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RESEARCHARTICLE ValidationoftheUnesp-Botucatupigcompositeacutepainscale(UPAPS)inpigletsundergoingcastration IRobles 1 ,S.P.L.Luna 2 ,P.H.E.Trindade 2 ,MLopez-Soriano 1 ,V.R.Merenda 1 ,A.V.Viscardi ID 3 ,ETamminga 4 ,M.E.Lou ID 3 ,M.D.Pairis-Garcia ID 1 * 1DepartmentofPopulationHealthandPathobiology,CollegeofVeterinaryMedicine,NorthCarolinaStateUniversity,Raleigh,NC,UnitedStatesofAmerica,2DepartmentofVeterinarySurgeryandAnimalReproduction,SchoolofVeterinaryMedicineandAnimalScience,SãoPauloStateUniversity(Unesp),Botucatu,SãoPaulo,Brazil,3DepartmentofAnatomyandPhysiology,CollegeofVeterinaryMedicine,KansasStateUniversity,Manhattan,KS,UnitedStatesofAmerica,4PrairieSwineCentre,Saskatoon,SK,Canada*mpairis@ncsu.edu Abstract

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CollegeofVeterinaryMedicine,KansasStateUniversity,Manhattan,KS,UnitedStatesofAmerica,4PrairieSwineCentre,Saskatoon,SK,Canada*mpairis@ncsu.edu Abstract Toaccuratelyassesspainandsupportbroadly-basedanalgesicprotocolstomitigateswinepain,itisimperativetodevelopandvalidateaspecies-specificpainscale.Theobjectiveofthisstudywastoinvestigatetheclinicalvalidityandreliabilityofanacutepainscale(UPAPS)adaptedfornewbornpigletsundergoingcastration.Thirty-ninemalepiglets(fivedaysofage,1.62±0.23kgBW)servedastheirowncontrol,wereenrolledinthestudyandunderwentcastrationinconjunctionwithaninjectableanalgesicadministeredone-hourpost-castration(flunixinmeglumine2.2mg/kgIM).Anadditional10,non-painfulfemalepig-letswereincludedtoaccountfortheeffectofnaturalbehavioralvariationbydayonpainscaleresults.Behaviorofeachpigletwasvideorecordedcontinuouslyatfourrecordingperi-ods(24hpre-castration,15minpost-castration,3and24hpost-castration).Pre-andpost-operativepainwasassessedbyusinga4-pointscale(score0–3)includingthefollowingsixbehavioralitems:posture,interactionandinterestinsurroundings,activity,attentiontotheaffectedarea,nursing,andmiscellaneousbehavior.BehaviorwasassessedbytwotrainedblindedobserversandstatisticalanalysiswasperformedusingRsoftware.Inter-observeragreementwasverygood(ICC=0.81).Thescalewasunidimensionalbasedontheprinci-palcomponentanalysis,allitemsexceptfornursingwererepresentative(r s

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r-observeragreementwasverygood(ICC=0.81).Thescalewasunidimensionalbasedontheprinci-palcomponentanalysis,allitemsexceptfornursingwererepresentative(r s 0.74)andhadexcellentinternalconsistency(Cronbach’salpha 0.85).Thesumofscoreswerehigherincastratedpigletspost-procedurecomparedtopre-procedure,andhigherthaninnon-painfulfemalepigletsconfirmingresponsivenessandconstructvalidity,respectively.Scalesensi-tivitywasgoodwhenpigletswereawake(92.9%)andspecificitywasmoderate(78.6%).Thescalehadexcellentdiscriminatoryability(areaunderthecurve>0.92)andtheoptimalcut-offsumforanalgesiawas4outof15.TheUPAPSscaleisavalidandreliableclinicaltooltoassessacutepainincastratedpre-weanedpiglets. PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,20231/26 a1111111111a1111111111a1111111111a1111111111a1111111111

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stratedpre-weanedpiglets. PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,20231/26 a1111111111a1111111111a1111111111a1111111111a1111111111 OPENACCESSCitation:RoblesI,LunaSPL,TrindadePHE,Lopez-SorianoM,MerendaVR,ViscardiAV,etal.(2023)ValidationoftheUnesp-Botucatupigcompositeacutepainscale(UPAPS)inpigletsundergoingcastration.PLoSONE18(4):e0284218.https://doi.org/10.1371/journal.pone.0284218Editor:I.AnnaS.Olsson,UniversidadedoPortoInstitutodeBiologiaMoleculareCelular,PORTUGALReceived:January19,2022Accepted:March23,2023Published:April13,2023PeerReviewHistory:PLOSrecognizesthebenefitsoftransparencyinthepeerreviewprocess;therefore,weenablethepublicationofallofthecontentofpeerreviewandauthorresponsesalongsidefinal,publishedarticles.Theeditorialhistoryofthisarticleisavailablehere:https://doi.org/10.1371/journal.pone.0284218Copyright:©2023Roblesetal.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.DataAvailabilityStatement:AllrelevantdataarewithinthepaperanditsSupportingInformationfiles.

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Introduction

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Introduction IntheUnitedStates(US),approximately94millionpigsarecastratedannuallytopreventunwantedbreeding[1],reduceaggression[2]andimprovemeatquality[3].Despitetheacknowledgmentthatcastrationispainful[4–7],andtheeconomicbenefitsassociatedwithpainreliefandlong-termweightgaininpigs[8],thisprocedureisnotcommonlyperformedinconjunctionwithpharmaceuticalpaincontrolintheUS[9–11].FromaEuropeanperspec-tive,manycountrieshavepassedlegislationrequiringmandatorypainreliefforcastratedpig-lets.However,nocurrentstudiestodatehaveassessedactualimplementationofpainreliefon-farm,andolderstudiesdatingbackto2016notedonly5%ofmalepigletsinsomeEuro-peancountriesarecastratedwithpainrelief[12].Currently,theUShasnoFoodandDrugAdministration(FDA)approveddrugspecificallylabelledforpainmanagementinswineifveterinariansweremandatedtoprovidepaincontrol[1,13].InordertoobtainFDAapprovalfortheuseofpainreliefinpigs,aproductmustfirstdemonstrateefficacyassupportedthroughclinicallyvalidatedend-points[1].Painassociatedwithcastrationcanbeassessedusingavarietyofmeasures,includingquan-tifyingdeviationsofapiglet’stypicalbehavioralrepertoire[14].Deviationsinmaintenancebehaviors(e.g.,sleeping,nursing,walking,drinking)aswellasincreasesinpain-associatedbehaviors(e.g.,tailwagging,rumpscratchingandprostration)havebeenmostfrequentlyusedtoquantifycastrationpain[15–17].Morerecently,agrimacepainscaleincastratedpigshasbeendevelopedandshowsgreatpotentialasafuturetoolforevaluatingpigletpainsensitivity[16,18].Toaccuratelyassesspainstatesandsupportbroadly-basedpainmanagementprotocolstomitigatecastrationpain,thedevelopmentandvalidationofobjectivemethodsisthenextlogi-calstep.Painassessmentscalesforuseinanimalsshowpromisegiventhesetoolsaredesignedtodirectlyquantifyindividualanimalresponsestopainfulstimuli,yieldreliabilityandvaliditywithinandbetweenobservers[14].Whenreviewingthepainliteratureinotherspecies,painassessmentscaleshavebeenwidelyadoptedasobjectivetoolstoquantifypainstatesinhumans[19],domesticateddogs[20–22],cats[23–25],ruminants[26,27],horses[28–30]andweanedpigs[31].Inaddition,painassessmentscalesofferadvantagesforuseinlivestockasthesescalescanbeappliednon-invasivelyondiversefarmsettings,withlittletonoover-headcostrequired[14,31].In2020,theUnesp-Botucatupigcompositeacutepainscale(UPAPS)wasdevelopedspecif-icallyforweanedpigs(age:38±3days)experiencingpainassociatedwithcastration[31].TheUPAPShasgoodtoverygoodintraandinter-observeragreement,excellentpredictiveandconcurrentcriterionvalidity,andresponsiveness[31].Althoughthisscalewasvalidatedasareliabletooltoquantifypaininpigs,castrationperformedoncommercialfarmsystemsistypi-callyconductedonpre-weanedpiglets(lessthan7daysofage)houseddirectlywiththesowandlittermates.Consequently,clinicalvalidationofthisscaleinamoreappropriateagecate-goryandenvironmentalsettingisnecessaryasageandsocialdynamicsinfluencethebehav-ioralrepertoireofpigs(i.e.pre-weanedpigswillspendmoretimenursingandsleepingthanolderpigs[32]).Therefore,theobjectiveofthisstudywastoinvestigatethereliabilityandvalidityoftheUPAPSinassessingpostoperativepaininpre-weanedpigletsundergoingcastration.

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erefore,theobjectiveofthisstudywastoinvestigatethereliabilityandvalidityoftheUPAPSinassessingpostoperativepaininpre-weanedpigletsundergoingcastration. Materialsandmethods AnimaluseandprocedureswereapprovedbyNorthCarolinaStateUniversityAnimalCareandUseCommittee(AnimalUtilizationProtocol#19–796). Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,20232/26 Funding:MPGandAVreceivedfundingfromtheNationalPorkBoard#19-065(pork.org).Thefunderhasnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.Competinginterests:Theauthorshavedeclaredthatnocompetinginterestsexist.

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Studyanimalsandhousing

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Studyanimalsandhousing Atotalof39Yorkshire-LandracexDurocpigletsfromninelitters(fivedaysofage,1.62±0.23kgBW;Fig1)wereenrolledinthestudy.Allmalepigletsineachlitterwereenrolledineachlitterforthestudy(range:1–10malesenrolledperlitter),and,onaverage,twofemalepigletsperlitterwereenrolledinaddition.Givenindividualvariationinpainsensitivity,theexperimentwasdesignedtoallowmalepigletstoserveastheirowncontrol,thusassessingandcomparingpainscorespriortoandfollowingapainfulevent.Malepigletswereweighedandindividuallyidentifiedusingapermanentmarkeronthebackofthepig.TheagecategoryselectedforthisstudywasfivedaysofagetosimulatetypicalcommercialproductionpracticesintheUS[33–35].Concurrently,10femalepigletswereenrolledsimultaneouslyonthestudyandunderwentasimulatedcastration(shamprocedure)inwhichtheyweretransportedandhandledinthesamemannerasmales,butnoincisionsweremade.Femalepigletsinnon-painstateswereenrolledtoaccountfortheeffectofnaturalbehavioralvariationbydayonpainscaleresults.Thus,femalepigletswerenotconsideredtruecontrols.Sowsandpigletswerehousedinindividualfarrowingcrates(0.8x2.3m)onfullyslattedfloorswithinonefarrowingroomatNorthCarolinaStateUniversity(NCSU)SwineEduca-tionalUnit(Raleigh,NorthCarolina,US).Thefarrowingroomwasmechanicallyventilated,andthetemperaturewasmaintainedat22˚±1.0˚C.Lightwasprovideddailyfrom0630to1630h.Creepareaforpigletswasheatedtoapproximately30–35˚Cusingheatinglamps.Sowshadadlibitumaccesstofeedandwaterviaawaternippleandstainlesssteelfeeder(dietmetorexceededNationalResearchCouncil[36]nutrientrequirementsforlactatingsows)whilehousedinthefarrowingsystem.Castrationprocedure.OnetrainedpersonfromNCSUperformedcastration(IG)onallenrolledmalepiglets(n=39)onthesamedaybetween0800-1000h.Pigletsweremovedtothealleywayviaatransportcartandindividuallysuspendedbythehindlegswhiletheprocedurewasperformed.Oncethepigletwassecure,two,2.5cmincisionsweremadeintothescrotalskinovereachtesticleusingascalpelblade.Spermaticcordsweretorn,testiclesremoved,andiodinewassprayedovertheincisionsitetoreduceinfectionrisk.GeneralorlocalanesthesiawasnotadministeredatthetimeofcastrationandisstandardpracticeintheUS.CurrentlyintheUS,therearenoFDAapproveddrugsspecificallylabeledtomanagepaininswine.There-fore,theuseofanyanalgesic/anestheticprotocoltomitigatecastrationpainmustbeadminis-teredunderthedirectsupervisionofaveterinarianinaccordancewiththeAnimalMedicinalDrugUseClarificationActof1994.Pigletsenrolledonthisstudywerecastratedaccordingtothecurrentstandardoperatingprocedure(SOP)approvedonfarmbytheattendingveterinar-ianandtheprotocoldidnotincludeextra-labeladministrationofananalgesic/anestheticatcastration.Allmalepigletslocatedatthisfacilityarerequiredtoundergocastrationpriortoweaning,thereforethecastrationprocedurewouldhaveoccurredregardlessoftheresearchprojectbeingconducted.Itshouldbenotedthatalthoughpainmitigationwasnotprovided

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g,thereforethecastrationprocedurewouldhaveoccurredregardlessoftheresearchprojectbeingconducted.Itshouldbenotedthatalthoughpainmitigationwasnotprovided Fig1.Flowoftheexperimentaldesign.https://doi.org/10.1371/journal.pone.0284218.g001 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,20233/26

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pre-operativelytocomplywithfarmSOP,allpigletsenrolledinthisresearchprojectreceivedpost-operativepainmanagementasapprovedbytheIACUCresearchprotocolone-hourpost-castration(2.2mg/kgflunixinmeglumineIM;MerckAnimalHealth,Millsboro,DE,US).Pig-letsenrolledonthestudyhadnootherprocedureperformedduringthetrialperiod(i.e.,earnotching,injection,ortaildocking). Behavioraldatacollection

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,DE,US).Pig-letsenrolledonthestudyhadnootherprocedureperformedduringthetrialperiod(i.e.,earnotching,injection,ortaildocking). Behavioraldatacollection Pigletswererecordedusinghigh-definitionvideocameras(AMCRESTIP3M-943B;Houston,TX,US;onecamerapercrate)mounted2.4mabovethegroundandangled45˚abovethefar-rowingcrates.Behaviorforeachpigletwasrecordedcontinuously1hindurationforthefol-lowingfourrecordingperiods:M1(24hpre-procedure),M2(15minpost-procedure),M3(3hpost-procedure),M4(24hpost-procedure;Fig2).Attheendofthetrial,oneindividual(MPG),notinvolvedinthebehavioralanalysis,downloadedvideos.Foreachvideo,four-min-utevideoclipsweretrimmedfromeachrecordingperiod(M1-M4)tobeevaluatedbyobserv-ersforpainscalevalidation.Videoclipswereobtainedatthe16-minmarkforeachrecordingperiod(i.e.,16minafterthestartofeachrecordingperiod)untilthe20-minmark.The16-minmarkwasrandomlyselectedtoeliminateobserverbiasinpotentiallyselectingavideosegmentwithspecificbehavioraldisplaysandfrequencies.Oncethecontinuous4-minvideoclipsweredownloaded,clipswererandomizedandmaskedforidentifyinginformation(i.e.,timestamp,dateetc.)priortoinitiationofpainscalevalidation.Observersassessedonepigletatatimeforeachobservation.Potentialbiasmayhaveoccurredifobserverswereabletodif-ferentiatepigletsex.However,camerapositionandanglemadeitdifficulttodifferentiatespe-cificdetailsonpigsandgenitalswerenoteasilydiscernablewhenreviewingvideospriortoanalysis,therefore,assumedbiaslevelwasminimum.Observertraining.Thevideoclipsfromthisstudywereanalyzedbytwofemaleobservers(IR,MEL).Priortodatacollectioninitiation,thetwoobserversunderwentthreetraining Fig2.Timelineoftheperiodsofobservationsusedforpainscalevalidation.https://doi.org/10.1371/journal.pone.0284218.g002 Painscalevalidationinpre-weanedpigletsundergoingcastration

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sofobservationsusedforpainscalevalidation.https://doi.org/10.1371/journal.pone.0284218.g002 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,20234/26

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sessions(2h/trainingsession)whereobserversstudiedcomponentsofthescale,observed,anddiscussedexamplesofeachbehavioralscore/criteriaandpracticedscoringfor90min.Subse-quently,inter-observerreliabilitywasassessed,andintra-classcorrelationcoefficient(ICC)wascalculatedbyhavingeachobserverscoretwelveanimalsindividually.Videoswereobservedinrandomorderandobserversweremaskedtodayandtimeofday.Scaleadaptations.Thepainscalevalidatedinthisstudyevaluatedsixbehavioralitems,witheachitemsub-categorizedintofourdescriptivelevels.Anumericalscorewasdesignatedfrom“0”to“3”,witha“0”representingnormalbehavior(freeofpain)and“3”correspondingtopronouncedbehavioraldeviation(Table1).Forthepurposesofthisstudy,twoadaptations Table1.TheUnesp-Botucatucompositepainscaleforassessingpostoperativepaininpiglets(UPAPS).ItemScoreScore/criterionLinkstovideosPosture0Normal 1 (anyposition,apparentcomfort,relaxedmuscles)orsleepinghttps://youtu.be/ojvbFAgCWo01Changesposture,withdiscomfort 2 https://youtu.be/SpaWsFCrPxE2Changesposture,withdiscomfort,andprotectstheaffectedarea 3 https://youtu.be/VjSlsRrG8yA3Quiet,tense,andbackarched 4 https://youtu.be/gHUWWf5836MInteractionandinterestinthesurroundings0Interactswithotheranimals;interestedinthesurroundings 5 orsleepinghttps://youtu.be/6s8xEn3QR3c1Onlyinteractsifstimulatedbyotheranimals;interestedinthesurroundings.https://youtu.be/9pMULbCO_WE2Occasionallymovesawayfromtheotheranimals,butacceptsapproaches 6 ;showslittleinterestinthesurroundingshttps://youtu.be/hp4wqdcHanA3Movesorrunsawayfromotheranimalsanddoesnotallowapproaches;disinterestedinthesurroundingshttps://youtu.be/se70oYXcWFwActivity0Movesnormally 7 orsleepinghttps://youtu.be/cC75t7L5-YA1Moveswithlessfrequency 8 https://youtu.be/lQo9wq8LAn82Movesconstantly,restless 9

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YXcWFwActivity0Movesnormally 7 orsleepinghttps://youtu.be/cC75t7L5-YA1Moveswithlessfrequency 8 https://youtu.be/lQo9wq8LAn82Movesconstantly,restless 9 https://youtu.be/YQRJjijLvpk3Reluctanttomoveordoesnotmove 10 https://youtu.be/Zyx0G3Wpt8oNursing*0Activelynursinghttps://youtu.be/6e2iYNKVFNY1Notnursingwhenlessthan50%ofthepigletsarenursinghttps://youtu.be/3xJuMo2t7b42Unsuccessfulattemptatnursing(oneormoreattempts)https://youtu.be/3PLgcf9ceQ83Notnursingwhen50%ormoreofthepigletsarenursinghttps://youtu.be/TZcB5PbsTT8AttentiontotheaffectedareaA.Elevatespelviclimboralternatesthesupportofthepelviclimbhttps://youtu.be/UD99ftO7HE0B.Scratchesorrubsthepainfularea 11 https://youtu.be/7idfFk1harEC.Movesand/orrunsawayand/orjumpsafterinjuryoftheaffectedareahttps://youtu.be/u-Pqubom278D.Sitswithdifficultyhttps://youtu.be/ETNEOCVV4h00Alltheabovebehaviorsareabsent1Presenceofoneoftheabovebehaviors2Presenceoftwooftheabovebehaviors3Presenceofthreeoralltheabovebehaviors(Continued) Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,20235/26

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Table1.(Continued)ItemScoreScore/criterionLinkstovideosMiscellaneousbehaviorsA.Wagstailcontinuouslyandintensely 12 https://youtu.be/pU5dGZFNRHcB.Bitesthebarsorobjectshttps://youtu.be/cF3dsq7gMtkC.Theheadisbelowthelineofthespinalcolumn.https://youtu.be/ZcIgngclRpID.Presentsdifficultyinovercomingobstacles(example:otheranimal)https://youtu.be/HlvdOI3lGuY0Alltheabovebehaviorsareabsent1Presenceofoneoftheabovebehaviors2Presenceoftwooftheabovebehaviors3Presenceofthreeoralltheabovebehaviors 1 Definedasanypositioninwhichthepigletdemonstratesapparentcomfortandrelaxedmuscles.Posturesconsiderednormalwhichdemonstratepigletcomfortincludebutarenotlimitedtopigletslayinginlateralorsternalrecumbencyeitherseparatelyoramongstthelitter.Normalposturalchangesincludeaquicktransitionfromstandingtolyingandlyingtostandingandtypicallyresultintheentirebodydescendingontoorawayfromtheflooratrelativelythesametime.Posturalpositionsaremostlikelydrivenbyexternalstimuli(e.g.,littermatestandsuporoverpigletswhilelying).Whilestanding,thepiglet’sheadisparalleltothespineandthepigwillfrequentlyexploretheenvironmentbychangingheadpositions(elevatingheadtoexploresurroundingareaandplacingheadtowardsfloortoexploreground). 2 Definedasapigletdemonstratingadeviationfromnormalbehaviorinwhichmusclesmaybetensed,posturalchangetransitionsoccurmoreslowlyandmayoccurmorefrequently.Pigletsdemonstratingchangingposturewithdiscomfortmayoftenextendtherearlegstoelevatetherumpoffthegroundwhilemaintainingphysicalcontacttothefloorwiththefrontlegsandhead.Transitionfromstandingtolyingorlyingtostandingalsooccursmoreslowlyinwhichthepigletmayremainmotionlessforshorttimeperiodsandchangepostureintheabsenceofexternalstimulisuchasotherlittermates. 3

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andingalsooccursmoreslowlyinwhichthepigletmayremainmotionlessforshorttimeperiodsandchangepostureintheabsenceofexternalstimulisuchasotherlittermates. 3 Demonstratedbehaviorallyasapigletperformingatuckedtailpostureinwhichtherearlegsareslightflexed,andthepigletorientsthecastrationsitetowardsthewallsorcornerofthefarrowingstallorrunsawaywhenanotherpigtouchestheaffectedarea. 4 Typicallyaccompaniedwithlittletonomovementoractivity.Backhasadefinedarch,headofthepigletisbelowthelevelofthespineandpigletspendsmajorityofthetimenotmovingorwithinasmallphysicalspaceofthepen. 5 Animalinteractionincludessnouttosnoutcontactwithlittermatesbutmayalsoincluderootingornosingbodypartsoflittermatesincludingtheflank,neck,andrearofthelittermateofinterest.Interestinthesurroundingenvironmentincludesactivelyexploringarea(i.e.,walking,runningtovariouspartsofthestall)andactivelyrootingwiththesnoutormouthingitemswithinthestallincludingbutnotlimitedtoheatmat,bars,andfloor. 6 Acceptsapproachistoreciprocateinteractionwithanotherlittermatewithoutrunningawayfromlittermate. 7 Definedasexhibitingforwardmotionwithallfourlegsincontactwiththefloortypicallyatawalkingpace.Normalmovementexamplesforpigletsinclude,butisnotlimitedto,exploringthestallfloorbywalkingtoandfromareassurroundingthesow(withandwithoutthepresenceofotherlittermates),attemptingtonursesowbyrootingatudder,andinteractingwithlittermatesonheatmat. 8 Definedasthepigletstillshowingactivitybutislesslikelytomoveforlongperiodsorcoverlargerareasofthestallandmaynotattempttoexploreapartofthestallinwhichnootherlittermatesarepresent,maynotparticipateinsocialinteractionswithlittermates,ortheirbehaviordeviatesfromthatoflittermates. 9

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partofthestallinwhichnootherlittermatesarepresent,maynotparticipateinsocialinteractionswithlittermates,ortheirbehaviordeviatesfromthatoflittermates. 9 Pigletswilllikelydemonstrateactivityatafastwalkorrunningpaceforthemajorityoftheobservation.Restlesspigletsmayalsodemonstratestartlebehaviorsinwhichthepigletwillquicklyfreeze(standingwithheadelevatedbutnotmoving)andthenresumefast-pacedmovement. 10 Pigletsarelikelytobeinalyingpositionforthemajorityoftheobservationanddemonstratebehaviorsimilartomoribundanimals.Pigletsreluctanttomovewillnotbeeasilystimulatedbylittermatesandmayisolatethemselvesfromlittermatesandresideinthecornerofthestall.Somepigletsmayshowthisbehaviorwhiledemonstratingaposturescoreof3andwillshowverylittletonoreactivityofactivityoccurringinthepen.Posturalchangesmayoccurbutwillbelessfrequentinnatureandwilllikelyresultinlittletonophysicaldistancechangedfromoriginalposturelocation. 11 Requiresphysicalcontactofthesurgicalsitewiththefarrowingenvironment(i.e.,floor,penning,mat)orlittermate.Pigletsoftenperformscratchingbehaviorinwhichtherearlegisextendedtotheear,butthisisnotconsideredforpurposesofscoringthisitemasitdoesnotinvolvethepainfularea. 12 Tailwaggingwithintensityisdescribedbothbytheforceinwhichthetailmovesandthefrequency.Intensetailwaggingwillinvolvemovementoftheentiretailandmovementwilloccurinshortrapidbursts(3+moretailwagsconsecutively).Tailwaggingcanoccurwhilethepigletisstanding,lying,orsitting.*Nursingbehaviorwasobservedandanalyzedinthestudybutwasexcludedfromthefinalpainscaletableasitdidnotmeetanyofthevalidationcriteria(principalcomponentanalysis,specificity,internalconsistency,item-totalcorrelationandresponsiveness).https://doi.org/10.1371/journal.pone.0284218.t001 Painscalevalidationinpre-weanedpigletsundergoingcastration

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y,item-totalcorrelationandresponsiveness).https://doi.org/10.1371/journal.pone.0284218.t001 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,20236/26

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tothepainscalewereadoptedandincluded:1)replacementoffeedingbehaviorwithnursingbehaviorand2)additionofthebehaviorsleepingintothe0sub-categoriesfortheitemspos-ture,interactionandinterestinthesurroundings,andactivity.Attheinitiationofdataobser-vation,pigletstatewasevaluatedandcategorizedaseitherawakeorasleep.Pigletstatewasthenincludedinthestatisticalmodeltofacilitatethediscriminationbetweenapigletsleeping(classifiedasnormal)comparedtoanawakepigletexpressingnormalnon-painfulbehavior. Statisticalanalyses Allstatisticalanalyseswereperformedoneperson(PHET)insoftwareRusingtheintegrateddevelopmentenvironmentRStudio(Version4.0.2[2020-06-22],RStudio,Inc.).Thefunctionsandpackageswerepresentedintheformat“function{package}”andαwasconsidered5%inalltests.Aminimumsamplesizeof6wouldbenecessarybasedontheoriginalUPAPSstudy[31]differenceoftotalpainsumsbeforeandaftercastrationbasedonalpha0.01andpowerof0.9(http://biomath.info/power/).StatisticalproceduresarepresentedinTable2,andthescoresoftwoobserverswereincluded.Litterwasincludedasarandomeffectforallanalyses.Forsomeanalyses,differentstratificationsofthedatabasewereusedasspecifiedinTable2.Toexcludetheeffectofsleepinginthescalevalidationperformance,scorespertainingonlytoawakepigletswereanalyzedforsomestatisticaltests(Table2). ResultsPainscaleadaptations ThefinalUPAPSscaleispresentedinTable1.TheUPAPS+nursing(nursingscoredfrom0–3scaledescribedinTable1)wasnotretainedafteranalysesasitdidnotmeetanyofthevalida-tioncriteria(principalcomponentanalysis,specificity,internalconsistency,item-totalcorrela-tion,andresponsivenessdescribedinTable2.ResultsforeachtestcompletedfromTable2utilizingtheoriginalUPAPS+nursingpainscalecanbelocatedinsupportinginformation(S1,S2FigsandS1–S7Tables). Distributionofscores

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achtestcompletedfromTable2utilizingtheoriginalUPAPS+nursingpainscalecanbelocatedinsupportinginformation(S1,S2FigsandS1–S7Tables). Distributionofscores Incastratedpigletsthedistributionofscores“1”,“2”,and“3”forallitemsoccurredmorefrequentlyattheM2andM3recordingperiodcomparedtoM1andM4(Fig3).Scoresformostitemswereproportionallygreaterpost-castration(M2)whencomparedtoaftertherescueanalgesiawasadmin-istered(M3),exceptfortheitems“attentiontotheaffectedarea”and“miscellaneousbehaviors”.ScoresatM4tendedtoreturntopre-castrationvaluespresentedattheM1recordingperiod(Fig3).Thebehaviorsincludedintheitem“attentiontotheaffectedarea”wereperformedmorefrequentlypost-castration(M2)comparedtopre-castration(M1).Themostfrequentbehaviordisplayedimme-diatelypost-castration(M2)fortheitem“attentiontotheaffectedarea”was“sitswithdifficulty”and“wagstailcontinuouslyandintensely”fortheitem“miscellaneousbehaviors”(Figs3and4). Inter-observerreliability Inter-observeragreementforallitemsofthepainscalerangedfrommoderatetoverygood(Table3),exceptfornursingwhichrangedfromreasonabletogood.TheagreementontotalsumofUPAPSwasverygood(0.81;Table3). Multipleassociation TheHorn’sParallelanalysis[38]wasusedtodeterminetheoptimalnumberofdimensionsforretentionandindicatedonedimensionforthePCA,providingstatisticalrationalebehindwhy Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,20237/26

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Table2.Statisticalmethodsusedforrefinement( R )andvalidation( V )oftheUnesp-Botucatupigacutepainscale(UPAPS)inthepigletspost-castration.Typeofanalysis*DescriptionGroupsanalyzed ¥ StatisticaltestInternalconsistency RV Theconsistency(interrelation)ofthescoresofeachitemofthescalewereestimated.Theanalysiswasperformedforallgroupedmoments.Allcastratedpiglets,andawakecastratedpigletsCronbach’salphacoefficient(α;“cronbach{psy}”)andMcDonald’somegacoefficient(ω;“omega{psy}”)wereused.Cronbach’sαinterpretation:0.60–0.64,minimallyacceptable;0.65–0.69acceptable;0.70–0.74good;0.75–0.80verygood;and>0.80excellent[37].McDonald’somegainterpretation:0.70–0.84isacceptableand>0.85isstrong[38]Distributionofscores V 1-Pigletswerecategorizedasawake“0”,awake 30seconds“1”orsleeping“2”.2-Percentageofthefrequencydistributionofthepresenceofthescores“0”,“1”,“2”and“3”ofeachitemoftheUPAPSateachmomentandallmomentsgrouped(MG).Allcastratedandawakepigletsforallmomentsgrouped(MG=M1+M2+M3+M4)Descriptiveanalysis.Inter-observerreliability RV Reproducibility(agreementmatrix)—thelevelofagreementbetweenthetwoobservers,usingthescoresforeachitemandthetotalsumofthescalewasassessed.AllcastratedpigletsForthescoresoftheitemsoftheUPAPS,theweightedkappacoefficient(k w )wasused;thedisagreementswereweightedaccordingtotheirdistancetothesquareofperfectagreement(“cohen.kappa{psych}”).The95%confidenceinterval(CI)k w basedin1,001replicationsbythebootstrapmethodwasestimated(“boot.ci{boot}”).ForthesumoftheUPAPS,theconsistencytypeintraclasscorrelationcoefficient(ICC;“icc{irr}”)andits95%CIbasedin1,001replicationsbythebootstrapmethod(“boot.ci{boot}”)wasused.Interpretationofk w

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ypeintraclasscorrelationcoefficient(ICC;“icc{irr}”)andits95%CIbasedin1,001replicationsbythebootstrapmethod(“boot.ci{boot}”)wasused.Interpretationofk w andICC:verygood0.81–1.0;good:0.61–0.80;moderate:0.41–0.60;reasonable:0.21–0.4;poor<0.2.Thek w andICC>0.50wereusedasacriteriontorefinethescale[24,39,40].Multipleassociation RV Themultipleassociationofthescaleitemswitheachotherwasanalyzedatallmomentsgroupedusingprincipalcomponentanalysis,todefinethenumberofdimensionsdeterminedbydifferentvariablesthatestablishthescaleextension.Allcastratedpiglets,andawakecastratedpiglets.Principalcomponentanalysisbasedoncorrelationmatrix(“princomp{stats”}andget_pca_(“var{factoextra}”).Horn’sParallelanalysiswasperformedasthemethodtodeterminetheoptimalnumberofdimensionstoberetainedinthePCA[41].Loadingvaluegreaterthan0.50orlesserthan—0.50wereconsideredforsignificantassociation.Forthebiplotconfidenceellipseswerebuiltwithsignificantlevelof95%showingthedensityofpiglets’scoresateachmoment.Item-totalcorrelation RV Thecorrelationofeachitemofthescale,afterexcludingtheevaluateditem,wasestimatedtoanalyzehomogeneity,theinflationaryitems,andtherelevanceofeachitemofthescale.Analysiswasperformedforallgroupedmoments.AsnursingwasnotincludedinUPAPS,nursingwascorrelatedwiththetotalsumoftheUPAPSwithoutanyexclusion.Allcastratedpiglets,andawakecastratedpigletsSpearmanrankcorrelationcoefficient(r 2 ;“rcorr{Hmisc}”).Interpretationofitem-totalcorrelation[39].Interpretationofcorrelationr:suitablevalues0.3–0.7Specificity RV andsensitivity RV

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ient(r 2 ;“rcorr{Hmisc}”).Interpretationofitem-totalcorrelation[39].Interpretationofcorrelationr:suitablevalues0.3–0.7Specificity RV andsensitivity RV ThescoresofeachitemofthescaleatM1(forspecificity)andatM2(forsensitivity)weretransformedintodichotomous(score“0”—absenceofpainexpressionbehaviorforagivenitem;scores“1”,“2”and“3”—presenceofpain).Forthetotalscoreofthescale,thepercentageofpigletsthathadscore<4atM1and 4(cut-offpoint)atM2wasconsideredspecificityandsensitivity,respectively.Allcastratedpiglets,andawakecastratedpiglets.Specificity,sensitivityandits95%CIwerecalculatedaccordingtothebootstrapmethoddescribedbelow(“epi.tests{epiR}”).Interpretation:excellent95–100%;good85–94.9%;moderate70–84.9%;notspecificornotsensitive<70%[30].(Continued) Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,20238/26

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thepainscaleappliedtopre-weanedpigletswasunidimensional.Allpainscaleitemscon-tainedhighloadingvaluesinthe1 st dimension,explaining63%ofthevarianceoftheentiredataset(Figs5and6;Table4).InFigs5and6,theellipsescorrespondingtopost-castrationpain(M2andM3)areintherightsideofthefigures.Ontheleftsidearetheellipsescorre-spondingtonopainandmildpain(M1andM4respectively).Pain(M2andM3)influenceallitemsonthescalebecausetheirvectorsaredirectedtotheseellipses. Item-totalcorrelation Item-totalcorrelationrangedfrom0.62to0.71forallmalepiglets,andfrom0.75to0.83forawakemalepigletstherefore,allitemswererelevant(Table5). Specificityandsensitivity Whenallcastratedpigletscoreswereincludedintheanalyses,theUPAPSitemsandtotalscorehadmoderatetogoodspecificitybutnosensitivity(Table6).Whenonlyawakepigletswereconsidered,UPAPStotalscoreanditemshadmoderatetogoodspecificityandgood Table2.(Continued)Typeofanalysis*DescriptionGroupsanalyzed ¥ StatisticaltestResponsivenessandconstructvalidity RV

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shadmoderatetogoodspecificityandgood Table2.(Continued)Typeofanalysis*DescriptionGroupsanalyzed ¥ StatisticaltestResponsivenessandconstructvalidity RV Responsiveness—thescoresofeachitem,andtotalsumofthescalewerecomparedovertime(M1vsM2vsM3vsM4).Interpretation:differencesinscoresareexpectedtooccurasfollows:M2>M4 M3>M1.Constructvaliditywasdeterminedusingfourmethods:1.Thethreehypothesistests:i)ifthescalereallymeasurespain,thescoreaftersurgery(M2)shouldbehigherthanthepreoperativescore(M1<M2),ii)ifanalgesiawasadequatethescoreshoulddecreaseafteranalgesia(M2>M3),iii)andovertime(M2>M4).2.Known-groupvalidity.TheShamfemalepain-freepigletsshouldhavelowerscoresthancastratedmalepigletssufferingpainatM2.3.Internalrelationshipsamongitemsaccordingtoallcriteriausedinstatisticalanalysis(multipleassociation,internalconsistencyanditem-totalcorrelation)4.Relationshipswiththescoresofotherinstruments,asdescribedforcriterionvalidityAllcastratedpiglets,Awakecastratedpiglets.AwakecastratedpigletsandAwakenon-painfulfemalepigletsThemodelresiduals(“residuals{stats}”)forallthedependentvariablesdidnotfitintoGaussiandistributionaccordingtothequantile-quantileandhistogramsgraphs(“qqnorm{stats}”and“histogram{lattice}”),thus,generalizedmixedlinearmodels(“glmer{lme4}”)wereapplied.Forthedichotomousvariablesbasedonbehaviorsofattentiontotheaffectedareaandmiscellaneousbehaviors,logisticregressionanalysis(“glm{stats}”)wasapplied.Momentsandobserverswereincludedasfixedeffectsandthepigletsnestedintheirlittersasrandomeffectinallthemodels.Tukeytestwasusedasaposthoctest("lsmeans{lsmeans}"and"cld{multcomp}")[27].Optimumcut-offpoint RV

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igletsnestedintheirlittersasrandomeffectinallthemodels.Tukeytestwasusedasaposthoctest("lsmeans{lsmeans}"and"cld{multcomp}")[27].Optimumcut-offpoint RV Thedatarelativetotheindicationofrescueanalgesiawasusedtodeterminetheoptimalcut-offpoint,thatis,theminimumscoresuggestiveoftheneedforanalgesicrescueorinterventionM1freeofpain(preoperative),andM2pain(postoperative)wereusedtodetermineoptimalcut-offpoint.AwakecastratedpigletsCut-offpointwasbasedontheYoudenindex(YI=[Sensitivity+Specificity]–1),whichdeterminesthehighestsensitivityandspecificityvalueconcurrentlyfromtheReceiverOperatingCharacteristic(ROC)curve(“roc{pROC}”and“ci.sp{pROC}”),providingagraphicimageoftherelationbetweenthe“truepositives”(sensitivity)andthe“falsepositives”(specificity).Thediscriminatorycapacityofthescalewasdeterminedbytheareaunderthecurve(AUC).AUCvaluesabove0.90representhighaccuracydiscriminatorycapacityofthescale[42].Inaddition,the95%CIwascalculatedfromtheYoudenindexbyreplicatingtheoriginalROCcurve1,001timesaccordingtothebootstrapmethod(“ci.coords{pROC}”and“ci.auc{pROC}”).*Allanalyses[37–42]includedbothadaptationsofthescale:UPAPSUnespBotucatupigcompositeacutepainscale;UPAPS+nursingbehavior. ¥ Totalpigletnumbersincluded:Malepiglets(n=39total,n=14awake),Femalenon-painfulpiglets(n=10total,n=6awake)M1(24-hpre-procedure),M2(15minpost-procedure),M3(3-hrpost-procedure),M4(24-hpost-procedure)https://doi.org/10.1371/journal.pone.0284218.t002 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,20239/26

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sensitivity,exceptforthefollowingitems:attentiontotheaffectedarea(nospecificity)andposture(nosensitivity;Table6). Constructvalidityandresponsiveness ThetotalsumofUPAPSwerehigherpost-castration(M2andM3)comparedtopre-castration(M1)and24hpost-castration(M4).ThetotalsumatM4wereintermediatebetweenM1and Fig3.DistributionofUPAPSitemsscores(percentageofoccurrence)incastratedpiglets(n=39).Legend:M1(24hpre-procedure),M2(15-minpost-procedure,beforerescueanalgesia),M3(3hpost-procedure,afterrescueanalgesia),M4(24hpost-procedure)GM—dataofthegroupedmoments(M1+M2+M3+M4).https://doi.org/10.1371/journal.pone.0284218.g003 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202310/26 Fig4.DistributionofUPAPSitemsscores(percentageofoccurrence)inawakecastratedpiglets(n=14).Legend:M1(24hpre-procedure),M2(15-minpost-procedure,beforerescueanalgesia);GM—dataofthegroupedmoments(M1+M2).https://doi.org/10.1371/journal.pone.0284218.g004 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202311/26

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M2/M3.Resultsdemonstratedscaleresponsivenesstopainandtimeofdayanddistinguishedmoderate(M4)tointensepain(M2).However,therescueanalgesicwasnoteffective,asdem-onstratedbytheunresponsivenessofthescaletotherescueanalgesiaprotocol(Fig7A;Table7).Awakemalepigletsservedastheirowncontrolanddemonstratedhigherscoresaftercas-trationthanbeforecastration(Fig7b;Table8).Aftercastration(M2),castratedpigletsobtainedhigherscoresthanfemalenon-painfulpiglets(Fig8;Table9),confirmingconstructvalidityandresponsiveness. Internalconsistency TheCronbach’sαcoefficientof0.85andtheMcDonald’sωof0.88indicatesexcellentandstronginternalconsistencyofUPAPSrespectively(Table9).Valuesofα=0.89andω=0.92increasedforcastratedpigletsawake(Table10).ThefindingthattheCronbach’sαofallindi-vidualitemswasbelow0.85(allpiglets)or0.89(pigletsawake),andtheMcDonald’sωofallindividualitemswasbelow0.88(allpiglets)or0.92(pigletsawake)wheneachitemwasexcludedforcalculation,indicatethatallitemsarerelevantandcontributetothetotalscoreofUPAPS. Optimalrescueanalgesiacut-offpoint Theoptimalcut-offforUPAPSinthisstudywas 4andAUCwas0.92demonstratingexcel-lentdiscriminatorycapacityoftheinstrument(Table11;Fig9). Discussion Mitigatingcastrationpainforpigletsisacriticalwelfareissueandpainnegativelyimpactstheaffectivestateofthepigandproductivityfortheproducer.Currently,theUShasnoFDAapproveddrugspecificallylabeledforpainmanagementinswine[1,13]andproductapprovalcanonlybecarriedoutbydemonstratingdrugefficacyassupportedthroughclinicallyvali-datedendpoints,suchasbehaviorscales.In2020,Lunaandcolleagues[31]developedandvali-datedanacutepainscaletoassesspainincastratedpigspost-weaning(age:38±3days). Table3.Weightedkappa(k w

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ales.In2020,Lunaandcolleagues[31]developedandvali-datedanacutepainscaletoassesspainincastratedpigspost-weaning(age:38±3days). Table3.Weightedkappa(k w )orintra-classcorrelationcoefficient(ICC)andconfidenceintervalofthe95%frominter-observermatrixagreementofeachitemandthesumoftheUPAPSappliedforallcastratedpiglets(n=39)overallmoments.Observer1vsObserver2ItemsonthescaleItemsonthescalek w Posture0.74(0.59–0.84)Interactionandinterest0.75(0.60–0.85)Activity0.68(0.51–0.81)Nursing0.57(0.30–0.76)Attentiontotheaffectedarea0.70(0.57–0.80)Miscellaneousbehaviors0.60(0.45–0.72)ScalesICCUPAPS0.81(0.72–0.87)UPAPS—Unesp-Botucatupigacutecompositepainscale.k w −weightedkappacoefficient;ICC—intraclasscorrelationcoefficient;Interpretationofreliability:verygood0.81–1.0;good0.61–0.80;moderate0.41–0.60;reasonable0.21–0.4;poor<0.2.https://doi.org/10.1371/journal.pone.0284218.t003 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202312/26

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Albeitpromising,thistoolrequiredadaptationforusewithpre-weanedpiglets,sincecastra-tionisperformedwithinthefirstweekoflifeoncommercialfarmsystemsandpigletsaregrouphousedwiththesowandlittermates.Thus,theobjectiveofthisstudywastoassessthereliabilityandvalidityoftheUPAPSinassessingpostoperativepaininpre-weanedpigletsundergoingcastration.ThisstudyutilizedtheUPAPSscaleoriginallydevelopedforweanedpigswiththeadapta-tionsoftwobehaviors(sleepandnursing).Analysisoftheadaptedscale(UPAPS+nursing)revealedthatnursingbehaviorasanitemlackedinternalconsistency,wasnothomogenousanddidnotinterrelatewellwithotheritems.Whenassessedindependently,nursingbehaviordidnotchangesignificantlyforanyofthemomentspre-andpost-castrationandbetweencas-tratedpigletsandnon-painfulfemalepiglets,indicatingalackofresponsivenessand Fig5.BiplotoftheprincipalcomponentanalysisoftheUPAPSitemsappliedtoallcastratedpiglets(n=39).UPAPS–Unesp-Botucatupigacutecompositepainscale.Confidenceellipsesindicateperioperativemomentsandpainscores.Moments:M1(24hpre-procedure),M2(15-minpost-procedure,beforerescueanalgesia),M3(3hpost-procedure,afterrescueanalgesia),M4(24hpost-procedure).Ellipseswereconstructedaccordingtotheassessment.Eachcirclecorrespondstothescorefromoneobserverattributedtoeachpigletateachmoment.https://doi.org/10.1371/journal.pone.0284218.g005 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202313/26

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nal.pone.0284218.g005 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202313/26 specificity.Theseresultsareincontrastwithpreviousstudiesutilizingnursingasanindicatorofpainfulstates[14].Nursingbehaviorisoftendifficulttocaptureduringshortobservationperiods,asboutsareshortindurationandsocialdynamicsinfluencethelengthandfrequencyofnursing.Futureuseofnursingbehaviorasanindicatorofpainstatemaybemoreeffectiveifobservationperiodsarelongerandevaluatedmorefrequently,therefore,theremainingdis-cussionofthisworkwillbebasedontheUPAPSexcludingnursingbehavior.TheUPAPSscaleadaptedforpre-weanedpigletshasbeenvalidatedasaneffectivetooltoassesspainincastratedpigletsutilizingtherequiredvalidationassessments[23,27,43,44].Thisscale,aswellastheUPAPSforpigs[31]andcattle[26],wereconsideredunidimensionalandallitemsinthescalehadindividualrelevanceandpresentedhomogeneity.When Fig6.BiplotoftheprincipalcomponentanalysisoftheUPAPSitemsappliedtoawakepiglets(n=14castratedpiglets).UPAPS–Unesp-Botucatupigacutecompositepainscale.Confidenceellipsesindicateperioperativemomentsandpainscores.Moments:M1(24hpre-procedure),M2(15-minpost-procedure,beforerescueanalgesia).Ellipseswereconstructedaccordingtotheassessment.Eachcirclecorrespondstothescorefromoneobserverattributedtoeachpigletateachmoment.https://doi.org/10.1371/journal.pone.0284218.g006 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202314/26

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comparedtotheUPAPSscaleforpigs,UPAPSforpre-weanedpigletshadlessspecificitybutbettersensitivitywiththemajorityofitemsachievingover92%.TheUPAPSscaledemonstratedbehavioraldeviationsindicativeofpainwhencomparingmalepigletbehaviorpre-castration(M1)topost-castration(M2).Givenindividualvariationinpainsensitivity,malepigletsservedastheirowncontrolbycomparingpainscalescorespriortocastrationandatthreemomentspost-castration,selectedspecificallytocapturevary-ingpainintensitylevels.Post-castratedpigletsdemonstratedgreaterpainscoresforallscaleitems(posture,interactionandinterest,activity,attentiontotheaffectedareaandmiscella-neous)atM2comparedtoM1.Behavioralchangespost-castrationincludedposturalchanges,decreasedactivityandinteractionwithsurroundingenvironmentsaswellasincreasedatten-tiontoaffectedarea,sittingwithdifficulty,tailwaggingandprostration(e.g.headbelowthespinalcolumn).Theseresultsagreewithpreviousworkconductedusingbothpainscalesandtraditionalbehavioralobservationwithcontinuousrecording.Pigletspost-castrationwillmorefrequentlydisplaytenseandstiffpostures[14,16,17],decreasedactivity[45–47],andincreasedsleepingbehavior[31].Incontrasttopreviouswork,theUPAPSscaledidnotdemonstratechangesinelevationofpelviclimbandscratchingbehaviorwhencomparingM1andM2periodsandisinagreementwithViscardiandTurner’sworkin2018[16].Scratchingbehaviorhasbeencommonlyusedinpreviouscastrationstudiestoquantifylocalsensitizationoftheaffectedarea,andhasoftenbeenanecdotallyassociatedwiththewoundhealingprocesswithsomestudiesreporting

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scastrationstudiestoquantifylocalsensitizationoftheaffectedarea,andhasoftenbeenanecdotallyassociatedwiththewoundhealingprocesswithsomestudiesreporting Table4.Loadingvalues,eigenvaluesandvarianceoftheUPAPSitemsappliedtocastratedpiglets(n=39)andcastratedpigletsawake(n=14)basedonprincipalcomponentsanalysis.LoadingValuesUPAPSUPAPS(awake)ItemsonthescaleDim.1Dim.2Dim.1Dim.2Posture0.840.190.860.16Interaction0.74-0.550.82-0.52Activity0.78-0.400.86-0.31Attentiontotheaffectedarea0.800.410.820.46Miscellaneousbehaviors0.790.290.840.21Eigenvalue3.140.743.540.64Variance62.7814.8870.6712.80UPAPS–Unesp-Botucatupigacutecompositepainscale.Thestructurewasdeterminedconsideringitemswithaloadingvaluegreaterthan0.50orlesserthan-0.50(inbold)andtheonlyfirstdimensionretainedbyHorn’sParallelanalysis[38].https://doi.org/10.1371/journal.pone.0284218.t004 Table5.Item-totalcorrelationoftheUPAPSappliedforallcastratedpiglets(n=39).ExcludingeachitembelowItem-total(Spearman)AllcastratedpigletsCastratedpigletsawakePosture0.680.77Interactionandinterest0.620.74Activity0.710.83Attentiontotheaffectedarea0.650.75Miscellaneousbehaviors0.640.76UPAPS:Unesp-Botucatupigacutecompositepainscale.InterpretationofSpearman’srankcorrelationcoefficient(r):0.3–0.7=acceptablevalues[39].https://doi.org/10.1371/journal.pone.0284218.t005 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202315/26

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scratchingbehaviortobeginwithinthefirsthourspostcastrationandcontinuingupto4dayspostcastration[48].However,thelackofchangeinscratchingbehaviorinthisstudymaybeduetothefactthatscratchingbehaviorisperformedinfrequently(3.56±0.83bouts;[15]),inclusters,andinshort-duration.Giventhisstudyonlyevaluatedbehaviorforfourconsecutiveminutespermoment,capturingpelviclimbalterationsandscratchingfrequencymaybediffi-culttoachieve.Nevertheless,theremainingbehaviorscategorizedwithin“attentiontoaffectedarea”(moves/runsaway,sitswithdifficulty)weredifferentwhencomparingpainfulandnon-painfulstates.Giventhepresenceofanyofthebehaviorswithinthecategorywereusedforassessment,painsensitivitycanstillbeaccuratelyassessed.Theinternalconsistencyofthisscalewasexcellentandcomparabletopig[31],cattle[26]andcat[43,44]scales.Thescaledemonstratedconstructvaliditywhencomparingtotalscalescoreincastratedmalepigletscomparedtonon-painfulfemalepiglets.Non-painfulfemalepigletswereincludedinthestudytoassesstheeffectofsex,handling,time,andsocialdynam-icsonthepainscaleduringanon-painstateandwerenotconsideredtruecontrols.Inadditiontoconstructvalidity,theUPAPSscalewasresponsivetopainstate,timeofday,anddistin-guishedmoderatetointensepain.Theoptimalcut-offpointcallingforrescueanalgesiawasthesameasreportedintheoriginalUPAPSstudy[31]demonstratingexcellentreproducibilityforanalgesictreatmentdecision-makingusingthistool.However,whenassessingresponsive-nesstorescueanalgesicprotocol,thescalewasnotresponsiveandsupportspreviousworkdemonstratingthatasingleadministrationofanNSAIDpost-castrationdoesnoteffectivelymitigatecastrationpain[14].Althoughcontinuousbehavioralassessmentiscurrentlyconsideredthegoldstandardforpainevaluation[49],theUPAPSscalefulfilledasimilartaskwithinashortassessmenttimeperpiglet(only4minutesperperiod).Thus,thisisapracticalclinicaltoolthatcanbeusedinaveterinaryhospitaland/oron-farm.Itisimportanttoacknowledgethat,althoughresultsarepromising,theUPAPSpainscaleshouldbefurtherinvestigatedasvalidationisacontinuousprocessandshouldnotbeconsideredtheoneandonlystandardforpainassessmentinpigs

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ising,theUPAPSpainscaleshouldbefurtherinvestigatedasvalidationisacontinuousprocessandshouldnotbeconsideredtheoneandonlystandardforpainassessmentinpigs Table6.Specificityandsensitivityand95%confidenceinterval(CI)oftheUPAPSappliedforallcastratedpiglets(n=39)andawakecastratedpiglets(n=14).Specificity(%)atM1Sensitivity(%)atM2AllcastratedpigletsItemsEstimatedCIEstimatedCIPosture84.6274.6791.7939.7428.8351.46Interactionandinterest88.4679.2294.5943.5932.3955.30Activity82.0571.7289.8356.4144.7067.61Attentiontotheaffectedarea75.6464.6084.6533.3323.0644.92Miscellaneousbehaviors78.2167.4186.7647.4436.0159.07UPAPS( 4)85.9076.1792.7446.1534.7957.82OnlyawakecastratedpigletsPosture85.7167.3395.9760.7140.5878.50Interactionandinterest82.1463.1193.9478.5759.0591.70Activity75.0055.1389.3192.8676.5099.12Attentiontotheaffectedarea64.2944.0781.3692.8676.5099.12Miscellaneousbehaviors78.5759.0591.7092.8676.5099.12UPAPS( 4)78.5759.0591.7092.8676.5099.12UPAPS—Unesp-Botucatupigacutecompositepainscale.Interpretationofspecificityandsensitivity:excellent95–100%;good85–94.9%;moderate70–84.9%;notspecificorsensitive<70%;boldvalues 70%[30].Specificityandsensitivityforthetotalscoreswereconsideredastherelationbetweenthenumberofpigletsthathadpainscores<4atM1and 4atM2andthetotalnumberofpiglets,respectively.M1(24-hpre-procedure),M2(15minpost-procedure,beforerescueanalgesia)https://doi.org/10.1371/journal.pone.0284218.t006 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202316/26

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forfuturework,givenseverallimitationsassociatedwiththemethodandthemannerinwhichbehaviorwascollectedmayhaveinfluencedtheresults.Pigletpainbehaviorsareoftenper-formedinfrequently,subtlyandcanbesignificantlyinfluencedbyexternalfactorssuchasthepresenceofhumans,facilitydesignandsocialsyncing(isolationanddesynchronization)[50]. Fig7.Violinplotofthetotalsum(median/amplitude)ofthea)UPAPS(allcastratedpiglets;n=39)andb)UPAPS(awakecastratedpiglets;n=14)beforeandaftercastrationinpiglets.Theviolincontourrepresentsthedispersionofdatadensity,withthewidercontourrepresentinggreaterdatadensity.Thetopandbottomboxlinesrepresenttheinterquartilerange(25to75%),thelinewithintheboxrepresentsthemedian,theextremesofthewhiskersrepresenttheminimumandmaximumvalues,blacklozenges(♦)representthemean,blackcircles(•)representoutliers.UPAPS:Unesp-Botucatupigacutecompositepainscale.Differentlettersexpresssignificantdifferencesbetweenmomentswherea>b>c,accordingtothegeneralizedmixedlinearmodels[27].M1(24hpre-procedure),M2(15-minpost-procedure,beforerescueanalgesia),M3(3hpost-procedure,afterrescueanalgesia),M4(24hpost-procedure).https://doi.org/10.1371/journal.pone.0284218.g007 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202317/26

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Forexample,workinhorses[51],rabbits[52]andcats[53]demonstratedthathumanpres-encecaninfluencepainbehaviorexpressedbyanimals.Inadditiontotheexternalfactorofhumanpresence,socialdynamicswithinthelittercanalsoinfluencebehavior.Malepigletnumberperlitterinthisstudyvaried,andallavailablemalepigletswereenrolled.Giventhisvariation,theexperimentwasnotdesignedtocontrolforsocialcohesion[50]andfuturecon-siderationsshouldevaluatethepainscaleusingcontrolledpignumberbylitterandcontrolfor Table7.Median(amplitude)oftheUPAPSscoresbeforeandaftercastrationinallcastratedpiglets(n=39).BehavioralpainscaleitemsM1M2M3M4Posture0 b (0–2)0 a (0–3)0 a (0–3)0 b (0–2)InteractionandInterest0 d (0–2)0 a (0–3)0 b (0–3)0 c (0–3)Activity0 c (0–3)1 a (0–3)0 ab (0–3)0 bc (0–3)Attentiontotheaffectedarea0 d (0–3)0 b (0–3)0 a (0–3)0 c (0–3)Elevatespelviclimb0 b (0–1)0 b (0–1)0 a (0–1)0 b (0–1)Scratchesthepainfularea0(0–1)0(0–1)0(0–1)0(0–1)Movesafterinjury0 b (0–1)0 a (0–1)0 a (0–1)0 a (0–1)Sitswithdifficulty0(0–1)0(0–1)0(0–1)0(0–1)Miscellaneousbehavior0 b (0–2)0 a (0–3)0.5 a (0–3)0 b (0–3)Wagstail0 b (0–1)0 a (0–1)0 a (0–1)0 b (0–1)Bitesobjects0(0–0)0(0–0)0(0–1)0(0–1)Headisbelowthespinalcolumn0 b (0–1)0 a (0–1)0 a (0–1)0 b (0–1)Difficultyovercomingobstacles0 ab (0–1)0 b (0–1)0 a (0–1)0 b (0–1)UPAPS0 c (0–10)3 a (0–12)4 a (0–12)1 b (0–11)UPAPS—Unesp-Botucatupigacutecompositepainscale.Differentlettersexpresssignificantdifferencesbetweenmomentswherea>b>c>d,accordingtothemixedlinearmodel[27].M1(24hpre-procedure),M2(15-minpost-procedure,beforerescueanalgesia),M3(3hpost-procedure,afterrescueanalgesia),M4(24hpost-procedure)https://doi.org/10.1371/journal.pone.0284218.t007

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post-procedure,beforerescueanalgesia),M3(3hpost-procedure,afterrescueanalgesia),M4(24hpost-procedure)https://doi.org/10.1371/journal.pone.0284218.t007 Table8.Median(amplitude)oftheUPAPSscoresbeforeandaftercastrationincastratedpigletsawake(n=14)duringtheassessment.ItemsM1M2Posture0 b (0–2)2 a (0–3)InteractionandInterest0 b (0–2)2 a (0–3)Activity0 b (0–3)3 a (0–3)Attentiontotheaffectedarea0 b (0–2)2 a (0–3)Elevatespelviclimb0 b (0–1)0 a (0–1)Scratchesthepainfularea0(0–1)0(0–1)Movesafterinjury0 b (0–1)0 a (0–1)Sitswithdifficulty0 b (0–1)1 a (0–1)Miscellaneousbehavior0 b (0–2)2 a (0–3)Wagstail0 b (0–1)1 a (0–1)Bitesobjects0(0–0)0(0–0)Headisbelowthespinalcolumn0 b (0–1)1 a (0–1)Difficultyovercomingobstacles0(0–1)0(0–1)UPAPS0 b (0–10)9.5 a (1–12)UPAPS—Unesp-Botucatupigacutecompositepainscale.Differentlettersexpresssignificantdifferencesbetweenmomentswherea>b,accordingtothemixedlinearmodel[27].M1:24hpre-castration;M2:15-minpost-castration.https://doi.org/10.1371/journal.pone.0284218.t008 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202318/26

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anequalmale:femaleratio.Anotherpossibleinvestigationistousemachinelearningalgo-rithmstoprovidestatisticalweightingsanddefinetheimportanceofeachbehaviorlikeper-formedinsheep[54].Futureworkshouldnotonlyaddresstheseexternalfactorsintheexperimentaldesignbutshouldalsoincludedirectcomparisonofpainscaledatatoothervalidatedmeasurestoassessphysiologicalpainresponseandpainbehaviorsusingcontinuoussamplingbehavioralmeth-odologies.Forexample,previousswinecastrationresearchhasidentifiedimportantpainbehaviorsspecifictoswinecastrationandinclude,prostrate,tailwagandscratching(summa-rizedin[50]).Forthisstudy,thesepainspecificbehaviorswerecategorizedwithintheitems“miscellaneous’and“attentiontoaffectedarea’andscoreswerebasedonthecumulativepres-ence/absenceofeachbehavior.Thisapproachdidnotallowdatatobeevaluatedacrossbehav-iorsbutbyacategoricalscore,thusassumingeachbehaviorincludedineithercategoryhavethesameweight,regardlessofthefrequencyanddurationofindividualbehaviors.Lastly,futureimplementationofthistoolshouldfocusonadoptingthescaletobeusedoncommercialswinefarmstoallowveterinarians,producers,andcaretakerstheabilitytoobjec-tivelyquantifypainandprovidepainreliefwhenneeded.Futureworkdevelopinganddissem-inatingcurrenttrainingprograms(https://animalpain.org/en/home-en/-pain.com)tothoseworkingdirectlywithpigsisneeded.

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eeded.Futureworkdevelopinganddissem-inatingcurrenttrainingprograms(https://animalpain.org/en/home-en/-pain.com)tothoseworkingdirectlywithpigsisneeded. Fig8.Violinplotofthetotalsum(median/amplitude)ofthea)UPAPSforcastratedpiglets(n=14)andnon-painfulfemalepiglets(n=6)aftercastration(M2)andbeforerescueanalgesiainpigletsawakeduringtheassessment.Theviolincontourrepresentsthedispersionofdatadensity,withthewidercontourrepresentinggreaterdatadensity.Thetopandbottomboxlinesrepresenttheinterquartilerange(25to75%),thelinewithintheboxrepresentsthemedian,theextremesofthewhiskersrepresenttheminimumandmaximumvalues,blacklozenges(♦)representthemean,blackcircles(•)representoutliers.Sub-UPAPS:Unesp-Botucatupigacutecompositepainscale.Differentlettersexpresssignificantdifferencesbetweenmomentswherea>b>c,accordingtothegeneralizedmixedlinearmodels[27].M2(15-minpost-procedure,beforerescueanalgesia).https://doi.org/10.1371/journal.pone.0284218.g008 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202319/26

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Limitations Thecurrentstudyhadsomelimitationsandconstraints.Theanimalsusedtodeterminecon-structvaliditywerenon-painfulfemalepiglets.Thislimitationwasdrivenbythesmallnumberofmalepigletsavailabletocompletethestudyand,althoughunlikely,sexualdifferencesinbehaviormayhaveinfluencedresults.Behavioraldifferencesaretypicallynotpresentuntilpigsreachsexualmaturityandtheauthorsareunawareofotherstudiesreportingasexeffect,asisdemonstratedinotherspeciessuchastherat[55,56].Despitethesmallnumberofnon-painfulpigletsenrolled,samplesizerequirementswerecalculatedandmet,andsignificanceandvalidationofthetoolwasachieved.Ofgreaterimportance,malepigletsservedastheirowncontrol,thusthestatisticaltestsanalyzedutilizedthetruecontrolofthemalepigletpriortocastrationprovidingbetterinsightintobehavioraldeviationspresentedafterundergoing Table9.Median(amplitude)oftheUPAPSscoresforcastrated(n=14)andnon-painfulfemalepiglets(n=6)awakepigletsatM2.ItemsFemaleCastratedPosture0 b (0–0)2 a (0–3)InteractionandInterest0 b (0–0)2 a (0–3)Activity0 b (0–2)3 a (0–3)Attentiontotheaffectedarea0 b (0–2)2 a (0–3)Elevatespelviclimb0(0–1)0(0–1)Scratchesthepainfularea0(0–1)0(0–1)Movesafterinjury0 b (0–1)0 a (0–1)Sitswithdifficulty0 b (0–1)1 a (0–1)Miscellaneousbehavior0.5 b (0–2)2 a (0–3)Wagstail0 b (0–1)1 a (0–1)Bitesobjects0(0–0)0(0–0)Headisbelowthespinalcolumn0(0–1)1(0–1)Difficultyovercomingobstacles0(0–1)0(0–1)UPAPS1 b (0–6)9.5 a (1–12)UPAPS—Unesp-Botucatupigacutecompositepainscale;Differentlettersexpresssignificantdifferencesbetweenmomentswherea>b,accordingtothemixedlinearmodel[27].M2:15-minpost-procedure,beforerescueanalgesia.https://doi.org/10.1371/journal.pone.0284218.t009

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weenmomentswherea>b,accordingtothemixedlinearmodel[27].M2:15-minpost-procedure,beforerescueanalgesia.https://doi.org/10.1371/journal.pone.0284218.t009 Table10.InternalconsistencyoftheUPAPSappliedforcastratedpigletsawake(n=14).AllitemsInternalconsistencyAllcastratedpiglets(n=39)Castratedpigletsawake(n=14)Cronbach’sαMcDonald’sωCronbach’sαMcDonald’sω0.850.880.890.92ExcludingeachitembelowPosture0.800.850.860.90Interactionandinterest0.830.880.870.91Activity0.820.870.860.90Attentiontotheaffectedarea0.810.860.870.91Miscellaneousbehaviors0.810.870.870.90UPAPS:Unesp-Botucatupigacutecompositepainscale.InterpretationoftheCronbach’sαcoefficientvalues:0.60–0.64minimallyacceptable;0.65–0.69acceptable;0.70–0.74good;0.75–0.80verygood;>0.80excellent[42].McDonald’somegainterpretation:0.70–0.84isacceptableand>0.85isstrong[41].https://doi.org/10.1371/journal.pone.0284218.t010 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202320/26

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Table11.Specificity,sensitivity,andYoudenindexofoptimalcut-offforUPAPS.UPAPSSpecificitySensitivityYoudenIndexEstimatedICEstimatedICEstimatedIC00.000.000.001.001.001.000.000.000.0010.570.390.751.001.001.000.570.390.7520.710.540.860.930.821.000.640.360.8630.750.570.890.930.821.000.680.390.8940.790.610.930.930.821.000.710.430.9350.790.610.930.790.640.930.570.250.8660.860.710.960.710.570.860.570.290.8270.890.791.000.680.500.820.570.290.8280.960.891.000.680.500.820.640.390.8290.960.891.000.540.360.710.500.250.71100.960.891.000.500.320.680.460.210.68111.001.001.000.360.180.540.360.180.54121.001.001.000.210.070.390.210.070.39https://doi.org/10.1371/journal.pone.0284218.t011 Fig9.ReceiverOperatingCharacteristic(ROC)curveandareaunderthecurve(AUC)oftheUPAPSappliedtocastratedpigletsawake(n=14).AUCvaluesabove0.90representhighdiscriminatorycapacity(accuracy)ofthescale[37].Thegrayregionrepresentsthe95%confidenceinterval.https://doi.org/10.1371/journal.pone.0284218.g009 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202321/26

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thispainfulprocedure.Exceptforsurgery,femalesunderwentthesamemanagementandhan-dlingtosimulatethesamenon-painfulbutpotentiallystressfulconditionsasmales.Theassessmentofallcastratedpiglets24hpriortocastrationservedasabaselineformalepigletbehaviorpriortocastration.Thevalidationofthistoolattheresearchlevelandbyremoteassessmentdoesnotautomat-icallyguaranteethatitmaybeapplicableforliveobservations.BeforeUPAPSisdeemedavalidinstrumentforuseinbothclinicalandcommercialsettingsdevoidofremoteassessment,thistoolwillneedtobevalidatedforliveobservation.Lastly,pre-weanedpigletsspendsignifi-cantperiodsoftheirtimebudgetsleeping.Randomlyselectingvideoclipscanbechallengingifpigletsareasleep.Thus,futureworkmusttakeintoaccounthowtorandomlyselectvideoclipstoassesspigletbehavior,whilemaximizingopportunitiestoviewactivepiglets. Conclusion TheUPAPSisavalidandreliabletoolforremoteassessmentofpost-castrationpaininpre-weanedpiglets.Furtherworkwiththistoolisneededtovalidateandrefinethescaleforliveobservationsoncommercialswinefarmsutilizingcaretakersandveterinariansresponsibleforanimalcareandmanagement. Supportinginformation

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refinethescaleforliveobservationsoncommercialswinefarmsutilizingcaretakersandveterinariansresponsibleforanimalcareandmanagement. Supportinginformation S1Fig.BiplotoftheprincipalcomponentanalysisoftheUPAPS+nursingitemsappliedtoallcastratedpiglets(n=39).UPAPS–Unesp-Botucatupigacutecompositepainscale.Confidenceellipsesindicateperioperativemomentsandpainscores.Moments:M1—pre-operative;M2—postoperative,beforerescueanalgesia;M3—postoperative,afterrescueanalgesia;M4-24hafterorchiectomy.Ellipseswereconstructedaccordingtotheassess-ment.Eachcirclecorrespondstothescorefromoneobserverattributedtoeachpigletateachmoment.(TIF)S2Fig.Violinplotofthetotalsum(median/amplitude)ofthea)UPAPS+nursing(allcas-tratedpiglets;n=39)andb)UPAPS+nursing(awakecastratedpiglets;n=14)beforeandafterorchiectomyinpiglets.Thetopandbottomboxlinesrepresenttheinterquartilerange(25to75%),thelinewithintheboxrepresentsthemedian,theextremesofthewhiskersrepre-senttheminimumandmaximumvalues,blacklozenges(♦)representthemean,blackcircles(•)representoutliersandwidthofthefiguresrepresentthedistributionofdata(widersectionsrepresentalargernumberofdata).UPAPS:Unesp-Botucatupigacutecompositepainscale.Differentlettersexpresssignificantdifferencesbetweenmomentswherea>b>c,accordingtothegeneralizedmixedlinearmodels[24,26].M1—preoperative;M2—postoperativebeforerescueanalgesia.(TIF)S1Table.Loadingvalues,eigenvaluesandvarianceoftheUPAPS+nursingandUPAPSitemsappliedtoallcastratedpiglets(n=39)andonlytoawakecastratedpiglets(n=14)basedonprincipalcomponentsanalysis.(TIF)S2Table.Specificityandsensitivityandconfidenceinterval(CI)ofthe95%oftheUPAPSappliedforallcastratedpiglets(n=39)andcastratedpigletsawake(n=14).(TIF) Painscalevalidationinpre-weanedpigletsundergoingcastration

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I)ofthe95%oftheUPAPSappliedforallcastratedpiglets(n=39)andcastratedpigletsawake(n=14).(TIF) Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202322/26

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S3Table.Median(amplitude)oftheUPAPSscoresbeforeandafterorchiectomyinallcas-tratedpiglets(n=39).(TIF)S4Table.Median(amplitude)oftheUPAPSscoresbeforeandafterorchiectomyincas-tratedpigletsawake(n=14)duringtheassessment.(TIF)S5Table.Median(amplitude)oftheUPAPSscoresforcastrated(n=14)andnon-painfulfemalepiglets(n=6)awakepiglets,afterorchiectomyduringtheassessment.(TIF)S6Table.Item-totalcorrelationandinternalconsistencyoftheUPAPS+nursingappliedforallcastratedpiglets(n=39).(TIF)S7Table.Item-totalcorrelationandinternalconsistencyoftheUPAPS+nursingappliedforcastratedpigletsawake(n=14).(TIF)S1Dataset.(XLSX) AuthorContributions Conceptualization:M.D.Pairis-Garcia.Datacuration:IRobles,S.P.L.Luna,P.H.E.Trindade,A.V.Viscardi,ETamminga,M.E.Lou,M.D.Pairis-Garcia.Formalanalysis:IRobles,S.P.L.Luna,P.H.E.Trindade.Fundingacquisition:V.R.Merenda.Investigation:M.D.Pairis-Garcia.Methodology:S.P.L.Luna,P.H.E.Trindade,MLopez-Soriano,ETamminga,M.E.Lou,M.D.Pairis-Garcia.Software:P.H.E.Trindade.Supervision:M.D.Pairis-Garcia.Validation:S.P.L.Luna,P.H.E.Trindade.Visualization:P.H.E.Trindade,MLopez-Soriano,V.R.Merenda.Writing–review&editing:S.P.L.Luna,MLopez-Soriano,V.R.Merenda,A.V.Viscardi,ETamminga,M.E.Lou,M.D.Pairis-Garcia. References 1.WagnerB,RoyalK,ParkR,Pairis-GarciaM.IdentifyingBarrierstoImplementingPainManagementforPigletCastration:AFocusGroupofSwineVeterinarians.Anim.2020;10:1202.https://doi.org/10.3390/ani10071202PMID:326797772.RydhmerL,ZamaratskaiaG,AnderssonHK,AlgersB,GuillemetR,Lundstro¨mK.Aggressiveandsex-ualbehaviorofgrowingandfinishingpigsrearedingroups,withoutcastration.ActaAgricScandAAnimSci.2006;56:109–119.3.PovodM,LozynskaI,SamokhinaE.Biologicalandeconomicaspectsoftheimmunologicalcastrationincomparisonwithtraditional(surgical)method.BulgJAgricSci.2019;25:403–409.

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ozynskaI,SamokhinaE.Biologicalandeconomicaspectsoftheimmunologicalcastrationincomparisonwithtraditional(surgical)method.BulgJAgricSci.2019;25:403–409. Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202323/26

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4.PrunierA,BonneauM,VonBorellEH,CinottiS,GunnM,FredriksenB,etal.Areviewofthewelfareconsequencesofsurgicalcastrationinpigletsandtheevaluationofnon-surgicalmethods.AnimWelf.2006;15:277.5.LlamasMoyaS,BoyleLA,LynchPB,ArkinsS.Effectofsurgicalcastrationonthebehavioralandacutephaseresponsesof5-day-oldpiglets.ApplAnimBehavSci.2008;111:133–145.6.VanBeirendonckS,DriessenB,VerbekeG,GeersR.Behaviorofpigletsaftercastrationwithorwithoutcarbondioxideanesthesia.JAnimSci.2011;89:3310–3317.https://doi.org/10.2527/jas.2010-3104PMID:215318487.YunJ,OllilaA,ValrosA,Larenza-MenziesP,HeinonenM,OlivieroC,etal.Behavioralalterationsinpigletsaftersurgicalcastration:Effectsofanalgesiaandanaesthesia.ResVetSci.2019;125:36–42.8.TellesFG,LunaSPL,TeixeiraG,BertoDA.Long-termweightgainandeconomicimpactinpigscas-tratedunderlocalanaesthesia.VetAnimSci.2016;1–2:36–39.https://doi.org/10.1016/j.vas.2016.11.003PMID:327340229.HanssonM,LundeheimN,NymanG,JohanssonG.Effectoflocalanaesthesiaand/oranalgesiaonpainresponsesinducedbypigletcastration.ActaVetScand.2011;53:34.https://doi.org/10.1186/1751-0147-53-34PMID:2162779710.RaultJ-L,LayDC,Marchant-FordeJN.Castrationinducedpaininpigsandotherlivestock.ApplAnimBehavSci.2011;135:214–225.11.SutherlandMA,DavisBL,BrooksTA,CoetzeeJF.Thephysiologicalandbehavioralresponseofpigscastratedwithandwithoutanesthesiaoranalgesia.JAnimSci.2012;90:https://doi.org/10.2527/jas.2011-4260PMID:2226698912.DeBriyneN,BergC,BlahaT,TempleD.Pigcastration:willtheEUmanagetobanpigcastrationby2018?PorcineHealthManag.2016;2:29.https://doi.org/10.1186/s40813-016-0046-xPMID:2840545513.BatesJL,KarrikerLA,StockML,PertzbornKM,BaldwinLG,WulfLW,etal.Impactoftransmammary-deliveredmeloxicamonbiomarkersofpainanddistressinpigletsaftercastrationandtaildocking.PLoSOne.2014;9:e113678.https://doi.org/10.1371/journal.pone.0113678PMID:2543786614.IsonSH,CluttonRE,DiGiminianiP,RutherfordKMD.AReviewofPainAssessmentinPigs.FrontVetSci.2016;3:108.https://doi.org/10.3389/fvets.2016.00108PMID:2796596815.ParkRM,CramerMC,WagnerBK,TurnerP,MoraesLE,ViscardiAV,etal.Acomparisonofbehavioralmethodologiesutilisedtoquantifydeviationsinpigletbehaviorassociatedwithcastration.AnimWelf.2020;29:285–292.16.ViscardiAV,TurnerPV.Efficacyofbuprenorphineformanagementofsurgicalcastrationpaininpiglets.BMCVetRes.2018;14:318.https://doi.org/10.1186/s12917-018-1643-5PMID:3035258617.ViscardiAV,TurnerPV.Useofmeloxicamorketoprofenforpigletpaincontrolfollowingsurgicalcastra-tion.FrontVetSci.2018;5:299.https://doi.org/10.3389/fvets.2018.00299PMID:3053455218.ViscardiAV,HunnifordM,LawlisP,LeachM,TurnerPV.DevelopmentofaPigletGrimaceScaletoevaluatepigletpainusingfacialexpressionsfollowingcastrationandtaildocking:Apilotstudy.FrontVetSci.2017;4:51.https://doi.org/10.3389/fvets.2017.00051PMID:2845905219.McLaffertyE,FarleyA.Assessingpaininpatients.NursStand.2008;22:42–46.https://doi.org/10.7748/ns2008.02.22.25.42.c6422PMID:1837663320.FirthAM,HaldaneSL.Developmentofascaletoevaluatepostoperativepainindogs.JAmVetMedAssoc.1999;214:651–659.PMID:1008801221.HoltonL,ReidJ,ScottEM,PawsonP,NolanA.Developmentofabehavior-basedscaletomeasureacutepainindogs.VetRec.2001;148:525–531.22.ReidJ,NolanAM,HughesJML,LascellesD,PawsonP,ScottEM.Developmentoftheshort-formGlasgowcompositemeasurepainscale(CMPS-SF)andderivationofananalgesicinterventionscore.AnimWelf.2007;16:97–104.23.BrondaniJT,LunaSPL,PadovaniCR.Refinementandinitialvalidationofamultidimensionalcompos-itescaleforuseinassessingacutepostoperativepainincats.AmJVetRes.2011;72:174–183.https://doi.org/10.2460/ajvr.72.2.174PMID:2128119124.BrondaniJT,MamaKR,LunaSPL,WrightBD,NiyomS,AmbrosioJ.ValidationoftheEnglishversionoftheUNESP-Botucatumultidimensionalcompositepainscaleforassessingpostoperativepainincats.BMCVetRes.2013;9:1.25.BelliM,deOliveiraAR,deLimaMT,TrindadePHE,SteagallPV,LunaSPL.ClinicalvalidationoftheshortandlongUNESP-Botucatuscalesforfelinepainassessment.PeerJ.2021;9:e11225.https://doi.org/10.7717/peerj.11225PMID:33954046

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PL.ClinicalvalidationoftheshortandlongUNESP-Botucatuscalesforfelinepainassessment.PeerJ.2021;9:e11225.https://doi.org/10.7717/peerj.11225PMID:33954046 Painscalevalidationinpre-weanedpigletsundergoingcastration PLOSONE|https://doi.org/10.1371/journal.pone.0284218April13,202324/26

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