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Citation:Söbbeler,F.J.;Wendt,S.;Briese,A.;Tünsmeier,J.;Waldmann,K.-H.;Kästner,S.B.R.;vonAltrock,A.ComparativeStudyofPain-RelatedResponsesofMalePigletsuptoSevenDaysofAgetotheApplicationofDifferentLocalAnaestheticsandSubsequentCastration.Animals2022,12,2833.https://doi.org/10.3390/ani12202833AcademicEditor:RobertE.MeyerReceived:23August2022Accepted:14October2022Published:19October2022Publisher’sNote:MDPIstaysneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffil-iations. Copyright:©2022bytheauthors.LicenseeMDPI,Basel,Switzerland.ThisarticleisanopenaccessarticledistributedunderthetermsandconditionsoftheCreativeCommonsAttribution(CCBY)license(https://creativecommons.org/licenses/by/4.0/). ArticleComparativeStudyofPain-RelatedResponsesofMalePigletsuptoSevenDaysofAgetotheApplicationofDifferentLocalAnaestheticsandSubsequentCastrationFranzJosefSöbbeler1,*,†,SörenWendt2,†,AndreasBriese3,JuliaTünsmeier1,Karl-HeinzWaldmann2,‡,SabineBeateRitaKästner1,†

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naestheticsandSubsequentCastrationFranzJosefSöbbeler1,*,†,SörenWendt2,†,AndreasBriese3,JuliaTünsmeier1,Karl-HeinzWaldmann2,‡,SabineBeateRitaKästner1,† andAlexandravonAltrock2,*,†1ClinicforSmallAnimals,UniversityofVeterinaryMedicineHannover,Hannover,Foundation,30559Hannover,Germany2ClinicforSwine,SmallRuminants,ForensicMedicineandAmbulatoryService,UniversityofVeterinaryMedicineHannover,Foundation,30173Hannover,Germany3eduToolbox,31157Sarstedt,Germany*Correspondence:franz.josef.soebbeler@tiho-hannover.de(F.J.S.);alexandra.von.altrock@tiho-hannover.de(A.v.A.)†Theseauthorscontributedequallytothiswork.‡Valuablecontributiontothispaperbeforehedeparted.SimpleSummary:Since2021,surgicalpigletcastrationmustbeperformedwithcompletepaineliminationaccordingtotheAnimalProtectionLawinGermany.Generalanaesthesiabyisofluraneinhalation,whichcanbeperformedbythefarmer,orbyinjectionofketamineandazaperone,whichmustbeperformedbyaveterinarian,aretheoptionsavailable.Atpresent,localanaesthesiaisstillunderdebatebecauseofthelackofproofofcompletepaineliminationandthepainoninjection.Wetestedthreelocalanaesthetics(procaine,lidocaine,andmepivacaine)attwodifferentdoseseach.Becausepainresponsescanbemaskedbyreactionscausedbyhandling,thepigletsweregivensuperficialisofluraneanaesthesia.Thepainoninjectiontothetesteswascomparedwithintramuscularinjection,andtheeffectivenessduringcastrationwascomparedamongthelocalanaesthetics.Nocifensivemovements,respiratoryrate,bloodpressure,heartrateanditsvariabilityaswellaselectroencephalography(EEG)changeswerestudiedinrelationtothepainfulinterventions.Mostindicatorsofnociceptionpointtotesticularinjectionpainbeingbeyondintramuscularinjectionpainwhenaneffectiveamountoflocalanaestheticwasused.However,completepaineliminationcouldnotbeachievedduringcastrationunderlocalanaesthesia.Abstract:Toevaluatepainresponsestointratesticularandsubscrotalinjectionofthreelocalanaes-theticsandtheirefficacyduringcastrationarandomizedcontrolledstudywasconducted.Ingroupsof20piglets,procaine(2%),lidocaine(2%),ormepivacaine(2%)wereadministeredsubscrotalandintratesticularlyintwodifferentdosages:0.5mLoftheoriginalsubstancesorthemaximumrec-ommendeddosageaccordingtobodyweightdilutedwithisotonicsalinetoavolumeof0.3mLpereachinjectionsite.Twoplacebogroupsreceivedtheequivalentvolumeofisotonicsaline.Acontrolgroupwasinjectedintramuscularlywith0.5mLisotonicsalineforinjectionpaincomparison.Electroencephalographicchanges,respiratoryrate,heartrateanditsvariability,bloodpressure,andnocifensivemovementswereassessedinsuperficialisofluraneanaesthesia.WhileEEG-changesandlinearmeasuresofheartratevariabilitydidnotappearconclusive,thelowfrequency/highfrequency(LF/HF)ratiocorrespondedbestwiththeotherpainindicatorsrecorded.Theinjectionof0.3mLdilutedlocalanaestheticperinjectionsiteelicitedsignificantfewersignsofpaincomparedtointramuscularinjectionofsaline.However,painreduction,butnotcompletepainelimination,duringcastrationcouldonlybeachievedwith0.5mLofthe2%localanaestheticsperinjectionsite,wherebylidocaineandmepivacainewerethemosteffective.Keywords:castration;piglet;localanaesthesia;painassessment;heartratevariability(HRV);lidocaine;procaine;mepivacaine;minimalalveolarconcentration(MAC);isoflurane

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castration;piglet;localanaesthesia;painassessment;heartratevariability(HRV);lidocaine;procaine;mepivacaine;minimalalveolarconcentration(MAC);isoflurane Animals2022,12,2833.https://doi.org/10.3390/ani12202833https://www.mdpi.com/journal/animals

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Animals2022,12,2833 2of24

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Animals2022,12,2833 2of24 1.IntroductionSurgicalcastrationofmalepigletsisapainfulprocedure.Primarily,castrationpreventsboartaint,whichaffectstheconsumeracceptabilityofporkandporkproducts.Therefore,castrationisnotaproducer’sdecisionbutamarket-drivenchoice[1].In2010,representa-tivesofEuropeanfarmers,themeatindustry,retailers,scientists,veterinariansandanimalwelfareorganisationsvoluntarilycommittedtoabandoningpigletcastrationasof1January2018[1].WhilesurgicalcastrationisstillpracticedinmanyEUcountries,anaesthesiabe-comesmoreandmoremandatory.Since1January2021,inGermany,thesurgicalcastrationofmalepigsagedlessthaneightdaysisnolongerallowedwithouteffectivepainelimi-nation(GermanAnimalProtectionLaw(Tierschutzgesetz)).IncomparableregulationsofotherEuropeanUnion(EU)MemberStates,painattenuationisconsideredassufficient[2].Anaesthesiaforpigletscanbeeithergeneralorlocal.InGermany,generalanaesthesiainpigletsmaybeperformedbyinhalationofisofluraneorbyintramuscular(i.m.)injectionofketamineincombinationwiththeneurolepticdrugazaperone.Ketamine-azaperoneanaes-thesiamustbecarriedoutbyaveterinarian.Themaindisadvantageofthistechniqueistheprolongedrecoveryperiod,duringwhichthepigletsmustbeseparatedtopreventthemfrombeingcrushedbythesow.Since2020anaesthesiawithisofluraneforcastrationofpigletscanbeperformedbythefarmerandpermitsasafeandrapidanaestheticinductionaswellasabriefrecovery[3].Inhalationanaesthesiawithisofluranebythefarmerrequiresananaestheticdevice,whichdeliversisofluraneoverasetperiodoftime.Duetothelackofindividualadjustment,sufficientdepthofanaesthesiaisnotalwaysachieved[4,5].Addi-tionally,isofluraneisagreenhousegas,affectingtheenvironmentandtheoperator.Foreconomicandanimalwelfareaspectsanaesthesiaforroutinepigletcastrationshouldmeetthefollowingcriteria:shortinductionandrecoveryperiod,quickandeasytobeperformed,causeminimalstress,cost-effective,noresiduals,andalargetherapeuticrangeofuseddrugs[6,7].Therefore,localanaesthesiaseemstobeagoodoptiontoeliminatepainduringcastration.Topicalanaesthesiahasfailedtoprovidetherequiredanalgesiaforallsurgicalstepsofcastration[8],theeffectofinfiltrationlocalanaesthesiainconnectionwithpigletcastrationisthesubjectofcontroversialdiscussion.Localanaestheticsreversiblyblockvoltagegatedsodiumchannelsinhibitingneuralconduction.Accordingtotheirstructure,theyarecategorizedintoestersoramides,andintoshort-(e.g.,procaine),intermediate(e.g.,lidocaine,mepivacaine)—andlong-acting(e.g.,bupivacaine)compounds[9].DependingontheirpKalocalanaestheticsdifferintheironsetofaction,makinglidocaine(pKa=7.8)andmepivacaine(pKa=7.9)fasteractingthanprocaine(pKa=9.0)[10].ProcainehasbeenassessedbytheEuropeanMedicinesAgency(EMA)asalocalanaesthetic,whichcanbeusedinfoodproducinganimalsandiscurrentlytheonlysubstanceapprovedforpigsinGermany.LidocainehasbeenlicensedinswineforcutaneousandepilesionalusesinceNovember16,2020[11].However,injectablelidocaineandmepivacainecanonlybeusedforpigletsinaccordancewiththecascaderule.Lidocaineisuptotwiceaseffectiveasprocaine[12].Studiesontheefficacyofbothsubstancesduringcastrationofpigletshaveshownapainreducingeffect[6,13–17].Theinjectionitselfwasrepeatedlyfoundtoresultinpainreactions[18–21].Directcomparisonofstudyresultsisalmostimpossible,astheinjectionsite,injectedvolumeanddrugconcentrationdiffer,asdothevariablesusedforpaindetection.Intworecentstudiestheeffectoffourlocalanaesthetics(4%procaine,2%lidocaine,0.5%bupivacaine,2%mepivacaine)wascompared[18,22].Itwasshownthatallfourlocalanaestheticsreducedsignsofnociceptionduringcastration,butitwasalsodemonstratedthattheintratesticularinjectioncausedvisiblenociceptionindicatedbyincreasedlimbmovements[18].Theevaluationofnocifensivemovementsforpainrecognitionisusefulinsevereacuteevents[23].Sincenociceptivewithdrawalresponsescanvarybetweenpigs,itisreasonabletolookatbehaviourandphysiologyincombina-tiontofullyassesstheimpactofapainfuleventontheindividual[23].Parameters,likerespiratoryrate(RR)andheartrate(HR),bloodpressure(BP)andprocessedEEGvariables

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Animals2022,12,2833 3of24

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Animals2022,12,2833 3of24 likeNarcotrend-Index(NI),totalpower(PTOT),medianfrequency(MF)and95%spectraledgefrequency(SEF95),areobjectivelymeasurableandmostoftheparametersmentionedwerealreadyusedinnumerousstudiestoidentifypainreactionsinpigs[6,15,18,21,24,25].PhysiologicalresponsesofHRandBPresultfromactivationoftheautonomousnervoussystemandmaybetriggeredbystressfromhandlingandrestraining[26].Toassociatephysiologicalreactionswithnociceptionduringcastrationgeneralanaesthesiamodelscanbeused[6,18].Heartratevariability(HRV)isapromisingclinicaltooltoevaluatetheautonomicresponseofdifferentstressorsinpigs[27,28].Itindicatesthevariationinthetimeintervalbetweenconsecutiveheartbeatsandrepresentstheinterplayoftheparasympatheticnerves,whichslowHR,andthesympatheticnerves,whichaccelerateit.Arecentsystematicreviewconcluded,thatHRVisagoodmeasureofautonomicreactivitytonociceptivestimulationinman[29].InvestigationsintoHRVtoprovepainreactionsinpigcastrationwerealreadyconducted[30,31].Themainaimofthestudywastoassessthesuitabilityoflocalanaesthesiaforpigletcastrationunder8daysofagefulfillingtherequirementsoftheGermanAnimalProtectionLawwithrespecttothecastrationprocedureandpainofintratesticularandsubscrotalinjection.Inordertoreducestress-inducedresponsescausedbyhandlingandrestrain-ingpigletswereheldinastandardized,minimumalveolarconcentration(MAC)-basedsubanaestheticisofluraneanaesthesia.Ourmainhypothesiswasthatlocalanaesthesiawithmepivacaine,lidocaineorpro-cainewillleadtosignificantreductioninSEF95%changesinresponsetocastration.Secondly,wehypothesisedthattesticularinjectionleadstomorenocifensiveresponsesthani.m.injectionandthirdly,localanaesthesiawithlidocaineandmepivacaineleadstolessnocifensiveandautonomicresponsestocastrationthanprocaine.2.MaterialsandMethodsThisstudywasreviewedandapprovedbytheethicalcommitteeforanimalexperimen-tationoftheFederalStateOfficeforConsumerProtectionandFoodSafetyofLowerSaxony,Germany(33.9-42502-04-19/3218).AllanimalprocedureswereperformedaccordingtotheGermanAnimalProtectionLaw(Tierschutzgesetz).2.1.AnimalsAllpigletsincludedinthisstudyweremale,twotosevendaysofage,andweighingonetothreekgofbodyweight.Theywereacquiredfromfourdifferentproducerfarms(MainStudy:FarmA:41piglets,FarmC:88piglets,FarmD:51piglets;nopigletfromFarmBwasincludedinthemainstudy)inLowerSaxony,Germany,andhavenotbeenpretreatedatthefarmoforigin.Nopigletwassubjectedtoteethclippingortaildocking.Healthstatusanddescendanceofbothtesticlesintothescrotumwasconfirmedbyaclinicalexamination.Pigletswithherniascrotalisoringuinaliswereexcluded.Aftercompletionofthestudythepigletswereraisedmotherlessinanartificialrearingsystem(rescuedeck).Basedonelectroencephalogram(EEG)responsetonociceptiondatafromKulka,etal.[32],anaprioripowercalculationresultedinasamplesizeof20pigletspergrouptodetecttheabsenceofachangeof20%inSEF95%withaneffectsizeof0.83,analphaerrorof5%andapowerof95%(G*Power3.1.9.4;HeinrichHeineUniversity,Düsseldorf,Germany).2.2.StudyDesignandProcedure2.2.1.Pre-TrialInapre-trial,distributionofalocalanaestheticafterintratesticularandintrafunicularinjectionwasevaluatedintenpigletsviacomputedtomographyAnaesthesiawasinducedviaafacemaskwith5vol%isofluranein100%oxygenandafreshgasflowof4Lmin 1.AfterreachingasufficientdepthofanaesthesiathepigletswereorotracheallyintubatedwithaMurphystylecuffedendotrachealTubeI.D.

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Animals2022,12,2833 4of24

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Animals2022,12,2833 4of24 2.5mm(Rüschelit®SuperSafetyClear,2.5mm,TeleflexMedicalSdn.Bhd.,Kamunting,Malaysia).Accordingtopositioningincommercialcastrationracksthepigletswerefixedinsupinepositioninaradiolucentfoampositioningdevice,sothatthetesticleswereeasilyaccessible.Asasurrogateforthecorrespondingvolumeofalocalanaesthetic,a1:7dilutionofanon-ionic,water-solubleX-raycontrastmedium(iobitridol,Xenetix®300,Guerbet,Villepinte,France)withlidocainehydrochloride(Lidor®20mg/mL,WDT,Garbsen,Germany)wasusedinordertoobtainacomparableviscosityandpHvalueasthepurelocalanaestheticsolution.Ateachtesticularsideatotalvolumeof0.3mLwasinjectedwitha25G 5/8needle(Neoject®,DispomedWittoHG,Gelnhausen,Germany).Fivedifferentinjectiontechniqueswereinvestigated:intrafunicular(0.3mL—palpationandfixationofthefuniculuswithonehandwhileinjectionwasperformedinanapprox.45 anglefromcaudalwiththeotherhand),intratesticular(0.3mL—fixationofthetesticlewithonehandwhileinjectionwasperformedina90 angle),subcutaneousinthesubscrotaltissue(subscrotal)(0.3mL—liftingaskinfoldwithonehandwhileinjectionwasperformedinanapprox.45 anglefromcaudalwiththeotherhand),acombinationofsubscrotal(0.15mL)andintrafunicularinjection(0.15mL)andacombinationofsubscrotal(0.15mL)andintratesticularinjection(0.15mL)wasperformed.Injectionswereperformedwithoutaspirationpriortoinjection.Twoanimalswereusedperinjectiontechnique.A64-multi-detector-rowCTscanner(PhillipsBrilliance64,PhilipsGmbH,Hamburg,Germany)wasusedforimageacquisition.CTscanswithaslicethicknessof0.64mmwereacquiredtwominutesafterinjectionandrepeated4,6,8and10minafterinjection.Acquiredimageswerevisuallyanalysedfordistributionofcontrastmedia.Forevalua-tionoftemporaldistribution,changesindistributionpatternovertimewereevaluatedvisually.2.2.2.MainStudyThemainstudywasdesignedasaplacebocontrolled,blinded,randomizedstudywith9parallelgroups(Table1).Randomizationwasperformedwiththeaidofwww.randomizer.org(accessedon11February2020).Table1.Treatmentgroups,injectedvolume,totaldoseandconcentrationofthelocalanaesthetic.

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erformedwiththeaidofwww.randomizer.org(accessedon11February2020).Table1.Treatmentgroups,injectedvolume,totaldoseandconcentrationofthelocalanaesthetic. GroupP0.3P0.5L0.3L0.5M0.3M0.5S0.3S0.5IM drugprocaine2%procaine2%lidocaine2%lidocaine2%mepivacaine2%mepivacaine2%saline0.9%saline0.9%saline0.9% volumepersite(mL)0.30.50.30.50.30.50.30.50.5 totaldose5mgkg 1dilutedinsaline40mgundiluted4mgkg 1dilutedinsaline40mgundiluted0.4mgkg 1dilutedinsaline40mgundiluted totalvolume(mL)1.221.221.221.220.5 Thethreelocalanaestheticsprocaine(Procamidor®20mg/mL,WDT,Garbsen,Ger-many),lidocaine(Lidor®20mg/mL,WDT,Garbsen,Germany)andmepivacaine(Mepidor®20mg/mL,WDT,Garbsen,Germany)andtheplacebo(NaCl0.9%,B.BraunMelsungenAG,Melsungen,Germany)werecomparedeachin2differentvolumesanddoses.Accordingtotheresultsofthepreliminaryinvestigation,eachlocalanaestheticorsalinewasadminis-teredatfourlocations(intratesticularleft,subscrotalleft,intratesticularrightandsubscrotalright)withavolumeofeither0.5mLor0.3mLresultinginatotalvolumeof2mLor1.2mLperpiglet.Ingroupswhere0.5mLwereadministeredateachlocationundilutedlocalanaestheticataconcentrationof2%wasinjected(procaine—groupP0.5,lidocaine—groupL0.5,mepivacaine—groupM0.5andtheplacebogroupwithnormalsaline—groupS0.5).Ingroupswhere0.3mLwereinjectedateachlocationthemaximallyrecommendeddoseaccordingtosummaryofproductcharacteristics”(SPC)(https://vetidata.de/accessed11February2020)wascalculatedforeachindividualpigletanddilutedwithnormalsaline

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Animals2022,12,2833 5of24

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Animals2022,12,2833 5of24 toatotalvolumeof1.2mL(procaine—groupP0.3,lidocaine—groupL0.3,mepivacaine—groupM0.3andplacebonormalsaline—groupS0.3).Themaximallyrecommendeddoseswere5mgkg 1forprocaine,4mgkg 1forlidocaineand0.4mgkg 1formepivacaine.Thedoseformepivacainewascalculatedfromadosestatedforhorses.Tocomparethenociceptiveresponsecausedbyintratesticularandsubscrotalinjectionwithacommonclin-icalnociceptivestimulusonegroupwasincludedthatreceivedanintramuscularinjectionof0.5mLnormalsaline(groupIM).AnaesthesiaandInstrumentationAnaesthesiawasinducedbymaskwith5vol%isoflurane(IsofluranBaxter®,BaxterDeutschlandGmbH,Unterschleißheim,Germany)in100%oxygenwithaflowof4Lmin 1.Uponlossofconsciousnessa4Frfeedingtube(ErnährungssondeCH4,B.BraunMelsun-genAG,Melsungen,Germany)wasinsertedintotheventralmeatusnasitodetermineinspiratoryandexpiratoryisofluraneconcentrationandrespiratoryratewiththemulti-gasmoduleofamultiparameteranaesthesiamonitor(DatexOhmedaS5,GEHealthcareFinlandOY,Helsinki,Finland).IsofluranewasreducedtomaintainanFeISOof1Vol%[representing0.8MACdeterminedinasimilarpopulationofpiglets[33].Twoattemptsweremadetocatheterizethesaphenousarterywithanovertheneedlecatheter(Introcan®22G,B.Braun,Melsungen,Germany).Invasivemeasurementofarterialbloodpressureandpulseratewasperformedwiththemultiparameteranaesthesiamonitorandapressuretransducer(MeritransDTXPlus®DisposableTransducer,MeritMedicalGmbH,Eschborn,Germany)connectedviafluidfilledlowcomplianceextensionlinestothearterialcatheter.Nointravenousfluidsweregiven.Incaseofunsuccessfulcatheterization,theprocedurewascontinuedwithoutinvasivemeasurementofbloodpressureandpulserate.Adhe-siveelectrodeswereattachedtoeachlegtorecordaleadIIelectrocardiogram(Televet®100.Rösch&AssociatesInformationengineeringGmbH,FrankfurtamMain,Germany).RecordingoftheEEGsignalwasperformedasdescribedbyWaldmann,etal.[21]withtheNarcotrend®-Compact-MonitorVersion5.0.Finally,thepigletswereplacedindorsalrecumbencyaccordingtopositioninginacastrationcradle.Animalswerewarmedwiththeaidofaheatinglamptomaintainbodytemperatureabove38 C.Afterinstrumentationandanequilibrationperiodof10minataFeISOof1Vol%,theinjectionofthetestdrugswasperformed.InjectionTechniqueInGroupIMoneintramuscularinjectionof0.5mLsalinewasperformedwitha18G 2”canula(BDMicrolance3,BDGmbH,Heidelberg,Germany)behindthebaseoftheear.ThepigletsinGroupIMwererecoveredwithoutcastration.Inallothergroupshalfofthetotalinjectionvolumewasdrawnupinasyringe,thescrotalskinwaspunctured,andhalfofthevolumewasadministeredintratesticularlyandhalfsubscrotalwhentheneedlewaswithdrawn(0.5mLor0.3mLofthesolutionperlocalisation).Injectionswereperformedineachpigletalwaysstartingontherightside,followedbytheleftside.Forintratesticularinjection,thetesticlewasfixatedwiththumbandindexfingerofonehandandtheinjectionwasperformedwitha25G 5/8needle(Neoject®,DispomedWittoHG,Gelnhausen,Germany)intothemiddleofthetesticlewithoutprioraspiration.CastrationThecastrationwasperformed5to15minmaximumafterintratesticularandsubscrotalinjection,dependingonthelossofsensitivityofthescrotalskin.Twoincisionsparalleltotheraphescrotiweremadeandthetesticleswereexteriorized.Bothtesticleswereremovedatthesametimebyuseofanemasculator.Toensureanadequatehaemostasiscompres-sionoftheemasculatorwasmaintainedfor20s.Meloxicam0.4mgkg 1(Melosolute®5mg/mL,CPPharma,Burgdorf,Germany)wasadministeredatleast20minbeforethesurgicalprocedurei.m.Afterdataacquisition,thepigletswererecoveredandplacedinanursingpen.

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Animals2022,12,2833 6of24

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Animals2022,12,2833 6of24 DataRecording NocifensivemovementsNocifensivemovementsduringinjectionateachlocationwerescoredas(0)—nomovement,(1)—minorpurposefulmovements(minormovementsofoneor2limbs)or(2)—majorpurposefulmovements(morethan2limbsorhead).Asanypurposefulmove-ment(movementassociateddirectlyrelatedtothestimulusduringinjectionorcastration)wasconsiderednotconformwiththeAnimalProtectionLaw,forstatisticalanalysisonlypurposefulmovementyesornowasdifferentiated.Allscoresfornocifensivemovementswereperformedlivebythesameinvestigatorblindedtotreatment.Oneminuteafterinjec-tionfollowedbyevery2minsensitivityofthescrotalskinintheareawheretheinjectionswereperformedwasexamined.Apeanclamp(Peha®instrumentPeanKlemme,Hartmann,HeidenheimGermany)equippedwitharubberhose-toavoidseveretissuetrauma-wasclampedtothefirstratchetlockfor1s.Castrationwasperformedifnoaversiveresponsewasnoted,oramaximumof15minhadpassed.Aperiodof5minafterinjectionwasalwaysawaitedtoensureanadequatetimeforonsetofactionoftheinjectate.Duringcastrationnocifensivemovementswerescoredasaboveforthetimepoints“skinincision”,“exteriorizationofthetesticles”and“emasculation”whichwereperformedwithanintervalof15s. RespiratoryrateandbloodpressureThefollowingtimepoints(Figure1)weredefinedandanalyzed:Baselineinjection(BL-INJ)valueswererecordedevery15soveraperiodof2minbeforeinjectionandthemeanwascalculated.Baselinecastration(BL-CA)valuewasassinglevaluerecordeddirectlybeforeskinincision.Ascardiorespiratorychangesinresponsetowardsashort-lastingnociceptivestimulusonlypersistforashortperiodthehighestvalueduringaone-minuteperiodafterstartofinjection(INJ-Max)andduringcastration(CA-Max)wererecorded.Aftercastration,respiratoryrateandmeanarterialbloodpressurewererecordedevery15sfor2min(POST-CA)andthemeanwascalculatedfromthe8individualvalues.Achangeof10%incardiorespiratoryparameterscomparedtobaselinewascalculatedforabsolutevaluesofrespiratoryrate,heartrateandMAP,asthisamountofchangewouldimplypresenceofnociceptioninaclinicalsetting.

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mparedtobaselinewascalculatedforabsolutevaluesofrespiratoryrate,heartrateandMAP,asthisamountofchangewouldimplypresenceofnociceptioninaclinicalsetting. Figure1.Timelineoftheexperimentalprocedure.Dottedarrowsindicatetimesegmentswithvariablelength.Abbreviationsofanalyzedtimepointsaregreen. HeartratevariabilityAnECGwasrecordedwiththeTelevet®100,SoftwareVersion6.2.0(EngelEngineeringServicesGmbH,Heusenstamm,Germany)andRRintervalsectionswereextractedandthefollowingtimeperiodsdefinedforanalysis:BL-INJebaseline2minbeforeinjectionINJe—1minafterstartofinjectionBL-CAe”baseline2minbeforecastration(skinincision)CAe—castration,startofskinincisionuntil20safterstartofemasculationPOST-CAepostcastration,2minafter“CAe”.Thesuperscript“e”wasaddedtodifferentiatethesetimeperiods/episodesfromthetimepointsforMAPandrespiratoryrate.ThisalsoreferstoEEGdata.

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Animals2022,12,2833 7of24

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Animals2022,12,2833 7of24 TheRR-intervalsectionsweretransferredtoKubios®RVversion2.0(BiosignalAnalysisandMedicalImagingGroup,UniversityofKuopio,Kuopio,Finland).TheECGrecordingswerecheckedvisually,errorswereeditedmanually,andthedatawereanalysedafterwards.TimedomainHRVanalysisincludedmeanheartrate,standarddeviationoftheheartrate(SDHR),meanRR-Interval(MeanRR)andthestandarddeviationoftheRR-intervals(SDRR).TheLF/HFratiowascalculatedduringfrequencydomainanalysiswiththelowfrequencyband(LF)definedat0.02–0.15Hzandthehighfrequencyband(HF)accordingtorespiratoryrateat0.25–1.4Hz. EEGVariablesThefollowingvariableswererecordedandvaluesforevery5stimeintervalwereexported:Narcotrend-Index(NI),totalpower(PTOT),medianfrequency(MF)and95%spectraledgefrequency(SEF95).Analysedtimepoints(epochs)were: BL-INJe:baselinebeforeinjectionfor2min INJe:1minafterstartofinjection BL-CAe:baseline2minbeforecastration(skinincision) CAe:castration,startofskinincisionuntil20safterstartofemasculation. POSTCAe:postcastration,2minaftertheendofemasculation2.3.StatisticalAnalysisDatawereanalysedusingRversion3.4.4.(TheRFoundationforStatisticalCom-puting,Vienna,Austria).ThedistributionofdatawastestedwithShapiro–WilkTestandHistograms.Normallydistributeddatawereanalysedwithingroupswithapairedt-testandanANOVAforrepeatedmeasurements.WilcoxonsignRanktestwasusedtocom-parenonparametricdatawithingroups.InbetweengroupsanANOVAforindependentvariableswasusedfornormallydistributeddataandtheKruskal–Wallis-TestfollowedbyWilcoxon’stwosampletestincaseofsignificantdifferenceswasusedtocomparenonpara-metricdata.Levelofsignificancewassetat5%.StatisticalanalysisofordinaldatawasperformedwiththeChi-square-testfollowedbyFisher’sexacttestincaseofcellvaluesbelow5.3.Results3.1.Pre-TrialThe10pigletsusedwerebetween2and7daysoldandhadabodyweightbetween1.9and2.4kg.Figure2A-Cshowexamplesofthedistributionofthesurrogatewithin2minafterinjectionattherespectivesites.Afterintratesticularinjection(n=8)thesurrogatewasvisibleinthetestisandalongthespermaticcordintotheabdominalcavity.Onlyinonepigletthelocalanaestheticwasfalselyinjectedwhichresultedinamainlyintrascrotaldistribution.Intrafunicularinjectionresultedinlessconsistentdistributioninthespermaticcord,inthescrotumandintheabdominalcavityorinthesubcutaneoustissueintheinguinalarea.Allsubscrotalapplications(n=12)resultedinasubcutaneousdepot.Inalllocalizationsthedistributionofthesurrogatedidnotchangeovertime.3.2.MainStudyMeanageofthe170pigletsincludedinthisstudywas4dayswithameanbodyweightof1.95 0.31kg.3.2.1.EEGBaselinevaluesforNIbeforeinjection(BL-INJe)werecomparableamongallinvesti-gationgroups(Figure3).ChangesinEEGparametersNI,PTOT,MF,SEF95inresponsetoinjectionorcastrationdidnotfollowaconsistentpatternanddidnotshowanysignificantdifferencesamongthegroups.

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Animals2022,12,2833 8of24 Figure2.3D-reconstructionoftheCT-imagesshowingthedistributionofthesurrogateafterintrates-ticular(A),intrafunicular(B)andsubscrotal(C)injection.Orangecapitalletters:A-anterior,L-left,R-right,H-head,F-feet.3.2.2.ResponsetoInjection NocifensivemovementsComparedtointramuscularinjection,intratesticularinjectioningroupP0.5resultedinmarkedlystrongernocifensivemovements(score2).Intratesticularinjectionofthesmallervolumeof0.3mLledtostatisticallysignificantlylessnocifensivemovementsthantheadministrationofavolumeof0.5mLevenincomparisontothecontrolgroup(Figure4).

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lervolumeof0.3mLledtostatisticallysignificantlylessnocifensivemovementsthantheadministrationofavolumeof0.5mLevenincomparisontothecontrolgroup(Figure4). Animals2022,12,2833 9of24 Figure3.BaselineNarcotrendIndex.BaselinebeforeinjectionofNarcotrendIndex(NI)forgroupsP0.3,P0.5,L0.3,L0.5,M0.3,M0.5,S0.3,S0.5andIM.Theboxcoverstheinterquartileinterval,where50%ofthedataarefound.Thewhiskersindicateminimumandmaximumandthebandinsidetheboxrepresentsthemedian. Figure4.NocifensivemovementstointratesticularinjectionforthegroupsIM,P0.3,P0.5,L0.3,L0.5,M0.3,M0.5,S0.3andS0.5.Numberofpigletswithascoreof0aredisplayedingrey,ascoreof1inlightblueandascoreof2indarkblue.The#representsasignificantdifferencecomparedtogroupIM. CardiorespiratorychangesNosignificantdifferenceswereseenbetweenBL-INJandINJ-Maxafterinjectionoflocalanaestheticinrespiratoryrate,heartrateandRR-intervals,butgroupswithlargervolumesadministeredshowedahigherdifferencecomparedtoIM.DifferencevaluesinMAPwerestatisticallysignificantlygreaterinP0.3andS0.5comparedtoIM.A10%increaseinrespiratoryratewasseeninallgroupsbutIMandP0.3andinMAPforalltreatmentsbutIM.L0.3andM0.3.Therewasnogroupwithanincreaseinheartrateabove10%.ForP0.5differenceofSDHRwassignificantlyhighercomparedtoIManddifferenceofSDRRwashighercomparedtoIMandM0.5whereastheLF/HF-ratioincreasedstatisticallyhigherduringinjectioningroupP0.5andS0.5comparedtoIM(Figure5A–G). Animals2022,12,2833 10of24 Figure5.Cont.

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Animals2022,12,2833 11of24 Figure5.(A–G)Difference(D)inrespiratoryrate,meanarterialbloodpressure,heartrate,RR-Interval,SDHR,SDRRandLF/HF-ratiobetweenbaselineandinjectionintheexperimentalgroups.Bardiagramsof(A)meandifferenceinrespiratoryrate[breathsminute 1]betweenbaselineandmaximumrespiratoryrateduringinjection,(B)meandifferenceinmeanarterialbloodpressure(MAP)[mmHg]betweenbaselineandmaximumMAPduringinjection,(C)meandifferenceinheartrate[beatsminute 1]betweenbaselineandduringinjection,(D)meandifferenceinRR-Interval[ms]betweenbaselineandduringinjectionwiththemeanatthetopofthebarandthewhiskersasstandarddeviation.Boxplotsof(E)differenceinstandarddeviationofheartrate(SDHR)[beatsminute 1]betweenbaselineandduringinjection,(F)differenceinstandarddeviationofRR-Interval(SDRR)[ms]betweenbaselineandduringinjectionand(G)differenceinlowfrequency/highfrequency(LF/HF)ratiobetweenbaselineandduringinjection.Theboxesrepresentingthefirstandthirdquartileandthewhiskersrangingfromminimumtomaximum.Themedianisindicatedbythebandinsidethebox.Differentletters(a,b,c)showsignificantdifferencesbetweentheexperimentalgroups(p<0.05).Thesuperscript“e”differentiatestimeperiods/episodes(“e”)fromtimepoints(MAPandrespiratoryrate).Theredasterisk(*)marksanincreaseabove10%frombaseline.3.2.3.ResponsetoCastration OnsetofactionMedian(Minimum;Maximum)valuesofthetimeperiodbetweentheintratesticu-lar/subscrotalapplicationandcastrationforgroupsP0.3,P0.5,L0.3,L0.5,M0.3,M0.5,S0.3andS0.5were5(5;15),5(5;7),5(5;10),5(5;5),11(5;15),5(5;5),11(5;15),and5(5;15)minutes,respectively(Figure6). NocifensivemovementsGroupP0.5,M0.5andL0.5resultedoverallinlessnocifensivemovementsduringcastrationcomparedtoplacebogroups(S0.3andS0.5)andM0.3.DuringemasculationlessnocifensivemovementswereseeningroupsM0.5andL0.5comparedtoP0.5(Table2).Table2.Numberofpigletsshowingnocifensivemovementsduringskinincision,exteriorizationoftesticlesandemasculation(n=20/group).

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dL0.5comparedtoP0.5(Table2).Table2.Numberofpigletsshowingnocifensivemovementsduringskinincision,exteriorizationoftesticlesandemasculation(n=20/group). P0.3P0.5L0.3L0.5M0.3M0.5S0.3S0.5 skinincision6a,c1a9b,c4a12c,d1a10c,d14d exteriorization10b1a4a2a16c1a18c15b,c emasculation16c8b9b1a19c3a18c17c noreaction21061501722 a,b,c,d:Differentletters(a,b,c,d)showsignificantdifferencesbetweentheexperimentalgroups(p<0.05).

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Animals2022,12,2833 12of24 Figure6.Scatterdotplotofthetimeperiodinminbetweentheintratesticular/subscrotalapplicationandcastrationforgroupsP0.3,P0.5,L0.3,L0.5,M0.3,M0.5,S0.3andS0.5.Theblackbarrepresentsthemedian. CardiorespiratorychangesOverallthefewestchangesofcardiorespiratoryvariableswerenotedingroupsL0.5andM0.5duringcastration.ThedifferenceinrespiratoryratewashigherinS0.5comparedtoL0.5(Figure7A).Furthera10%increaseinabsolutevaluesofrespiratoryratewasobservedinM0.3,S0.3andS0.5forBL-CA-CA-MaxandforM0.3,S0.3andS0.5forBL-CA-POST-CA.DifferenceinMAPwassignificantlyloweringroupsL0.5andM0.5comparedtoS0.3,S0.5andP0.3andingroupL0.5alsocomparedtoM0.3(Figure7C).A10%increaseinabsoluteMAPvalueswasobservedinP0.3,M0.3,S0.3andS0.5fromBL-CAtoCA-MaxandfromBL-CAtoPOST-CA.L0.5wastheonlygroupwithadecreaseinheartrateduringcastration.Therewasnogroupwithanincreaseinheartrateabove10%duringoraftercastration.DifferenceinheartratewasloweranddifferenceinRR-intervalswerehigheringroupsP0.5,L0.3,L0.5andM0.5comparedtoS0.3andS0.5.IngroupsP0.3andM0.3differenceinheartratewashighercomparedtogroupM0.5andRR-intervalsshortercomparedtoM0.5andL0.5(Figure8A,C).ThedifferenceinSDHRandSDRRwerelowestforM0.5andL0.5.GroupsS0.3,S0.5andM0.3hadsignificantlyhigherdifferencesinSDHRcomparedtoM0.5,L0.3andL0.5anddifferenceinSDRRcomparedtoM0.5andL0.5(Figure8E,G).ThedifferenceinLF/HFratiowaslowestforL0.5andM0.5withastatisticallysignificantdifferencetoS0.3,S0.5andM0.3.IngroupL0.5thedifferencewasalsosignificantlylowercomparedtoP0.3andL0.3(Figure8I).

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orL0.5andM0.5withastatisticallysignificantdifferencetoS0.3,S0.5andM0.3.IngroupL0.5thedifferencewasalsosignificantlylowercomparedtoP0.3andL0.3(Figure8I). Animals2022,12,2833 13of24 Figure7.(A–D)Mean(SD)difference(D)inrespiratoryrateandmeanarterialbloodpressurebetweenbaselineandduringcastrationandbetweenbaselineandpostcastrationintheexperimentalgroups.Bardiagramsof(A)meandifference(D)inrespiratoryrate[breathsminute 1]betweenbaselineandmaximumrespiratoryrateduringcastrationand(B)betweenbaselineandpostcastrationaswellasbardiagramsof(C)meandifferenceinmeanarterialbloodpressure(MAP)[mmHg]betweenbaselineandmaximumMAPduringcastrationand(D)betweenbaselineandpostcastration.Thebarindicatesthemeanandstandarddeviationisrepresentedbythewhiskers.Differentletters(a,b,c,d,e)showsignificantdifferencesbetweentheexperimentalgroups(p<0.05).Theredasterisk(*)marksanincreaseabove10%frombaseline. Animals2022,12,2833 14of24 Figure8.Cont.

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nificantdifferencesbetweentheexperimentalgroups(p<0.05).Theredasterisk(*)marksanincreaseabove10%frombaseline. Animals2022,12,2833 14of24 Figure8.Cont. Animals2022,12,2833 15of24 Figure8.(A-J)Meandifferenceinheartrate,RR-Interval,SDHR,SDRRandLF/HF-ratiobetweenbaselineandduringcastrationandbetweenbaselineandpostcastrationintheexperimentalgroups.Bardiagramsof(A)meandifferenceinheartrate[beatsminute 1]betweenbaselineandduringcastrationand(B)betweenbaselineandpostcastrationaswellasbardiagramsof(C)meandifferenceinRR-Interval[ms]betweenbaselineandduringcastrationand(D)betweenbaselineandpostcastration.Thebarindicatesthemeanisdisplayedasthetopofthebarandstandarddeviationisrepresentedbythewhiskers.Boxplotsof(E)differenceinstandarddeviationofheartrate(SDHR)[beatsminute 1]betweenbaselineandduringcastrationand(F)betweenbaselineandpostcastra-tion,(G)differenceinstandarddeviationofRR-Interval(SDRR)[ms]betweenbaselineandduringcastrationand(H)betweenbaselineandpostcastrationand(I)differenceinlowfrequency/highfrequency(LF/HF)ratiobetweenbaselineandduringcastrationand(J)betweenbaselineandpostcastration.Theboxesrepresentthefirstandthirdquartileandthewhiskersrangefromminimumtomaximum.Themedianisindicatedbythebandinsidethebox.Differentletters(a,b,c,d)showsignificantdifferencesbetweentheexperimentalgroups(p<0.05).Thesuperscript“e”differentiatestimeperiods/episodes(“e”)fromtimepoints(MAPandrespiratoryrate).

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Animals2022,12,2833 16of24 3.2.4.Post-CastrationPhase CardiorespiratorychangesDifferencesinrespiratoryratebetweenBL-CAandPOST-CAwerelowerinP0.5,L0.3,L0.5andM0.5comparedtoM0.3,S0.3andS0.5(Figure7B).GroupsP0.5,L0.5andM0.5showedstatisticallysignificantlowerdifferencesinMAPcomparedtoallothergroupsexceptL0.3(Figure7D).ThedifferencesinheartratewerelowerandinRR-intervalsgreateringroupsL0.3,L0.5andM0.5comparedtoS0.3andS0.5.ThedifferenceinheartrateincreasedfurtheraftercastrationandtheRR-intervalsbecameshorterinbothprocainegroups.3.3.MissingDataCatheterplacementforarterialbloodpressuremeasurementwassuccessfulinGroupP0.3inn=14,ingroupP0.5inn=14,ingroupL0.3inn=14,ingroupL0.5inn=14,ingroupM0.3inn=16,ingroupM0.5inn=16,ingroupS0.3inn=16,ingroupS0.5inn=15,andingroupIMinn=15piglets.Duetotechnicalproblemsandmovementofpigletsduringinjectionandcastrationwithdisplacementofthenasalfeedingtube,measurementofrespiratoryratewasnotdeterminedduringcastrationingroupL0.5inn=1,duringexteriorizationoftesticlesingroupS0.3inn=1andingroupS0.5inn=1,duringemasculationingroupS0.5inn=1,andpostoperativelyingroupP0.5inn=1aswellasinonepigletingroupIMduringthewholeprocedure.Furthermore,ECGrecordingsfromtwoanimalsingroupP0.3anddataforbaselinecastration,castrationandPOST-CAeinoneanimalingroupM0.3wereexcludedduetotechnicalerrorsandartifacts.3.4.SupplementraryMaterialAbsolutevaluesforrespiratoryrateandmeanarterialbloodpressureforthetime-pointsbaselineinjection(BL-INJ),injection(INJ-MAX),baselinecastration(BL-CA),cas-tration(CA)andpostcastration(POST-CA)aswellasabsolutevaluesforheartrate,RR-Interval,SDHR,SDRRandLF/HF-ratioforthetimepointsbaselineinjection(BL-INJe),injection(INJe),baselinecastration(BL-CAe),castration(CAe)andpostcastration(POST-CAe)canbefoundinthesupplementarymaterials.4.DiscussionOurmainhypothesiswasthatlocalanaesthesiawithmepivacaine,lidocaineorpro-cainewillleadtosignificantreductioninEEGresponses(SEF95%changes)duringcastra-tion.Atthebeginningoftheexamination,theNIcouldconfirmthatallpigletswereinaconsistent,lightplaneofanaesthesiaacrossthetreatmentgroups.However,incontrasttoobservationsmadebyWaldmannetal.[21]andHagaandRanheim[6]inpigsaswellasOttoandMally[34]andOtto[35]insheep,wewerenotabletodetectconsistentarousalre-actionsinresponsenoxiousstimulibydesynchronisationorsynchronisationwithincreaseordecreaseinEEGparameters.Onecausemaybethetime-delayeddisplayofthestagesandindexvaluesoftheNarcotrend®monitor.Pilgeetal.[36]describedatimedelayof30–65sandKlesperetal.[37]of20–175s.Inthepresentstudy,stimuliweresetinaperiodof60sto120s;accordingly,analmostsimultaneousadjustmentofthedisplayisnecessaryforinterpretation.Inotherstudiesindogs[38],cattle[39]andpigs[25,40]consistentEEGchangescouldalsonotbeobservedafterpainfulstimuli.Therefore,EEGindicesarenotfurtherdiscussed.IndependentlyoftheEEGexaminations,itwaspossibletodetectthepainfulinterventionswiththehelpoftheothervariablescollected.Oursecondandthirdhypothesis,thattesticularinjectionleadstomorenocifensiveresponsesthani.m.injectionandthat,localanaesthesiawithlidocaineandmepivacaineleadstolessnocifensiveandautonomicresponsestocastrationthanprocainewereconfirmed.4.1.Sub-MACIsofluraneConcentrationforPainAssessmentInthisstudy,thebasisforstudyingthepainresponsesofthepigletsduringcastra-tionwasaconstant,reproducible,superficialanaesthesiathatpreventedstress-induced

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Animals2022,12,2833 17of24 responsesduetohandlingandfixation,butstillallowednociceptioninducedchangesinvitalsignstobedetected,aswellasnocifensivemovements.Itwasessentialtokeepallpigletsunderthesameconditionstoavoidbiasintheresults.Toquantifyandstandardizedepthofanaesthesia,theconceptofminimumalveolarconcentration(MAC)isusefulinanimalstudiesbecauseofasmallinter-individualvariance[41].TheMACofinhaledanaestheticsisdefinedasthealveolargasconcentrationatsealevelrequiredtopreventpurposefulmovementsin50%ofpatientsinresponsetosurgicalincision.AgehasbeenshowntoaffectMAC[42].Therefore,MACdeterminedinapre-trialinpigletsofthesameagegroup(age:2–7daysoflife)wasusedtodefinesuperficialanaesthesia.Inthispre-trial,MACof1.2 0.3vol%isofluranewasdeterminedbyelectricstimuli[33],asimilarvalue(1.20 0.43vol%)wasdeterminedbytheclampingtechniquein2–17dayoldpiglets[43].AhigherMACvalueof1.41–2.00%isgivenbythemanufactureroftheisofluraneprepara-tionused[44].Toallownociceptionassessment,end-tidalconcentrationwasadjustedto0.8MAC,whichcorrespondsto1.0vol%isofluraneaccordingtoourpreviousstudy[33].TheEEGrecordingsshowedthatallanimalswereatcomparabledepthsofanaesthesiaatbaseline.Subanaestheticisofluraneconcentrationshavelittleornoantinociceptiveef-fect[45]andwithdrawalmovementstriggeredbynoxiousstimulationcanbeelicitedatsub-MACanaestheticconcentrations[46],whilereactionsduetohandlingaresuppressed.Tomeasurenociceptionofpigletsduringinjectionandcastration,Salleretal.[18]choseindividualMACisofluraneanaesthesiafortheirminimalanaesthesiaprotocoldeterminedbysinglereactionstoatoepinch.Althoughtheappliedmeanend-tidalconcentration,whichwasfinallyused,wasnotmentioned,itseemstobehigherthantheMACwechose,astheystartedwith1.69 0.3vol%withaflowof3L/minoxygenandadaptedanaestheticdepthinstepsof0.2%.Similartoourownobservationstheycouldseeinbloodpressureandheartrateaswellaslimbmovements,associatedwithnociceptivestimuli.HagaandRanheim[6]madecorrespondingobservationsusing1.4xMACofhalothaneanaesthesia.4.2.InjectionofLocalAnaestheticsandOnsetofActionThelocalanaestheticswereinjectedintothetestisandsubscrotaltissue.There-sultsofthepreliminarystudydemonstrated,thattheapplicationatthesesitesshowedawidespreaddistributionofamixtureofcontrastmediumandlidocainebeneaththescrotalskin,inthetestis,andinthespermaticcord[47].However,radiolabelledlidocaineinjectedintothetestisdidnotreadilydiffusethroughthetunicavaginalisandintothecremastermuscle.Therefore,RanheimandHaga[48]assumedincompleteblockofthesensoryinnervationresultingininsufficientanalgesiaduringcastration.Thisisconfirmedbytheresultsofourstudy,sinceregardlessofthetypeanddosageofthelocalanaestheticused,acompleteabsenceofchangesintheobservedpainindicatorscouldnotbeachieved,evenintheM0.5groupbeingthemosteffectivegroupwiththeleastdeviationsfromthebaselinevalues.Whenusinglocalanaesthetics,themaximumamountthatcanbeappliedtoapigletmustbeconsideredtoavoidtoxicity[9].Awidevarietyofdosesandconcentrationsoflocalanaestheticsusedinpigletcastrationhavebeendescribed,buttoxicsideeffectswererarelymentioned.Nevertheless,inthepresentstudy,therecommendeddrugassociatedmaximumdosagesgivenbyVETIDATA,aweb-basedveterinaryinformationsystem(www.vetidata.de(accessedon11February2020),andaneasytohandlevolumeofcommerciallyavailableproducts(0.5mL)werecompared.Notoxicsideeffectswereobserved,despiteexceedingthemaximumrecommendeddosebyamultiplewhenusing0.5mLofthelocalanaestheticsperinjectionsite(2mLperpiglet).Duetothecloserelationshipbetweensystemictoxicityandtheplasmaconcentrationoflocalanaesthetics[49],themaximumplasmalevelisdecisivefortheonsetofsideeffects.Scottetal.[50]demonstratedthatthemaximumplasmaconcentrationoflidocaineoccurredbetween10and20minafterinjectioncomparing4injectionsites(intercostal,subcutaneousvaginal,lumbarepidural,subcutaneousabdominal).Duringthistime,however,theinfiltratedtestishasalreadybeen

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Animals2022,12,2833 18of24 removed,sothatcompleteabsorptioncouldnottakeplace,whichmayhelptoprotectthepigletfromsystemicsideeffects.Comparingthemedianonsetofactionofthelocalanaesthetics,nosignificantdiffer-encesexceptformepivacaineinthelowapplicationvolume(M0.3)couldbedetected,M03andS03hadthelongestmedianwith11min.Interestingly,quiteafewanimals,whichgotsaline0.9%,didnotshowanyaversivereactionstopinchingofthescrotalskinwithinthe15controlminutes.Despitethestandardizedprocedure,thelackofreactionmayhavebeencausedbytheinhalationanaesthesiaorreflectstheindividualvariationofpainperception[51].4.3.PainAssessmentduringInjectionWecomparedpaininducedreactionsduringintratesticular/subscrotalinjectionwithintramuscularinjection,becausethelatterisaroutinerouteofdrugadministrationinpigs.Therefore,theinflictionofsuchpainisgenerallyacceptedalsobytheGermanAnimalWelfareAct.Thediameterofthecanulaforthei.m.injectionswascomparabletoroutinelyusedcannulasinthefield.Toreducethepaincausedbyneedleinsertionintothetestis,asmallerneedlewasused,asrecommended[52,53].Painassociatedreactionswereonlyassessedinconnectionwithinjectionofthelocalanaestheticorsalinesolution.However,painresponsestothesolepunctureoftheskinandthetestismightalsobeimportantintheassessmentofanimalwelfareaspectsinpigletcastration.Regardlessofthelocalisationandthecontent,everyinjectionhascausednociception,butthereweremarkedvariationsinthepainresponse.Volunteersrecognizeddifferencesafterintradermalandsubcutaneousinfiltrationoffivelocalanaesthetics,whichwerenotrelatedtotheacidity,ionization,proteinbinding,sodiumchlorideconcentrationorosmolality,butarelationtolipidsolubilitywasassumed[54].Accordingly,theinjectionofmepivacainewasperceivedasmorepainfulthanlidocaine[54].Thecurrentresultsindicatemoreinfluenceoftheinjectedvolumethanlipidsolubility,sincenodifferenceswerefoundafterinjectionofmepivacaineorlidocaine.Infact,aftersmallerintratesticularandsubscrotalvolumesfewerpigsshowednocifensivemovementscomparedtothecontrolgroupinwhichthepigsreceived0.5mLsalinei.m.Atthesametime,RRaswellasMAProseabovethe10%ofbaselineinthosegroupswhichreceivedavolumeof0.5mLperinjectionsite,regardlessoftheagent.Anincreaseof10%frombaselinewasdefinedasapositiveresponsetotheinvasiveinterventionaccordingtoOttoetal.[55]andRoizenetal.[56].Additionally,althoughnotstatisticallysignificant,thepaininducedreactionsafterthelargervolumeofsalineledtostrongerresponses.Therefore,itisassumedthatthepaininducedreactioniscausedinparticularbythepressureinthetissue,asthevolumeofthetesticlescannotexpandduetothefirmlayers.Incontrast,Coutantetal.[57]couldnotobserveanydifferencesinvocalisationandforelegmovementsafterintratesticularapplicationof0,3mLor0.5mLprocaine2%.Accordingtopreviousstudies,procaineinjectioncausesthegreatestdiscomfort[13,19,58].TissueirritationduetothesignificantlylowerpHofprocaine(pHof3.7)comparedtolidocaine(pHof5.0)andmepivacaine(pHof5.5)cannotberuledoutasacause.ThesensationofnociceptionwasconfirmedbythechangesintheLF/HFratiooftheHRV.IncreaseinLFandLF/HFratioreflectssympatheticbaroreflexactivity[59,60],whileHFreflectsparasympatheticinfluencesandcorrespondstotheHRvariationdependingontherespiratorycycle(respiratorysinusarrhythmia)[61].TheincreaseintheLF/HFratioisaccompaniedbyanincreaseinMAP,oneofthemostsensitivenociceptiveindicatorsinpigs[25].Thesimultaneouslyobservedincreasesintimedomainindices(SDHR,SDRR)wereunexpectedaswithanincreasedheartrateacceleratedbythesympatheticnerves,timebetweenheartbeats(RRintervals)decreasedandlesstimeforvariabilityoccur,whichmeans,HRVshouldactuallydecrease.Additionally,theheartratedidnotchangeasexpected.Wecouldnoticeasmalldecreaseinheartrate(<3.5%frombaseline)aswellasanincreaseinRR-intervalsduringtheminuteafterstartofinjectionintheL0.3,M0.3andS0.3

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Animals2022,12,2833 19of24 group.Clementetal.[62]reportedsuddenandrelativelysustainedfallsinbotharterialpressure(upto56%frombaseline)andheartrate(upto30%frombaseline)afternoxiousdeepsomaticandnoxiousvisceralmanipulations.HagaandRanheim[63]alsoobservedadecreaseinthepulserateduringinjectionoflidocaineintothefuniculusspermaticusinpiglets,whileSalleretal.[18]reportedadecreaseinbloodpressureandheartrateinresponsetothecastration.Thisparadoxicaleffectistheresultofavasovagalresponse,wherebybradycardiaiscausedbyasuddenincreaseinvagalactivityandhypotensionresultsfromasuddenreductioninsympatheticactivityandrelaxationofarterialresistancevessels[64].AsHRVreflectstheactivityofthesympatheticandparasympatheticnervoussystems,changesintherecordedparametersinthosethreegroupscouldbeexpectedbyincreasingHRVduetotheprolongedRRinterval.However,SDHRandSDRRincreasedinallgroups.Burtonetal.[65]observedatemporaryincreaseintheheartrate(7.0 2.0%)afterinducingpainbyintramuscularorsubcutaneousinjectionsofhypertonicsalinewhichreturnedtobaselinewithin60safterinducingpainreflectingonlybriefarousalresponses,butcausinganincreaseintheLF/HFratioofHRV.Radeisen[31]alsocouldrelatechangesinfrequencydomainindicestointraoperativepainassociatednocifensivemovementsduringcastrationofboarsinthepubicregion,especiallyduringincisionoftheskinandthevaginalprocessandduringthetractionofthespermaticcord,wherebytimedomainHRVdidnotreflectthosesinglepainevents.UsingtheLF/HFratioRaueetal.[66]coulddetectnociceptivestimulationincatsinonly60spoststimulationepochmeasurements,whilelongertimeperiodsrevealedareadjustmenttothebasalvalues.Generally,short-termmeasuresofHRVrapidlyreturntobaselineaftertransientdisturbances[28],thereforetherecordingtimehasasignificantinfluenceespeciallyontimedomainvalues[30]andarapidrecoveryoftheheartrateaftertheinjectionmightbethereasonfortheriseinSDHRandSDRR.4.4.PainAssessmentduringCastrationIngroupM0.5(n=17;85.0%)thelowestnumberofanimalsshowednocifensivemovementsduringcastrationfollowedbygroupL0.5(n=15;75.0%).Evenineachoftheplacebogroupsthereweretwoanimals(10.0%)thatshowednowithdrawalmovementsduringcastrationpossiblyasaresultofisofluraneanaesthesia.Salleretal.[18]alsonoticedthreeoutofninepigletsofapositivecontrolgroup,whichreceivedsodiumchloride(withoutpainrelief)withoutlimbmovementsduringcastrationusingalightisofluraneanaesthesiamodel.AutonomicresponsestocastrationweremostpronouncedintheplacebogroupsaswellasintheM0.3groupduringcastration.Duetothehighdilutionofmepivacaine(dependingonbodyweightofthepiglet1:23–1:45),whichwasbasedonthetoxicitylimitforthehorse,asnocorrespondingvaluewasavailableforthepig,effectivelocalanaesthesiacouldhardlybeexpected.AnincreaseinRRandMAPofover10%abovethebaselinevaluewasnoted,whichwasnotseenintheothergroups,sothatamarkedperceptionofpainmustbeassumed.ThegroupsP0.5,L0.5andM0.5differedsignificantlyfromtheplacebogroupsindifferencesofMAP,HR,andRRintervalrecordedduringcastration,whileonlyintheL0.5grouptheHRslightlydroppedandconsequentlytheRRintervalsincrease.Nevertheless,SDHRandSDRRincreasedaccordingtothechangesduringtheinjection,butthiscouldberelatedtothealternatinginfluencesoftheparasympatheticandsympatheticnervoussystemsduringthedifferentstepsofcastrationandthepaininflictedinthesequenceofskinincision,exteriorizationofthetesticlesandemasculation.Especiallytheinfluenceoftheparasympatheticnervoussystemleadstorapidchangesintherangeofmilliseconds,whiletheeffectsofthesympatheticnervoussystemareratherslow(timescaleofseconds)[67].Besidesthedifferencesmentionedabove,L0.5andtheMepi0.5differedsignificantlyfromtheplacebogroupsinthedifferentialvaluesoftheLF/HFratio.Thissuggeststhat

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Animals2022,12,2833 20of24 bothsubstancesarebestatblockingsympatheticbaroreflexactivity,butonlyifitisgivenintheappropriatedosage.Comparedtobasallevels,theM0.5groupshowedtheleastchangesinHRvariabilityparametersandthereforeappearssuperiorinefficacytolidocaineandprocaine.4.5.PainAssessmentPostCastrationIntheimmediatepostoperativephase,thevaluesofRRandMAPingroupP0.3,M0.3,S0.3,andS0.5remainedatasimilarlevelorshowedafurtherincreasecomparedtothevaluesrecordedduringcastration,indicatingongoingnociceptionduetothelackofanalgesiaduringandaftercastration.Thisobservationalsoillustrates,thatdespitethetimelyadministrationoftheNSAID,acutepostoperativepainisnoteffectivelyeliminated.SDHR,SDRRandLF/RFratioreturnedtobaselinelevelsfrombeforecastrationinallgroups,accordingtothestatementofvonBorelletal.[28],thatshort-termmeasuresofHRVrapidlyreturntobaselineaftertransientdistress.Overall,duringcastration,thelowestdeviationsinautonomousresponsesfromthebaselinevalueswereobservedintheL0.5andM0.5groups,sothatthemosteffectivepainreductionisalsoassumedforthepostoperativephasewiththesetwoagents.4.6.LimitationsLimitationsofthisstudyincludethechangeinadministeredvolumeanddosebetweenthe0.3and0.5groupssothattheindividualeffectsofeitherdoseorvolumecannotbedifferentiatedbetweentreatments.Isofluranewasusedtofacilitateimmobilityofthepiglets.AsitinfluencedelectricalbrainactivityitmayhavebluntedtheEEGparametersdespitetheverylowconcentrationofisofluraneused.Moreover,arterialcannulisationwasnotpossibleinallpigletssothatdataforMAPwerenotavailableforallpiglets.5.ConclusionsContrarytoourhypotheses,theEEG-ParametersNI,PTOT,MF,andSEF95didnotshowanychangesinconnectionwiththepainfulproceduresandarethereforenotassessedassuitableforpaindetectionduringinjectionorcastrationofpiglets.Incontrast,theconcordanceofchangesintheLF/HFratiowiththefurtherpainindicatorscollecteddemonstratestheapplicabilityandutilityoffrequency-basedHRVforthedetectionofpainresponsesduringsucklingpigletcastration.Incomparisontotheintramuscularinjection,morepronouncedpainresponsesinpigletswereobservedwithintratesticularapplicationofthelargervolumeof0.5mLoflocalanaestheticsaswellassalinethanwiththeuseof0.3mL,althoughregularstatisticalevidenceislacking.Pain-relatedresponsesduringcastrationwerereducedusingthehighervolumeandthusthehighercontentofmepivacaineandlidocaine,whileprocaineappearsunsuitableforlocalanaesthesiainpigletcastrationTheagreementregardingpainassessmentwithacombinationofseveralphysiologicvariablesandbehaviouralobservationsleadstotheconclusionthatacompletelypain-freecastrationisnotfeasiblebymeansofintratesticularandsubscrotalinjectionofprocaine,lidocaineormepivacaineintheappliedconcentrationof2%.Whetherahigherconcen-trationofmepivacaineandlidocaine,withareducedvolume,wouldleadtosufficientdistributioninthetissuesandcompleteanalgesiaduringcastrationneedstobeinvestigatedinfurtherstudies.SupplementaryMaterials:Thefollowingsupportinginformationcanbedownloadedat:https://www.mdpi.com/article/10.3390/ani12202833/s1,TableS1:Meanandstandarddeviationofrespiratoryrate[breathsmin 1],meanarterialbloodpressure(MAP[mmHg]),andpulserate[pulsesmin 1])forgroupsP0.3,P0.5,L0.3,L0.5,M0.3,M0.5,S0.3,S0.5andIMforthetimepointsbaselineInjection(BL-INJ),injection(INJ),baselinecastration(BL-CA),castration(CA)andpostcastration(POST-CA);TableS2:Heartratevariability.Meanandstandarddeviationofheartrate[beatsmin 1],RR-Interval[ms],andmedian,minimumandmaximumofStandarddeviationofheartrate(SDHR

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Animals2022,12,2833 21of24 [beatmin 1]),StandarddeviationofRR-Interval(SDRR[ms])andLowFrequencyHighFrequencyratio(LF/HF-ratio)forgroupsP0.3,P0.5,L0.3,L0.5,M0.3,M0.5,S0.3,S0.5andIMforthetimeperiodsbaselineInjection(BL-INJe),injection(INJe),baselinecastration(BL-CAe),castration(CAe)andpostcastration(POST-CAe).AuthorContributions:F.J.S.andS.W.carriedouttheclinicalstudiesanddraftedthemanuscript;A.B.conductedthestatisticalanalysis;J.T.andK.-H.W.(deceased)participatedinthedesignofthestudy,S.B.R.K.andA.v.A.conceptualized,designed,andsupervisedthestudyandeditedthemanuscript.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.Funding:ThisresearchwasfundedbytheMinistryofFood,Agriculture,andConsumerProtection,LowerSaxony,Germany(ML),ProjectNo.206/204.1-04032-165.ThisOpenAccesspublicationwasfundedbytheDeutscheForschungsgemeinschaft(DFG,GermanResearchFoundation)—491094227“OpenAccessPublicationFunding”andtheUniversityofVeterinaryMedicineHannover,Foundation.InstitutionalReviewBoardStatement:ThestudywasconductedinaccordancewiththeDeclarationofHelsinki.AnapplicationforpermissionofthisprojectwassubmittedtotheLowerSaxonyStateOfficeforConsumerProtectionandFoodSafety(LAVES),Hannover,Germanyinaccordancewith§8(1)oftheGermanAnimalHealthandWelfareActandapprovedunderthepermitnumber33.9-42502-04-19/3218.DataAvailabilityStatement:ThedatapresentedinthisstudyareavailableinSupplementaryMaterials.Additionaldataareavailableonrequestfromthecorrespondingauthors.Acknowledgments:TheauthorsthankMartinBeyerbachforprovidinghelpwithstatisticalanalysis.ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.Thefundershadnoroleinthedesignofthestudy;inthecollection,analyses,orinterpretationofdata;inthewritingofthemanuscript,orinthedecisiontopublishtheresults.References1.EuropeanCommission.EuropeanDeclarationofalternativestosurgicalcastrationofpigs.Availableonline:https://food.ec.europa.eu/system/files/2016--10/aw_prac_farm_pigs_cast-alt_declaration_en.pdf(accessedon23September2020).2.FederalMinistryofFoodandAgriculture.Debateonpigletcastration.Availableonline:https://www.bmel.de/EN/topics/animals/animal-welfare/debate-piglet-castration.html(accessedon20December2020).3.Hodgson,D.S.Aninhalerdeviceusingliquidinjectionofisofluraneforshorttermanesthesiainpiglets.Vet.Anaesth.Analg.2006,33,207–213.[CrossRef]4.Rüdebusch,J.;Kästner,S.;Waldmann,K.H.;Wendt,M.;VonAltrock,A.Investigationintotheoptimizationofautomatedisofluraneanesthesiafortheperformanceofsafe,painlesscastrationofmalesucklingpigs.BerlMunchTierarztlWochenschr2022,135,1–13.[CrossRef]5.Schwennen,C.;Kolbaum,N.;Waldmann,K.H.;Holtig,D.Evaluationoftheanaestheticdepthduringpigletcastrationunderanautomatedisoflurane-anaesthesiaatfarmlevel.BerlMunchTierarztlWochenschr2016,129,40–47.6.Haga,H.A.;Ranheim,B.Castrationofpiglets:Theanalgesiceffectsofintratesticularandintrafunicularlidocaineinjection.Vet.Anaesth.Analg.2005,32,1–9.[CrossRef]7.Jäggin,N.;Gerber,S.;Schatzmann,U.Generalanaesthesia,analgesiaandpainassociatedwiththecastrationofnewbornpiglets.ActaVetScand2006,48,S12.[CrossRef]8.Schwennen,C.;Dziuba,D.;Schön,P.C.;Kietzmann,M.;Waldmann,K.H.;vonAltrock,A.Localanaesthetictechniquesforpainreductionduringcastrationofsucklingpiglets.BerlMunchTierarztlWochenschr2020,133,1–14.[CrossRef]9.Lirk,P.;Picardi,S.;Hollmann,M.W.Localanaesthetics:10essentials.Eur.J.Anaesthesiol.2014,31,575–585.[CrossRef]10.Richter,A.Lokalanästhetika.InLehrbuchderPharmakologieundToxikologiefürdieVeterinärmedizin,4thed.;Löscher,W.,Richter,A.,Eds.;Enke:Stuttgart,Germany,2016.11.CommissionImplementingRegulation(EU)2020/1712of16November2020amendingRegulation(EU)No37/2010toclassifythesubstancelidocaineasregardsitsmaximumresiduelimit.Off.J.Eur.Union2020,L384/3.12.Löscher,W.Lokalanästhetika.InPharmakotherapiebeiHausundNutztieren;Löscher,W.,Ungemach,F.R.,Kroker,R.,Eds.;Enke:Stuttgart,Germany,2010;pp.134–140.13.Hofmann,K.;Rauh,A.;Harlizius,J.;Weiss,C.;Scholz,T.;Schulze-Horsel,T.;Escribano,D.;Ritzmann,M.;Zols,S.Painanddistressresponsesofsucklingpigletstoinjectionandcastrationunderlocalanaesthesiawithprocaineandlidocaine-Part1:Cortisol,chromograninA,woundhealing,weights,losses.Tierarztl.Prax.Ausg.GGrosstiereNutztiere2019,47,87–96.[CrossRef]14.Hansson,M.;Lundeheim,N.;Nyman,G.;Johansson,G.Effectoflocalanaesthesiaand/oranalgesiaonpainresponsesinducedbypigletcastration.ActaVet.Scand.2011,53,34.[CrossRef]

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40.Martin-Cancho,M.F.;Lima,J.R.;Luis,L.;Crisostomo,V.;Ezquerra,L.J.;Carrasco,M.S.;Uson-Gargallo,J.Bispectralindex,spectraledgefrequency95%,andmedianfrequencyrecordedforvariousconcentrationsofisofluraneandsevofluraneinpigs.Am.J.Vet.Res.2003,64,866–873.[CrossRef][PubMed]41.Aranake,A.;Mashour,G.A.;Avidan,M.S.Minimumalveolarconcentration:Ongoingrelevanceandclinicalutility.Anaesthesia2013,68,512–522.[CrossRef][PubMed]42.Lerman,J.;Robinson,S.;Willis,M.M.;Gregory,G.A.Anestheticrequirementsforhalothaneinyoungchildren0-1monthand1-6monthsofage.Anesthesiology1983,59,421–424.[CrossRef]43.Schieber,R.A.;Namnoum,A.;Sugden,A.;Shiu,G.K.;Orr,R.A.;Cook,D.R.Hemodynamiceffectsofisofluraneinthenewbornpiglet:Comparisonwithhalothane.Anesth.Analg.1986,65,633–638.[CrossRef][PubMed]44.Anonymous.FachinformationinFormderZusammenfassungderMerkmaledesTierarzneimittelsIsofluranBaxter®vet1000mg/gFlüssigkeitzurHerstellungeinesDampfeszurInhalationfürHunde,Katzen,PferdeundSchweine(Ferkel).TechnicalinformationinGerman.2018.Availableonline:https://www.baxter.de/sites/g/files/ebysai1301/files/2020-11/isofluran_vet_fs.pdf(accessedon15December2020).45.Petersen-Felix,S.;Arendt-Nielsen,L.;Bak,P.;Roth,D.;Fischer,M.;Bjerring,P.;Zbinden,A.M.Analgesiceffectinhumansofsubanaestheticisofluraneconcentrationsevaluatedbyexperimentallyinducedpain.Br.J.Anaesth.1995,75,55–60.[CrossRef]46.Jinks,S.L.;Dominguez,C.L.;Antognini,J.F.Drasticdecreaseinisofluraneminimumalveolarconcentrationandlimbmovementforcesafterthoracicspinalcoolingandchronicspinaltransectioninrats.Anesthesiology2005,102,624–632.[CrossRef]47.Wendt,S.;Söbbeler,F.J.;Waldmann,K.-H.;Kästner,S.B.R.;vonAltrock,A.Localpainduringpigletcastration—Investigationonthedistributionoflocalanestheticsfromdifferentinjectionsites.InProceedingsofthe12thEuropeanSymposiumofPorcineHealthManagement,Online,14–16April2021;p.384.48.Ranheim,B.;Haga,H.A.Localanaesthesiaforpigssubjecttocastration.ActaVet.Scand.2006,48(Suppl.SI),S13.[CrossRef]49.Riff,C.;LeCaloch,A.;Dupouey,J.;Allanioux,L.;Leone,M.;Blin,O.;Bourgoin,A.;Guilhaumou,R.LocalAnestheticPlasmaConcentrationsasaValuableTooltoConfirmtheDiagnosisofLocalAnestheticSystemicToxicity?AReportof10YearsofExperience.Pharmaceutics2022,14,708.[CrossRef]50.Scott,D.B.;Jebson,P.J.;Braid,D.P.;Ortengren,B.;Frisch,P.Factorsaffectingplasmalevelsoflignocaineandprilocaine.Br.J.Anaesth.1972,44,1040–1049.[CrossRef]51.DiGiminiani,P.;Brierley,V.L.;Scollo,A.;Gottardo,F.;Malcolm,E.M.;Edwards,S.A.;Leach,M.C.TheAssessmentofFacialExpressionsinPigletsUndergoingTailDockingandCastration:TowardtheDevelopmentofthePigletGrimaceScale.Front.Vet.Sci.2016,3,100.[CrossRef]52.McLeod,I.K.LocalAnesthetics.Availableonline:https://emedicine.medscape.com/article/873879-overview(accessedon2April2021).53.Gutzwiller,A.KastrationvonFerkelnunterLokalanästhesie.Agrarforschung2003,10,10–13.54.Morris,R.;McKay,W.;Mushlin,P.Comparisonofpainassociatedwithintradermalandsubcutaneousinfiltrationwithvariouslocalanestheticsolutions.Anesth.Analg.1987,66,1180–1182.[CrossRef][PubMed]55.Otto,K.A.;Gerich,T.Comparisonofsimultaneouschangesinelectroencephalographicandhaemodynamicvariablesinsheepanaesthetisedwithhalothane.Vet.Rec.2001,149,80–84.[CrossRef][PubMed]56.Roizen,M.F.;Horrigan,R.W.;Frazer,B.M.Anestheticdosesblockingadrenergic(stress)andcardiovascularresponsestoincision–MACBAR.Anesthesiology1981,54,390–398.[CrossRef]57.Coutant,M.;Malnkvist,J.;Kaiser,M.;Foldager,L.;Herskin,M.S.Piglet´sacuteresponsestoprocaine-basedlocalanestheticsinjectionandsurgicalcastration:Effectsoftwovolumesofanesthetic.Front.PainRes.2022,3,943138.[CrossRef]58.Rauh,A.;Hofmann,K.;Harlizius,J.;Weiss,C.;Numberger,J.;Scholz,T.;Schulze-Horsel,T.;Otten,W.;Ritzmann,M.;Zols,S.Painanddistressresponseofsucklingpigletstoinjectionandcastrationunderlocalanaesthesiawithprocaineandlicocaine-Part2:Defencebehaviour,catecholamines,coordinationofmovements.Tierärztl.Prax.Ausg.GGroßtiereNutztiere2019,47,160–170.[CrossRef]59.Koenig,J.;Jarczok,M.N.;Ellis,R.J.;Hillecke,T.K.;Thayer,J.F.Heartratevariabilityandexperimentallyinducedpaininhealthyadults:Asystematicreview.Eur.J.Pain2014,18,301–314.[CrossRef]60.Moak,J.P.;Goldstein,D.S.;Eldadah,B.A.;Saleem,A.;Holmes,C.;Pechnik,S.;Sharabi,Y.Supinelow-frequencypowerofheartratevariabilityreflectsbaroreflexfunction,notcardiacsympatheticinnervation.HeartRhythm2007,4,1523–1529.[CrossRef]61.McCraty,R.;Shaffer,F.Heartratevariability:Newperspectivesonphysiologicalmechanisms,assessmentofself-regulatorycapacity,andhealthrisk.Glob.Adv.HealthMed.2015,4,46–61.[CrossRef]62.Clement,C.I.;Keay,K.A.;Owler,B.K.;Bandler,R.Commonpatternsofincreasedanddecreasedfosexpressioninmidbrainandponsevokedbynoxiousdeepsomaticandnoxiousvisceralmanipulationsintherat.J.Comp.Neurol.1996,366,495–515.[CrossRef]63.Haga,H.A.;Lykkjen,S.;Revold,T.;Ranheim,B.Effectofintratesticularinjectionoflidocaineoncardiovascularresponsestocastrationinisoflurane-anesthetizedstallions.Am.J.Vet.Res.2006,67,403–408.[CrossRef][PubMed]64.vanLieshout,J.J.;Wieling,W.;Karemaker,J.M.;Eckberg,D.L.Thevasovagalrespon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Animals2022,12,2833 24of24 66.Raue,J.F.;Tarvainen,M.P.;Kastner,S.B.R.ExperimentalstudyontheeffectsofisofluranewithandwithoutremifentanilordexmedetomidineonheartratevariabilitybeforeandafternociceptivestimulationatdifferentMACmultiplesincats.BMCVet.Res.2019,15,258.[CrossRef]67.Levy,M.N.Neuralcontrolofcardiacfunction.Bailliere’sClin.Neurol.1997,6,227–244.