribution,anindependentsamplet-testwasused.Forthedistributionscore,theintraclasscorrelationcoefﬁcients(ICCs)andtheir95%conﬁ-denceintervalswerecalculatedusingIBMSPSSStatisticsforMacversion1.0.0.1508.Basedonthemeanrating,absoluteagreementand2-waymixed-effectsmodel,theICCvaluesrangedbetween0and1,withvaluescloserto1indicatingstrongerreliability.Statisticalsigniﬁcancewasconsideredatp 0.05.StatisticalanalyseswereperformedusingRversion3.6.1(2019-07-05)andGraphPadPrism9.0.2.3.Results3.1.DistributionThedistributionofacontrastagentdilutedin2%lidocainehydrochlorideinjectedviatwodifferentapplicationmethodswasvisualizedusingCT(Figure2A,B).Theone-stepFandtwo-stepinjectionachievedawidespreaddistributionofthelidocaine/contrastagentmixtureinthescrotalskin,testicles,testicularsheathsandspermaticcorddirectlyafteradministration.Nofurtherspreadingofthecontrastagentwasobservedduringthe20minafterinjection(FigureA1).ThedistributionandaccumulationofthecontrastmediumwereevaluatedbasedonaCTdistributionscore(Table1).TheICCfortheevaluationofthedistributionofthecontrastagentswas0.89(conﬁdenceinterval(CI):0.76–0.96).Nosigniﬁcantdifferencewasobservedbetweentheone-stepFandtwo-stepinjectiongroups(Figure2C).Bothinjectiontechniquesproducedsimilardistributionpatternsandreachedcomparabledistributionscores. Figure2.Distributionofthelidocaine/contrastmediummixturedirectlyafterinjection,usingtheone-stepFandtwo-stepmethods.Exemplarythree-dimensionalreconstructionofapigletinjectedwithonestepF(A)andapigletinjectedusingthetwo-stepmethod(B).Bonestructuressuchaspartsofthespine,pelvisandfemurarecoloredbrown.Theinjectedlidocaine/contrastagentmixtureiscoloredgreen.(C)CTdistributionscoringinpigletsinjectedwiththeone-stepFortwo-stepmethod. Animals2022,12,1028 9of19 3.2.InjectionPressureandInjectionTimeFigure3showstheinjectionpressuresduringinjectionsintotheairandtesticu-lartissueusingtheone-stepFandtwo-stepinjectionmethods.A0.6mLintratestic-ularinjectionof0.9%sodiumchlorideusingtheone-stepFmethodgeneratedamax-imumpressureof1572.67 381.53mmHg.Theinjectionof0.4mLintratesticularlyand0.2mLsubcutaneouslyusingthetwo-stepmethodcreatedamaximumpressureof1486.64 461.46mmHg.One-stepFinjectionexceededthemaximalmeasurablepres-sureinfourofsevenanimals,anditwasobservedinoneoffouranimalsusingthetwo-stepmethod.Measuringtheinjectionpressureinairresultedinanaveragepressureof1215.87 308.11mmHgwiththeone-stepFmethodand1083.33 714.24mmHgwiththetwo-stepinjectionmethod.Themeanexecutiontimefortheone-stepFinjection,includingbothtesticles,was14 1.6s.Thetwo-stepmethodwasmoretimeconsuming,withameanof23 2.3s. Figure3.Injectionpressureproﬁlesofthetwoinjectionmethodsintothetestisandair.i.t.=intratesticular.3.3.GroupHomogeneityTwopigletsfromtheLido1groupwereexcludedduetoincompletedataacquisition.Thetreatmentgroupswerehomogeneouslydistributedinage,weightandend-tidalisoﬂu-raneconcentrationduringinjectionandcastration(Table3)anddidnotsigniﬁcantlydifferbetweengroups.Themeanend-tidalisoﬂuraneofallpigletsinthisstudywas1.4 0.35%withaﬂowof3Lmin 1oxygen.Thelocalanestheticdosagewasthesameforeachpiglet,regardlessoftheindividualpigletweight.Therefore,theadministeredlocalanestheticdoseperweightvariedbetweentheanimals.Thehighestlidocainedose(19.29mg/kg)wasadministeredintheLido2group,andthelowestdosewasadministeredintheLido1group(7.74mg/kg).Formepivacaine,thehighestdosageperweightwasgivenintheMepi1group(17.14mg/kg),andthelowestdosage(7.87mg/kg)wasgivenintheMepi2group.Noneofthepigletsinjectedwithalocalanestheticshowedsignsofincompatibilityorsideeffects.Table3.Meanvaluesandstandarddeviationofage,bodyweight,localanestheticdosing,andend-tidalisoﬂuraneconcentrationduringinjectionandcastration. HandlingNaClLido1Lido2Mepi1Mepi2 Age(days)4.78 0.795.44 1.174.57 1.364.22 1.135 1.334.89 1.45 Animals2022,12,1028 10of19 Table3.Cont. HandlingNaClLido1Lido2Mepi1Mepi2 Weight(kg)2.01 0.512.32 0.692.37 0.412.04 0.431.98 0.382.21 0.50Localanestheticdosing(mg/kg)*10.4 1.6513.8 3.0712.01 2.8012.61 2.5End-tidalIsoﬂurane–injection(Vol%)1.46 0.381.32 0.261.4 0.201.39 0.441.38 0.181.57 0.38End-tidalIsoﬂurane–castration(Vol%)1.52 0.481.31 0.261.46 0.221.38 0.441.39 0.161.62 0.38 *Thevaluesreferonlytotheamountsappliedtothetesticularregionanddonotincludeinﬁltrationoftheneckwithapproximately0.3mLof2%lidocaineforthecatheterimplementation.3.4.BloodPressureandHeartRate3.4.1.InterdigitalPinchThemeasuredbaselinemeanMAPforallpigletswas49.94 6.04mmHg.Thedepthofanesthesiawasindividuallyadjustedusinganinterdigitalpinch.Theobservedchangesinbloodpressureasanociceptivereactiontotheinterdigitalpinchweresimilarbetweengroups,andnosigniﬁcantdifferenceswereobserved(FigureA2).Thesameresultswereobtainedforchangesinheartrateduringinterdigitalpinch.3.4.2.InjectionInjectionsofalocalanestheticorsodiumchlorideprovokednosigniﬁcantchangesinMAPorHRcomparedtothatofthehandlinggroup,inwhichtheinjectionwasonlysimulated(Figure4). Figure4.Injection.PercentchangeinMAP(A)andHR(B)duringinjection.Valuesshownaremeans SD.3.4.3.CastrationFigure5AshowstheMAPchangesduringskinincision.Cuttingofthescrotalskinwithoutpainrelief(NaCl)provokeda17.24%maximalchangeinMAPfrombaseline.Incontrast,thehandlinggroupshowedasigniﬁcantlylowerMAP(p=0.037).Administration Animals2022,12,1028 11of19 ofalocalanesthetic,exceptforLido2,causedsigniﬁcantlyreducedchangesinMAPcomparedtoNaCl(Lido1:p=0.034,Mepi1:p=0.034,Mepi2:p=0.034)andwascomparabletothehandlinggroup.Lido1(p=0.038)andLido2(p=0.044)achievedasigniﬁcantreductioninHRchangescomparedtoNaCl(Figure5B).Amongtheothergroups,nosigniﬁcantdifferencesinHRwereobservedduringtheskinincision. Figure5.Castration.PercentchangeinMAP(A,C)andHR(B,D)duringskinincision(A,B)andcuttingofthespermaticcord(C,D).Valuesshownaremeans SD.Statisticalsigniﬁcanceisindicatedas*p 0.05,**p<0.01,***p<0.001. Animals2022,12,1028 12of19 Duringcuttingofthespermaticcord,theNaClgrouprespondedwiththehighestchangesinMAP(36.10%)andwassigniﬁcantlyincreasedcomparedtothehandlingpiglets(p=0.016)(Figure5C).ComparedtoNaCl,theMAPofthefourlocalanestheticgroups(Lido1:p=0.044,Lido2:p=0.002,Mepi1:p=0.001,andMepi2:p=0.001)wassigniﬁcantlydecreased.TheMAPofthegroupsreceivingalocalanestheticwascomparabletothehandlinggroup.NosigniﬁcantdifferencesinHRdeviationweredetectedbetweenthetreatmentandcontrolgroupsaftercuttingthespermaticcord(Figure5D).3.5.NocifensiveMovementsAsshowninTable4,onlyMepi1showedsignificantlymorenocifensivemovementsduringinjectionthanLido2(p=0.031)andHandling(p=0.031).ThehighestproportionofpigletsshowingnosignsofnocifensivemovementsaccordingtoinjectionwereobservedintheHandlingandLido2groups.Nosignificantdifferenceswereobservedduringskinincisionsbetweenthegroups.TheNaClgrouphadthehighestnumberofpigletsrespondingtoskinincisionsandachievedthehighestscore(5.1 6.7)amongtheothergroups.HandlingandLido1pigletsdidnotmoveatallduringtheskinincision.Forcuttingthespermaticcord,NaClproducedthehighestmeanscore(10.9 8.7),andmostpiglets(eightofnine)showednocifensivemovementsinthisgroup.Significantlylowernocifensivescores(p 0.001)werefoundforallexperimentalgroupsandthehandlinggroup.HandlingandMepi2pigletsshowednonocifensivemovements,butonlyonepigletineachoftheotherlocalanesthesiagroupsreactedtocuttingofthespermaticcord.Table4.Nocifensivescoresmean SDandnumberofpigletswithoutnocifensivemovementsduringinjection,skinincisionandcuttingofthespermaticcord. HandlingNaClLido1Lido2Mepi1Mepi2 InjectionScoremean0.1a0.81.30.8a7.1b2.7SD0.31.62.12.26.53.2Animalswithoutnocifensivemovementsn8/95/94/78/93/94/9 SkinincisionScoremean05.101.73.12.1SD06.703.18.84.0Animalswithoutnocifensivemovementsn9/95/97/76/98/96/9 CuttingofthespermaticcordScoremean0a10.9b0.9a0.1a0.7a0aSD08.72.10.31.90Animalswithoutnocifensivemovementsn9/91/96/78/98/99/9 a,bDifferingsuperscriptswithinonelineindicatesigniﬁcant(p<0.05)differencesbetweengroups.3.6.FosProteinExpressionFigure6summarizestheFosproteinexpressioninthelumbarandsacralsegmentsofthespinalcordsofthesixtreatmentgroupsusingasemiquantitativescorebasedonthestainingintensity.SigniﬁcantlyreducedFosproteinexpressioncomparedtotheNaClcontrolwasfoundonlyinLido2(p=0.016). Animals2022,12,1028 13of19 Figure6.Fosproteinexpressioninthelumbarandsacralsegmentsofthespinalcord90minaftercastration.*p 0.05.4.DiscussionLocalanesthesiaforpigletcastrationhasbeeninvestigatedinmanystudies,butdivergentoutcomeshavebeenreported.Itisdifﬁculttocomparetheresultsoftheseinves-tigationsduetotheirverydifferentstudydesigns.Differentlocalanesthetics,evaluationcriteria,injectionmethodsandvolumeswereused.Moststudiesusedanintratesticularinjection[1,2,15,21–29],ortheinjectionintothetestiswascombinedwithasubcutaneousdepot[1,10–12,14,17–20,26,30–33].Otherstudies[2,10,14,17,18]choseanintrafunicularandsubcutaneousapproach,andZankletal.andWhiteetal.injectedlocalanestheticsonlyin-trascrotally[15,34].Sutherlandetal.examinedaneedle-freeinjectionthroughtheskin[35].Notably,theexactinjectionprocedureisnotdescribedindetailinmanypublications,whichmayexplainthepoorreproducibilityofsomestudies.Thepresentstudyinvestigatedtwodifferentinjectionmethods:thetwo-stepmethod,modiﬁedaccordingtoHanssonetal.[11],andtheone-stepFmethod,whichusedanewlydevelopedcannulawithadditionallateralopenings.Thefocusofbothmethodswasachievingpracticabilityforfarmersinpracticewhilemaintaininghighefﬁcacy.Thecomplexinnervationofthetesticularregionandthevarietyofanatomicalstruc-turesthatmustundergosensoryblockforpainlesscastrationarechallengingintheadminis- Animals2022,12,1028 14of19 trationoflocalanesthesia.Sourcesofporcinetestisinnervationarepelvic,sensory,pre-andparavertebralganglia.Themainnervesupplyisprovidedbytheanteriorpelvicganglionandthecaudalmesentericganglion[36].Originatingfromthecaudalmesentericganglion,thetesticularplexusrunstogetherwiththetesticulararteryandinnervatesthetestisandepididymis[37].Theinnervationofthescrotumoriginatesfromthenerviscrotalesdorsales,whichareendbranchesofthepudendalnerve,andthetunicavaginalisandthecremastermusclearesuppliedbybranchesofthegenitofemoralnerve[37].CTimagingshowedthattheone-stepFandtwo-stepinjectionmethodsresultedinanevendistributionwithinthescrotalskin,testicularsheath,testisandspermaticcorddirectlyafterinjection.AlthoughonlythedistributionofthecontrastagentcanbefollowedwithCT,wepresumedthatthelocalanestheticwasdistributedinasimilarmanner.ThispresumptionissupportedbyRanheimetal.[33],whereintratesticularlyappliedradiolabeledlidocainewasrapidlytransportedintothespermaticcordandmaximumenrichmentwasmeasuredafterthreeminutes.Forbettercomparability,a20minwaitingtimebeforecastrationwasused,basedonourpreviousstudies[19,20].Fastertimesofonsetofseveralminuteswerereportedforlidocaineandmepivacaine[38],whichsuggeststhatashorterwaitingperiodtoperformcastrationmaybesufﬁcient.Ashorteningofthewaitingperiodhasbeenappliedinotherstudiesandmayleadtomoreﬂexibilityintheimplementationofthemethodinpractice[14].Thetunicaalbugineaisaﬁbroustissuecapsulethatcoversthetestisandpreventsthetesticlefromexpanding[39].Thisroughcapsulelimitsintratesticularlyinjectedﬂuids,andtheresultinghighpressureinthetesticlepushestheﬂuidtowardthespermaticcord[33].Theinjectionpressuresachievedwithbothmethodsintothetestisarecomparabletointraneuralorintradermalinjections[40,41].However,thesedatashouldbeinterpretedwithcautionbecausethemeasurementsystemonlydetectedpressuresupto2121mmHg.Thepressurelimitwasreachedﬁvetimes,fourtimesintheone-stepFgroupandonceinthetwo-stepgroup.Therefore,wedonotknowexactlytheintensityofthepressure.Theinjectionsintotheairreachedsimilarvalues,whichindicatesthattheresistancewithinthesyringesystemandthecannulawasalreadyhigh.Thesmalldiametersofthecannulasandtheinjectionspeedmayleadtothishighbasepressure.Injectionspeed(mLpersecond)wasnotsupervisedinthisstudy,butthesametwopersonsperformedinjectionstostandardizetheinjectionasmuchaspossible.Otherfactors,suchasneedlediameterandlength,injectionvolume,temperatureoftheﬂuidanddrugformulation,inﬂuencetheperceptionofpainduringinjections[42].Theinjectionoflocalanestheticsprovokednocifensivemovementsinawakepiglets[20].Therefore,wefurthertriedtominimizeinjectionpainbyreducingthetotalinjectionvolumefrom1mLto0.6mLperside.AlthoughonlyMepi1reachedsigniﬁcantlyhighernocifensivemovementscoresthanLido2andHandlinginourstudy,manypigletsintheNaCl,Mepi2andLido1groupsshowednocifensivereactions.However,thesereactionswerenotsigniﬁcant.Toimprovetheobservedreactions,thetotalvolumemaybefurtherreduced.However,asufﬁcientanalgesiceffectmustbeensured.Previousstudiesdidnotfullypreventthediscomfortofintratesticularinjectionsbyvolumereduction[10].Furthereffortstominimizeinjectionpainmayincludetheuseofthinnerneedlesortobufferlocalanesthetics[43].Bufferingoftenautomaticallyincreasestheinjectionvolumewhentherearenomoreconcentratedproductsonthemarket.Theadditionofbicarbonateisamedicinalproductpreparationthatmaynotbeperformedbythefarmer.Whetheraninjectionintothetestismayoccurwithoutanysensationofpainandwhethertheinjectionshouldbeincludedintheevaluationoflocalanesthesiaasalegalalternativeforthecastrationofconsciouspigletsmustbediscussed.Forthetimerequirement,acomparisonofbothtechniquesrevealedthattheone-stepinjectiontookmuchlesstimethanthetwo-stepinjection.Theimpressionoftheveterinarianoperatorswasthatthepracticalimplementationofthetwo-stepinjectionwasmorechallengingthanthatoftheone-stepF.Theone-stepFinjectionrequiredonlyonesimpleplacementofthecannulaintothetestis.Duetotheadditionallateralholes,norepositioningofthecannulaforsubcutaneousinﬁltrationwasnecessary.Oneissue Animals2022,12,1028 15of19 thatoccurredduringtheperformanceofthesecondstepofthetwo-stepmethodwasthedifﬁcultyofinjectinganexactvolumeofonly0.2mLwhilewithdrawingthecannula.Thisdifﬁcultyoccurredbecauseanautomaticself-ﬁllingsyringewithapresetapplicationvolumeof0.4mLwasusedfortheﬁrststep.Asecondvolumecouldnotbepreset,whichmadetheexactdosingforthesecondstepmoredifﬁcult.Skadeetal.[10]comparedtwoinjectionmethodsandfavoredafasterandlesscomplicatedmethodforpracticalapplication.Asanextstep,theinjectionmethodsmustbeevaluatedintheﬁeldofawakeanimalstodrawaﬁnalconclusion.Localanestheticsolutionsdonotcontainepinephrinebecausevasoconstrictorscouldaffectbloodpressureandheartratemeasurements[19].BothinjectiontechniquesandbothlocalanestheticsachievedasigniﬁcantreductioninnocifensivemovementsandMAPduringcuttingofthespermaticcord,whichisthemostpainfulpartofcastration[44].Onlythecombinationoflidocaineandthetwo-stepmethoddidnotachieveasigniﬁcantreductioninMAPduringskinincisions.Fornocifensivemovementsduringskinincision,therewasnosigniﬁcantdifferencebetweenpigletscastratedwithoutpainreliefandpigletsthatreceivedalocalanesthetic.ThefactthatnoteverypigletfromtheNaClgroupshowednocifensivemovementscouldbebecausethepigletswereunderlightisoﬂuraneanesthesia.Althoughisoﬂuranewasindividuallytitratedforeachpiglettoreachastateofhypnosis,asindicatedbythepresenceofapedalwithdrawalreﬂex,anesthesiacouldmaskpossiblemovements.Thereasonwhyindividualpigletsofthelocalanesthesiagroupsstillmovedduringskinincisionandcuttingofthespermaticcord,despitelocalanesthesia,maybethatnociceptionwasnotfullyblockedinsomepiglets.Anotherexplanationmaybethatexternalinﬂuences,suchasﬁxationofthetestes,affectedthepiglet’sreactionsbecauseoneanimalinthehandlinggroupalsoshoweddefensivereactionsduringsimulatedinjection.Fosproteinisexpressedinneuronsofthedorsalhornofthespinalcordafternoci-ceptivestimulationanditisusedasamarkerforpain[45].Fosproteinexpressionreﬂectsonlythesumofthepainexperiencedandcannotbeassignedtoonespeciﬁcpainevent.Nyborgetal.[46]showedthatinjectionofalocalanestheticbeforecastrationreducedthenumberofFos-positiveneuronsinthedorsalhorn.Reiseretal.foundasigniﬁcantreductionintheFosstainingscoreinpigletscastratedafteraninjectionoflidocaine,mepi-vacaineandprocaine[47].OnlyMepi1achievedasigniﬁcantreductioninFosproteinexpressioncomparedtotheNaClcastratedpigletsinthepresentstudy.PigletsthatdidnotexperienceinjectionorcastrationalsoexpressedtheFosprotein.Thisexpressionmaybeexplainedbythefactthatstressorunpleasantstimulation,suchasﬁxationofthepigletitselfandespeciallythetesticles,alsoincreasesFosproteinexpressioninthedorsalhorn.Theneonatalageofthepigletsmayexplainthisresult.Forexample,aFosresponsetoinnocuousstimulationwasobservedinneonatalratsuntilpostnatalday21[48].Therefore,Fosproteinexpressionshouldnotbeusedasthesoleparametertoassessnociceptioninpigletsfollowingcastration,anditshouldalwaysbeevaluatedincombinationwithotherparameters.ThesamplesizeforthisstudywascalculatedbasedonMAP,whichisthemostsen-sitivecardiovascularparameterassociatedwithnoxiousstimuliduringcastration[19,49].Therefore,thepowerofthestudymaybetoolowtodrawdeﬁniteconclusionsonnocifen-sivemovementsandFosexpression,andadirectcomparisonoftheinjectionmethodsandlocalanestheticsisdifﬁcult.Thepresentstudyonlyevaluatednociceptivereactionsinre-sponsetoacutestimuliunderlightisoﬂuraneanesthesia.Chroniceffectsandpracticabilityinawakepigletsmustbeevaluatedinfurtherongoingstudies.5.ConclusionsInconclusion,bothinjectiontechniques,regardlessofthelocalanestheticused,re-ducednociceptionduringcastrationofmalepigletsunderstandardizedconditions.Onechallengeremainsthehandlingofthediscomfortoftheinjectionitself,whichcontinuedtoelicitnocifensivemovementsinseveralpigletsfromthedifferentexperimentalgroups.Forspeedandmanageability,theone-stepFmethodwasbeneﬁcial.However,compared Animals2022,12,1028 16of19 tothetwo-stepmethod,theappliedpressurewashigherandmorefrequentlyexceededthemaximalmeasurablepressure.Bothinjectiontechniquesmustbefurtherevaluatedinconsciouspigletstodrawﬁnalconclusionsabouttheireffectivenessandfeasibilityinpracticaluse.AuthorContributions:Conceptualization,S.Z.andC.B.;Datacuration,J.W.andA.M.S.;Formalanalysis,Y.Z.;Fundingacquisition,S.Z.andC.B.;Investigation,J.W.,A.M.S.,J.R.,S.S.,P.D.,N.A.,J.F.andK.S.;Methodology,J.W.,A.M.S.,J.R.,S.S.,P.D.,N.A.,J.F.,A.G.,R.M.,K.S.,S.B.,M.H.E.,M.R.,S.Z.andC.B.;Projectadministration,S.Z.andC.B.;Resources,M.R.,S.Z.andC.B.;Supervision,M.H.E.,M.R.,S.Z.andC.B.;Validation,K.S.,M.H.E.,M.R.,S.Z.andC.B.;Visualization,J.W.andA.M.S.;Writing—originaldraft,J.W.;Writing—review&editing,A.M.S.,J.R.,S.S.,P.D.,N.A.,J.F.,A.G.,R.M,S.B.,Y.Z.,K.S.,M.H.E.,M.R.,S.Z.andC.B.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.Funding:ThisresearchwasfundedbytheGermanFederalMinistryofFoodandAgriculture(BMEL,https://www.bmel.de(accessedon20March2022)basedonadecisionoftheParliamentoftheFederalRepublicofGermany,grantedbytheFederalOfﬁceforAgricultureandFood(BLE,https://www.ble.de(accessedon20March2022),grantnumber:2817HS016,2817HS013).InstitutionalReviewBoardStatement:ThestudywasperformedincompliancewiththeEUDirec-tive2010/63/EUforanimalexperimentsandtheGermanAnimalWelfareAct[27].AllprocedureswereapprovedbytheEthicsCommitteeforAnimalExperimentsoftheGovernmentofUpperBavaria,Munich,Germany(ReferenceNumberROB-55.2-2532.Vet_02-19-11).InformedConsentStatement:Notapplicable.DataAvailabilityStatement:Notapplicable.Acknowledgments:TheauthorsthankAndreasKnipfer,whosupportedthecomputedtomographyscans,andTanjaGroll,MarionMielkeandOlgaSeelbach,whoperformedparafﬁnembedding,cuttingandimmunohistologicalstaining.ConﬂictsofInterest:Theauthorsdeclarenoconﬂictofinterest.Thefundershadnoroleinthedesignofthestudy,inthecollection,analyses,orinterpretationofdata,inthewritingofthemanuscript,orinthedecisiontopublishtheresults.AppendixA FigureA1.Distributionofthelidocaine/contrastmediummixture20minafterinjectionusingtheone-stepFandtwo-stepmethods.Exemplarythree-dimensionalreconstructionofapigletinjectedusingtheone-stepF(A)andapigletinjectedusingthetwo-stepmethod(B).Bonestructuressuchaspartsofthespine,pelvisandfemurarecoloredbrown.Theinjectedlidocaine/contrastagentmixtureiscoloredgreen. Animals2022,12,1028 17of19 AppendixB FigureA2.Interdigitalpinch.Percentchangein(A)meanarterialbloodpressure(MAP)and(B)heartrate(HR)duringtheinterdigitalpinchfortheadjustmentofanesthesiadepth.Valuesshownaremeans 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,AnnaM.Saller1,JudithReiser1,SteffanieSenf2 ,PaulineDeffner2,NoraAbendschön2,JohannesFischer1,AndreaGrott3,ReginaMiller3,YuryZablotski2 ,KatjaSteiger4 ,ShanaBergmann3,MichaelH.Erhard3,MathiasRitzmann2,SusanneZöls2andChristineBaumgartner1,*1CenterofPreclinicalResearch,TechnicalUniversityofMunich,81675Munich,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.)2ClinicforSwine,CenterforClinicalVeterinaryMedicine,LMUMunich,85764Oberschleissheim,Germany;s.senf@med.vetmed.uni-muenchen.de(S.S.);p.deffner@med.vetmed.uni-muenchen.de(P.D.);nora.abendschoen@gmail.com(N.A.);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.)3ChairofAnimalWelfare,Ethology,AnimalHygieneandHusbandry,LMUMunich,80539Munich,Germany;a.schoerwerth@tierhyg.vetmed.uni-muenchen.de(A.G.);r.miller@tierhyg.vetmed.uni-muenchen.de(R.M.);s.bergmann@tierhyg.vetmed.uni-muenchen.de(S.B.);m.erhard@tierhyg.vetmed.uni-muenchen.de(M.H.E.)4InstituteofPathology,SchoolofMedicine,TechnicalUniversityofMunich,81675Munich,Germany;katja.steiger@tum.de*Correspondence:christine.baumgartner@tum.deSimpleSummary:Paineliminationduringthesurgicalcastrationofmalepigletsisanimportantanimalwelfareissue.Bansoncastrationwithoutpainreliefarependinginmostpork-producingEuropeancountries.SurgicalcastrationofsucklingpigletswithoutanesthesiahasbeenprohibitedinGermanysinceJanuary2021.Boarfattening,gonadotropin-releasinghormone(GnRH)vaccinationandsurgicalcastrationunderisoﬂuraneorinjectionanesthesiaarepermittedaslegalalternatives.Thesealternativeshaveadvantagesanddisadvantages,andafeasibleandsimplemethodisstillneeded.Thelegalbasisforanotheralternative,i.e.,theapplicationofalocalanestheticbythefarmerpriortocastration,hasbeencreatedinGermany,butevidenceofitsefﬁcacyremainscontroversial.Thepresentstudydevelopedafeasibleandeffectivemethodforlocalanesthesiainpigletcastration.Twodifferentinjectiontechniquesincombinationwiththelocalanestheticslidocaineandmepivacainewereinvestigatedusingnociceptiveparameters,injectionpressureandtissuedistribution.Bothinjectiontechniquessigniﬁcantlyreducedthenociceptiveparametersregardlessofthelocalanestheticusedandachievedsimilardistributionpatternsandinjectionpressures.Onemethodperformedslightlybetterintheexperimentalsetup,basedoninjectiondurationandhandling.Abstract:Thepresentstudyevaluatedtheeffectsoftwoinjectiontechniquesincombinationwithlidocaineormepivacaineforpigletsundergoingcastration.Toimproveeaseofuse,acannulawithsideholes(one-stepfenestrated(F))wasinventedtosimultaneouslydeliveralocalanestheticintothetestisandscrotumandwascomparedtoatwo-stepinjectiontechnique.Thedistributionofalidocaine/contrastagentmixtureusingthetwomethodswasexaminedusingcomputedtomography.Pigletswererandomlydividedintotreatmentgroups:handling,castrationwithoutpainreliefandcastrationafterlidocaineormepivacaineinjectionusingtheone-stepFortwo-stepmethod.Acutephysiologicalresponsestonoxiousstimuliwereevaluatedbymeasuringthemeanarterialbloodpressure(MAP),heartrate(HR)andnocifensivemovements.Fosproteinexpressioninthespinaldorsalcordwassemi-quantitativelyanalyzed.Bothinjectiontechniquesachievedsimilardistributionpatterns.Theone-stepFmethodwasfasterandeasier.InjectionwasnotassociatedwithsigniﬁcantchangesinMAPorHR,butMepi1andNaClelicitedsigniﬁcantlyincreasednocifensivemovements.BothtechniquessigniﬁcantlyreducedMAPandnocifensivemovementswhenthespermaticcords

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orHR,butMepi1andNaClelicitedsigniﬁcantlyincreasednocifensivemovements.BothtechniquessigniﬁcantlyreducedMAPandnocifensivemovementswhenthespermaticcords Animals2022,12,1028.https://doi.org/10.3390/ani12081028https://www.mdpi.com/journal/animals

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Animals2022,12,1028 2of19 werecut,regardlessofthelocalanesthetictype.ComparedtoNaCl,onlythelidocainetreatmentssigniﬁcantlyreducedHRduringskinincision.Lido2signiﬁcantlyreducedFosproteinexpression.Keywords:piglet;castration;localanesthesia;pain;bloodpressure;heartrate;defensivemovements;fosprotein;distribution;injection

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ducedFosproteinexpression.Keywords:piglet;castration;localanesthesia;pain;bloodpressure;heartrate;defensivemovements;fosprotein;distribution;injection 1.IntroductionSurgicalcastrationofmalepigletswithoutpainreliefisamajoranimalwelfareconcern.Basedonthecurrentknowledge,surgicalcastrationisconsideredapainfulprocedureregardlessofthepiglet’sage[1,2].Themainreasonsforthecastrationofmalepigletsaretoavoidboartaintincarcasses,toreduceaggressiveandsexualbehaviorsthatarespeciﬁctointactmales,andtoensureconstantmeatquality[3–6].Europeanlegislationallowssurgicalpigletcastrationwithoutanestheticuptoanageof7days[7].Thereisagrowingconsensustocompletelybansurgicalpigletcastrationbyimplementingthepracticeofboarfatteningorimmunocastration.However,surgicalcastrationremainsacommonhusbandryprocedureinEurope,butitishandledverydifferentlyinindividualmemberstates.Bansoncastrationwithoutpainreliefarestillpendinginmostpork-producingEuropeancountries[8].SeveralEuropeancountrieshaverecentlycommittedtoabandoningthepracticeofsurgicalcastrationwithoutanesthesia.SurgicalcastrationdoesnotplayamajorroleinPortugal,Spain,Ireland,theUnitedKingdomandtheNetherlandsbecausetheproductionofboarsiscommon[8,9].Commonanestheticapproachesforsurgicalpigletcastrationareinhalationalanesthe-siawithisoﬂurane(Switzerland,Germany)orCO2/O2(Netherlands)orinjectionanesthesia(Croatia,Bulgaria,Germany,Portugal,Switzerland)[9].Anotheralternativeistheappli-cationofalocalanestheticpriortocastration,whichispracticedinsomeScandinaviancountries[8,10].ThelegalrequirementsfortheuseoflocalanesthesiaforpigletcastrationarestricterinGermanybecausetheynecessitatenotonlypainreliefbuteffectivepaineliminationduringcastration.LocalanesthesiaisnotpermittedforpigletcastrationinGermanyforthefollowingreasons:evidenceofefﬁcacyiscontroversial,andnoapprovedveterinarylocalanestheticproductforpigletcastrationiscurrentlyavailableontheGermanmarket.Severalstudieshaveinvestigatedlocalanesthesiaforpigletcastrationandreporteddivergentresults[10–18].Ameaningfulcomparisonofthedataisdifﬁcultduetodifferentstudyprotocols,suchasthechoiceoflocalanesthetics,injectiontechniquesandoutcomemeasures.Furtherstudiesandapproachesareneededtoconﬁrmeffectivepaineliminationduringcastration.Thepresentstudyisbasedontheresultsoftwopreviousstudies,whichevaluatedtheeffectsoffourlocalanestheticsinisoﬂurane-anesthetizedandawakepigletsundergoingcastration[19,20].Onlylidocaineandmepivacainewerefurtherinvestigatedinthepresentstudybecausetheseagentsshowedthemostpromisingresultsforpainreductionduringcastration.Bothlocalanestheticsachievedsigniﬁcantreductionsinmeanarterialbloodpressure(MAP)duringallcastrationstepsunderlightisoﬂuraneanesthesiaandproducedadecreaseinheartrate(HR)changesduringcastration,exceptforlidocaineafterskinincision[19].Bothlocalanestheticsreduceddefensivebehaviorduringskinincisionsandcuttingofthespermaticcordinconsciouspiglets[20].However,theinjectiontechnique(intratesticularinjectionof0.5mL,retractingcannulaintosubscrotaltissue,makingaskinfoldandinjectionof0.5mLsubscrotally)usedbySalleretal.[19]andAbendschönetal.[20]appearedtobechallenginginawakepiglets.Theinjectiontechniqueinducednocifensivebehaviorinresponsetotheinjectionofalocalanesthetic.Therefore,thepresentstudyinvestigatedamorepracticalandlesspainfulmethodfortheinjectionoflocalanesthesiawhilemaintainingtheefﬁcacydemonstratedinSallerandAbendschön[19,20].Theinjectionvolumewasreducedfrom1mLto0.6mLperside,andtwodifferentinjectionmethodswereevaluated.Onetechniqueuseda

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Animals2022,12,1028 3of19

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Animals2022,12,1028 3of19 two-stepinjectionprocessmodiﬁedbyHanssonetal.[11].Theothertechniqueusedaspeciallydesignedcannulatosimultaneouslyapplyalocalanestheticintratesticularlyandsubcutaneouslyinonlyonestep(theone-stepfenestrated,orF,method).Thepresentstudyevaluatedandcomparedtwodifferentinjectiontechniquesandtwolocalanestheticsforeffectivenessduringpigletcastrationandimprovementofinjectionpainbasedoncardiovascularchanges,nocifensivemovementsandFosexpression.Tissuedistributionbycontrast-enhancedcomputedtomography(CT)andinjectionpressureofbothapplicationmethodswerealsocompared.2.MaterialsandMethods2.1.AnimalsFifty-twoclinicallyhealthyhybridGermanLandrace/GermanLargeWhitexPietrainmalepigletsaged3to7days(4.8 1.3days)wereincludedinthecastrationstudy.TwelvepigletswereusedforsemiquantitativedistributionexperimentsusingCTimaging.SowsandpigletswerehousedintheanimalhusbandryunitoftheClinicforSwine(Oberschleißheim,Bavaria,Germany),accordingtotheGermanOrderfortheKeepingofProductiveAnimalsandtheEUDirective2010/63/EUforanimalexperiments.Forallpiglets,thefollowingadditionalinclusioncriteriawereapplied:abodyweightover1.4kg(mean:2.1kg 0.5kg)andtwotesticleswithoutanydeviationfromthenormalanatomicalcondition.Pigletsreceivedanoralbolusofiron(1mLperpiglet;Ursoferran®150mg/mL,SerumwerkBernburgAG,Bernburg,Germany)duringtheﬁrst10hoflife.Notoothclipping,eartaggingortaildockingwasperformed.ThestudywasperformedincompliancewithEUDirective2010/63/EUforanimalexperimentsandtheGermanAnimalWelfareAct(2020).TheEthicalCommitteeforAnimalExperimentsoftheGovernmentofUpperBavaria,Munich,Germany,approvedtheexperiments(ReferenceNumberROB-55.2-2532.Vet_02-19-11).2.2.DistributionStudyToinvestigatethedistributionofalocalanestheticappliedusingthetwodifferentinjectiontechniques,12isoﬂurane-anesthetizedpigletswererandomlyassignedtothetwoinjectionmethods(n=6).Atotalvolumeof0.6mLcomprisedofamixtureofcontrastagent(Imeron®300M,300mgIod/mL,InjektionslösungIomeprol,BraccoImagingDeutschlandGmbH,Konstanz,Germany)diluted1:10inlidocainehydrochloride2%(Xylocitin®2%,MibeGmbHArzneimittel,Sandersdorf-Brehna,Germany)wasinjectedwitheithertheone-stepFortwo-stepmethodasdescribedbelow.Thesametwopeopleperformedﬁxationandinjection.Pigletsreceivedanintramusculardoseof50mg/kgmetamizole(METAMIZOLWDT,500mg/mL,InjektionslösungfürPferde,Rinder,SchweineundHunde,WDT—WirtschaftsgenossenschaftdeutscherTierärzteeG,Hanover,Germany)30minpriortotheinductionofanesthesia.CTwasperformedusingadual-layerspectralCT(IQonSpectral-CT,PhilipsHealthcare,Best,TheNetherlands)everytwominutesuntil20minafterinjection.ThefollowingevaluationparameterswereusedforallCTscans:120kV,74mAand2-mmthickness.ImageanalysiswasperformedusingastandardDICOMViewer(LARAView,DEKOMMedical,Hamburg,Germany),and3Dreconstructionwasperformedusingsemiautomatedsoftware(IntelliSpacePACS,PhilipsHealthcare,Best,theNetherlands).ThreeindependentandblindedratersperformedsemiquantitativeanalysesusingaCTdistributionscoringsystem(Table1).Directlyafterinjectionandafter20min,CTscanswereevaluatedforthedistributionandaccumulationofthecontrastagentinthetesticulararea.2.3.InjectionPressureandInjectionTimeTheinjectionpressureofthetwomethodsusedintheexperimentswasmeasuredinfourpigletsinjectedusingthetwo-stepmethodandsevenpigletsinjectedusingtheone-stepFmethod.Immediatelyaftereuthanasiainjectionwith0.6mL0.9%sodiumchloride(B.Braun,Melsungen,Germany)wasperformedviatheone-stepFortwo-stepmethod,as

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Animals2022,12,1028 4of19 described.Beforeinjection,thesystemwasﬂushedwith0.9%sodiumchloride,andallairbubbleswereremovedfromthehoses.Pressurewasmeasuredviaapressuresensor(Xtrans,CodanpvbMedicalGmbH)andrecordedeveryfourseconds(EIM-B,EIM-A,HAEMO-DYNsoftware,HugoSachsElektronik—HarvardApparatusGmbH,March-Hugstetten,Germany).Injectionsintotheairwereperformedtodeterminethebaselinesystempressureduetotheequipment.Themeanmaximumpressuresofbothinjectiontechniquesintothetesticulartissueandairwerecalculated.Todeterminethedurationofinjection,thetimefrominjectionintotheﬁrsttestistowithdrawalofthecannulafromthesecondtestiswasmeasured.Table1.CTdistributionscoringsystem. AnatomicalStructuresDistributionAccumulationScore Scrotalskinn.a.no0n.a.yes1 Testis 0++1+++2+++++++3 Testicularsheath 0++1+++2+++++++3 Spermaticcord 0++1++++2 Maximumscorepertestis9Maximumscoreperpiglet18 n.a.=notassessed, =notpresent,+=moderate,++=intense.2.4.CastrationStudyThiswasarandomized,double-blindexperimentalstudy.Acomputerprogramforsimplerandomizationwasusedtodividethe52pigletsfrom17Lamongthesixexperimentalgroups(n=9):(1)handling:shamhandlingwithoutinjectionandcastration;(2)NaCl:injectionwith0.9%sodiumchlorideusingtheone-stepFmethodandcastrationwithoutpainrelief;(3)Lido1:2%lidocainehydrochlorideusingtheone-stepFmethod;(4)Lido2:2%lidocainehydrochlorideusingthetwo-stepmethod;(5)Mepi1:2%mepivacainehydrochlorideusingtheone-stepFmethod;and(6)Mepi2:2%mepivacainehydrochlorideusingthetwo-stepmethod(Table2).Table2.Classiﬁcationofstudygroups. HandlingNaClLido1Lido2Mepi1Mepi2

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e-stepFmethod;and(6)Mepi2:2%mepivacainehydrochlorideusingthetwo-stepmethod(Table2).Table2.Classiﬁcationofstudygroups. HandlingNaClLido1Lido2Mepi1Mepi2 npergroup997999InjectionmethodShamOne-stepFOne-stepFTwo-stepOne-stepFTwo-stepInjectionsolution-NaClLidocaine2%Lidocaine2%Mepivacaine2%Mepivacaine2%Injectionvolume-0.6mL0.6mL0.4+0.2mL0.6mL0.4+0.2mLCastrationShamYesYesYesYesYes Shaminjection:Thepigletwasﬁxedforinjection.Thetesticleswereﬁxedandmerelytouchedwithaneedlecap.Shamcastration:Thepigletwasﬁxedforcastration.Thetesticleswereﬁxed,andaskinincisionwassimulatedwiththebackofthescalpelhandle.

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Animals2022,12,1028 5of19

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Animals2022,12,1028 5of19 2.5.AnesthesiaEachpigletwasanesthetizedwiththeindividuallydeterminedminimumalveolarconcentrationofisoﬂurane.Maskinductionwasperformedwith5%isoﬂurane(IsoﬂuraneBaxtervet.,BaxterDeutschlandGmbH,Unterschleißheim,Germany)inoxygen.Themain-tenancedoseofisoﬂuranewasindividuallyevaluatedforeachpigletusinganinterdigitalpinchtomaintainlightanesthesia.Thedepthofanesthesiawasconsideredsufﬁcientwhenanimalsstillshowedapedalwithdrawalreﬂexinresponsetoaninterdigitalpinch,butthestimulusdidnotawakentheanimals.Ifnowithdrawalreactionwasobserved,theisoﬂuraneconcentrationwasreducedby0.2%.Prolongedpaddlingand/ormovementoftheforelimbs,backorheadwerejudgedassignsthatthestateofnarcosiswastoolight,andtheinspiredisoﬂuraneconcentrationwasincreasedby0.2%.Afterathree-minutestabilizationperiod,anotherinterdigitalpinchwasappliedtotheotherhindlimbandtheconcentrationofinspiratoryisoﬂuranewasmaintainedorfurtherincreaseddependingonthemovementintensity.Afteranotherstabilizationperiod,theinjectionwasperformed.Thepigletsbreathedspontaneouslyduringtheentireperiodofnarcosis.Inspiratoryandexpiratoryisoﬂuranelevelswerecontinuouslymonitored(Vamos®plus,DrägerMed-icalDeutschlandGmbH,Unterhaching,Germany).AmoredetaileddescriptionoftheexperimentalsetupandthemethodusedtoadjustthedepthofanesthesiamaybefoundinSalleretal.[19].Aftercastration,anesthesiawasmaintainedforanother90minbeforethepigletswereeuthanizedwithanintravenousoverdoseofpentobarbital(Euthadorm500mg/mLInjektionslösung,CPPharma,Hanover,Germany),andthespinalcordofeachanimalwasthenremovedforFosanalysis.2.6.NociceptiveMeasurementsItwaspresumedthatallpigletsexperiencedsimilarstressfromthebasicexperimentalsetup,suchasinductionofanesthesiaandestablishmentofmeasurements.Fortheapplicationofthemeasurementdevicesandtheﬁnalexperimentalprocedure,pigletswereplacedinasupineposition.Warmwaterbottleswerearrangedaroundthepiglettomaintainbodytemperatureandpreventcooling.Eyeointment(BepanthenAugen-undNasensalbe,BayerVitalGmbH,Leverkusen,Germany)wasapplied,andcottonwoolwasplacedintheexternalauditorycanaltominimizetheimpactofbackgroundnoiseonthemeasurementresults.Alocalanestheticcream(Emla®,AstraZenecaGmbH,Wedel,Germany)wasappliedtotheventralskinofthethroatfor20minbeforeskinincision.Theregionforvascularaccesswasinﬁltratedwithamaximumof0.3mLlidocaine(2%lidocaine,bela-pharmArzneimittelfabrik,Vechta,Germany)subcutaneouslypriortotheskinincision.Withgentlepreparation,avascularaccesssheath(3F,Baltextrusion,Montmorency,France)wasintroducedintotheleftcarotidartery,andinvasivebloodpressurewasmeasuredusingamicrotipcatheter(FISO-LSFiberOpticPressureCatheter,FOP-LS-2FR-10,FISOTechnologiesInc.,Quebec,QC,Canada).Beforeeachmeasurement,thecatheterwascalibratedtoroomair.Systolicbloodpressure,diastolicbloodpressure,MAPandanelectrocardiogram(ECG)wererecordedcontinuously(PLUGSYSmodule,EIM-B,EIM-A,heartratemodule,HAEMODYNsoftware,HugoSachsElektronik—HarvardApparatusGmbH,March-Hugstetten,Germany,FFP-LSandEvolutionSoftware,FISOTechnologiesInc.,Quebec,QC,Canada).BaselinevaluesforMAPandHRweredeterminedoveroneminutepriortoinjectionandcastration.MeanMAPandHRvalueswerecalculated.Withinoneminuteaftereachevent,themaximumchangefrombaselinewasidentiﬁed,andthepercentdeviationfrombaselinewasdetermined.Duringtheentireperiodofanesthesia,oxygensaturation,HR(2500AVET,NoninMedicalInc.,Plymouth,MN,USA),bodytemperature(PLUGSYSThermocoupleAmpliﬁerModule(TCAM),HAEMODYNsoftware,HugoSachsElektronik—HarvardApparatusGmbH,March-Hugstetten,Germany),respiratoryfrequencyandend-tidalCO2weremonitored(Vamos®plus,DrägerMedicalDeutschlandGmbH,Unterhaching,Germany).

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Animals2022,12,1028 6of19

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Animals2022,12,1028 6of19 Nocifensivemovementswereassessedineverypigletduringinjection,skinincisionandcuttingofthespermaticcord.Thepersonwhoimmobilizedthepigletassessedthemovementofthefourlimbsandthetensionofthespinewhileblindedtothetreatments.Forthehandlinggroup,noblindingwaspossiblebecausethedifferenceswereobvious(i.e.,shaminjectionandshamcastration).ThequalityofthenocifensivemovementswasevaluatedusingaspeciﬁcscoreadoptedfromSalleretal.[19].Allfourlimbswereassessedindividuallyforeachtesticleasfollows:score1=onemovement,score2=twotothreemovements,andscore3=morethanthreemovements,longlasting.Theback/spinewasevaluatedseparatelyforeachtesticle:score1=musclecontractionandscore2=movement.Amaximumscoreof28points,with14pointspertesticle,couldbereachedperevent(injection,skinincisionandcuttingofthespermaticcord).2.7.InjectionThefollowinglocalanestheticswereused:2%lidocainehydrochloride(Xylocitin®2%,MibeGmbHArzneimittel,Brehna,Germany)intheLido1andLido2groupsand2%mepivacainehydrochloride(Mepidor®20mg/mLsolutionforinjectioninhorses,RichterPharmaAG,Wels,Austria)intheMepi1andMepi2groups.TheNaClgroupwasinjectedwith0.9%sodiumchloride(B.Braun,Melsungen,Germany)usingtheone-stepFmethod.Theinjectionwasmerelysimulatedinthehandlinggroupbyﬁxingandgentlytouchingthetesteswithaneedlecap.Forbothinjectiontechniques,anautomaticself–filling1mLsyringe(HSWECO–MATIC®,Henke-Sass,WolfGmbH,Tuttlingen,Germany)wasused,andneedleswerechangedbe-tweeneachpiglet.Tostandardizetheprocedureasmuchaspossible,thesametwopersonsalwaysperformedtheinjectionandcastrationthroughoutthestudy.Fortheinjectionandcastrationprocedures,onepersonconstrainedthepigletsinthesupinepositionwithbothpalmsunderthebackwhilekeepingthehindlimbscraniallyusingthethumbs.Thepersonperformingtheinjectionandcastrationpressedthetesticlescaudallyintothescrotalsacandﬁxedthembetweenthethumbandindexﬁnger.Theone-stepFinjectionwasperformedusingaspecialcannulainventedbytheauthors.Theobjectiveindevelopingthisdevicewastocreateacannulaforadministeringalocalanestheticforcastrationofapigletthatiseasytohandleandfacilitatesefﬁcientandproperlocalanesthetizationofthetesticlesandscrotalskin.Thenewlyinvented25Gcannula(0.5 10mm)containedtheusualdistalopeningandtwoproximalandtwodistallateralopeningsforsimultaneousreleaseofthelocalanesthetic.Thespecializedcannulasweremanufacturedindividually.Thelateralholesweredrilledusinglasertechnology.Theneedlewasinsertedcaudallytoitsfulllengthsothatthedistalopeningandthedistallateralopeningwerepositionedinthetesticleandtheproximallateralopeningwasarrangedinthesubcutaneoustissueofthescrotum(Figure1).Avolumeof0.6mLofalocalanesthetic(Lido1,Mepi1)orsodiumchloride(NaCl)wasadministeredperside.Thetwo-stepinjectionwasmodiﬁedbyHanssonetal.[11].Intheﬁrststep,acom-merciallyavailable25Gneedle(0.5 16mm,B.BraunTravaCareGmbH,Hallbergmoos,Germany)wasinsertedcaudallyforapproximatelythree-fourthsofitslengthsothatthetipoftheneedlewaspositionedintratesticularly.Avolumeof0.4mLofthelocalanestheticwascarefullyinjected.Inthesecondstep,theneedlewaswithdrawn,andavolumeofap-proximately0.2mLwasevenlydistributedovertheinjectioncanaltothesurface(Figure1).Intotal,approximately0.6mLwasinjectedpertestis.2.8.CastrationSurgicalcastrationwasperformed20minafterinjectiontoensurethatbothlocalanestheticswerefullyeffective.Twoverticalskinincisionsweremadeparalleltotheraphescrotiusingascalpelandthetesticlesweregentlypressedout.Toevaluatenoxiousstimulationoftheskinincisionindependently,aperiodoftwominuteswasallowedtopassforstabilizationoftheparametersbeforethespermaticcordswereseveredusingan

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Animals2022,12,1028 7of19 emasculator.Castrationwasmerelysimulatedinthehandlinggroupbyholdingtheintacttesticlesinpositionandgentlytouchingtheskinwiththebluntbackofthescalpelhandle.

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culator.Castrationwasmerelysimulatedinthehandlinggroupbyholdingtheintacttesticlesinpositionandgentlytouchingtheskinwiththebluntbackofthescalpelhandle. Figure1.Schematicoverviewoftheone-stepFandtwo-stepmethods.a—Scrotalskin,b—testis,c—Spermaticcord.OnestepFmethod:theneedlewasinsertedcaudallytoitsfulllengthsothatthedistalopeningandthedistallateralopeningwerepositionedinthetesticleandtheproximallateralopeningwasarrangedinthesubcutaneoustissueofthescrotum.Atotalof0.6mLofalocalanestheticwasadministered.Two-stepmethod:Step(1)theneedlewasinsertedforapproximatelythree-fourthsofitslengthandavolumeof0.4mLofthelocalanestheticwasintratesticularlyinjected.Step(2)Theneedlewaswithdrawn,andavolumeof0.2mLwasevenlydistributedovertheinjectioncanal.2.9.FosProteinExpressionNinetyminutesafterthecastrationwasﬁnished,thepigletwaseuthanized,andlum-barandsacralsegmentsofthespinalcordwerecarefullyremovedfromthevertebralcanal.Usingthevertebraeaslandmarks,thespinalcordwasdividedintolumbar(L1,L2,L3)andsacral(S1–S3)spinalcordsegments,ﬁxedin38%(w/w)neutralbufferedformaldehydeforatleast48handembeddedinparafﬁn(LeicaASP300S,Leica,Wetzlar,Germany).Crosssections(2 m)wereproducedstartingfromthecranialcutsurface.Serialsectionswerestainedwithhematoxylinandeosinusingastandardprotocol.ImmunohistochemistrywasperformedtoinvestigateFosproteinexpression(anti-c-Fosantibody,ab209794,Abcam,Cambridge,UK,dilutedinantibodydiluent1:100)usingaLeicaBondRXmsystem.Brieﬂy,afterdeparafﬁnization,epitoperetrieval1wasperformedusingcitratebuffer(pH6)for30min.Forprimaryantibodybindingdetectionandvisualization,aPolymerReﬁneDetec-tionkitwith3,3’-diaminobenzidine(DAB)andnosecondaryantibodywasused.SlidesweredigitizedusingaLeicaAT2scanningsystem.ThespinaldorsalhornwasidentiﬁedasthegraymatterdorsaltoalinedrawnfromthecentralcanaltothelateralborderofthegraymatterusingAperioImagescopeSoftwareversion12.4.0.7081(LeicaBiosystems,Wetzlar,Germany).AGermanboard-certiﬁedpathologistblindedtothesampleidenti-tiesperformedsemiquantitativeanalysisofthestainingintensityofFos-positiveneurons.Fourdifferentsegmentsofeachanimalwereevaluated.Scoreswereassignedasfollows:0=nostaining,1=slightstaininginsingleneurons( 25%),2=slightstaininginsomeneurons( 50%),3=moderatestaininginsomeneurons( 50%),4=strongstaininginsomeneurons( 50%),5=strongstaininginallneurons.2.10.DataandStatisticalAnalysisForthestatisticalanalysis,groupsweretestedforchangesinMAP,HRandFosproteinexpression,andsigniﬁcantdifferencesinage,weightandend-tidalisoﬂuraneconcentrationandnocifensivemovementswereobserved.ThedistributionofallcontinuousparameterswastestedusingtheShapiro–Wilknormalitytest.One-wayANOVAwasperformedfordatawithnormallydistributedresiduals.Ifsigniﬁcant,pairwiseStudent’st-testsorGames–HowelltestswithBenjamini

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Animals2022,12,1028 8of19 andHochbergp-valueadjustmentformultiplecomparisonswereperformed.TheKruskal–Wallistestwasperformedfornon-normallydistributedresiduals.Ifsigniﬁcant,Dunn’stestofmultiplecomparisonsusingranksumswithBenjaminiandHochbergp-valueadjustmentformultiplecomparisonswasperformed.Forthestatisticalanalysisofthedistribution,anindependentsamplet-testwasused.Forthedistributionscore,theintraclasscorrelationcoefﬁcients(ICCs)andtheir95%conﬁ-denceintervalswerecalculatedusingIBMSPSSStatisticsforMacversion1.0.0.1508.Basedonthemeanrating,absoluteagreementand2-waymixed-effectsmodel,theICCvaluesrangedbetween0and1,withvaluescloserto1indicatingstrongerreliability.Statisticalsigniﬁcancewasconsideredatp 0.05.StatisticalanalyseswereperformedusingRversion3.6.1(2019-07-05)andGraphPadPrism9.0.2.3.Results3.1.DistributionThedistributionofacontrastagentdilutedin2%lidocainehydrochlorideinjectedviatwodifferentapplicationmethodswasvisualizedusingCT(Figure2A,B).Theone-stepFandtwo-stepinjectionachievedawidespreaddistributionofthelidocaine/contrastagentmixtureinthescrotalskin,testicles,testicularsheathsandspermaticcorddirectlyafteradministration.Nofurtherspreadingofthecontrastagentwasobservedduringthe20minafterinjection(FigureA1).ThedistributionandaccumulationofthecontrastmediumwereevaluatedbasedonaCTdistributionscore(Table1).TheICCfortheevaluationofthedistributionofthecontrastagentswas0.89(conﬁdenceinterval(CI):0.76–0.96).Nosigniﬁcantdifferencewasobservedbetweentheone-stepFandtwo-stepinjectiongroups(Figure2C).Bothinjectiontechniquesproducedsimilardistributionpatternsandreachedcomparabledistributionscores.

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weentheone-stepFandtwo-stepinjectiongroups(Figure2C).Bothinjectiontechniquesproducedsimilardistributionpatternsandreachedcomparabledistributionscores. Figure2.Distributionofthelidocaine/contrastmediummixturedirectlyafterinjection,usingtheone-stepFandtwo-stepmethods.Exemplarythree-dimensionalreconstructionofapigletinjectedwithonestepF(A)andapigletinjectedusingthetwo-stepmethod(B).Bonestructuressuchaspartsofthespine,pelvisandfemurarecoloredbrown.Theinjectedlidocaine/contrastagentmixtureiscoloredgreen.(C)CTdistributionscoringinpigletsinjectedwiththeone-stepFortwo-stepmethod.

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Animals2022,12,1028 9of19 3.2.InjectionPressureandInjectionTimeFigure3showstheinjectionpressuresduringinjectionsintotheairandtesticu-lartissueusingtheone-stepFandtwo-stepinjectionmethods.A0.6mLintratestic-ularinjectionof0.9%sodiumchlorideusingtheone-stepFmethodgeneratedamax-imumpressureof1572.67 381.53mmHg.Theinjectionof0.4mLintratesticularlyand0.2mLsubcutaneouslyusingthetwo-stepmethodcreatedamaximumpressureof1486.64 461.46mmHg.One-stepFinjectionexceededthemaximalmeasurablepres-sureinfourofsevenanimals,anditwasobservedinoneoffouranimalsusingthetwo-stepmethod.Measuringtheinjectionpressureinairresultedinanaveragepressureof1215.87 308.11mmHgwiththeone-stepFmethodand1083.33 714.24mmHgwiththetwo-stepinjectionmethod.Themeanexecutiontimefortheone-stepFinjection,includingbothtesticles,was14 1.6s.Thetwo-stepmethodwasmoretimeconsuming,withameanof23 2.3s.

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jectionmethod.Themeanexecutiontimefortheone-stepFinjection,includingbothtesticles,was14 1.6s.Thetwo-stepmethodwasmoretimeconsuming,withameanof23 2.3s. Figure3.Injectionpressureproﬁlesofthetwoinjectionmethodsintothetestisandair.i.t.=intratesticular.3.3.GroupHomogeneityTwopigletsfromtheLido1groupwereexcludedduetoincompletedataacquisition.Thetreatmentgroupswerehomogeneouslydistributedinage,weightandend-tidalisoﬂu-raneconcentrationduringinjectionandcastration(Table3)anddidnotsigniﬁcantlydifferbetweengroups.Themeanend-tidalisoﬂuraneofallpigletsinthisstudywas1.4 0.35%withaﬂowof3Lmin 1oxygen.Thelocalanestheticdosagewasthesameforeachpiglet,regardlessoftheindividualpigletweight.Therefore,theadministeredlocalanestheticdoseperweightvariedbetweentheanimals.Thehighestlidocainedose(19.29mg/kg)wasadministeredintheLido2group,andthelowestdosewasadministeredintheLido1group(7.74mg/kg).Formepivacaine,thehighestdosageperweightwasgivenintheMepi1group(17.14mg/kg),andthelowestdosage(7.87mg/kg)wasgivenintheMepi2group.Noneofthepigletsinjectedwithalocalanestheticshowedsignsofincompatibilityorsideeffects.Table3.Meanvaluesandstandarddeviationofage,bodyweight,localanestheticdosing,andend-tidalisoﬂuraneconcentrationduringinjectionandcastration. HandlingNaClLido1Lido2Mepi1Mepi2 Age(days)4.78 0.795.44 1.174.57 1.364.22 1.135 1.334.89 1.45

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Animals2022,12,1028 10of19 Table3.Cont. HandlingNaClLido1Lido2Mepi1Mepi2 Weight(kg)2.01 0.512.32 0.692.37 0.412.04 0.431.98 0.382.21 0.50Localanestheticdosing(mg/kg)*10.4 1.6513.8 3.0712.01 2.8012.61 2.5End-tidalIsoﬂurane–injection(Vol%)1.46 0.381.32 0.261.4 0.201.39 0.441.38 0.181.57 0.38End-tidalIsoﬂurane–castration(Vol%)1.52 0.481.31 0.261.46 0.221.38 0.441.39 0.161.62 0.38 *Thevaluesreferonlytotheamountsappliedtothetesticularregionanddonotincludeinﬁltrationoftheneckwithapproximately0.3mLof2%lidocaineforthecatheterimplementation.3.4.BloodPressureandHeartRate3.4.1.InterdigitalPinchThemeasuredbaselinemeanMAPforallpigletswas49.94 6.04mmHg.Thedepthofanesthesiawasindividuallyadjustedusinganinterdigitalpinch.Theobservedchangesinbloodpressureasanociceptivereactiontotheinterdigitalpinchweresimilarbetweengroups,andnosigniﬁcantdifferenceswereobserved(FigureA2).Thesameresultswereobtainedforchangesinheartrateduringinterdigitalpinch.3.4.2.InjectionInjectionsofalocalanestheticorsodiumchlorideprovokednosigniﬁcantchangesinMAPorHRcomparedtothatofthehandlinggroup,inwhichtheinjectionwasonlysimulated(Figure4). Figure4.Injection.PercentchangeinMAP(A)andHR(B)duringinjection.Valuesshownaremeans SD.3.4.3.CastrationFigure5AshowstheMAPchangesduringskinincision.Cuttingofthescrotalskinwithoutpainrelief(NaCl)provokeda17.24%maximalchangeinMAPfrombaseline.Incontrast,thehandlinggroupshowedasigniﬁcantlylowerMAP(p=0.037).Administration

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kinwithoutpainrelief(NaCl)provokeda17.24%maximalchangeinMAPfrombaseline.Incontrast,thehandlinggroupshowedasigniﬁcantlylowerMAP(p=0.037).Administration Animals2022,12,1028 11of19 ofalocalanesthetic,exceptforLido2,causedsigniﬁcantlyreducedchangesinMAPcomparedtoNaCl(Lido1:p=0.034,Mepi1:p=0.034,Mepi2:p=0.034)andwascomparabletothehandlinggroup.Lido1(p=0.038)andLido2(p=0.044)achievedasigniﬁcantreductioninHRchangescomparedtoNaCl(Figure5B).Amongtheothergroups,nosigniﬁcantdifferencesinHRwereobservedduringtheskinincision. Figure5.Castration.PercentchangeinMAP(A,C)andHR(B,D)duringskinincision(A,B)andcuttingofthespermaticcord(C,D).Valuesshownaremeans SD.Statisticalsigniﬁcanceisindicatedas*p 0.05,**p<0.01,***p<0.001.

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Animals2022,12,1028 12of19 Duringcuttingofthespermaticcord,theNaClgrouprespondedwiththehighestchangesinMAP(36.10%)andwassigniﬁcantlyincreasedcomparedtothehandlingpiglets(p=0.016)(Figure5C).ComparedtoNaCl,theMAPofthefourlocalanestheticgroups(Lido1:p=0.044,Lido2:p=0.002,Mepi1:p=0.001,andMepi2:p=0.001)wassigniﬁcantlydecreased.TheMAPofthegroupsreceivingalocalanestheticwascomparabletothehandlinggroup.NosigniﬁcantdifferencesinHRdeviationweredetectedbetweenthetreatmentandcontrolgroupsaftercuttingthespermaticcord(Figure5D).3.5.NocifensiveMovementsAsshowninTable4,onlyMepi1showedsignificantlymorenocifensivemovementsduringinjectionthanLido2(p=0.031)andHandling(p=0.031).ThehighestproportionofpigletsshowingnosignsofnocifensivemovementsaccordingtoinjectionwereobservedintheHandlingandLido2groups.Nosignificantdifferenceswereobservedduringskinincisionsbetweenthegroups.TheNaClgrouphadthehighestnumberofpigletsrespondingtoskinincisionsandachievedthehighestscore(5.1 6.7)amongtheothergroups.HandlingandLido1pigletsdidnotmoveatallduringtheskinincision.Forcuttingthespermaticcord,NaClproducedthehighestmeanscore(10.9 8.7),andmostpiglets(eightofnine)showednocifensivemovementsinthisgroup.Significantlylowernocifensivescores(p 0.001)werefoundforallexperimentalgroupsandthehandlinggroup.HandlingandMepi2pigletsshowednonocifensivemovements,butonlyonepigletineachoftheotherlocalanesthesiagroupsreactedtocuttingofthespermaticcord.Table4.Nocifensivescoresmean SDandnumberofpigletswithoutnocifensivemovementsduringinjection,skinincisionandcuttingofthespermaticcord. HandlingNaClLido1Lido2Mepi1Mepi2 InjectionScoremean0.1a0.81.30.8a7.1b2.7SD0.31.62.12.26.53.2Animalswithoutnocifensivemovementsn8/95/94/78/93/94/9

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ord. HandlingNaClLido1Lido2Mepi1Mepi2 InjectionScoremean0.1a0.81.30.8a7.1b2.7SD0.31.62.12.26.53.2Animalswithoutnocifensivemovementsn8/95/94/78/93/94/9 SkinincisionScoremean05.101.73.12.1SD06.703.18.84.0Animalswithoutnocifensivemovementsn9/95/97/76/98/96/9 CuttingofthespermaticcordScoremean0a10.9b0.9a0.1a0.7a0aSD08.72.10.31.90Animalswithoutnocifensivemovementsn9/91/96/78/98/99/9 a,bDifferingsuperscriptswithinonelineindicatesigniﬁcant(p<0.05)differencesbetweengroups.3.6.FosProteinExpressionFigure6summarizestheFosproteinexpressioninthelumbarandsacralsegmentsofthespinalcordsofthesixtreatmentgroupsusingasemiquantitativescorebasedonthestainingintensity.SigniﬁcantlyreducedFosproteinexpressioncomparedtotheNaClcontrolwasfoundonlyinLido2(p=0.016).

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Animals2022,12,1028 13of19 Figure6.Fosproteinexpressioninthelumbarandsacralsegmentsofthespinalcord90minaftercastration.*p 0.05.4.DiscussionLocalanesthesiaforpigletcastrationhasbeeninvestigatedinmanystudies,butdivergentoutcomeshavebeenreported.Itisdifﬁculttocomparetheresultsoftheseinves-tigationsduetotheirverydifferentstudydesigns.Differentlocalanesthetics,evaluationcriteria,injectionmethodsandvolumeswereused.Moststudiesusedanintratesticularinjection[1,2,15,21–29],ortheinjectionintothetestiswascombinedwithasubcutaneousdepot[1,10–12,14,17–20,26,30–33].Otherstudies[2,10,14,17,18]choseanintrafunicularandsubcutaneousapproach,andZankletal.andWhiteetal.injectedlocalanestheticsonlyin-trascrotally[15,34].Sutherlandetal.examinedaneedle-freeinjectionthroughtheskin[35].Notably,theexactinjectionprocedureisnotdescribedindetailinmanypublications,whichmayexplainthepoorreproducibilityofsomestudies.Thepresentstudyinvestigatedtwodifferentinjectionmethods:thetwo-stepmethod,modiﬁedaccordingtoHanssonetal.[11],andtheone-stepFmethod,whichusedanewlydevelopedcannulawithadditionallateralopenings.Thefocusofbothmethodswasachievingpracticabilityforfarmersinpracticewhilemaintaininghighefﬁcacy.Thecomplexinnervationofthetesticularregionandthevarietyofanatomicalstruc-turesthatmustundergosensoryblockforpainlesscastrationarechallengingintheadminis-

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Animals2022,12,1028 14of19 trationoflocalanesthesia.Sourcesofporcinetestisinnervationarepelvic,sensory,pre-andparavertebralganglia.Themainnervesupplyisprovidedbytheanteriorpelvicganglionandthecaudalmesentericganglion[36].Originatingfromthecaudalmesentericganglion,thetesticularplexusrunstogetherwiththetesticulararteryandinnervatesthetestisandepididymis[37].Theinnervationofthescrotumoriginatesfromthenerviscrotalesdorsales,whichareendbranchesofthepudendalnerve,andthetunicavaginalisandthecremastermusclearesuppliedbybranchesofthegenitofemoralnerve[37].CTimagingshowedthattheone-stepFandtwo-stepinjectionmethodsresultedinanevendistributionwithinthescrotalskin,testicularsheath,testisandspermaticcorddirectlyafterinjection.AlthoughonlythedistributionofthecontrastagentcanbefollowedwithCT,wepresumedthatthelocalanestheticwasdistributedinasimilarmanner.ThispresumptionissupportedbyRanheimetal.[33],whereintratesticularlyappliedradiolabeledlidocainewasrapidlytransportedintothespermaticcordandmaximumenrichmentwasmeasuredafterthreeminutes.Forbettercomparability,a20minwaitingtimebeforecastrationwasused,basedonourpreviousstudies[19,20].Fastertimesofonsetofseveralminuteswerereportedforlidocaineandmepivacaine[38],whichsuggeststhatashorterwaitingperiodtoperformcastrationmaybesufﬁcient.Ashorteningofthewaitingperiodhasbeenappliedinotherstudiesandmayleadtomoreﬂexibilityintheimplementationofthemethodinpractice[14].Thetunicaalbugineaisaﬁbroustissuecapsulethatcoversthetestisandpreventsthetesticlefromexpanding[39].Thisroughcapsulelimitsintratesticularlyinjectedﬂuids,andtheresultinghighpressureinthetesticlepushestheﬂuidtowardthespermaticcord[33].Theinjectionpressuresachievedwithbothmethodsintothetestisarecomparabletointraneuralorintradermalinjections[40,41].However,thesedatashouldbeinterpretedwithcautionbecausethemeasurementsystemonlydetectedpressuresupto2121mmHg.Thepressurelimitwasreachedﬁvetimes,fourtimesintheone-stepFgroupandonceinthetwo-stepgroup.Therefore,wedonotknowexactlytheintensityofthepressure.Theinjectionsintotheairreachedsimilarvalues,whichindicatesthattheresistancewithinthesyringesystemandthecannulawasalreadyhigh.Thesmalldiametersofthecannulasandtheinjectionspeedmayleadtothishighbasepressure.Injectionspeed(mLpersecond)wasnotsupervisedinthisstudy,butthesametwopersonsperformedinjectionstostandardizetheinjectionasmuchaspossible.Otherfactors,suchasneedlediameterandlength,injectionvolume,temperatureoftheﬂuidanddrugformulation,inﬂuencetheperceptionofpainduringinjections[42].Theinjectionoflocalanestheticsprovokednocifensivemovementsinawakepiglets[20].Therefore,wefurthertriedtominimizeinjectionpainbyreducingthetotalinjectionvolumefrom1mLto0.6mLperside.AlthoughonlyMepi1reachedsigniﬁcantlyhighernocifensivemovementscoresthanLido2andHandlinginourstudy,manypigletsintheNaCl,Mepi2andLido1groupsshowednocifensivereactions.However,thesereactionswerenotsigniﬁcant.Toimprovetheobservedreactions,thetotalvolumemaybefurtherreduced.However,asufﬁcientanalgesiceffectmustbeensured.Previousstudiesdidnotfullypreventthediscomfortofintratesticularinjectionsbyvolumereduction[10].Furthereffortstominimizeinjectionpainmayincludetheuseofthinnerneedlesortobufferlocalanesthetics[43].Bufferingoftenautomaticallyincreasestheinjectionvolumewhentherearenomoreconcentratedproductsonthemarket.Theadditionofbicarbonateisamedicinalproductpreparationthatmaynotbeperformedbythefarmer.Whetheraninjectionintothetestismayoccurwithoutanysensationofpainandwhethertheinjectionshouldbeincludedintheevaluationoflocalanesthesiaasalegalalternativeforthecastrationofconsciouspigletsmustbediscussed.Forthetimerequirement,acomparisonofbothtechniquesrevealedthattheone-stepinjectiontookmuchlesstimethanthetwo-stepinjection.Theimpressionoftheveterinarianoperatorswasthatthepracticalimplementationofthetwo-stepinjectionwasmorechallengingthanthatoftheone-stepF.Theone-stepFinjectionrequiredonlyonesimpleplacementofthecannulaintothetestis.Duetotheadditionallateralholes,norepositioningofthecannulaforsubcutaneousinﬁltrationwasnecessary.Oneissue

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Animals2022,12,1028 15of19 thatoccurredduringtheperformanceofthesecondstepofthetwo-stepmethodwasthedifﬁcultyofinjectinganexactvolumeofonly0.2mLwhilewithdrawingthecannula.Thisdifﬁcultyoccurredbecauseanautomaticself-ﬁllingsyringewithapresetapplicationvolumeof0.4mLwasusedfortheﬁrststep.Asecondvolumecouldnotbepreset,whichmadetheexactdosingforthesecondstepmoredifﬁcult.Skadeetal.[10]comparedtwoinjectionmethodsandfavoredafasterandlesscomplicatedmethodforpracticalapplication.Asanextstep,theinjectionmethodsmustbeevaluatedintheﬁeldofawakeanimalstodrawaﬁnalconclusion.Localanestheticsolutionsdonotcontainepinephrinebecausevasoconstrictorscouldaffectbloodpressureandheartratemeasurements[19].BothinjectiontechniquesandbothlocalanestheticsachievedasigniﬁcantreductioninnocifensivemovementsandMAPduringcuttingofthespermaticcord,whichisthemostpainfulpartofcastration[44].Onlythecombinationoflidocaineandthetwo-stepmethoddidnotachieveasigniﬁcantreductioninMAPduringskinincisions.Fornocifensivemovementsduringskinincision,therewasnosigniﬁcantdifferencebetweenpigletscastratedwithoutpainreliefandpigletsthatreceivedalocalanesthetic.ThefactthatnoteverypigletfromtheNaClgroupshowednocifensivemovementscouldbebecausethepigletswereunderlightisoﬂuraneanesthesia.Althoughisoﬂuranewasindividuallytitratedforeachpiglettoreachastateofhypnosis,asindicatedbythepresenceofapedalwithdrawalreﬂex,anesthesiacouldmaskpossiblemovements.Thereasonwhyindividualpigletsofthelocalanesthesiagroupsstillmovedduringskinincisionandcuttingofthespermaticcord,despitelocalanesthesia,maybethatnociceptionwasnotfullyblockedinsomepiglets.Anotherexplanationmaybethatexternalinﬂuences,suchasﬁxationofthetestes,affectedthepiglet’sreactionsbecauseoneanimalinthehandlinggroupalsoshoweddefensivereactionsduringsimulatedinjection.Fosproteinisexpressedinneuronsofthedorsalhornofthespinalcordafternoci-ceptivestimulationanditisusedasamarkerforpain[45].Fosproteinexpressionreﬂectsonlythesumofthepainexperiencedandcannotbeassignedtoonespeciﬁcpainevent.Nyborgetal.[46]showedthatinjectionofalocalanestheticbeforecastrationreducedthenumberofFos-positiveneuronsinthedorsalhorn.Reiseretal.foundasigniﬁcantreductionintheFosstainingscoreinpigletscastratedafteraninjectionoflidocaine,mepi-vacaineandprocaine[47].OnlyMepi1achievedasigniﬁcantreductioninFosproteinexpressioncomparedtotheNaClcastratedpigletsinthepresentstudy.PigletsthatdidnotexperienceinjectionorcastrationalsoexpressedtheFosprotein.Thisexpressionmaybeexplainedbythefactthatstressorunpleasantstimulation,suchasﬁxationofthepigletitselfandespeciallythetesticles,alsoincreasesFosproteinexpressioninthedorsalhorn.Theneonatalageofthepigletsmayexplainthisresult.Forexample,aFosresponsetoinnocuousstimulationwasobservedinneonatalratsuntilpostnatalday21[48].Therefore,Fosproteinexpressionshouldnotbeusedasthesoleparametertoassessnociceptioninpigletsfollowingcastration,anditshouldalwaysbeevaluatedincombinationwithotherparameters.ThesamplesizeforthisstudywascalculatedbasedonMAP,whichisthemostsen-sitivecardiovascularparameterassociatedwithnoxiousstimuliduringcastration[19,49].Therefore,thepowerofthestudymaybetoolowtodrawdeﬁniteconclusionsonnocifen-sivemovementsandFosexpression,andadirectcomparisonoftheinjectionmethodsandlocalanestheticsisdifﬁcult.Thepresentstudyonlyevaluatednociceptivereactionsinre-sponsetoacutestimuliunderlightisoﬂuraneanesthesia.Chroniceffectsandpracticabilityinawakepigletsmustbeevaluatedinfurtherongoingstudies.5.ConclusionsInconclusion,bothinjectiontechniques,regardlessofthelocalanestheticused,re-ducednociceptionduringcastrationofmalepigletsunderstandardizedconditions.Onechallengeremainsthehandlingofthediscomfortoftheinjectionitself,whichcontinuedtoelicitnocifensivemovementsinseveralpigletsfromthedifferentexperimentalgroups.Forspeedandmanageability,theone-stepFmethodwasbeneﬁcial.However,compared

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Animals2022,12,1028 16of19 tothetwo-stepmethod,theappliedpressurewashigherandmorefrequentlyexceededthemaximalmeasurablepressure.Bothinjectiontechniquesmustbefurtherevaluatedinconsciouspigletstodrawﬁnalconclusionsabouttheireffectivenessandfeasibilityinpracticaluse.AuthorContributions:Conceptualization,S.Z.andC.B.;Datacuration,J.W.andA.M.S.;Formalanalysis,Y.Z.;Fundingacquisition,S.Z.andC.B.;Investigation,J.W.,A.M.S.,J.R.,S.S.,P.D.,N.A.,J.F.andK.S.;Methodology,J.W.,A.M.S.,J.R.,S.S.,P.D.,N.A.,J.F.,A.G.,R.M.,K.S.,S.B.,M.H.E.,M.R.,S.Z.andC.B.;Projectadministration,S.Z.andC.B.;Resources,M.R.,S.Z.andC.B.;Supervision,M.H.E.,M.R.,S.Z.andC.B.;Validation,K.S.,M.H.E.,M.R.,S.Z.andC.B.;Visualization,J.W.andA.M.S.;Writing—originaldraft,J.W.;Writing—review&editing,A.M.S.,J.R.,S.S.,P.D.,N.A.,J.F.,A.G.,R.M,S.B.,Y.Z.,K.S.,M.H.E.,M.R.,S.Z.andC.B.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.Funding:ThisresearchwasfundedbytheGermanFederalMinistryofFoodandAgriculture(BMEL,https://www.bmel.de(accessedon20March2022)basedonadecisionoftheParliamentoftheFederalRepublicofGermany,grantedbytheFederalOfﬁceforAgricultureandFood(BLE,https://www.ble.de(accessedon20March2022),grantnumber:2817HS016,2817HS013).InstitutionalReviewBoardStatement:ThestudywasperformedincompliancewiththeEUDirec-tive2010/63/EUforanimalexperimentsandtheGermanAnimalWelfareAct[27].AllprocedureswereapprovedbytheEthicsCommitteeforAnimalExperimentsoftheGovernmentofUpperBavaria,Munich,Germany(ReferenceNumberROB-55.2-2532.Vet_02-19-11).InformedConsentStatement:Notapplicable.DataAvailabilityStatement:Notapplicable.Acknowledgments:TheauthorsthankAndreasKnipfer,whosupportedthecomputedtomographyscans,andTanjaGroll,MarionMielkeandOlgaSeelbach,whoperformedparafﬁnembedding,cuttingandimmunohistologicalstaining.ConﬂictsofInterest:Theauthorsdeclarenoconﬂictofinterest.Thefundershadnoroleinthedesignofthestudy,inthecollection,analyses,orinterpretationofdata,inthewritingofthemanuscript,orinthedecisiontopublishtheresults.AppendixA

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adnoroleinthedesignofthestudy,inthecollection,analyses,orinterpretationofdata,inthewritingofthemanuscript,orinthedecisiontopublishtheresults.AppendixA FigureA1.Distributionofthelidocaine/contrastmediummixture20minafterinjectionusingtheone-stepFandtwo-stepmethods.Exemplarythree-dimensionalreconstructionofapigletinjectedusingtheone-stepF(A)andapigletinjectedusingthetwo-stepmethod(B).Bonestructuressuchaspartsofthespine,pelvisandfemurarecoloredbrown.Theinjectedlidocaine/contrastagentmixtureiscoloredgreen.

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Animals2022,12,1028 17of19 AppendixB

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Animals2022,12,1028 17of19 AppendixB FigureA2.Interdigitalpinch.Percentchangein(A)meanarterialbloodpressure(MAP)and(B)heartrate(HR)duringtheinterdigitalpinchfortheadjustmentofanesthesiadepth.Valuesshownaremeans SD.References1.Horn,T.;Marx,G.;vonBorell,E.Behaviorofpigletsduringcastrationwithandwithoutlocalanesthesia.Dtsch.Tierarztl.Wochenschr.1999,106,271–274.[PubMed]2.McGlone,J.J.;Hellman,J.M.LocalandGeneralAnestheticEffectsonBehaviorandPerformanceofTwo-andSeven-Week-OldCastratedandUncastratedPiglets.J.Anim.Sci.1988,66,3049–3058.[CrossRef][PubMed]3.Aluwe,M.;Tuyttens,F.A.;Millet,S.Fieldexperiencewithsurgicalcastrationwithanaesthesia,analgesia,immunocastrationandproductionofentiremalepigs:Performance,carcasstraitsandboartaintprevalence.Animal2015,9,500–508.[CrossRef][PubMed]4.Weiler,U.;Isernhagen,M.;Stefanski,V.;Ritzmann,M.;Kress,K.;Hein,C.;Zols,S.PenileInjuriesinWildandDomesticPigs.Animals2016,6,25.[CrossRef]5.Bunger,B.;Schrader,L.;Schrade,H.;Zacharias,B.Agonisticbehaviour,skinlesionsandactivitypatternofentiremale,femaleandcastratedmaleﬁnishingpigs.Appl.Anim.Behav.Sci.2015,171,64–68.[CrossRef]6.Bartongade,P.A.MeatandFatQualityinBoars,CastratesandGilts.Livest.Prod.Sci.1987,16,187–196.[CrossRef]7.CouncilDirective2008/120/ECof18December2008LayingDownMinimumStandardsfortheProtectionofPigs.2009,pp.5–13.Availableonline:https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32008L0120&from=EN(accessedon13March2022).8.DeBriyne,N.;Berg,C.;Blaha,T.;Temple,D.Pigcastration:WilltheEUmanagetobanpigcastrationby2018?Porc.HealthManag.2016,2,29.[CrossRef]9.CASTRUMConsortium;EuropeanCommission,Directorate-GeneralforHealthandFoodSafety.PigCastration:MethodsofAnaes-thesiaandAnalgesiaforallPigsandOtherAlternativesforPigsUsedinTraditionalProducts;PublicationsOfﬁce:Luxembourg,2017.[CrossRef]10.Skade,L.;Kristensen,C.S.;Nielsen,M.B.F.;Diness,L.H.Effectoftwomethodsandtwoanaestheticsforlocalanaesthesiaofpigletsduringcastration.ActaVet.Scand.2021,63,1.[CrossRef]11.Hansson,M.;Lundeheim,N.;Nyman,G.;Johansson,G.Effectoflocalanaesthesiaand/oranalgesiaonpainresponsesinducedbypigletcastration.ActaVet.Scand.2011,53,34.[CrossRef]12.Kluivers-Poodt,M.;Houx,B.B.;Robben,S.R.;Koop,G.;Lambooij,E.;Hellebrekers,L.J.EffectsofalocalanaestheticandNSAIDincastrationofpiglets,ontheacutepainresponses,growthandmortality.Animal2012,6,1469–1475.[CrossRef]13.Kluivers-Poodt,M.;Zonderland,J.J.;Verbraak,J.;Lambooij,E.;Hellebrekers,L.J.Painbehaviouraftercastrationofpiglets;effectofpainreliefwithlidocaineand/ormeloxicam.Animal2013,7,1158–1162.[CrossRef][PubMed]

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Animals2022,12,1028 18of19

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Animals2022,12,1028 18of19 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Animals2022,12,1028 19of19 40.Vermeylen,K.;Hermans,M.;Soetens,F.;Vereecke,E.;Steinfeldt,T.;Groen,G.;Hadzic,A.;VandeVelde,M.OpeningInjectionPressureIsHigherinIntraneuralComparedWithPerineuralInjectionsDuringSimulatedNerveBlocksoftheLowerLimbinFreshHumanCadavers.Reg.Anesth.PainMed.2017,42,362–367.[CrossRef][PubMed]41.Gupta,J.;Park,S.S.;Bondy,B.;Felner,E.I.;Prausnitz,M.R.Infusionpressureandpainduringmicroneedleinjectionintoskinofhumansubjects.Biomaterials2011,32,6823–6831.[CrossRef]42.Strazar,R.;Lalonde,D.Minimizinginjectionpaininlocalanesthesia.CMAJ2012,184,2016.[CrossRef]43.Hockett,D.;Kress,L.;MacDonald,R.;Krenzischek,D.A.;Maheshwari,A.EffectivenessofBufferedLidocaineforLocalAnesthesiaDuringLiverBiopsy.Gastroenterol.Nurs.2021,44,172–176.[CrossRef]44.Taylor,A.A.;Weary,D.M.Vocalresponsesofpigletstocastration:Identifyingproceduralsourcesofpain.Appl.Anim.Behav.Sci.2000,70,17–26.[CrossRef]45.Ahmad,A.H.;Ismail,Z.c-fosanditsConsequencesinPain.Malays.J.Med.Sci.2002,9,3–8.46.Nyborg,P.Y.;Sørig,A.;Lykkegaard,K.;Svendsen,O.Nociceptionefterkastrationafjuvenilegrisemåltvedkvantitativbestemmelseafc-Fosudtrykkendeneuronerirygmarvensdorsalhorn.Dan.Veterinærtidsskrift2000,83,6–17.47.Reiser,J.;Kreuzer,M.;Werner,J.;Saller,A.M.;Fischer,J.;Senf,S.;Deffner,P.;Abendschön,N.;Groll,T.;Weiß,C.;etal.Pain-inducedchangesinelectroencephalographicactivityandFOSproteinexpressioninpigletsundergoingcastrationunderisoﬂuraneanesthesia.2022,tobesubmitted.48.Jennings,E.;Fitzgerald,M.C-foscanbeinducedintheneonatalratspinalcordbybothnoxiousandinnocuousperipheralstimulation.Pain1996,68,301–306.[CrossRef]49.Haga,H.A.;Tevik,A.;Moerch,H.Electroencephalographicandcardiovascularindicatorsofnociceptionduringisoﬂuraneanaesthesiainpigs.Vet.Anaesth.Analg.2001,28,126–131.[CrossRef][PubMed]

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