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ArticleEfficacyofIntra-OperativeTopicalWoundAnaesthesiatoMitigatePigletCastrationPain—ALarge,Multi-CentredFieldTrialMeredithSheil1,* ,GiuliaMariaDeBenedictis2 ,AnnalisaScollo3 ,SuzanneMetcalfe4,GilesInnocent5,AdamPolkinghorne6,7andFlavianaGottardo2 Citation:Sheil,M.;DeBenedictis,G.M.;Scollo,A.;Metcalfe,S.;Innocent,G.;Polkinghorne,A.;Gottardo,F.EfficacyofIntra-OperativeTopicalWoundAnaesthesiatoMitigatePigletCastrationPain—ALarge,Multi-CentredFieldTrial.Animals2021,11,2763.https://doi.org/10.3390/ani11102763AcademicEditor:AlisonSmallReceived:4August2021Accepted:15September2021Published:22September2021Publisher’sNote:MDPIstaysneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffil-iations.

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d:15September2021Published:22September2021Publisher’sNote:MDPIstaysneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffil-iations. Copyright:©2021bytheauthors.LicenseeMDPI,Basel,Switzerland.ThisarticleisanopenaccessarticledistributedunderthetermsandconditionsoftheCreativeCommonsAttribution(CCBY)license(https://creativecommons.org/licenses/by/4.0/).1AnimalEthicsPty.Ltd.,YarraGlen,VIC3775,Australia2DepartmentofAnimalMedicine,ProductionandHealth,PadovaUniversity,Vialedell’Università16,35020Legnaro,Italy;giuliamaria.debenedictis@unipd.it(G.M.D.B.);flaviana.gottardo@unipd.it(F.G.)3DepartmentofVeterinarySciences,UniversityofTorino,LargoBraccini2,10095Grugliasco,Italy;annalisa.scollo@unito.it4KnoellAnimalHealthLtd.,BankBarn,HowMill,BramptonCA89JY,UK;smetcalfe@knoell.com5BiomathematicsandStatisticsScotland,EdinburghEH93FD,Scotland,UK;giles.innocent@bioss.ac.uk6DepartmentofMicrobiologyandInfectiousDiseases,NSWHealthPathology,NepeanHospital,Penrith,NSW2750,Australia;adam@majormitchellconsulting.com.auoradam.polkinghorne@health.nsw.gov.au7MajorMitchellConsulting,Buderim,QLD4556,Australia*Correspondence:mlksheil@me.comSimpleSummary:Pigletcastrationcausespainandstresstotheanimal.Althoughdesperatelyneeded,therearecomplexitiesdevelopingsafeandeffectivemethodsofpainalleviationapplicableforon-farmuse.Topicalanaesthesia,instilledtothewoundduringsurgery,isanewlyevolvingon-farmmethodtomitigatecastrationpain.Inthecurrentstudy,weinvestigatedtheuseofTri-Solfen®(AnimalEthicsPtyLtd,Melbourne,Australia),atopicallocalanaestheticandantisepticformulation,instilledtothewoundduringtheprocedure,toalleviatesubsequentcastration-relatedpainexperiencedinpiglets.Weperformedalarge,blind,multi-centredtrialcomparingpaininpigletscastratedwithorwithoutTri-Solfen®.PigletstreatedwithwoundinstillationofTri-Solfen®,30spriortosubsequentcastration,showedsignificantlylowerpain-inducedmotorandvocalresponsesduringtheprocedure.Acutepost-operativepain-relatedbehaviours,evidentinpigletsinthefirst30minfollowingcastration,werealsosignificantlyreducedintreatedpigletscomparedwithuntreatedpiglets.Usingthismethod,Tri-Solfen®providesaneffectiveon-farmmethodtomitigateacutecastration-relatedpaininyoungpiglets.Abstract:Pigletcastrationresultsinacutepainandstresstotheanimal.Thereisacriticalneedforeffectiveon-farmmethodsofpainmitigation.LocalanaesthesiausingTri-Solfen®(AnimalEthicsPtyLtd.,Melbourne,Australia),atopicallocalanaestheticandantisepticformulationinstilledtothewoundduringsurgery,isanewlyevolvingon-farmmethodtomitigatecastrationpain.ToinvestigatetheefficacyofTri-Solfen®,instilledtothewoundduringtheprocedure,toalleviatesubsequentcastration-relatedpaininneonatalpiglets,weperformedalarge,negativelycontrolled,randomisedfieldtrialintwocommercialpigfarmsinEurope.Piglets(173)wereenrolledandrandomisedtoundergocastrationwithorwithoutTri-Solfen®,instilledtothewoundimmedi-atelyfollowingskinincision.A30swaitperiodwasthenobservedpriortocompletingcastration.Efficacywasinvestigatedbymeasuringpain-inducedmotorandvocalresponsesduringthesub-sequentprocedureandpost-operativepain-relatedbehaviourintreatedversusuntreatedpiglets.Therewasasignificantreductioninnociceptivemotorandvocalresponseduringcastrationandinthepost-operativepain-relatedbehaviourresponseinTri-Solfen®-treatedcomparedtountreatedpiglets,inthefirst30minfollowingcastration.Althoughnotaddressingpainofskinincision,Tri-Solfen®iseffectivetomitigatesubsequentacutecastration-relatedpaininpigletsundercommercialproductionconditions.

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oughnotaddressingpainofskinincision,Tri-Solfen®iseffectivetomitigatesubsequentacutecastration-relatedpaininpigletsundercommercialproductionconditions. Animals2021,11,2763.https://doi.org/10.3390/ani11102763https://www.mdpi.com/journal/animals

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Animals2021,11,2763 2of20 Keywords:lidocaine;bupivacaine;adrenaline;peri-operative;topical;anaesthetic;relatedbehaviour;vocalisation

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Animals2021,11,2763 2of20 Keywords:lidocaine;bupivacaine;adrenaline;peri-operative;topical;anaesthetic;relatedbehaviour;vocalisation 1.IntroductionThereisanurgentwelfareimperativetodevelopeffectiveanalgesicstrategiesforuseon-farmtoalleviatepainassociatedwithlivestocksurgicalhusbandryprocedures,includingpigletcastration[1–5].Pigletcastrationisacommonprocedureconductedincommercialpigindustriesaroundtheworldwiththeaimofreducingboartaint[6],aggressionandmatingbehavioursinmalepiglets[1].Mostcommonlyperformedon-farminthefirstweekoflifeinpigletsintendedtobekeptpastsexualmaturity,surgicalcastration,traditionallyperformedwithoutanaesthesiaoranalgesia[1,4]inducesevidenceofpainandstressinpigletsbasedonphysiologicalresponses,motorandvocalresponsesandalterationofpigletbehaviourduringandfollowingtheprocedure[1–5,7–17].Concernforthewelfareofpigletsundergoingcastrationisdrivingeffortstodevelopeffectivemethodsofpainmitigation.Althoughdesperatelyneeded,therearecomplexitiesdevelopingsafeandeffectivemethodsofpainalleviationapplicableforon-farmuse.Al-thoughreportsvary[18–21],analgesicssuchasNon-SteroidalAnti-InflammatoryDrugs(NSAIDs)maybeofusetoaddressinflammatory-relatedpainthatdevelopsinthepost-operativeperiod[8,22],butgenerallylackefficacytoamelioratepainduringtheprocedureandintheearlypost-operativeperiodwhenpainismostacute[4,23,24].Standardmethodsforaddressingsurgicalproceduralpain,suchastheuseofgeneralanaesthesia[18,25–29]orinjectedlocalanaesthetics,althougheffectivetomitigateacuteintra-operativepain,usedalone,theyprovidelittlepost-operativepainmitigation[22,24,25,28,30–40].Furthermore,theirusemaybeimpracticaland/orimpededon-farmbycost,logistics,occupationalhealthandsafety,foodsafetyorwelfareconcerns.Generalorinjectedlocalanaestheticinterventions,forexample,generallyrequireskilledveterinaryadministration,mayre-quirespecialiseddeliveryandmonitoringequipment,and/orextralabourwhichmaybecostprohibitive[1,3,5].Generalanaestheticsandsedativesmayfurthermorecausepost-operativesedation,interferingwithtemperatureregulationandfeedingandincreasingtheriskofcrushinjuryormortality[1,41].Additionally,anaestheticand/oranalgesictherapiesmayrequiretimetotakeeffect,requiringprolongedordoublehandlingwithassociatedincreasedstress[37].Together,suchfactorsmayreducetheoverallwelfarebenefit[1–8,41,42].TopicallocalanaesthesiawithTri-Solfen®,(AnimalEthicsPtyLtd.,Melbourne,Vic,Australia)aregisteredtopicallocalanaestheticandantisepticformulation,appliedintra-operativelybywoundinstillation,hasrecentlyemergedasanalternativefarmer-appliedmethodtodeliverlocalanaesthesiaandmitigateacutecastration-relatedpaininlambsandcalves,thatisalsosafeandtechnicallysimpletoadministerinproductioncondi-tions[35,36,43–46].Tri-Solfen®isaviscousliquidformulationcontainingarapidonsetlocalanaesthetic(Lignocainehydrochloride5%),along-actinglocalanaesthetic(Bupiva-cainehydrochloride0.5%),alongwithavasoconstrictor(Adrenalinacidtartrate(45 g/L))andanantiseptic(Cetrimide0.5%).Itwasdevelopedtoprovidesingledoserapidonsetlo-calwoundanaesthesiawithaprolongedeffectfollowingapplicationtosurgicalhusbandrywoundsinfarmanimals[43–48]andhasregulatoryapprovalforusetoalleviatepainfollowingcastrationand/orotherhusbandryproceduresinlambsandcalvesinAustraliaandNewZealand.Recentstudiessuggestthatitmaybesimilarlyeffectivetomitigateacutecastration-relatedpaininpiglets[44,48,49].Lomaxetal.[44],forexample,reportedintraoperativewoundinstillationofTri-Solfen®waseffectivetoreducewoundhyperal-gesicresponsesinpigletsfromwithin1minupto4hfollowingcastration.Amidelocalanaestheticsworkbyblockingpainsignalconductioninnervefibres.Appliedtopicallytointactskin,theyareofslowonsetandlimitedefficacyduetoinabilitytopenetratekeratinisedtissuestoreachunderlyingnervetissue.Theymayberapidlyeffective(within

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Animals2021,11,2763 3of20

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Animals2021,11,2763 3of20 1min)whenappliedtoopenwounds,whereskinlayersaredisruptedandtraumatisednervefibresareexposed.Theymaybesimilarlyrapidlyeffective,(within30s)[50,51],whenappliedtonon-keratinisedmucousmembranes,whichtheycanreadilypenetrate.Inthesettingofcastration,Tri-Solfen®isappliedviawoundinstillationimmediatelyfollow-ingskinincision,thuscoatingthenon-keratinisedmucosaltissuesofthespermaticcord,andthecutskinedge.Providinga30sdwelltimemaythusallowtimefortheanaestheticactivestopenetratemucosaltissuesofthespermaticcord,priortoapplyingtractiononthetestisandseveringthespermaticcords.Theselatterstepsareconsideredthemostpainfulpartoftheprocedure[14,15,52].Consistentwiththis,reducednociceptivemotorandvocalresponsesduringcastration,(aswellasreducewoundhyperalgesicresponsesinthefirst2hfollowingcastration)wererecentlyreportedinpigletsreceivingintraoperativewoundinstillationofTri-Solfen®withasubsequent30sdwelltime[49].Tomeetregula-toryrequirementsandachieveapprovalforuse,veterinaryproductsmustmeetproofofefficacyrequirementstointernationallyharmonised(VICH),GoodClinicalpractice(GL9)guidelines[53].Thecurrent,follow-upregulatorydrugfieldefficacytrialwasundertakentoaddresstheserequirements.Proofofanaestheticefficacyischallenginginneonatalpiglets,particularlyinthe“field”or“on-farm”settingaspertheVICHGL9standard[53]requiredforveterinarydrugregulatoryapprovals.Thereisnoone“goldstandard”orvalidatedmeasureof“pain”inneonatalpiglets.Instead,arangeofpredominantlyindirectmeasureshavebeenusedinpreviousstudiesincludingphysiological,motorandvocalresponses,mechanicalsensorytestingandpost-operativebehaviouraldisturbances[54].Apainscalehasrecentlybeenvalidatedfordocumentingpost-operativepaininolderpiglets[55],however,atthetimeofthesestudies,wasnotavailableforneonatalpiglets.Followingadetailedreviewoftheliterature[54],pigletmotorandvocalresponsesduringcastration,aswellasdirectwoundsensorytestingusingvon-Freyandneedlestickstimulation,alongwithpost-operativepain-relatedbehaviourfollowingcastration,wereidentifiedasmethodsthathadmostconsistentlyachievedtheseobjectives.Arangeofothermeasures(includingcortisollevelsandphysiologicalparameters)wereexcludedaslackingsensitivityorspecificforpain,beinginconsistentand/orunreliablemeasuresofpainincastratedneonatalpiglets,particularlyinthesettingofexaminingpainmitigationusinglocalanaesthesiawithadrenaline[54].Withregardtoassessmentofpost-operativepain,thetwoselectedvariablesareunabletobemeasuredinthesamecohortaswoundsensorytestingrequiresfrequentpost-operativehandling,whichinterfereswiththequietobservationrequiredforbehaviouralassessment[44].Astheresponsetowoundsensorytestingpost-castrationwaspreviouslyreported[44,49],thecurrentstudywasdesignedtofollow-uptoinvestigatepain-relatedbehaviourasthepost-operativeefficacyparameter.Thedetectionofpost-operativepain-relatedbehaviourinneonatalanimalsisparticu-larlychallenging.Arecentreviewoftheliterature[54]alongsideothers[56],revealedthatbehaviouralchangespost-castrationinneonatalpigletsmaybesubtle,transientand/orvariablyexpressed,suchthatfindingsarenotalwaysreproducible,and,insomecases,contradictoryresultshavebeenreported.Behaviouralassessmentsusuallyinvolveeitherdirectquietobservationandscoringofpigletbehavioursbytrainedblindedobservers,orcontinuoustime-lapsevideorecordingwithoff-linescoringeitherusingeventmonitoringsoftwareorblindedobservers.Assessmentstypicallyincludeobservationsofpiglet(i)posture(e.g.,lying,standing,sittingetc),(ii)location(e.g.,underheatlamp,incontactwiththesoworpenmatesversusinisolation),and(iii)activities,includingnon-specificbehaviours(e.g.,sucking,sleeping,walking,playing,exploratoryoraggressivebehaviour,etc.)and“pain-specific”behaviours.Thislattercategory,firstdetailedbyHayetal.[12]andbasedonpain-specificbehavioursreportedinotherspecies,includes“prostration”(e.g.,standingorsittingwithheaddownbelowshoulderheight),“huddledup”(i.e.,ventrallyingwiththreeormorelegstuckedup),“tremorsortrembling”,“spasms(i.e.,localisedmusclespasm)”,“tailwagging”and“scratching”(i.e.,rubbingtherumpalongthefloororwalls,alsocalled“scooting”).Inviewoftherelativelylowincidenceandvariabilityof

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Animals2021,11,2763 4of20

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Animals2021,11,2763 4of20 pain-relatedbehaviour,aggregationof“pain-specificbehaviours”iscommonlyemployedtoderivea“total”or“global”painscoreforeachpigletoverspecifictimeperiods[54].Basedonthisrecentlypublishedreview[54],themostconsistentlyreportedvariationsinpigletbehaviourfollowingcastrationareanincreaseintotal“pain-specific”behaviours.Thesearetypicallyonlyevidentduringtheearliesttimeperiodmeasuredfollowingcastra-tion(e.g.,upto180min[54]),althoughmuchshorterdurations(e.g.,30min)havebeenreported[35].Themostconsistentlyreportedvariationinbehaviourinlatertimeperiods(e.g.,8–24h+)isanincreaseintailwaggingandscratching.Thecurrentstudywasundertakentoaddressdrugregulatoryproofofefficacyrequire-mentstointernationallyharmonised(VICH),GoodClinicalpractice(GL9)guidelines[53].Basedonthedataabove,itwashypothesisedthatTri-Solfen®applicationbywoundin-stillationfollowedbya30sdwelltimewouldresultinasignificantreductioninacutepain-inducedresponsesduringsubsequentcastrationandintheearlypost-operativepe-riodascomparedwithuntreatedcastrated(control)piglets.Nociceptivemotorandvocalresponsestocastrationwereselectedastheprimaryandsecondaryefficacyvariablesforassessmentofpainmitigationduringcastration,whiletheprimaryefficacyvariableforevaluationofpost-operativepain-mitigationwasasignificantreductionintotal“pain-specific”behaviourinthefirst30minfollowingcastration,asassessedviaacombinationoffocalassessmentsandscansampling.2.MaterialsandMethods2.1.StudyDesignThestudywasamulti-centred,negativelycontrolled,randomisedandblindedstudyconductedbetweenJuneandAugust2018totesttheclinicalefficacyofTri-Solfen®(50g/LLignocainehydrochloride,5g/LBupivacainehydrochloride,0.048g/LAdrenaline(asacidtartrate),5g/LCetrimide).Allstudyprocedureswereconductedundercountry-specifictrialapprovals,includingethicalapproval.InGermany,approvalwasobtainedfromtheSchleswig-HolsteinauthoritywhileapprovalfortheItalianstudywasprovidedbytheMin-isterodellaSalute.Thestudywasconductedbyindependentcontractresearchproviders,inaccordancewithVICHguidelinesforconductofregulatorysafetyandefficacyfieldtrialswiththeprinciplesofGoodClinicalPractice(GCP)aslaiddownintheCouncilDirective2001/82/ECandguidelineCVMP/VICH/595/98VICHTopicGL9Step7-GuidelineonGoodClinicalPractices(CVMPapprovedJuly2000).Theanimalstudywasperformed,withownerconsent,attwocommercialpigfarmsinGermanyandItalydedicatedtothemanagementofbreedingsows.Together,173commerciallybredmalepiglets(DanishandLandrace LargeWhite)between3and7daysofageandingoodhealthwereconfirmedsuitableforenrolmentinthestudy.Priorpoweranalysisindicatedtherequirementforaminimumof80animalspergrouptoshowasignificantreductionatthe5%level.Litterswith6–12malepiglets(7average)wereselectedforthestudyateachsite.Pigletswithinalitterwereindividuallyidentifiedbyeartag(appliedonstudyDay 2)andspray/penmark.Pigletswithinalitterwererandomlyallocatedtotreatmentorcontrolgroups.Allstudypigletshadstandardbluespray-onpigletmarkerdyeappliedtothescrotumpriortoprocedures,toblindpost-operativeobserverstoanytranslucent-bluecolouringthatmayhaveremainedfollowingapplicationoftreatment.Thismethodofblindingwasusedratherthanaplacebosolution,toallowcomparisonwithstandardcastrationpractice,andpreventconfoundingduetopotentialimpacts(pain/inflammation/infection)fromwoundinstillationofaplacebosolution.Hence,thepersonperformingthetreatmentwasunblinded,however,thispersondidnotparticipateinanytrialassessmentsordataanalysis.Studyanimalsweremaintainedintheirnormalfarrowingpensasperstandardfarmpracticewiththeirdamandlittermates.Pigletshadadlibaccesstopotablewaterandcon-stantsucklingaccesstotheirdam.Routinemanagementpracticeswerefollowed,however,procedures(suchastaildocking)orothertreatmentswhichmayhaveinfluencedtheeffect

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Animals2021,11,2763 5of20 onpotentialpainresponsesoranalgesiaprovidedbytreatmentwerenotpermittedandwouldhaveresultedintheremovalofpigletsfromthestudy.ClinicalexaminationwasperformedonStudyDay 1aspartofpigletinclusion,priortopre-operativebehaviouralassessments,aswellasStudyDay1,afterthemorningbehaviouralassessments,andStudyDays6and12,inthemorning.Examinationsincludedmeasurementsofweightandrectaltemperaturesusingcalibratedinstrumentsandclinicalassessmentofgeneraldemeanourandvitality,intermsofnormal/abnormal,andanyevidenceofotherillness,injury,woundinfectionorinflammation.Additionallywoundhealingwasscoredonascalefrom1to6,basedonSutherlandetal.[48].Castrationandtreatmentprocedurestotesttheefficacyofthisformulationwereconductedasfollows.Briefly,afterthepigletswereremovedfromtheirdams,eachanimalwasrestrainedgentlybutfirmlyinaKerblpigletcastrationcradle(AlbertKerblGmbH,Buchbach,Germany)toexposetheano-genitalregionofthepiglet.Thescrotalskin,(includingunderlyingparietaltunica),wasthenincisedtorevealandexteriorisethetestis.Intreatedanimals,Tri-Solfen®wasthenappliedwithatotaldoseof1mL(pigletsupto2kg),and2mL(piglets2–4kg)deliveredviawoundinstillationusinga1mLballtippedapplicator(ProdigyInstrumentsPtyLtd.,Sydney,Australia).Halfofthetotaldosewasappliedtoeachsidewithcaretoensurethattheformulationcoatedtheexposedspermaticcordandcutedgesofthescrotalsack.Aperiodof30swasallowedtoelapsepriortothetestesthenbeingremovedbyseveringthecordasperroutineprocedure.Pigletsassignedtothecontrolgroupwerehandledinthesamewayasthetreatmentgroup,butwithoutthephysicaladministrationofTri-Solfen®.2.2.VideoRecordingduringCastrationandNociceptiveMotorResponseScoringVideorecordingwasperformedusingadeviceinfixedpositionabovethecradle,torecordpost-treatmentcastrationprocedureandresponses.Avideo-camerarecord-ingdevice(iPodTouch-AppleInc.Cupertino,CA,USA)wasusedwiththeapplication‘TimestampCamera’.Eachpigletwasclearlyidentifiedbyplacinganumbernexttothepigletcradlewithincameraview.Videorecordingcommencedfollowingskinincisionandproductadministration,approximately10–20sintothe30swaitperiodpriortoremovalofthetestesinanimalsfromeachgroup,documentingthespermaticcordseveranceandtesticleremovalphaseandonlyfinishingfollowingtheremovalofthesecondtesticle.Thenociceptivemotorresponsewasassessedoff-linebyablindedassessor,asrecentlydescribed[8].Briefly,pigletresponsesatfourdifferenttimepointswererecorded:(1)tractiononfirsttestis;(2)cuttingfirstspermaticcord;(3)tractiononsecondtestis;(4)cuttingthesecondspermaticcord.Anumericalratingscale(0–2)wasusedtogradepigletnociceptivemotorresponseacrossalltimepointstogiveatotalscoreofupto8foreachpigletusingthefollowinggradingincrements:0=nomotorresponse,1=mildmotorresponse,suchasshortlivedlegextensionorfrontlegpaddlingorkickingbutnomajorbodyresistancemovementinthecradle,2=markedmotorresponse,suchasprolongedlegmovementsand/ormarkedbodyresistancemovementinthecradle.2.3.AudioRecordingduringCastrationandVocalResponseAssessmentSoundwasrecordedwithavalidatedDigitalSoundLevelMeter,positioned60cmdirectlyinfrontofthesnout.Recordingcommencedapproximately10–20sintothe30swaitperiodpriortoremovalofthetestesinanimalsfromeachgroupthroughto1–10safterthecuttingofthesecondcord.Soundfileswereanalysedoff-linebyasoundconsultantblindedtopiglettreatment.SoundfileswereimportedtoProTools®(AvidTechnologyInc.,Burlington,MA,USA)andplacedontheProToolstimelineattheirtimestamppositiontoenablesynchronisationwiththeaudiofilesfromthevideorecording.Isolationandcomparativequantificationofpigletvocaloutputwasachievedfollowingannotationofthetimelinewithtimesforcommencementoftraction,andcompletionofcordseveranceforeachtesticle(Figure1).Quantificationwasfocussedontwotimeperiods:(1)commencingtractiontocompletionofseveranceofthefirstspermaticcord;(2)commencingtraction

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Animals2021,11,2763 6of20 to1spost-completionofseveranceofthesecondspermaticcord.Maximumvocalisation(decibels(dB),volume)foreachtimeperiodwasrecorded.Screenshotswerealsogeneratedwiththesametimedurationwindow(Xaxis)andsignalscale(Yaxis).TheAreaUnderthedecibel(dB)/timewaveformCurve(AUC)wasthencalculated(inpixels)usingimageanalysissoftware(Image-J®U.S.NationalInstitutesofHealth,Bethesda,MD,USA).Thisprovidesameasureoftotalvocalisationduringeachtimeperiod(Figure1).

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ingimageanalysissoftware(Image-J®U.S.NationalInstitutesofHealth,Bethesda,MD,USA).Thisprovidesameasureoftotalvocalisationduringeachtimeperiod(Figure1). Figure1.RepresentativescreenshotofdB/timewaveform,andyellowImageJtracingforAUC(areaunderthedB/timewaveform)analysis.Theimageshowstheblackhighlightedsectionencompassingthetimefromfirstcommencingtraction,untilcompletingremovalofthefirsttesticle.2.4.AssessmentofPost-OperativePain-RelatedBehaviourThepresenceofpain-relatedbehaviourinthepigletsfollowingcastrationwasrecordedbyfocalassessmentandscansamplingusingdifferentindependenttrainedobserversblindedtotreatmentgroups.Twoseparateobserverswereused,oneforfocalassessmentandoneforscanassessments,ateachtrialsite.Tominimisethepotentialforinter-observererror,thesameobserverperformedallscanorfocalassessments.FocalassessmentwasconductedforeachpiglettwiceonStudyDay 1(morningandafternoon),andpost-castration/treatment(StudyDay0andStudyDay1)atapproximately1min(+1min),15min 2min),30min( 5min)andthenatapproximately60and90min,aswellasat2,3,6,8,24and30h.The1minfocalassessmentcommencedfollowingthereturnoftheindividualpiglettothepenpost-castration,andincludedrecordingofthepresenceorabsenceof“pain-specific”behavioursusinganethogrambasedonHayetal.[12],Moyaetal.[13]andGottardoetal.[35],includinghuddledup,stiffness,prostrated,attemptstosuckle,scratching,tailwagging,tremors/trembling,panting(Table1).Focalassessmentswereperformedforadurationof1minwithallactivitiesrecorded.Pigletswerescored1=behaviourpresent;0=behaviourabsent.Onlyindividualsthatdemonstratednoneoftheseatanassessmentwererecordedasnotshowingpain-associatedbehaviouratthatassessment.

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Animals2021,11,2763 7of20 Scansamplingofanimalswasconductedoverathree-hourperiodinthemorningsandatwo-hourperiodintheafternoonsonthedaypriortocastration(StudyDay 1),thedayofcastrations(StudyDay0)andthemorningofthedayafter(StudyDay+1).Morningscansonthedayofcastration/treatmentwereperformedassoonaspossiblefollowingthecastrationproceduresbeingcompleted.Duringthescansampling,behaviourswererecordedat10minintervalswithbehaviouralassessmentconductedusinganethogrambasedonpreviousreports[12,13,35],includingthegeneralposition,posture,activityandcontactofeachpiglet,andincludingposturesandactivitiesdeemedtobe“pain-related”asperthedefinitionsoutlinedinTable2(e.g.,prostrated,stifforhunchedposture(standingorsitting),ventrallyinginisolation,andwalkingwithstifforabnormalgait).Forthescanassessmentswithineachperiod,ifananimalwasrecordedasdemonstratinganyofthefollowingbehavioursatanypointduringthatperiod,thentheanimalwasrecordedasdemonstratingpain-associatedbehaviourduringthatperiod.Table1.Descriptionofvariablesincludedinfocalassessmentofpain-relatedbehaviours. BehaviourDescription HuddledupLyingwithatleastthreelegstuckedunderStiffnessLyingorstandingwithextendedandtensedlegsProstratedAwake,sittingorstandingmotionless,withtheheaddown,lowerthanshoulderAttemptstosuckleAttemptstosucklebywalkingandpushingotherpigletswhilemostoftheothersaresucklingScratchingScratchingthescrotal/perinealareabyrubbingitagainstthefloororthepenwallTailwaggingTailmovementsfromsidetosideorupanddownTremors/TremblingShiveringaswithcold.Theanimalmaybelying,sittingorstandingPantingHigherrespiratoryrateandsuperficialrespiration Table2.Ethogramincludingdescriptionofvariablesincludedinscansamplingofgeneralandpain-relatedbehavioursinpigletspost-castrationinthisstudy. BehaviourDescription

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hogramincludingdescriptionofvariablesincludedinscansamplingofgeneralandpain-relatedbehavioursinpigletspost-castrationinthisstudy. BehaviourDescription IsolatedorincontactwithlittermatesIsolatedwasdefinedasbeingasidefromtheotherpigletsaloneorwithonepigletatmost.Adistanceofatleast40cm(approximatelythewidthoftwopiglets)separatedthepigletfromtheclosestlittermate.Incontactwasdefinedasbeingnearatleasttwopigletswithadistanceoflessthan40cm.Position:crateornestCrate(pigletisoutofthenestarea)Nest(undertheconeoflightofthelampwithatleasthalfofitsbody)Posture:standing,lyingorsittingStanding-Headdown(prostrate).Bodyweightsupportedbythefourlegs,pigletawake,standingmotionlessorwalingwiththeheaddown,lowerthanshoulderlevel;OR-Curvedspine(hunched).Bodyweightsupportedbythefourlegs,awake,standingmotionlessorwalkingwithacurvedspine(kyphosis);OR-Legstiffness.Standingorwalkingwithextendedandtensedlegs(standingorwalkingontiptoes);OR-Normal.Bodyweightsupportedbythefourlegs.Noheadprostrated,nocurvedspineandnolegstiffness.Lying-Group/Flock.Thepigletswerecloseanddisorderlyoverlappedwitheachotherandthelyingposturewasnotclassifiable;OR-Sternal/Ventral.Lyingsternal,bodyweightsupportedbybelly,sternumincontactwiththefloor;OR-Lateral.Lyinglateral,bodyweightsupportedbyside,shoulderandribsincontactwithfloor.

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Animals2021,11,2763 8of20 Table2.Cont. BehaviourDescription Sitting-Sittingheaddown(prostrate).Bodyweightsupportedbyhindquartersflexedwithischiumonthefloorandfrontlegsextended.Awake,sittingmotionless,withtheheaddown,lowerthanshoulderlevel;OR-Normal.Bodyweightsupportedbyhindquartersflexedwithischiumonthefloorandfrontlegsextended,headnotdown/lowerthanshoulderlevel.Activity(explorativebehaviours)Awakeinactive.Nospecificactivitybutawake.Lyingsittingorstanding;ORSuckling.Teatinmouth,vigorousandrhythmicsucklingmovements;ORLookingforteat.Attemptstofindateatandtosuck.Thepigletisdesynchronisedfromthesucklingbehaviouroftheotherpiglets;ORExploring/Playing.Pigletnosing/manipulating:thesnoutisclosetoorincontactwithasubstrateorlittermate.Snoutmovementsmaybeobserved.Playing.Headshaking,springing(suddenjumpingorleaping),runningwithverticalandhorizontalbouncymovements.Caninvolvelittermates(gentlenudgingorpushing,mounting,chasing);ORAggression.Forcefulfighting,pushingwithheadorbitinglittermatesinaviolentmanner;ORWalking.Slowlymovingforwardwithonelegatatime;ORSleeping.Lyingdown,eyesclosed.

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ion.Forcefulfighting,pushingwithheadorbitinglittermatesinaviolentmanner;ORWalking.Slowlymovingforwardwithonelegatatime;ORSleeping.Lyingdown,eyesclosed. 2.5.StatisticalMethodsRawdatawereenteredintoMicrosoftExcelusingdouble-entrytechniques,andtransferredintoRsoftware,withdatalabelled‘TreatmentGroup1’and‘TreatmentGroup2’maintainingblinding.Differencesbetweentreatmentgroupsandstudysites:anydifferencesinweightbetweentreatmentgroupandstudysite,alongwithanyevidenceofaninteractionbetweentreatmentgroupandstudysiteweretestedatthe5%levelusingageneralisedlinearmodel(GLM)withnormallydistributedoutcomeandlinearlink.2.5.1.NociceptiveMotorResponsetoPigletCastrationTheprimaryefficacyparameterforpaincontrolduringcastrationwasasignificantmitigationofthenociceptivemotorresponsetocastration.Thescoringprovidedforthepiglet’smotorresponserepresentedanordinalscore.Ateachofthefourpoints,tractiononthetestisandcuttingthespermaticcordforboththefirstandsecondtestis,themotorresponsewasgradedonascaleof0to2.Thus,thetotalscoreiswithintherangeof0–8.Inviewofthefactthatamotorresponseinapigletmayoccurinresponsetorestraintratherthanpain,itwasconsideredthattherewasnotaclearcut-offvalueindicativeof‘pain’.Instead,itwasassumedthathighervalueseitherrepresentmorepainorcanbeconsideredmorelikelywhenapigletexperiencesapainfulprocedure.Anordinalregressionwasthusconsideredappropriateforthesedata,specificallyanorderedprobitmodel.Thenociceptivemotorresponsewasanalysedusinganordinalmixedeffectsregressionmodel,ontheordinalbehaviouralresponsescaletoestimatetheeffectoftreatment,siteandtheinteractionbetweentreatmentandsite.Efficacywasdeterminedbydemonstratingstatisticallysignificantdifferencesatthe5%level.Litterwasfittedinthismodelasarandomeffect.2.5.2.VocalResponsetoPigletCastrationVocalresponsewasexaminedasasecondaryefficacyparameter.Thepeakauditoryresponse(peakdB)andtotalvocalisation(AUC)duringspermaticcordcuttingwereanalysedusingamixedeffectslinearmodeltoestimatetheeffectoftreatment,site,andtheinteractionbetweentreatmentandsite.Thenaturallogarithmofmaximumvolume(dB)andtotalvolumeofvocalisation(AUC)weretakentoensurenormalityinthemodelresiduals.Peakvocalisationdatawereallnegative,sotheseweremultipliedby 1beforetakingthenaturallogarithm.

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Animals2021,11,2763 9of20 2.5.3.Post-OperativePain-RelatedBehaviourTheprimaryefficacyparameterforpaincontrolfollowingcastrationwasbasedonthedemonstrationofasignificantreductionintotalspecific“pain-related”behaviourbypigletsobservedduringthefirst30minpost-castrationfollowingreturntopen.Thisinvolvedassessmentofallspecific“pain-related”behavioursrecordedviafocalassessmentsattimes,1,15and30min,and/orviascansamplingassessmentsattimes0–10,10–20and20–30min(i.e.,datafromallfocalandscanrecordingsinthefirst30min).Forthefocalassessmentsateachoftheseassessments,ananimalwasclassedasshowingpain-associatedbehaviourifitwasrecordedashavingahuddled-upposture,stiffness,prostration,scratchingtheperinealareaortrembling/tremors.Onlyindividualsthatdemonstratednoneoftheseatanassessmentwererecordedasnotshowingpain-associatedbehaviouratthatassessment.Forthescanassessmentswithineachperiod,ifananimalwasrecordedasdemonstratinganyoneofthefollowingbehavioursatanypointduringthatperiodthentheanimalwasrecordedasdemonstratingpain-associatedbehaviourduringthatperiod:“CurvedSpine(Hunched)”,“HeadDown(Prostrate)”,“LegStiffness”,“Scratchingscrotal/perinealareaonfloor”,orwasrecordedas“Sternal/Ventrallying”andisolatedwithactivity“AI”(awakeinactive).Usingthisapproach,thelargeamountofinformationfromthefocalandscanobservationswascondensedforeachindividualintosixbinaryobservations(threefocalresultsandthreescanresults)ofpain-associatedbehaviour(yes/no).Ageneralisedlinearmixedmodelwithbinomiallydistributedoutcomeandlogisticlinkwasfittedtothepresenceorabsenceofpain-relatedbehaviourdatatoestimatetheeffectoftreatment,site,theinteractionbetweentreatmentandsite,timeaftercastrationand,whereappropriate,theinteractionoftimeandtreatment.Forallmodels,effectsstatisticallysignificantatthe5%levelwerekeptinthefinalmodelswhileotherswereremovedbybackwardselimination.Efficacywasdeterminedbydemonstratingeitherastatisticallysignificantdifferencebetweentreatments,acrossalltimepointsatthe5%level,oradifferenceinreductioninpain-relatedbehaviourovertime,statisticallysignificantatthe5%level.2.5.4.Additional(Secondary)BehaviouralAssessmentsScansamplingdatawasanalysedusingmultivariateanalysisofvariance(MANOVA)toestimatetheeffectoftreatment,site,theinteractionbetweentreatmentandsite,timebefore/aftercastrationandtheinteractionoftimeandtreatment.Duetothelargenumberofobservations,thetimeofobservationswasconsideredinthreephases:pre-castration,0–30minfollowingcastration,morethan30minfollowingcastration.Allobservationsmadeoneachanimalwereretainedinthedataanalysed.Effectssignificantatthe5%levelwerekeptinthefinalmodelwhilstotherswereremovedbybackwardselimination.Forthisanalysis,behaviourwasnotcategorisedas“pain-associated”ornot,butthemultivariatenatureoftheobservationswasanalysed(i.e.,ananimalcoulddemonstrateseveraldifferentbehaviourswithinonetimesegment),hencetheuseofaMANOVAanalysis.Post-hocanalysesofpigletpain-relatedbehaviouralresponsespre-andpost-castration,wereconductedonscansamplingandfocalassessmentdataseparatelytoallowtimeandtimexgroupcomparisons.Alltimepointsbeforecastrationweresettoasingletimepoint(“ 1”forthescananalysis,“StudyDay 1”forthefocalassessments),allowingimprovedprecisionofestimatesof“normal”behaviour.ThisdatawasthenfittedtoamodelwithTimepointandTreatmentgroupasmaineffectsandaninteractiontermbetweenthetwo.ThisallowedtheinterpretationofthemaineffectofTreatmentgroupasthedifferencebetweenthetwotreatmentstopriortocastration,whichwasnotexpectedtobedifferent.TheTimepointeffectisthedifferencebetweenpre-castrationandthattimepointforGroup1(Tri-Solfen®)andtheinteractionterm,thedifferencebetweenthetwogroupsatthattimepoint,assumingnodifferenceatthepre-castrationtimepoint.InitiallyamixedeffectsmodelwithPigletIDasarandomeffectwasfitted,butthisfailedtoconverge.Therefore,aGLMignoringtheeffectofpigletwasfitted.Thiswasconsideredtobeaconservativeapproachsinceonesourceofvariabilityinthedata,thedifferencesbetweenindividuals,wasnotaccountedfor,thusincreasingtheresidualvarianceinthemodel.This

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Animals2021,11,2763 10of20 hadtheeffectofincreasingtheminimumeffectsizethatwasconsideredtobestatisticallysignificantatthe5%levelwithoutalteringtheestimatesofeffectsize.3.Results3.1.DemographicsandClinicalParametersFromthe173piglets,86pigletswereassignedtothetreatmentgroupand87pigletswereassignedtotheuntreatedcontrolgroup.Sevenanimalsweresubsequentlyremovedfromthetrial,including(a)fouranimalsfromthetreatmentgroup(2 intestinalpro-lapse;1 inadvertentvariationincastrationtechnique;1 treatmentoverdose);(b)threeanimalsfromtheuntreatedcontrolgroup(3 inadvertentvariationincastrationtech-nique).OneanimalinthecontrolgroupdiedonStudyDay2.Necropsyperformedbyanindependentpathologistrevealedthepiglethadahighgradediffuse,purulentfibrinousenterocolitis/peritonitiswithEscherichiacolidetected.Asmallnumberofindividualswereremovedfromanalysesofspecificparametersduetoincompletedatarecords(e.g.,videorecordingsduringcastrationfailedintwocontrolpiglets).Therewasasignificanteffectofstudysiteoninitialweightatthe5%level(p-value<0.001),withmeanbodyweightattheGermansite(2.72kg 0.047)generallyhigherthanthoseattheItaliansite(1.97kg 0.064),however,therewerenodifferenceinbodyweightsbetweentreatmentgroupsateithersite.Therewerenosignificantdifferencesbetweentreatmentgroupsindemeanourandvitalityparameters,rectaltemperature,weight,orclinicalwoundassessments.Therewasastatisticaldifferenceinwoundhealingscorewithfeweranimalsinthetreatmentversuscontrolgrouphavingascore<5atDay6(64/84versus80/85,respectively,p=0.001)and<3atday12(52/84and74/85,respectively,p<0.001).TherewasoneSeriousAdverseEventineachgroup,includingonedeathofacontrolpiglet(duetoperitonitis),andshockwithrecovery(duetoanaphylaxis)inapigletinthetreatmentgroup.Therewerenosignificantdifferencesinadverseeventsbetweengroups.3.2.PigletNociceptiveMotorResponsesduringCastrationMeannociceptivemotorresponsescores,werefoundtobehigherincontrolpigletscomparedtoTri-Solfen®-treatedpigletsateachstepofcastration(e.g.,traction,cutting),aswellasintotalscores(4.37versus2.86,Table3).Forthetotalnociceptivemotorresponsescores,treatmentgroupwasfoundtobestatisticallysignificantatthe5%level,withthepainresponsepredictedtobeelevatedintheuntreatedcontrolgroup(p<0.001).Theresultsfurthermoreindicatetheoddsratioofanociceptivemotorresponsescorefromananimaltakenatrandomfromthecontrolgroupbeinginanequalorgreatercategorytoarandomanimaltakenfromthetreatedgroupwas2.9 10−1.07.Table3.Mean( standarddeviation)nociceptivemotorresponseduringpigletcastrationbetweenTri-Solfen®treatedanduntreatedcontrolgroups.

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edgroupwas2.9 10−1.07.Table3.Mean( standarddeviation)nociceptivemotorresponseduringpigletcastrationbetweenTri-Solfen®treatedanduntreatedcontrolgroups. TreatmentGroupTractiononFirstTestisaCutFirstSpermaticCordaTractionon2ndTestisaCutSecondSpermaticCordaTotalMotorResponseScoreb Tri-Solfen®0.1( 0.4)1.3( 0.7)0.4( 0.6)1.1( 1.1)2.9( 1.5)Control0.6( 0.7)1.7( 0.5)0.5( 0.7)1.6( 1.6)4.4( 1.5) aMaximumresponsescoreof2;bMaximumresponsescoreof8.3.3.PigletVocalResponsesduringCastrationThepeakdBandtotal(AUC)vocalisationresultsforeachtreatmentgroupcanbevisualisedinFigures2and3withthedataforeachexperimentalsitepresentedseparately.Atbothsites,greaterpeakintensity(dB)vocalisationresultswererecordedintheuntreatedcontrolgroupcomparedtopigletstreatedwithTri-Solfen®, andthisresultwasstatisticallysignificantatthe5%level(p<0.0001)andconsistentacrosssites(Figure2).Whilediffer-encesinthepeakintensitywerenotedbetweensites,thesedifferenceswerenotstatistically

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Animals2021,11,2763 11of20 significantatthe5%level(p=0.064).AnalysisoftheAUCbytreatmentgrouprevealedthatvocalresponsesinthetreatmentgroupwerestatisticallysignificantlylowerthanthoseofuntreatedpiglets(p<0.0001;Figure3)duringthetractionandseveranceofeachspermaticcord.Thisreductionwasconsistentacrosssites. Figure2.Boxplotofpeakvocalresponses(peakdB)bytreatmentgroupandsite.(Note,peakdBisrecordedonanegativelogarithmicscalewhere0isthemaximumresponse).Whitecirclesareoutliers. Figure3.Boxplotoftotalvocalresponses(AUC)calculatedfromareaunderthedB/timewaveformusingimageanalysis(Pxls)(encompassingvocalresponsefromcommencingtractiononthefirstspermaticcorduntil1sfollowingremovalofthesecondtesticleineachpiglet),displayedbytreatmentgroupandsite.Whitecirclesareoutliers.3.4.Pain-RelatedBehaviourinPigletsFollowingCastration3.4.1.Total“Pain-Related”BehaviourduringtheFirst30minPost-CastrationTheGLMModelfittedtothepain-relatedbehaviourduringthefirst30minfollowingcastrationfoundadifferenceintheoddsofshowingpainthatwasstatisticallysignificantatthe5%level(p<0.0001)withthetreatedgroup,showingsignificantlylesstotalspecific“pain-related”behaviourthantheuntreatedcontrolgroup.Table4showsagreaternumberofTri-Solfen®-treatedpigletsshowednopainbehaviour(n=23)inthe30minpost-castrationversustheircontrolgroupcounterparts(n=4).Apigletinthecontrolgrouphadtheoddsofshowing“pain-related”behaviourwhichwas2.39 10−0.87timesthatofa

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Animals2021,11,2763 12of20 pigletinthetreatedgroup,acrossallobservations.Therewasnoevidenceforaninteractionbetweentreatmentandobservation.Inallobservations,ananimalintheuntreatedcontrolgrouphadhigheroddsofbeingobservedexhibitingpain-associatedbehaviourthanananimalinthetreatedgroup.Overall,thelevelofpain-associatedbehaviourdecreasedoverthe30min.Pain-relatedbehaviourwasmorelikelytobeobservedduringthefocalobservationsthanthescansampling,andthiseffectwasstatisticallysignificantatthe5%level(p<0.0001).Asummaryofthenumberofpigletsexhibitingatleastonepain-relatedbehaviourduringthefocaland/orscanassessmentsperformedoverthe24hpriortocastrationisalsoincludedinTable4.Aproportionofpigletsexhibitedatleastonepain-relatedbehaviourduringgeneralobservationspriortocastration,withthemostcommonbeing“huddlingup”and“tremors/trembling”.Notethatthesearenotdirectlycomparablewithresultsfrom0–30minaspre-operativeresultsincludedatafromagreaterscanassessmentperiod(5h).Proportionsofpigletsdemonstratingstiffness,prostrationorscratchingduringtheDay 1scansandfocalassessmentswereverylow(<5%).Therewerenostatisticallysignificantdifferencesbetweengroups.Table4.Numberofpigletsshowingspecificpain-relatedbehaviourspre-castrationoveratotal5hobservationperiodand0–30minpost-castration. Pre-Castrationa0–30MinPost-Castration Totalobservation(timeperiod)5h0.5h GroupTri-Solfen(n=84)n(%)Control(n=86)n(%)Tri-Solfen(n=83)n(%)Control(n=86)n(%) Huddled39(46.2)38(44.2)37(44.6)52(60.5) Stiffness1(1.2)0(0.0)22(26.5)27(31.4) Prostrated3(3.6)3(3.5)30(36.1)41(47.7) Scratching0(0.0)1(1.2)2(2.4)0(0.0) Tremors9(10.7)5(5.8)11(13.3)16(18.6) Pigletsshowingatleast1pain-relatedbehaviour:focalassessment46(54.8)46(53.5)59(71.1)78(90.7)

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.0)1(1.2)2(2.4)0(0.0) Tremors9(10.7)5(5.8)11(13.3)16(18.6) Pigletsshowingatleast1pain-relatedbehaviour:focalassessment46(54.8)46(53.5)59(71.1)78(90.7) Pigletsshowingatleast1pain-relatedbehaviour:scansampling24(28.6)25(29.1)12(14.5)33(38.4) Pigletsshowingatleast1pain-relatedbehaviour:focalAssessmentORScanSampling57(67.9)55(64.0)60(72.2)82(95.3) Pigletsnotexhibitinganypain-relatedbehaviour27(32.1)31(36.0)23(27.7)4(4.7) aNumbersshownarefromtheintentiontotreatpopulation.3.4.2.ScanSamplingDataThesecondarypost-operativeendpointexaminedinvolvedacomparisonofthebe-haviourintreatedandcontrolgroupsbyMANOVA,performedonthescansamplingdata.Althoughstatisticaldifferenceswerefoundbetweenscanresultsatthetwositesandbetweenthetimeperiods(pre-treatment,first30minaftercastrationand>30minaftercastration;p<0.01foreach),therewasnotreatmentandtimeinteraction(p=0.475)oroveralltreatmenteffect(p=0.39).Therefore,therewasnoeffectshownonthissecondaryvariable.Thescansamplingresults(Figure4)identifiedthat,inthe24hpriortocastration,pigletswereeithersleepingorsucklinginthemajorityofscanobservations(55–60%,and~20%,respectively).Smallerproportionswereawakeinactive,walkingorexploring(5–10%ofobservations),withonlyminorproportionssitting,scratchingorlookingforteat(<5%ofobservations).Therewasanincreaseininstancesofpigletssleepinginthefirst

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Animals2021,11,2763 13of20 24hfollowingcastrationascomparedwithpre-operativevalues.Thiswasparticularlyevidentoverthetwo-hourDay0PMscanperiod(i.e.,intheafternoonfollowingcastration).Consistentwithanincreaseinthenumberofpigletssleeping,therewasarelativedecreaseintheproportionofpigletsawakeinactive,walkingandexploring,aswellasarelativeincreaseinpigletslying,anddecreaseinpigletsstandingduringthissameperiod,however,theproportionofpigletssucklingwassimilaroverallobservationperiods.Theproportionofpigletsdisplayingabnormal(pain-related)posturesonscansampling(i.e.,showingprostration,standingorsitting,hunchedstanding,stiffnessorscratching)wasverylow(<5%ofobservations)otherthaninthefirst20minfollowingcastration,duringwhichtimeitwasmoreprominentinuntreatedcontrolthantreatedpiglets.Statisticalanalysisofpain-relatedbehaviourfocusedondatacollectedonStudyDay 1(pre-castration)andin10minintervalsupto2hpost-castrationandidentifiednostatisticallysignificantdifferenceinpain-relatedbehaviourbetweenthetwogroupspriortocastration(datanotshown).Therewasalowmagnitudestatisticallysignificantincrease(p<0.05)inpain-relatedbehavioursovertimeascomparedwithpre-operativevaluesuntilthe40mintimepoint.Thiswasmostprominent10–20minpost-castrationincontrolanimals,however,differencesbetweengroupsdidnotreachstatisticalsignificanceatanytimepoint.Theproportionofanimalsobservedtobeinthenest(underheat)orisolatedvariedbetweenthetwoinvestigationsites.Atalltimepoints,agreaterproportionofanimalswereobservedtobeinthenest(underheat)andalesserproportionisolated(asopposedtoincontactwiththesoworlittermates),attheGermanratherthantheItaliansite. Figure4.Resultsofscanassessmentsshowingproportionofpiglets(A)observedindifferentactivities(B)observedindifferentposturesinuntreatedcontrol(CON)andTri-Solfen(TS)treatmentgroups.

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Animals2021,11,2763 14of20 3.4.3.FocalAssessmentDataPost-hocanalysiswasperformedonthefocalassessmentdataseparatelyexaminingdifferencesbetweengroupsandovertime,includingpre-andpost-castration.Nosta-tisticallysignificantdifferenceswereobservedintotalpain-relatedbehavioursbetweentreatmentandcontrolgroupspriortocastration.Atthe1minobservation,theTri-Solfen®-treatedgroupshowedastatisticallysignificantincreaseintotalpain-relatedbehaviour(p<0.05)ascomparedwithpre-operativevalues.Atnoothertimepointwasthelevelofpain-relatedbehavioursignificantlydifferentfrompre-castrationlevelsinthetreatedgroup.Thecontrolgroupshowedmoretotalpain-relatedbehaviourpost-operativelycomparedwithpre-operativelyandascomparedwiththetreatedgroupatboth1and15mintimepointspost-castration.Thesedifferenceswerestatisticallysignificantatthe5%level.4.DiscussionThereisanurgentneedforeffectiveproductsforpainmitigationduringandfol-lowingpigletcastration,thatarepracticalforuseincommercialproductionsystems.Tomeetregulatoryrequirementsandachieveapprovalforuse,suchproductsmustmeetproofofefficacyrequirementstointernationallyharmonised(VICH)goodclinicalpractice(GL9)guidelines[53].InthisstudywereportresultsofaVICHGL9fieldefficacystudydemonstratingefficacyofatopicallocalanaestheticandantisepticproduct(Tri-Solfen®),administeredviaintraoperativewoundinstillation,witha30sdwelltime,tomitigatesubsequentcastration-relatedpaininpigletsbasedonasignificantreductionofnocicep-tivemotorandvocalresponsesduringtheprocedureandinpost-operativepain-relatedbehaviourevidentinthefirst30minfollowingtheprocedure.Thisbuildsonresultsfromapreviousstudyreportingefficacybasedonasignificantreductioninpigletmotorandvocalresponsesduringtheprocedureandwound-hyperalgesicresponsesinthepost-operativeperiod[49].Reducednociceptivemotorresponsetothecastrationprocedure(tractiononandseveringthespermaticcord)wastheprimaryefficacyparameterforpaincontroldur-ingtheprocedureinthisstudy.Studiesofpigletnociceptivemotorandvocalresponseidentifytractionon,andseveringthespermaticcords[7,14,15]asinducingthegreatestpain[7,14–16,52]oftheprocedure.Previousstudiesidentifiedpre-emptivelidocaineorbupivacaineinjectionaseffectivetomitigatethepain-induced(nociceptive)motorre-sponse[22,28,36,37].Mostrecently,Salleretal.[28]reportedthat,usedincombinationwithlightgeneralanaesthesiatominimisethemotorresponsetorestraintandhandlingstress,Lidocaine2%,Bupivacaine0.5%,Mepivacaine2%andProcaineadministeredviainjection20minpriortocastrationwerealleffectivetoblockthenociceptivemotorresponsetocastrationascomparedwiththoseinjectedwithnormalsaline(placebo).Limbmovementswereevidentduringinjections,however,thereafterwerevirtuallyabolishedinresponsetoskinincisionandtoseveringthecordsinlocalanaesthetictreated,butnotsalinetreatedpiglets,withthegreatesteffectinlidocaineandbupivacaine-treatedpiglets.Administeredbyinjectionsuchasintothetestis,ittakes3minormoreforlidocainetodiffusetothenervetissueinthespermaticcord,andbegintotakeeffect[57].Onsetmaybemuchmorerapid,within30s[50,51,58]however,whenlidocaineisappliedtopically,directlytonon-keratinisedmucosaltissuessuchasintheconjunctivaorgingivalsulcus.Thisappearsalsotoapplytothespermaticchordaltissueinneonatalpigletsas,usingdirectwoundinstillationfollowingskinincisionwitha30sdwelltime,ithasbeenshownthatTri-Solfen®waseffectivetomitigatethenociceptivemotorresponsetothesubsequentcas-trationprocedure,ascomparedwithplacebo-treatedpiglets,inaprevioustrialin3–7dayoldpigletsundergoingcastrationonacommercialpigfarminAustralia[49].Consistentwiththisprevioustrial,inthecurrentstudy,Tri-Solfen®similarlyadministered,resultedinasignificantreductioninmeannociceptivemotorresponsetotractionon,andexcisionofeachtestisduringsurgicalcastration.Theseresultswereconsistentacrossbothtrialsites,andtogetherwithourpreviousstudyresults[49],areconsideredtoconfirmtheefficacyof

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Animals2021,11,2763 15of20 theproductusedviathismethodtomitigateacuteproceduralcastrationpaininneonatalpiglets.Pigletvocalresponsetocastrationwasexaminedasasecondaryefficacyparameterforpainmitigationduringcastrationinthecurrentstudy,asintheprevioustrial[49].Althoughpigletsvocaliseinresponsetorestraintandhandling,vocalresponsesincastratedpigletshavepreviouslybeenshowntobesignificantlylouderandmorefrequentincontrolanimalscomparedtothosepre-treatedwithlocalanaesthetic[22,52]andsham-treatedanimals[14,59].Analysisofthespecificstepsinthecastrationprocessrevealedthatthehighestfrequencycallsareemittedwhenthespermaticcordsarepulledandsevered[15].Asnotedinotherpublicationsinvestigatingtheroleoflocalanaestheticusetoreducepainincastratedpiglets[22],areductioninvocalresponsesduringcastrationisconsideredtobeindicativeofsignificantpainmitigation.Sutherlandetal.[48]reportednosignificanteffectoftopicalanaesthetic(Tri-Solfen®orCetacaine®)instilledintothewoundonpigletvocalresponsetocastration,however,inthisprevioustrial,nodwelltimehadbeenobservedtoallowthelocalanaestheticstotakeeffect.Withinclusionofa30sdwelltime,Sheiletal.[49]reportedasignificantreductioninvocalresponsesinTri-Solfen®-treatedpigletsfromthefirsttractiononthefirstspermaticcordthroughtocordseverance,comparedtocontrols.Thisreductioninpigletvocalresponsewasobservedforboththemaximumvocalisationresult(peakdB)andthetotalvocalresponse,asmeasuredbyareaunderthedecibel/timewaveform.Inthisprevioustrial[49],atrendeffectwasalsoevidentduringtractionon,andseveringthesecondspermaticcord,howeveritdidnotreachstatisticalsignificance.Increasedvariability,associatedwithinadvertentprolongationoftheanalysedrecordingtime,(whichwascontinueduntilpigletvocalisationreturnedtobaselineratherthanceasingafterremovalofthesecondtesticle),wasconsideredtohavepotentiallyconfoundedtheresults.Inthecurrenttrial,thevocalresponsedataanalysiswasconfinedtothespecifictimeperiodsoftractiononandseveringeachspermaticcordandceased1sfollowingcompletionofremovalforeachtesticle.Inthissetting,andwithgreaterpowerprovidedbyalargersamplesize,asignificantreductioninpigletvocalresponsetoseveringbothspermaticcordswasevidentandconsistentwithareductioninnociceptivemotorresponseduringthesameproceduralevents.Therobustnessofthisobservationisevidencedbythefactthatthisresultwasrepeatableattwoindependentsites,workingpiggeriesinGermanyandItaly.TheseresultsarethusconsideredindicativeofsignificantproceduralpainmitigationinTri-Solfen®-treatedpiglets.ItshouldbenotedthatthenociceptivemotororvocalresponsetoskinincisionwasnotmeasuredasTri-Solfen®wasonlyappliedfollowingincisionandisnotexpectedtohaveapainmitigatingeffect.Ideally,inthefuture,methodsofconcurrentlyprovidingpre-incisionalskinanaesthesia,maybedevelopedthatarepractical,rapidandpainless.Onesuchoption,iscryo-anaesthesia,whichiscurrentlyunderinvestigationasamethodofpre-operativeskinanaesthesiaforhusbandryproceduresinpigletsandcalves[60,61].Topicalanaesthesiacanalsobeeffectivetoanesthetiseintactskinpriortominorprocedures,however,maytake20–30mintohaveeffectduetodifficultypenetratingkeratinisedtissues,whichmaynotbepracticalforon-farmuse.Subcutaneousinjectionofalocalanaestheticprovidesanotheroptionforpre-incisionalskinanaesthesia.Itisnotable,however,thattheinjectionsthemselvesarepainful,andmaybeequallyaspainfulasquickskinincision,basedonsimilarnociceptivemotor[28],andvocalresponsescores[62,63].Thismayalsoincreasethetotalstressoftheprocedureduetotheneedforeitherdoublinghandlingorprolongedrestraintfor3mintoreachskinanaestheticeffect,suchthatitisnotclearthatitwouldprovideanoverallwelfarebenefitinthissetting[37].Intheinterim,itisnotablethatskinincisionisconsistentlyidentifiedastheleastpainfulpartofthecastrationprocedure[14,15,36,63,64]andanaestheticonsetisveryswift(withinoneminute)followingTri-Solfen®applicationtopicallytotheincisionsite,asthelocalanaestheticsactivesareabletoactdirectlyonexposedtraumatisednerveendingsinthecutskinedge,asnotedabove.UsingVon-Freyandneedlestickassessmentatthecutskinedge,rapidityofonsetofanaestheticeffect(withinoneminute)hasbeendemonstratedfollowingTri-Solfen®

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Animals2021,11,2763 16of20 applicationtoarangeofwounds,includingthepigletcastrationwound[43,44],aswellashusbandrywoundsinlambsandcalves[43,45,65].Inthecurrentstudy,anacutepain-relatedbehaviourresponse,evidentincontrolpigletsinthefirst30minpost-castration,wassignificantlyreducedinTri-Solfen®-treatedpiglets.Theresultsofthebehaviouralanalysisappeartofurtherconfirmpreviousreportsfromdirectobservationalstudies[8,12,13,19,35,66]identifyingthatanacuteincreasein“pain-specific”behavioursoccurinneonatalpigletsintheearlytimeperiodsfollowingcastration.Thesebehavioursaresubtleandshort-lived,beingevidentatsignificantlevelsinthisstudyprimarilyonlyoverthefirst30min(scanandfocalassessmentsdatacombined)andat1and15minpost-castrationusingfocalassessmentdataalone.Focalassessmentsprovedtobemoresensitivethanscansamplingfordetectionofpain-associatedbehaviour.Scansamplingrevealedtheverylowproportionofpigletsexhibitingsuchbehaviours.Similarresultswerealsopreviouslynotedinpigletsreceivingpre-emptiveinjectableNSAIDs,orpre-andpost-castrationtopicalpreparationsoftetracaine[35],inwhichpigletscastratedwithouttreatmentshowedincreasedfrequencyofspecificpain-relatedbehaviourinthefirst30minincomparisonwithsham-handledandtreatedpiglets,however,nobehaviouraldifferenceswereapparentafter60min.Scandatarevealedthemajorityofpigletswereobservedtobesleepingorsucklingduringscanobservations,andtheproportionsleepingincreasedsignificantlytheafternoonfollowingcastration.Thismayexplainthedifficultyindetectingpain-associatedbehaviourinpigletsofthisageoverthistimeperiod.Increasedpigletsleeping,aswasevidentataround6–8hfollowingcastrationinthecurrenttrial,hasalsobeenreportedbyViscardiandTurner[63,67,68]whosimilarlycomparedpigletbehavioirpre-andpost-castration,andalsobyKluiversandPoodt[8],whoreported70–75%ofpigletssleepingduringscanas-sessmentstheafternoonfollowingcastrationorshamhandling,withnodifferencebetweengroups.Asleepresponsetoaversivestimulationisknowntooccurinneonates[69,70].Ashandlingandrestraintareaversivetopiglets,increasedsleepingfollowinghandlingandrestraintmaybecommontobothcastratedandsham-handledanimalsandrequiresfurtherinvestigation.Inthissetting,trendsforincreasedlying,withreducedstanding,walking,exploring,etc.,couldallbeconsequentuponanincreaseinpigletssleepingfollowinghandling,ratherthanbeingindicativeofpainpost-castration.Interestingly,however,theincreaseinsleepingbehaviourdidnotappeartoeffectsucklingornursingbehaviourpre-andpost-castration.Fromthefocalassessmentdata,therewerenosignificantdifferencesintailwaggingorscratchingpre-castrationorintheearlyhourspost-castrationinpigletsinthistrial.Therewasanincreaseofrelativelylowmagnitudeinbothgroupsatlatertimepointswithapeakat24h,however.ThisobservationisconsistentwithHayetal.[12]andViscardiandTurner[63,67].Tailwaggingandscratchingaregenerallyconsideredtoindicateirritationordiscomfortandhavebeenreportedtopeak24hfollowingcastration[12,63,67],andhavebeenreportedtoremainelevatedforseveraldaysinsomestudies[12,64].Others,however,havenotreportedanincreaseintail-waggingpost-castration[13].Paradoxically,tailwagginghasbeenreportedtobeincreasedinpigletstreatedwithinjectedlidocaineadministeredpre-castration[8]ortopicallidocainesprayappliedpost-castration[19],asopposedtountreatedcastratedcontrols.Authorsofthesestudieshypothesisedthatlidocainemayinduceasensationasitwearsoff(suchastingling)thatinducestailwagging,orthe80%alcoholcontentofthespray-formulationusemayhavesensitisedthetail.Tri-Solfen®contains5%lidocaine,alongwith0.5%Bupivacaine.Nosignificantincreaseintailwagginginpigletsinthefirsthoursaftercastrationascomparedwithpre-operativevalueswasobserved,norasignificantdifferenceintailwaggingbetweentreatedanduntreatedcontrolpigletsinthecurrenttrial.Bupivacainehasasloweronsetofactionthanlidocaine,generally5–10minwhenappliedtomucosaltissues,however,ithasamoreprolongeddurationofaction,lastinguptoseveralhours.Itispossiblethattheactionofbupivacainepreventsanysensationduetolidocainewearingoff,andthuspreventsincreasedtailwagginginthissituation.

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Animals2021,11,2763 17of20 Itshouldbenotedthatacutepain-relatedbehaviourwasonlyevidentinpigletsforthefirst30mininthisstudy.Thisisofsignificantlyshorterdurationthanacutepost-operativemechanicalhyperalgesia,whichhasbeendocumentedfrom2to4hpost-castrationinuntreatedneonatalpigletsusingresponsetovon-Freyandneedlestickstimulationofthewound[44,49].Thisisnotunexpectedasquantitativemechanicalsensorytestingexaminesevokedpainresponses,whichmayormaynotreflectthedegreeof“spontaneous”painananimalexperiencesfromawoundsiteintheabsenceofastimulus(e.g.,whilelyingquietlyorsleeping).Kawamataetal.[71],forexample,subjectedhumanvolunteerstoasmallincisioninthevolarforearm,anareafrequentlyusedforsensorytestinginhumans.Inthesevolunteers,painatrestdecreasedanddisappearedwithin2haftertheexperimentalincision.Painresponsetomechanicalstimuliattheincision(i.e.,primarymechanicalhyperalgesia),however,waspresentforseveraldays.Inthefield,safetywasevidentinthelackofimpactsonweight,demeanour,vitality,rectaltemperatureorincidenceofadverseeventsincludingwoundinfection.Differencesinwoundhealingscoressuggestpossibleminordelayinwoundscabresolutionintreatedpigletsinthistrial,however,thisdidnotappeartohaveasignificantimpactonclinicalrecovery.Initialwoundsizewasnotmeasuredinthisstudytoknowiftheremayhavebeenaninadvertentdifferenceatthetimeofsurgery.WoundhealingdifferenceswerenotapparentinTri-Solfen®-treatedpigletsinaprevioustrialreportedbySutherlandetal.[48],orinaregulatoryTargetAnimalSafetystudy(VICHGL43)[72],tobereportedseparately.Similarly,nosignificantimpactsonwoundhealingand/orimprovedwoundhealinghavebeenreportedfollowinguseofTri-Solfen®onotherwoundtypesinotherspecies[47,73–75].5.ConclusionsTheresultsofthisVICHGL9regulatorydrugfieldefficacystudyareconsideredtoconfirmthatintra-operativeadministrationofTri-Solfen®viawoundinstillationimmedi-atelyfollowingskinincisionandthena30swaitperiodresultsinsignificantmitigationofpigletpainduringthesubsequentcastrationprocedure,andtheearlypost-operativeperiod,whenpainismostacute.AstopicalwoundinstillationofTri-Solfen®(with30sdwelltime)maybefarmerappliedandinvolvesrelativelyminimalinterruptiontothestandardproceduresutilisedforpigletcastration,itisanticipatedthatitmayprovideanimportantandviablestrategytomitigateacutecastration-relatedpaininpigletsundercommercialproductionconditions,andcontributetoimprovepigletwelfarewheresurgicalcastrationisstillutilisedincommercialpigfacilitiesworldwide.AuthorContributions:Conceptualisation,M.S.;G.M.D.B.;A.S.;S.M.;F.G.;methodology,M.S.;G.M.D.B.;A.S.;S.M.;F.G;formalanalysis,G.I.;investigation,G.M.D.B.;A.S.;F.G;writing—originaldraftpreparation,A.P.;M.S.;writing—reviewandediting,A.P.,M.S.,S.M.;G.M.D.B.;A.S.;G.I.;F.G.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.Funding:FundingforthisstudywasprovidedbyAnimalEthicsPtyLtd.,YarraGlen,Victoria,NSW,Australia.InstitutionalReviewBoardStatement:ThestudywasconductedaccordingtotheguidelinesoftheDeclarationofHelsinki.Allstudyprocedureswereconductedundercountry-specifictrialapprovals,includingethicalapproval.InGermany,approvalwasobtainedfromtheSchleswig-HolsteinauthoritywhileapprovalfortheItalianstudywasprovidedbytheMinisterodellaSalute.InformedConsentStatement:Notapplicable.DataAvailabilityStatement:Relevantdataiscontainedwithinthearticle.Acknowledgments:Wethankthefollowingpeopleforassistancewiththisstudy,including(i)DirkHoffmanforassistancewiththeGermananimalstudies;(ii)TimothyPearceforthepigvocaldataanalysis;(iii)SabrinaLomaxforassistancewithvideoanalysis;(v)HelenCuppello,JoanneKnipeandLynneBraidwoodforassistancewithanimalmonitoring,qualityassuranceandregulatoryapproval.

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ConflictsofInterest:ResearchwassponsoredbyAnimalEthicsPtyLtd.,andcarriedoutbyinde-pendentveterinaryresearchcompanyTriveritasPtyLtd.,toGCPandVICHstandards,toaddressnationalandinternationalveterinarymedicinesregulatoryapprovalrequirements.SheilisaninventorofTri-Solfen®,aFoundingDirectorandindirectShareholderofAnimalEthicsPtyLtd.References1.Bonneau,M.;Weiler,U.ProsandConsofAlternativestoPigletCastration:Welfare,BoarTaint,andOtherMeatQualityTraits.Animals2019,9,884.[CrossRef]2.vonBorell,E.;Baumgartner,J.;Giersing,M.;Jaggin,N.;Prunier,A.;Tuyttens,F.A.;Edwards,S.A.Animalwelfareimplicationsofsurgicalcastrationanditsalternativesinpigs.Animal2009,3,1488–1496.[CrossRef][PubMed]3.CastrumConsortium.CASTRUM—PigcastrationforTraditionalandConventionalProducts:AReportonMethodsandTheirImpactsonAnimalWelfare,MeatQualityandSustainabilityofEuropeanPorkProductionSystems.FinalReport.2016.Availableonline:https://publications.europa.eu/en/publication-detail/-/publication/5fe8db00-dbb8-11e6-ad7c-01aa75ed71a1(accessedon18September2021).4.DeBriyne,N.;Berg,C.;Blaha,T.;Temple,D.Pigcastration:WilltheEUmanagetobanpigcastrationby2018?Porc.HealthManag.2016,2,29.5.Sutherland,M.A.Welfareimplicationsofinvasivepiglethusbandryprocedures,methodsofalleviationandalternatives:Areview.N.Z.Vet.J.2015,63,52–57.[CrossRef]6.Andresen,O.Boartaintrelatedcompounds:Androstenone/skatole/othersubstances.ActaVet.Scand.2006,48,S5.7.Rault,J.-L.;Lay,D.C.;Marchant-Forde,J.N.CastrationInducedPaininPigsandOtherLivestock.Appl.Anim.Behav.Sci.2011,135,214–225.8.Kluivers-Poodt,M.;Zonderland,J.J.;Verbraak,J.;Lambooij,E.;Hellebrekers,L.J.Painbehaviouraftercastrationofpiglets;effectofpainreliefwithlidocaineand/ormeloxicam.Animal2013,7,1158–1162.[CrossRef][PubMed]9.Prunier,A.;Bonneau,M.;VonBorell,E.H.;Cinotti,S.;Gunn,M.;Fredriksen,B.;Giersing,M.;Morton,D.B.;Tuyttens,F.A.M.;Velarde,A.Areviewofthewelfareconsequencesofsurgicalcastrationinpigletsandtheevaluationofnon-surgicalmethods.Anim.Welf.2006,15,277–289.10.McGlone,J.J.;Nicholson,R.I.;Hellman,J.M.;Herzog,D.N.Thedevelopmentofpaininyoungpigsassociatedwithcastrationandattemptstopreventcastration-inducedbehavioralchanges.J.Anim.Sci.1993,71,1441–1446.[CrossRef]11.Marchant-Forde,J.N.;Lay,D.C.,Jr.;McMunn,K.A.;Cheng,H.W.;Pajor,E.A.;Marchant-Forde,R.M.Postnatalpiglethusbandrypracticesandwell-being:Theeffectsofalternativetechniquesdeliveredseparately.J.Anim.Sci.2009,87,1479–1492.[CrossRef]12.Hay,M.;Vulin,A.;Génin,S.;Sales,P.;Prunier,A.Assessmentofpaininducedbycastrationinpiglets:Behavioralandphysiologicalresponsesoverthesubsequent5days.Appl.Anim.Behav.Sci.2003,82,201–218.[CrossRef]13.Moya,S.L.;Boyle,L.A.;Lynch,P.B.;Arkins,S.Effectofsurgicalcastrationonthebehavioralandacutephaseresponsesof5-day-oldpiglets.Appl.Anim.Behav.Sci.2008,111,113–145.14.Weary,D.M.;Braithwaite,L.;Fraser,D.Vocalresponsetopaininpiglets.Appl.Anim.Behav.Sci.1998,56,161–172.15.Taylor,A.;Weary,D.M.Vocalresponsesofpigletstocastration:Identifyingproceduralsourcesofpain.Appl.Anim.Behav.Sci.2000,70,17–26.[PubMed]16.Puppe,B.;Schon,P.C.;Tuchscherer,A.;Manteuffel,G.Castration-inducedvocalisationindomesticpiglets,Susscrofa:Complexandspecificalterationsofthevocalquality.Appl.Anim.Behav.Sci.2005,95,67–78.17.AmericanVeterinaryMedicalAssociation.LiteratureReviewontheWelfareImplicationsofSwineCastration.Availableonline:https://www.avma.org/resources-tools/literature-reviews/welfare-implications-swine-castration(accessedon8June2020).18.Sutherland,M.A.;Davis,B.L.;Brooks,T.A.;Coetzee,J.F.Thephysiologicalandbehavioralresponseofpigscastratedwithandwithoutanesthesiaoranalgesia.J.Anim.Sci.2012,90,2211–2221.[CrossRef][PubMed]19.Burkemper,M.C.;Pairis-Garcia,M.D.;Moraes,L.E.;Park,R.M.;Moeller,S.J.EffectsofOralMeloxicamandTopicalLidocaineonPainassociatedBehaviorsofPigletsUndergoingSurgicalCastration.J.Appl.Anim.Welf.Sci.2020,23,209–218.[CrossRef]20.O’Connor,A.;Anthony,R.;Bergamasco,L.;Coetzee,J.;Gould,S.;Johnson,A.K.;Karriker,L.A.;Marchant-Forde,J.N.;Martineau,G.S.;McKean,J.;etal.Painmanagementintheneonatalpigletduringroutinemanagementprocedures.Part2:Gradingthequalityofevidenceandthestrengthofrecommendations.Anim.HealthRes.Rev.2014,15,39–62.[CrossRef][PubMed]21.Dzikamunhenga,R.S.;Anthony,R.;Coetzee,J.;Gould,S.;Johnson,A.;Karriker,L.;McKean,J.;Millman,S.T.;Niekamp,S.R.;O’Connor,A.M.Painmanagementintheneonatalpigletduringroutinemanagementprocedures.Part1:Asystematicreviewofrandomizedandnon-randomizedinterventionstudies.Anim.HealthRes.Rev.2014,15,14–38.[CrossRef]22.Hansson,M.;Lundeheim,N.;Nyman,G.;Johansson,G.Effectoflocalanaesthesiaand/oranalgesiaonpainresponsesinducedbypigletcastration.ActaVet.Scand.2011,53,34.[CrossRef]23.Keita,A.;Pagot,E.;Prunier,A.;Guidarini,C.Pre-emptivemeloxicamforpostoperativeanalgesiainpigletsundergoingsurgicalcastration.Vet.Anaesth.Analg.2010,37,367–374.[CrossRef]24.Kluivers-Poodt,M.;Houx,B.B.;Robben,S.R.;Koop,G.;Lambooij,E.;Hellebrekers,L.J.EffectsofalocalanaestheticandNSAIDincastrationofpiglets,ontheacutepainresponses,growthandmortality.Animal2012,6,1469–1475.[Cros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26.Walker,B.;Jaggin,N.;Doherr,M.;Schatzmann,U.Inhalationanaesthesiaforcastrationofnewbornpiglets:Experienceswithisofluraneandisoflurane/NO.J.Vet.Med.APhysiol.Pathol.Clin.Med.2004,51,150–154.[CrossRef][PubMed]27.Kohler,I.;Moens,Y.;Busato,A.;Blum,J.;Schatzmann,U.Inhalationanaesthesiaforthecastrationofpiglets:CO2comparedtohalothane.Zent.Vet.A1998,45,625–633.[CrossRef]28.Saller,A.M.;Werner,J.;Reiser,J.;Senf,S.;Deffner,P.;Abendschon,N.;Weiss,C.;Fischer,J.;Schorwerth,A.;Miller,R.;etal.Localanesthesiainpigletsundergoingcastration-Acomparativestudytoinvestigatetheanalgesiceffectsoffourlocalanestheticsonthebasisofacutephysiologicalresponsesandlimbmovements.PLoSONE2020,15,e0236742.[CrossRef]29.Yun,J.;Ollila,A.;Valros,A.;Larenza-Menzies,P.;Heinonen,M.;Oliviero,C.;Peltoniemi,O.Behaviouralalterationsinpigletsaftersurgicalcastration:Effectsofanalgesiaandanaesthesia.Res.Vet.Sci.2019,125,36–42.[CrossRef]30.Marsalek,P.;Svoboda,M.;Smutna,M.;Blahova,J.;Vecerek,V.Neopterinandbiopterinasbiomarkersofimmunesystemactivationassociatedwithcastrationinpiglets.J.Anim.Sci.2011,89,1758–1762.[CrossRef]31.Zöls,S.;Ritzmann,M.;Heinritzi,K.EinflussvonSchmerzmittelnbeiderKastrationmännlicherFerkel[Effectofanalgesicsonthecastrationofmalepiglets].Berl.Munch.Tierarztl.Wochenschr.2006,119,193–196.[PubMed]32.Courboulay,V.;Hemonic,A.;Gadonna,M.;Prunier,A.Effectoflocalanesthesiaoranti-inflammatorytreatmentonpainassociatedwithpigletcastrationandonlabourdemand.JournessDeLaRech.Porc.EnFr.2010,42,27–34.33.Bonastre,C.;Mitjana,O.;Tejedor,M.T.;Calavia,M.;Yuste,A.G.;Ubeda,J.L.;Falceto,M.V.Acutephysiologicalresponsestocastration-relatedpaininpiglets:Theeffectoftwolocalanestheticswithorwithoutmeloxicam.Animal2016,10,1474–1481.[CrossRef][PubMed]34.Nyborg,P.Y.;Sørig,A.;Lykkegaard,K.;Svendsen,O.Nociceptionaftercastrationofjuvenilepigsdeterminedbyquantitativeestimationofc-Fosexpressingneuronsinthespinalcorddorsalhorn.Dan.Veterinærtidsskrift2000,83,16–17.35.Gottardo,F.;Scollo,A.;Contiero,B.;Ravagnani,A.;Tavella,G.;Bernardini,D.;DeBenedictis,G.M.;Edwards,S.A.Painalleviationduringcastrationofpiglets:Acomparativestudyofdifferentfarmoptions.J.Anim.Sci.2016,94,5077–5088.[CrossRef][PubMed]36.Horn,T.;Marx,G.;vonBorell,E.[Behaviorofpigletsduringcastrationwithandwithoutlocalanesthesia].Dtsch.Tierarztl.Wochenschr.1999,106,271–274.[PubMed]37.Leidig,M.S.;Hertrampf,B.;Failing,K.;Schumann,A.;Reiner,G.Painanddiscomfortinmalepigletsduringsurgicalcastrationwithandwithoutlocalanaesthesiaasdeterminedbyvocalizationanddefencebehavior.Appl.Anim.Behav.Sci.2009,116,174–178.[CrossRef]38.Haga,H.A.;Ranheim,B.Castrationofpiglets:Theanalgesiceffectsofintratesticularandintrafunicularlidocaineinjection.Vet.Anaesth.Analg.2005,32,1–9.[CrossRef]39.Hancock,T.M.;Caulkett,N.A.;Pajor,E.A.;Grenwich,L.Aninvestigationoftheeffectsofintratesticularalfaxaloneandlidocaineduringcastrationinpiglets.Vet.Anaesth.Analg.2018,45,858–864.[CrossRef]40.Scollo,A.;Contiero,B.;DeBenedictis,G.M.;Galli,M.C.;Benatti,D.;Gottardo,F.Analgesiaand/oranaesthesiaduringpigletcastration—PartI:Efficacyoffarmprotocolsinpainmanagement.Ital.J.Anim.Sci.2021,20,143–152.[CrossRef]41.Scollo,A.;Galli,M.C.;Contiero,B.;DeBenedictis,G.M.;Orlandi,B.;Gottardo,F.Analgesiaand/oranaesthesiaduringpigletcastration—PartII:Practicabilityoffarmprotocols„resourceefficiencyandeconomicimplications.Ital.J.Anim.Sci.2021,20,472–478.[CrossRef]42.Kluivers-Poodt,M.;Hopster,H.;Spoolder,H.A.M.CastrationunderAnesthesiaand/orAnalgesiainCommercialPigProduction;AnimalSciencesGroup:WageningenUR,Holland,2007.43.Lomax,S.;Dickson,H.;Sheil,M.;Windsor,P.A.Topicalanaesthesiaalleviatesshort-termpainofcastrationandtaildockinginlambs.Aust.Vet.J.2010,88,67–74.[CrossRef]44.Lomax,S.;Harris,C.;Windsor,P.A.;White,P.J.Topicalanaesthesiareducessensitivityofcastrationwoundsinneonatalpiglets.PLoSONE2017,12,e0187988.[CrossRef]45.Lomax,S.;Sheil,M.;Windsor,P.A.Durationofactionofatopicalanaestheticformulationforpainmanagementofmulesinginsheep.Aust.Vet.J.2013,91,160–167.[CrossRef][PubMed]46.Lomax,S.;Windsor,P.A.Topicalanesthesiamitigatesthepainofcastrationinbeefcalves.J.Anim.Sci.2013,91,4945–4952.[CrossRef]47.Lomax,S.;Sheil,M.;Windsor,P.A.Impactoftopicalanaesthesiaonpainalleviationandwoundhealinginlambsaftermulesing.Aust.Vet.J.2008,86,159–168.[CrossRef]48.Sutherland,M.A.;Davis,B.L.;Brooks,T.A.;McGlone,J.J.Physiologyandbehaviorofpigsbeforeandaftercastration:Effectsoftwotopicalanesthetics.Animal2010,4,2071–2079.[CrossRef]49.Sheil,M.L.;Chambers,M.;Sharpe,B.Topicalwoundanaesthesia:Efficacytomitigatepigletcastrationpain.Aust.Vet.J.2020,98,256–263.[CrossRef][PubMed]50.Akerman,S.B.Surfaceanaestheticpropertiesofthenewlocalanaestheticamidetidocaine(Duranest):Alaboratoryevaluation.Br.J.Anaesth.1975,47,923–927.[CrossRef][PubMed]51.Friskopp,J.;Nilsson,M.;Isacsson,G.Theanestheticonsetanddurationofanewlidocaine/prilocainegelintra-pocketanesthetic(Oraqix)forperiodontalscaling/rootplaning.J.Clin.Periodontol.2001,28,453–458.[CrossRef][PubMed]52.White,R.G.;DeShazer,J.A.;Tressler,C.J.;Borcher,G.M.;Davey,S.;Wani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