Espinoza et al 2015

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Evaluatingtheef ! cacyofatopicalanaestheticformulation andketoprofen,aloneandincombination,onthepain sensitivityofdehorningwoundsinHolstein-Friesiancalves CrystalA.Espinoza A ,DominiqueMcCarthy A ,PeterJ.White A ,PeterA.Windsor A andSabrinaH.Lomax A , B A FacultyofVeterinaryScience,TheUniversityofSydney,NSW2006,Australia. B Correspondingauthor.Email:sabrina.lomax@sydney.edu.au Abstract. Theaimofthisstudywastoinvestigatetheeffectofatopicallyappliedlocalanaestheticandthenon-steroidal anti-in ! ammatorydrugketoprofen,aloneandincombination,onthepainsensitivityresponseofcalvestodehorning (meanage2.2months).Calveswererandomlyallocatedandblockedbyagetooneoffourgroups.Groupswere:scoop dehorning(D, n =8),scoopdehorning+i.m.administrationof3mg/kgketoprofen(DK, n =8),scoopdehorning+ applicationoftopicalanaesthetic(DTA, n =7)andscoopdehorning+applicationoftopicalanaestheticandi.m. administrationofketoprofen(DKTA, n =7).Apressurealgometerwasusedtodeterminethemechanicalnociceptive threshold(MNT),beingthepressure(kg/f)atwhichcalveswithdrewfromthestimulus.Measurementsweretakenbefore dehorningandat1min,1,2,5and24hpost-dehorningatboththecutskinedgeofthewoundandtheperi-woundarea. Theeffectoftreatmentchangedovertime( P < 0.001).MNTwashighestbeforetreatment(MNT=5.03kg/f)andtended todecreaseovertime(MNT=1.16kg/f24hpost-treatment).Overall,DcalvesexhibitedthelowestMNTwithanaverage of1.77kg/f.DTAcalveshadthehighestMNT(3.89kg/f),followedcloselybyDKTAcalves(3.24kg/f).DKcalves exhibitedanintermediateMNTof2.61kg/f.MNTofthecutskinedgewasgenerallylowerthanthatoftheperi-wound area(2.01vs3.81kg/f,respectively, P =0.02).Thetopicalanaestheticformulationsigni " cantlyreducedthepainsensitivity ofdehorningwounds.Therewasnoobservedenhancedanalgesiceffectwithadditionofketoprofen.Thecutskinedge

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cformulationsigni " cantlyreducedthepainsensitivity ofdehorningwounds.Therewasnoobservedenhancedanalgesiceffectwithadditionofketoprofen.Thecutskinedge wasmoresensitivetopressurethantheperi-woundarea. Additional keywords: calf, non-steroidal anti-in ! ammatory drug, topical anaesthesia, welfare. Received 12 January 2014, accepted 15 February 2015, published online . B Z Introduction Dehorningisacommonprocedureinthedairyandbeefcattle industriesandiswelldocumentedasbeingpainful(Fisher etal . 2008 ).Itisanimportantmanagementtoolforproductionand safetyreasons.Apartfromincreasedprobabilityofinjuryand dif " cultyinhandling,hornsmayhaveadditionaleconomic impactsthroughbruisingandhidedamagetoothercattle,and increasedrequirementsforspaceduringfeedingandtransport (Vowles 1976 ;Marshall 1977 ;StookeyandGoonewardene 1996 ;StaffordandMellor 2005 ).Dehorningcattlereduces bruisetrimby~50%thushelpingtoreduceeconomiclosses (Prayaga 2007 ). Despitethejusti " cationsfordehorning,thereisgrowing publicconcernforproductionanimalwelfare,includingthe welfareofanimalsundergoingpainfulhusbandryprocedures (Fisher etal . 2008 ).Thisrequiresevaluationofalternative optionstodehorningormodi " cationofthedehorning proceduretoreduceoreliminatepain.Althoughthelong-term solutiontodehorningisthebreedingofpolledcattle,thisis currentlynotafeasibleorpracticalsolutionformanydairyand beefproducers.Intheinterim,painmanagementisonerealistic alternativetoallaywelfareconcernsatdehorning(Fisher etal . 2008 ).Althoughdehorningistraditionallyperformedwithout painreliefinmanycountries(HuxleyandWhay 2006 ;Fisher etal . 2008 ;Paull

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ehorning(Fisher etal . 2008 ).Althoughdehorningistraditionallyperformedwithout painreliefinmanycountries(HuxleyandWhay 2006 ;Fisher etal . 2008 ;Paull etal . 2009 ),changinglegislationespecially intheEUrecommendspainreliefbeusedforcalvesover 4weeksofage(CouncilofEurope 1988 ).Researchhasbeen conductedontheapplicationofanaesthesiaandanalgesiafor theprocedure,howeverthecost-effectivenessandpracticalityof thisapproachforroutineon-farmuseisquestionable(Petherick 2005 ).Studiesontheattitudesofproducersandpractitioners topainalleviationwhendehorning,haveshownthatthemain reasonsforlackofuseofanaesthesiaandanalgesiaweretime constraints,cost,andlackofinformationorskill(Huxleyand Whay 2006 ;Gottardo etal . 2011 ;Guatteo etal . 2012 ). Regardlessofexistingconstraintsandlimitations,thereis evidencethatlivestockindustriesarerespondingtowelfare concerns,withconsiderationofpainreliefforcalves undergoingdehorning.Legislationrequiringtheadministration oflocalanaesthesiabeforecastrationanddehorninghasbeen CSIRO PUBLISHING AnimalProductionScience http://dx.doi.org/10.1071/AN14012 Journalcompilation ! CSIRO2015www.publish.csiro.au/journals/an

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enactedbyseveralEuropeancountries(GrafandSenn 1999 ; Rollin 2004 ).TheFarmAnimalWelfareCouncilintheUK recommendsthatanalgesicsandlocalanaesthesiabeused whendehorningcalves,andthattheprocedurebeperformed before2monthsofage(Anonymous 1997 ). ‘ TheAustralian ModelCodeofPracticefortheWelfareofAnimals:Cattle ’ encouragesdehorningasyoungaspossibleandrecommends theuseofalocalanaestheticwhendehorningcalvesover 6monthsofage(Anonymous 2004 a ).TheAustralian VeterinaryAssociationalsostatesthatcattleshouldbe dehornedasyoungaspossibleandthatanalgesiashouldbe usedwhereappropriate(Anonymous 2004 b ). Issuesofimpracticalityandcostofpainrelieffordehorning canbeaddressedthroughtheintroductionofatopicalanaesthetic thatcanbeeasilyappliedon-farmbyproducers.Recently,a topicalanaestheticwounddressingwasshowntosigni " cantly reduceshort-termpainsensitivityinscoopdehorneddairycalves (Espinoza etal . 2013 ).Thismultipurposeformulationcontained lignocaineandbupivacaineforlocalanaesthesia,cetrimidefor antisepsis,andaluminiumchlorohydrateforastringencyofthe wound.Theseingredientswerecarriedinaviscousgelbaseto improveadhesiontothewoundandsurroundingtissue. Lignocaine,whenadministeredasacornualnerveblockora ringblock,providesrapidonsetofanaesthesiaat2 – 5minafter application,andtheanaestheticeffectsaredocumentedtolast upto3h(McMeekan etal . 1999 ;FaulknerandWeary 2000 ; StaffordandMellor 2005 ;Stewart etal . 2009 ;Coetzee 2011 ). Bupivacaineisalongeractinglocalanaestheticthatbeginsto takeeffect20 – 30minafteradministration,withanaesthesia lastingupto8h(FaulknerandWeary 2000 ;Staffordand Mellor 2005 ;Coetzee 2011 ).Althoughtheuseofinjected localanaestheticagentshasbeenwelldocumented,their topicalapplicationrequiresfurtherexamination. Non-steroidalanti-in ! ammatorydrugs(NSAID)have

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jected localanaestheticagentshasbeenwelldocumented,their topicalapplicationrequiresfurtherexamination. Non-steroidalanti-in ! ammatorydrugs(NSAID)have analgesiceffectsthatextendintothepost-operativeperiodby inhibitingcyclooxygenaseandprostaglandinproductionand reducingnociceptorsensitisation(Coetzee 2011 ).Subcutaneous in " ltrationwithalocalanaesthetic,incombinationwiththe NSAIDketoprofen,hasbeenshowntoreducethecortisol response,pain-relatedbehaviour,andheartrateincalves undergoingdehorningtoagreaterdegreethanlocal anaesthesiaalone(FaulknerandWeary 2000 ;Staffordand Mellor 2005 ;Coetzee 2011 ).Thus,ifusedincombination withatopicalanaesthetic,theinclusionofanNSAIDmay provideenhancedreliefofthepainassociatedwithdehorning. Algometryisaformofquantitativesensorytestingthat providesobjective,repeatableresults(Duarte etal . 2005 ) whenmeasuringsensation,paindetectionandpaintolerance (Clark etal . 2011 ).Analgometerappliesincreasingpressure ontoatargetsiteandmeasuresthepressure,usuallyinkilograms orpoundsofforce,atwhichtheanimalwithdrawsfromthe stimulus(Clark etal . 2011 ).Themaximumpressureappliedis interpretedasthepainthresholdresponse(ormechanical nociceptivethreshold;MNT)oftheanimal(Fitzpatrick etal . 2013 ).Algometryhasbeenusedtosuccessfullyprovidean indicationofpaininavarietyofconditionsincattleincluding lameness,digitaldermatitis,ocularpainincalvesaffectedwith bovinekeratoconjunctivitis,andmastitis(Dyer etal . 2007 ; Cutler etal . 2013 ;Fitzpatrick etal . 2013 ;Dewell etal . 2014 ). Algometryhasalsobeenpreviouslyvalidatedasatooltoquantify painsensitivityfollowingdehorning(Heinrich etal . 2010 ; Tapper 2011 ).Calvesundergoingcauterydisbuddingwere foundtohavelowerMNTthancalvesthathadnotbeen disbudded,suggestingthattheprocedurecausesapain response(Heinrich etal . 2010

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ergoingcauterydisbuddingwere foundtohavelowerMNTthancalvesthathadnotbeen disbudded,suggestingthattheprocedurecausesapain response(Heinrich etal . 2010 ).Itwasalsofoundthatcalves thatweredehornedfollowingadministrationofmeloxicam, hadhigherMNTthancalvesthatweredehornedwithout analgesia,suggestingthatmeloxicamhelpedtorelievethe painresponse(Heinrich etal . 2010 ). Thisexperimentaimedtoinvestigatethedurationandeffect ofatopicalanaestheticandtheNSAIDketoprofen,aloneandin combination,onthepainsensitivityofcalvesundergoingscoop dehorning.Itwashypothesisedthatcalvesreceivingthetopical anaestheticandketoprofenwouldexhibitlowerpainsensitivity thancalvesdehornedalone,andthattheircombinedusewould resultinanadditiveeffectonpainsensitivity. Materialsandmethods Animalsandhousing ThirtyHolstein-Friesianheifercalves(51 – 86daysold)were sourcedfrom ‘ Corstophine ’ DairyUnitatTheUniversityof Sydney(NewSouthWales,Australia)inMay2013.The calveshadbeenraisedundercommercialdairyoperational conditions.Theexperimentalprotocolwasconductedunder institutionalanimalethicscommitteeapproval(ApprovalNo. 5832). CalfweightswereestimatedusingaHolstein-Friesiandairy calfweightestimationtape(TheCoburnCo.,Inc.,Whitewater, WI,USA)witharangeof65.5 – 122.3kg.Hornbuddiameter (cm)foreachcalfwasmeasuredusingastandardmetricruler. Diameterwassetasthedistanceacrossthebaseofthehorn whereitattachedtotheskull.Calvesweregroup-housedin 50 · 20-mpaddocksfrom2daysofageandfortheduration oftheexperimentalperiod.Thecalveswerefedamilkrationat ~10%oftheirbodyweighttwicedailyat0730hoursand1530 hoursviaanarti " cialteatandhad adlibitum accesstowater andkikuyu-basedpasture. Experimentaldesignandtreatments Onthedayoftheexperiment,calveswererandomlyallocated tooneoffourtreatmentgroups:(1)scoopdehorning(D, n =8),

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sedpasture. Experimentaldesignandtreatments Onthedayoftheexperiment,calveswererandomlyallocated tooneoffourtreatmentgroups:(1)scoopdehorning(D, n =8), (2)scoopdehorningwithpre-operativeadministrationof ketoprofen(3mg/kg)(KeyInjection,ParnellTechnologiesNZ PtyLtd,Auckland)(DK, n =8),(3)scoopdehorningwithpost- operativeapplicationoftopicalanaesthetic(TA)(Technical ConsultancyServicesPtyLtd,Rockdale,NSW,Australia) (DTA, n =7)and(4)scoopdehorningwithpre-operative administrationofketoprofenandpost-operativeapplicationof TA(DKTA, n =7).Theexperimentwasconductedover2days, with16calvesbeingtreatedonthe " rstday(DandDKtreatment groups)and14calvesbeingtreatedonthesecondday(DTA andDKTAtreatmentgroups).Calveswerequietlymovedfrom theirhousingpaddockstoaholdingpen.Ketoprofenwas administeredintramuscularlyintheneck20minbefore dehorningatadosageof3mg/kg.Allcalvesthatdidnot receiveketoprofen(DandDTAcalves)wereadministereda placeboinjectionofbufferedsalineintramuscularlyinthe B AnimalProductionScience C.A.Espinoza etal .

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neckequivalenttothevolumeitwouldhavereceivedifgiven ketoprofen.Thiswasconductedtoeliminateanypossibleeffect theinjectionmayhaveonthecalves ’ painsensitivity.Individual calveswerethenmovedthroughtheraceandrestrainedina standinguprightpositioninacalfcradle(ArrowFarmquip, Tamworth,NSW,Australia)fortreatmentanddatacollection. Scoopdehorningwasperformedbyasingleexperienced technician.Thedehorningdevicewaspositionedoverthehorn budandthehandlespulledaparttoexcisethehornbudand surroundingtissue.TAwasadministeredimmediatelyafter dehorningwitha10-mLsyringeandasoftsiliconebrush. Approximately7mLofTAwasappliedperwound(~14mL percalf),orenoughtocompletelycoverthedehornedwound andtheperi-woundarea.Theperi-woundareacoveredbyTA was~1cmwideforthetotalcircumferenceofthedehorned wound.Thebrushwascleanedbetweencalvesinabucketof 100mL/Lchlorhexidinesolution(Hibitane,CoopersAnimal Health,Sydney,NSW,Australia).Followingtreatmentand datacollection,calveswerequietlyreturnedtotheirpaddocks untilsubsequentdatacollectionwasrequired,wheretheywere quietlymovedbacktotheholdingpenandcradle. Assessmentofpressure/painthreshold Pressurealgometrywasusedtomeasurechangesinpain sensitivityovertime,withtheamountofforcerequiredto elicitaheadwithdrawalresponsedenotedastheMNT(in kg/f).Thisvaluecanalsobeinterpretedastheminimum pressurerequiredtoinduceapainresponse.Forthis experiment,apainresponsewasde " nedasafullhead withdrawalre ! exawayfromthemechanicalstimulus.Testing wasperformedusingacalibratedhand-heldpressurealgometer (WagnerPainTestFPIXDigitalAlgometer,WagnerInstruments, Riverside,CT,USA)withamaximumthresholdof10kg force(to50gaccuracy).Measurementsweretakenaftereach calfhadsettledinthecalfcradleandtherewasminimalnoise orexternalactivitytodistracttheanimal.Thealgometer,

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to50gaccuracy).Measurementsweretakenaftereach calfhadsettledinthecalfcradleandtherewasminimalnoise orexternalactivitytodistracttheanimal.Thealgometer, equippedwitharoundrubbertip~1cmindiameter,was placedperpendiculartothetargetsiteforeachmeasurement. Increasingpressurewasslowlyapplieduntilthecalfwithdrew itshead,andthemaximumpressureappliedwasrecordedas theMNT(kg/f).Calvesthatdidnotrespondtopressuretesting haditsMNTrecordedas10kg/f(maximumthresholdofthe algometer).Thealgometerwasresettozeroaftereachpressure sensitivitytest. Pressuresensitivitywasmeasuredimmediatelybefore dehorning,and1min,1and,2,5and24hpost-dehorning. Thealgometerwasoperatedthroughouttheexperimentbya singleandtrainedtechnician.Pressuremeasurementswere takenfromeightsitesoneachcalfintotal:foursiteslocated onthecutskinedgeofthewoundandfoursiteslocatedonthe surroundingtissue(peri-woundarea~1cmfromthecutskin edge)(Fig. 1 ).Thecutskinedgesiteswerechosenasithas previouslybeenshownthatthesensitivityofcutskinedgesin mulesingwoundsaremoresensitivetopainduetodamaged nerveendings(Lomax etal . 2008 ).Theperi-woundsiteswere chosentoinvestigatesecondaryhyperalgesia(exaggeratedpain responseintissuesurroundingthewound).Thevisibilityofthe TAonthewoundsurfacemeantthatthetechnicianwasableto differentiatebetweencalvesthathadbeentreatedwithTAand thosethathadnot. Statisticalanalyses Datawasnormalisedbylog-etransformation.Pressurereadings wereanalysedbyconductingresidualmaximumlikelihoodusing thestatisticalsoftwarepackage,G ENSTAT (VSNInternationalLtd, HemelHempstead,UK).The " xedeffectsofthemodelwere treatment(D,DK,DTA,DKTA),time(pre-treatment,1min,1,2, 5,and24hpost-treatment),andtheirinteraction,areatested (peri-woundorcutskinedge),calfweightandage,andhorn buddiameter.Therandomeffectofthemodelwascalf.For

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d24hpost-treatment),andtheirinteraction,areatested (peri-woundorcutskinedge),calfweightandage,andhorn buddiameter.Therandomeffectofthemodelwascalf.For allstatisticalcalculations, P -values ! 0.05wereconsidered statisticallysigni " cant. Results Overall Therewasasigni " cantinteractiveeffectoftimeandtreatment ( P < 0.001),andareatestedandtreatment( P =0.02)onMNT. Therewasnosigni " canteffectofcalfage,weightorhornbud diameteronMNT. Timeandtreatmentinteractiveeffect Therewasasigni " canteffectoftimeandtreatment,anda signi " canttimeandtreatmentinteraction( P < 0.001).All calveshadanMNTbetween4.44and6.12kg/fbefore treatment(mean5.03kg/f)(Fig. 2 ).ThereafterMNTdecreased overtimereachingthelowestmeanMNTat24hpost-treatment. Overall,DcalvesexhibitedthelowestMNTwithanaverageof 1.77kg/f.DKcalveshadameanMNTof2.61kg/f.DTAcalves hadthehighestmeanMNT(3.89kg/f),followedcloselyby DKTAcalves(3.24kg/f). DversusDK At1minpost-dehorning,themeanMNTofDK calveswasgreaterthanthatofDcalves(5.22and3.65kg/f, respectively).At1and2hpost-treatment,DKcalveshada signi " cantlygreatermeanMNTthanDcalves( P < 0.05).Mean MNTofthesecalvesweresimilar5and24hpost-treatment. Fig.1. Sitessubjectedtopressuresensitivitytesting(peri-woundsites: blackstars;cutskinedgesites:greystars). Topicalanaesthesiafordehorningincalves AnimalProductionScience C

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DversusDTA ThemeanMNTofDTAcalveswas signi " cantlyhigherthanDcalvesatalltimepointspost- treatmentexcept24h( P < 0.05). DversusDKTA ThemeanMNTofDKTAcalveswasgreater thanthatofDcalvesatalltimepoints,withsigni " cantdifferences 2and5hpost-treatment( P < 0.05). DKversusDTA MNTwasnumericallyhigherinDTAthan DKcalvesatmosttimepoints,withasigni " cantdifference detected2hpost-treatment( P < 0.05). DKversusDKTA MNTofDKandDKTAcalvesweresimilar 1min,1and24hpost-treatment.DKTAcalveshadsigni " cantly greaterMNTthanDKcalves2and5hpost-treatment( P < 0.05). DTAversusDKTA MNTwasnumericallyhigherinDTA thanDKTAcalves1min,1,2and24hpost-treatment.MNT wasnumericallyhigherinDKTAthanDTAcalves5hpost- treatment.Nosigni " cantdifferencesweredetectedbetween groupsatanytimepoint. Areaandtreatmentinteractiveeffect Mechanicalstimulationofthecutskinedgearearesultedin lowermeanMNTthanstimulationoftheperi-woundtissue (2.01vs3.81kg/f,respectively)(Fig. 3 ).Differencesvaried dependingontreatmentwiththegreatestdifferenceseenin DTAcalves(2.09kg/f)andsimilardifferencesinD,DKand DKTAcalves(1.63 – 1.7kg/f; P =0.02). Discussion Thisstudyassessedtheef " cacyofatopicalanaesthetic formulationincombinationwiththeNSAIDketoprofen,for a a a a a a a ab b b ab a a b b c bc a a ab ab c c a 0 1 2 3 4 5 6 7 8 9 Pre1 min1 h2 h5 h24 h Mean mechanical nociceptive threshold (kg/f) Time Dehorned Dehorned + Ketoprofen Dehorned + Topical Anaesthetic Dehorned + Ketoprofen + Topical Anaesthetic Fig.2. Predictedmeanmechanicalnociceptivethresholdsfollowingalgometermechanical stimulationonvarioussitesovertimeontheperi-woundandcutskinedgeofdehornedcalves subjectedtothefollowingtreatments:dehornedalone( n =8),dehornedwithketoprofen( n =8) dehornedandtreatedwithtopicalanaesthesia( n =7),anddehornedwithketoprofenandtreatedwith

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gtreatments:dehornedalone( n =8),dehornedwithketoprofen( n =8) dehornedandtreatedwithtopicalanaesthesia( n =7),anddehornedwithketoprofenandtreatedwith topicalanaesthesia( n =7).Calvesnotsharingthesamesuperscriptwithineachtimepointare consideredstatisticallysigni " cant( P < 0.05). a a a a b b b b 0 1 2 3 4 5 6 7 DDKDTADKTA Mean mechanical nociceptive threshold (kg/f) Peri-wound Skin-edge Fig.3. Predictedmeanmechanicalnociceptivethresholdsfollowing algometermechanicalstimulationontheperi-woundandcutskinedgeof dehornedcalvessubjectedtothefollowingtreatments:dehornedalone(D) ( n =8),dehornedwithketoprofen(DK)( n =8),dehornedandtreatedwith topicalanaesthesia(DTA)( n =7),anddehornedwithketoprofenandtreated withtopicalanaesthesia(DKTA)( n =7).Calvesnotsharingthesame superscriptwithineachtreatmentareconsideredstatisticallysigni " cant( P < 0.05). D AnimalProductionScience C.A.Espinoza etal .

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thepainameliorationofscoopdehorningwoundsincalves.As hypothesised,calvestreatedwithTAweresigni " cantlyless pressure-sensitivethancalvesdehornedwithoutTA(exceptat 24hpost-treatment).Treatmentwithketoprofenreduced pressuresensitivitywithsigni " cantreductionsobserved1and 2hpost-treatment.ThecombineduseofTAandketoprofen resultedinsomereducedpressuresensitivitycomparedwith calvesdehornedwithoutanalgesia,howevernoadditiveeffect wasobservedwithDKTAcalvesdisplayingsimilarsensitivity toDTAcalves. PreviousresearchhasshownthatTAsigni " cantlyreduces painsensitivityforatleast1.5hpost-dehorning(Espinoza etal . 2013 ).However,asthepainofdehorningappearstobemost severeduringthe7 – 9hpost-proceduralperiod,andmaystillbe evident1 – 2dayslater,addressingchronicpainisconsidered important(Sylvester etal . 1998 , 2004 a ;StaffordandMellor 2005 ).Therefore,acombinationofdrugs,asusedinthepresent study,mayprovideamultimodalapproachtoanalgesia,which hasbeendemonstratedtobemoreef " cacious(Coetzee 2011 ). Thepost-operativeapplicationofTAondehorningwounds resultedinsigni " cantlygreatermeanMNTthancalvesdehorned withoutTA.ThisreducedpainsensitivityinDTAandDKTA calveswasobservedforupto5hpost-treatment,exceeding thedurationofef " cacyof1.5hdetectedpreviously(Espinoza etal . 2013 ).Thismaybeduetotheincreasedconcentrationof bupivacaineinthecurrentformulation(1%vs0.5%inEspinoza etal . 2013 ).ThequantityofTAmayalsobeimportantfor ef " cacy,withalargervolumeusedherethaninpreviouswork (7mLvs4mLperdehorningwound).Animportantconsideration howeveristhedifferentmodesofpainassessmentusedtogauge ef " cacyinthetwoexperiments.ThevonFreymono " laments usedinEspinoza etal .( 2013 )weredesignedtobendataneven, repeatableforce(10and300g/f).Thusthesameforcewasexerted

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oexperiments.ThevonFreymono " laments usedinEspinoza etal .( 2013 )weredesignedtobendataneven, repeatableforce(10and300g/f).Thusthesameforcewasexerted onthewoundandsurroundingskinofallcalvesinordertoinvoke apainresponse.Thisresponsetostimulationwasthenvisually scoredusinganumericalratingscalerangingfrom0to3(no, minor,moderateorsevereresponse).Thepresentstudyutilised algometrywherebytolerance(upto10kg/f)toincreasing mechanicalstimulationwasmeasuredandaquantitative outcomeobtained.Therefore,thetypeofpainresponsebeing measuredisdifferentbetweenthetwotrials,whichdoesnotallow fordirectcomparison. Theadministrationofketoprofenprovidedsigni " cantshort- termreductioninthepainsensitivityofdehorningwoundswhen comparedwithDcalves(upto2h).However,noreductionwas evident5and24hpost-dehorning.Theonsetofketoprofenhas beendocumentedat2hpost-dehorning(McMeekan etal . 1998 a ;StaffordandMellor 2011 )whereasothersources suggestahalf-lifeinmostanimalsoflessthan2h(Papich 2010 ).Hencethepharmacokineticsofketoprofenareunclear. DTAandDKTAcalvesexhibitedsimilarmeanMNTatall timepointsfollowingdehorning,whereasinotherstudies,the combineduseofketoprofenandlignocainehasbeenshowntobe effectiveatreducingthepainresponsetodehorning(McMeekan etal . 1998 a ;FaulknerandWeary 2000 ;Sutherland etal . 2002 ). Routeofadministrationcouldaccountfortheseresults.Although thedoseisthesameinthepresentstudyasintheliterature(3mg/ kg),ketoprofenwasadministeredintramuscularlyintotheneck ratherthanintravenouslyintothecalfjugularvein(McMeekan etal . 1998 a ;Sutherland etal . 2002 ),ororally(Faulknerand Weary 2000 ).Inaddition,localanaesthesiawasadministered topicallyratherthanviasubcutaneousin " ltration,allowingfor slowerabsorptionandconcentrationoftheactivesattheinjury site.

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alanaesthesiawasadministered topicallyratherthanviasubcutaneousin " ltration,allowingfor slowerabsorptionandconcentrationoftheactivesattheinjury site. TheeffectofanNSAIDonthepainsensitivityofdisbudding woundshasbeenpreviouslyinvestigated.Calvesthatwere cauterydisbuddedfollowingtheadministrationofmeloxicam andlignocaine(cornualnerveblock),displayedlowerpain sensitivity(asdeterminedthroughalgomtery)4hafter disbuddingthancalvesgivenlignocainealone(Heinrich etal . 2010 ).ThisindicatestheeffectivenessofanNSAIDasan ameliorantforacutedehorningpain.Resultsfromthepresent studyalsofoundsigni " cantlylowerpainsensitivityin ketoprofen-treatedcalves1and2hpost-dehorningcompared withcalvesnotgivenketoprofen.Anotherstudy,which investigatedtheeffectofmeloxicamonthepainsensitivityof disbuddingwounds,failedtodetectanyanalgesiceffectbut attributedthis " ndingtotheprobableinabilityofvonFrey mono " lamentsinexertingsuf " cientpressuretoinducea response(Mintline etal . 2013 ).Heinrich etal .( 2010 )and Mintline etal .( 2013 )studiedmeloxicam,believedtobea preferentialcyclooxygenase-2isoforminhibitorwithahalf-life between20and43h.Ketoprofenisanonspeci " ccyclooxygenase inhibitorwithahalf-lifeinmostanimalsoflessthan2hbut durationofactionupto24h(Papich 2010 ).Themuchshorter half-lifeofketoprofencomparedwithmeloxicammayexplain theseeminglydiminishingeffectfrom1to2hintheDKgroup. Methodofdehorningisanimportantconsiderationgiventhat cautery(usedinHeinrich etal . 2010 ;andMintline etal . 2013 ) andamputationdehorning(usedinthepresentstudy)elicit verydifferentwoundsandsubsequentpainresponses.Cautery disbuddingelicits " rst,secondandthirddegreeburnsresulting inthedamageanddestructionofnociceptorsleadingtoalteredor lossofsensation(Petrie etal . 1996 ).Thuscauterydisbudding

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secondandthirddegreeburnsresulting inthedamageanddestructionofnociceptorsleadingtoalteredor lossofsensation(Petrie etal . 1996 ).Thuscauterydisbudding hasbeensuggestedaslesspainfulthanamputationdehorning, whichresultsinasurgicalwoundresultinginbleeding,and exposedandseverednociceptors. Thedurationofnerveblockadeprovidedbylocalanaesthetics hasbeensuggestedashavinganimpactontheef " cacyof NSAIDfortheameliorationofdehorningpain(Staffordand Mellor 2005 ).Ketoprofenincombinationwithlignocaine administeredaroundthecornualnervehasbeenshownto virtuallyeliminatethecortisolresponsetodehorning,whereas theuseofbupivacaineappearstoproduceadelayedcortisol responseoncethebupivacainewearsoff(McMeekan etal . 1998 a ).Whileinthiscircumstanceitisnotclearwhyshorter- actinglocalanaesthesiaismoreeffectivethanlonger-actinglocal anaesthesia,thisoccurrencemayexplainwhytheketoprofenand TAcombinationdidnotappeartohaveanenhancedanalgesic effect. Importantinformationispresentedinthepresentstudywith respecttotheonsetoftopicalanaesthesiafordehorningwounds. Evidenceoflocalanaesthesiamaybeseen1minpost-dehorning andtreatmentwithTA.Someindicationoflocalanaesthesia wasseeninpreviousdehorningworkat1min,thoughitwasnot statisticallysigni " cantwhencomparedwithcontrolanimals andcouldbeduetothedifferenttoolusedforassessment:von Freymono " laments(Espinoza etal . 2013 ).Previousresearch Topicalanaesthesiafordehorningincalves AnimalProductionScience E

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onTAforcastrationandmulesinginlambsreportedevidence ofanaesthesiabetween1and3minpost-treatment,respectively (Lomax etal . 2008 , 2010 ).TAenablesafasteronsetoflocal anaesthesiathaninjectableadministration,(whichis3 – 5min foracornualnerveblockincalves)asagentsareapplied directlytoopenwoundsandmucosalsurfacesallowrapid absorptionandactionthroughexposednerveendingsand bloodvessels. Thecutskinedgeofthedehorningwoundwasfoundtobe moresensitivethantheperi-woundarea,regardlessoftreatment. Thissupportsprevious " ndingsofincreasedsensitivityofthe cutskinedgeofmulesingwoundsinlambs(Lomax etal . 2008 ). Thissensitivityislikelytobecausedbydirectinjuryto nociceptors(Meyer etal . 2005 ).Atthesiteofinjury(the dehorningwound),sensitisationofexposednerveendings leadstoanenhancedresponsefromthesenociceptors. Productionofin ! ammatorymediatorsandupregulationof proin ! ammatoryenzymes,alongwithchangesintissuepH andelectrolytecomposition(Tapper 2011 )canexplainthe signi " cantdecreaseinmeanMNTovertimeobservedinthe presentstudy.Therewasasigni " cantdecreaseinmeanMNT overtimeindicatingtheeffectofin ! ammatorymediatorson exposednerveendingsinadditiontouninjuredtissue(Meyer etal . 2005 ). TheTAformulationusedpreviouslyandinthisstudy,was modi " edfromtheoriginalformulationdesignedforthe mulesingofsheepinAustralia(Tri-Solfen,BayerAnimal Health,Pymble,NSW,Australia).Scoopdehorningwounds areusuallysigni " cantlysmallerthanmulesingwounds althoughthereisoftenincreasedhaemorrhage,particularlyif arterialbleedingoccurs(Espinoza etal . 2013 ).Aluminium chlorohydratewasincludedasavasoconstrictiveagentto promotehaemostasisandslowtherateofsystemicabsorption ofanaestheticactives,whichaidsinextendingthedurationof localanaesthesia(Lomax etal . 2013 ). Thereareseveraladvantagestothepost-operativeapplication

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n ofanaestheticactives,whichaidsinextendingthedurationof localanaesthesia(Lomax etal . 2013 ). Thereareseveraladvantagestothepost-operativeapplication oflocalanaesthetics.Inanimalproduction,routinehusbandry proceduresneedtobeperformedquicklyandef " cientlydueto timeandlabourconstraints.TAcanbeappliedeasily,hasarapid onset,andthereisnoneedfordouble-handlingofanimals. Post-operativeapplicationofTAallowsdirectcontactofthe anaestheticagents,lignocaineandbupivicaine,withdamaged tissueandnerveendings.Thisdirectcontactbetweenthesource ofpainandthepainrelievingactivesimproveef " cacyandspeed ofpainalleviation.Additionally,thehaemostaticandantiseptic agentsareapplieddirectlytothewoundallowingpain,blood lossandinfectiontobetreatedsimultaneously.Post-operative applicationofTAisthereforeaneffective,practicalandcost- effectiveoptionforfarmers,whichminimisestheamountof handlingrequired(Lomax etal . 2010 ). Thehighcostandimpracticalityofinjectablepainrelief methodshavepreventedthewidespreaduptakeofpainrelief bythemajorityofcattleproducers.Althoughasigni " cant amountofresearchhasfocussedontheuseofinjectedlocal anaesthesiaforamelioratingdehorningpain(Morisse etal . 1995 ;McMeekan etal . 1998 a , 1999 ;GrafandSenn 1999 ; Sutherland etal . 2002 ;StaffordandMellor 2005 ;Doherty etal . 2007 ;Stewart etal . 2009 ),thisstudyisoneofthe " rstto investigatetheuseofTA.TAhasapotentialwelfarebene " t foryoungcalvesundergoingscoopdehorning,aswellasa potentialbene " tforbeefanddairyindustriestoaddress welfareconcernswitharelativelypracticalapproach. Alldehornedcalvesdemonstratedincreasedsensitivityover time.Alimitationofthisstudyistheinabilitytocomparewith non-dehornedanimalsasnosham(positivecontrol)animals wereincludedinthestudy.Rather,areductioninpainfrom

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imitationofthisstudyistheinabilitytocomparewith non-dehornedanimalsasnosham(positivecontrol)animals wereincludedinthestudy.Rather,areductioninpainfrom thedehornedanimals(negativecontrol)withaknownpainful woundcanbeperformed.ItislikelythatthedecreaseinMNT overtimeobservedinallanimalsisduetotissuedamageandthe in ! ammatoryprocess,andnotanartefactofrepeatedtesting. Thepainfollowingdehorninghasbeenwelldocumented(Petrie etal . 1996 ;McMeekan etal . 1997 , 1998 b ;Sylvester etal . 2004 b ).Shamdehornedcalvessubjectedtomechanical stimulationwithvonFreymono " lamentsdidnotdisplay increasingsensitivitybutanunchangingresponseupto24and 75hpost-shamdehorning(Espinoza etal . 2013 ;Mintline etal . 2013 ).Whereasscoopdehornedcalvesshowedanincreased sensitivitytovonFreystimulationupto24hpost-dehorning (Espinoza etal . 2013 ).Shamdehornedcalvessubjectedto pressurealgometryshowedareductioninMNTovertime (Tapper 2011 )andthismaybeduetothereturnofsensation, orconsequence,fromtheadministrationoftwolignocaine cornualnerveblocksgiventoallanimalsthroughoutthe experimentalperiod.Repeatedmechanicalstimulationin sheephasproducedmixedresults.Healthysheepwithan algometerstrappedtotheirhindlegdidnotbecomesensitised duringthetestingperiod(Nolan etal . 1987 a , 1987 b , 1987 c ; WelshandNolan 1995 ).Anotherstudyusingahandheld algometertodeterminetheMNToftheforelimbofsheep observedadecreaseinMNTover3days(Stubsjoen etal . 2010 ).Thenumberofchosentimepointsmaybeanimportant considerationaseachsheepwastested30timesovera30-min period.Theinclusionofthisgroupinfutureworkwouldbe bene " cialtocomparesensitivityofintactanddehornedcalves. Pressurealgometryisanovelindicatorofthepaininduced bydehorningdespiteitswidespreaduseforthequanti " cation ofpainsensitivityforotherbovinediseases(Dyer etal . 2007 ; Cutler etal

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dicatorofthepaininduced bydehorningdespiteitswidespreaduseforthequanti " cation ofpainsensitivityforotherbovinediseases(Dyer etal . 2007 ; Cutler etal . 2013 ;Fitzpatrick etal . 2013 ;Dewell etal . 2014 ). Algometryisusefulfortheassessmentoflocalisedwound painandanaesthesiabutdoesnotquantifysystemicanalgesia. Algometrycanbeinvasiveasmechanicalpressureisbeingused tomeasurehyperalgesia.There-handlingandrestraintof animals,whichisrequiredforpressuretesting,canalsocause stressandpotentiallydisturbthewound. Algometrywaschosenasthesolepainassessmenttoolin thisstudyforseveralreasons.Algometryisaformofmechanical sensorytesting,whichinducesapainresponseinorderto establishmaximumnociceptivethreshold.Thisinductionof painwouldlikelyinduceacortisolresponse,thusinterfering withthemeasurementofcortisol.Inaddition,animalsshould beminimallyhandledforassessmentofcortisolsothat handlingandrestraintdonotinducestressandarisein cortisol(Hemsworth etal . 2011 ).Behaviourassessmentcould notbecombinedwithmechanicalsensorytestingasrepeated- measuresoneachanimalwasbeingperformedtodocument changingsensitivityovertime.Thisrequiredanimalstobere- handledandrestrainedatregularintervals,whichwould disturbandconfoundanybehaviourobservationssigni " cantly. F AnimalProductionScience C.A.Espinoza etal .

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Toobtainqualitydataitisvitalthatbehaviourobservationsbe unobtrusive(PlogerandYasukawa 2003 ),performedalone,and withminimaltonointerferenceorhandlingfortheobservation period.EvaluationofTAandketoprofenfordehorningpaincould beperformedusingphysiologicalandbehaviouralindicatorsbut inisolationandnotinconjunctionwithpressurealgometry. Otherlimitationstothepresentstudywouldrequirefurther examination.Infuturetrials,theinclusionofaplacebogelwould allowforeffectiveblindingtotreatment,inadditiontoevaluating awoundbarriereffectthathaspreviouslybeenobservedin sheep(Lomax etal . 2008 ).Althoughtheexperimentwas conductedinanidenticalmanneronbothdaysofthestudy, futureexperimentsshouldrepresentalltreatmentsgroupson eachdayofthestudy.Thiswouldeliminateanypotential confoundingby ‘ dayofexperiment ’ . Throughtheuseofpressurealgometry,itcanbeconcluded thatTAisabletoprovidepainreliefinunder3-month-olddairy calvesforupto5hpost-scoopdehorning.Thiswasevident throughreducedpressuresensitivityatboththecutskinedgeof thewoundandtheperi-woundarea.AcombinedeffectofTAand ketoprofenwasnotdetectedwiththemethodsusedandshouldbe furtherinvestigated.ThelowMNTobserved24hafterdehorning suggeststhatdehorningcausesprolongedpain.Extendingthe observationperiodbeyond24hwillhelpunderstandthe generationandextentofpainfollowingdehorning.TAoffersa practicalandeffectiveoptionforimprovingthewelfareofyoung calvesundergoingscoopdehorning. Acknowledgements TheauthorsgratefullyacknowledgeKimMcKeanforprovisionofcalves andequipment,BayerAnimalHealthforprovisionoftopicalanaesthetic andMarkHazeltonforpracticalassistance.ThestatisticaladviceofPeter Thomson,EvelynHallandMaryAbdelsayedisgreatlyappreciated. References Anonymous(1997)FAWCReportontheWelfareofDairyCattle.Available

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tisticaladviceofPeter Thomson,EvelynHallandMaryAbdelsayedisgreatlyappreciated. References Anonymous(1997)FAWCReportontheWelfareofDairyCattle.Available athttp://webarchive.nationalarchives.gov.uk/20121007104210/http:// www.fawc.org.uk/reports/dairycow/dcowr069.htm[Veri " ed9April 2015] Anonymous(2004 a )PrimaryIndustriesStandingCommitteeModelCode ofPracticefortheWelfareofAnimals:Cattle.Availableathttp://www. publish.csiro.au/nid/22/pid/4831.htm[Veri " ed2June2013] Anonymous(2004 b )Dehorningofcattle.Availableathttp://www.ava.com. au/policy/84-dehorning-cattle[Veri " ed2June2014] ClarkC,MendlM,JamiesonJ,ArnoneA,Waterman-PearsonA,MurrellJ (2011)Dopsychologicalandphysiologicalstressorsaltertheacutepain responsetocastrationandtaildockinginlambs? VeterinaryAnaesthesia andAnalgesia 38 ,134 – 145.doi: 10.1111/j.1467-2995.2011.00594.x CoetzeeJ(2011)Areviewofpainassessmenttechniquesand pharmacologicalapproachestopainreliefafterbovinecastration: practicalimplicationsforcattleproductionwithintheUnitedStates. AppliedAnimalBehaviourScience 135 ,192 – 213.doi: 10.1016/ j.applanim.2011.10.016 CouncilofEurope(1988)Recommendationconcerningcattle.TheStanding CommitteeoftheEuropeanConventionfortheProtectionofAnimalskept forFarmingPurposes.Availableathttp://www.coe.int/t/e/legal_affairs/ legal_co-operation/biological_safety_and_use_of_animals/farming/Rec %20cattle%20E.asp[Veri " ed9April2015] CutlerJHH,CramerG,WalterJJ,MillmanST,KeltonDF(2013) Randomizedclinicaltrialoftetracyclinehydrochloridebandageand pastetreatmentsforresolutionoflesionsandpainassociatedwith digitaldermatitisindairycattle. JournalofDairyScience 96 , 7550 – 7557.doi: 10.3168/jds.2012-6384 DewellRD,MillmanST,GouldSA,Tof ! emireKL,WhitleyRD,ParsonsRL, RoweEW,LiuF,WangC,O ’ ConnorAM(2014)Evaluatingapproaches

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,Petersson-WolfeCS,DeVriesTJ,KeltonDF, Duf " eldTF,LeslieKE(2013)Theeffectofmeloxicamonpain sensitivity,ruminationtime,andclinicalsignsindairycowswith endotoxin-inducedclinicalmastitis. JournalofDairyScience 96 , 2847 – 2856.doi: 10.3168/jds.2012-5855 GottardoF,NalonE,ContieroB,NormandoS,DalvitP,CozziG(2011) Thedehorningofdairycalves:practicesandopinionsof639farmers. JournalofDairyScience 94 ,5724 – 5734.doi: 10.3168/jds.2011-4443 GrafB,SennM(1999)Behaviouralandphysiologicalresponsesofcalves todehorningbyheatcauterizationwithorwithoutlocalanaesthesia. Appl.Anim.Behav.Sci. 62 ,153 – 171.doi: 10.1016/S0168-1591(98) 00218-4 GuatteoR,LevionnoisO,FournierD,GuemeneD,LatoucheK,LeterrierC, MormedeP,PrunierA,ServiereJ,TerlouwC,NeindreP(2012) Minimisingpaininfarmanimals:the3sapproach – ‘ suppress, substitute,soothe. Animal 6 ,1261 – 1274.doi: 10.1017/S1751731112 000262 HeinrichA,Duf " eldT,LissemoreK,MillmanS(2010)Theeffectof meloxicamonbehaviorandpainsensitivityofdairycalvesfollowing cauterydehorningwithalocalanesthetic. JournalofDairyScience 93 , 2450 – 2457.doi: 10.3168/jds.2009-2813 HemsworthPH,RiceM,KarlenMG,CallejaL,BarnettJL,NashJ,Coleman GJ(2011)Human-animalinteractionsatabattoirs:relationshipsbetween handlingandanimalstressinsheepandcattle. AppliedAnimalBehaviour Science 135 ,24 – 33.doi: 10.1016/j.applanim.2011.09.007 HuxleyJ,WhayH(2006)Currentattitudesofcattlepractitionerstopain andtheuseofanalgesicsincattle. VeterinaryRecord 159 ,662 – 668. doi: 10.1136/vr.159.20.662 LomaxS,SheilM,WindsorP(2008)Impactoftopicalanaesthesiaon painalleviationandwoundhealinginlambsaftermulesing. Australian VeterinaryJournal 86 ,159 – 168.doi: 10.1111/j.1751-0813.2008.00285.x LomaxS,DicksonH,SheilM,WindsorPA(2010)Topicalanaesthesia

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AustralianVeterinaryJournal 88 ,67 – 74.doi: 10.1111/j.1751-0813. 2009.00546.x LomaxS,SheilM,WindsorPA(2013)Durationofactionofatopical anaestheticformulationforpainmanagementofmulesinginsheep. AustralianVeterinaryJournal 91 ,160 – 167.doi: 10.1111/avj.12031 MarshallBL(1977)Bruisingincattlepresentedforslaughter. NewZealand VeterinaryJournal 25 ,83 – 86. McMeekanCM,MellorDJ,StaffordKJ,BruceRA,WardRN,GregoryNG (1997)Effectsofshallowscoopanddeepscoopdehorningonplasma cortisolconcentrationsincalves. NewZealandVeterinaryJournal 45 , 72 – 74.doi: 10.1080/00480169.1997.35994 McMeekanCM,StaffordKJ,MellorDJ,BruceRA,WardRN,GregoryNG (1998 a )Effectsofregionalanalgesiaand/oranon-steroidalanti- in ! ammatoryanalgesicontheacutecortisolresponsetodehorningin calves. ResearchinVeterinaryScience 64 ,147 – 150.doi: 10.1016/S0034- 5288(98)90010-8 McMeekanCM,MellorDJ,StaffordKJ,BruceRA,WardRN,GregoryNG (1998 b )Effectsoflocalanaesthesiaof4to8hours ’ durationontheacute cortisolresponsetoscoopdehorningincalves. AustralianVeterinary Journal 76 ,281 – 285.doi: 10.1111/j.1751-0813.1998.tb10160.x McMeekanCM,StaffordKJ,MellorDJ,BruceRA,WardRN,GregoryNG (1999)Effectsofalocalanaestheticandanon-steroidalanti-in ! ammatory analgesiconthebehaviouralresponsesofcalvestodehorning. New ZealandVeterinaryJournal 47 ,92 – 96.doi: 10.1080/00480169.1999. 36120 MeyerR,RingcampM,CampbellJ,RajaS(2005)Neuralmechanismsof hyperalgesiaaftertissueinjury. JohnsHopkinsAPLTechnicalDigest 26 , 56 – 66. MintlineEM,StewartM,RogersAR,CoxNR,VerkerkGA,StookeyJM, WebsterJR,TuckerCB(2013)Playbehaviorasanindicatorofanimal welfare:disbuddingindairycalves. AppliedAnimalBehaviourScience 144 ,22 – 30.doi: 10.1016/j.applanim.2012.12.008 MorisseJ,CotteJ,HuonnicD(1995)Effectofdehorningonbehaviourand

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sphenylbutazone,ketoprofenor adrenocorticotropichormonepriortodehorning. Researchin VeterinaryScience 73 ,115 – 123.doi: 10.1016/S0034-5288(02)00005-X SylvesterSP,MellorDJ,StaffordKJ,BruceRA,WardRN(1998) Acutecortisolresponsesofcalvestoscoopdehorningusinglocal anaesthesiaand/orcauteryofthewound. AustralianVeterinary Journal 76 ,118 – 122. SylvesterSP,StaffordKJ,MellorDJ,BruceRA,WardRN(2004 a ) Behaviouralresponsesofcalvestoamputationdehorningwithand withoutlocalanaesthesia. AustralianVeterinaryJournal 82 ,697 – 700. SylvesterSP,StaffordKJ,MellorDJ,BruceRA,WardRN(2004 b ) Behaviouralresponsesofcalvestoamputationdehorningwithand withoutlocalanaesthesia. AustralianVeterinaryJournal 82 ,697 – 700. doi: 10.1111/j.1751-0813.2004.tb12162.x TapperK(2011) ‘ Aninvestigationofpressurealgometryandthermal sensitivitytestsforassessingpainassociatedwithasowlameness modelandcalfdisbudding. ’ (IowaStateUniversity:Ames,IA) VowlesB(1976)Bruisingofcarcassescostsusmillions. Journalof Agriculture,Victoria 74 ,388 – 392. WelshEM,NolanAM(1995)Effectof ! unixinmeglumineonthe thresholdstomechanicalstimulationinhealthyandlamesheep. ResearchinVeterinaryScience 58 ,61 – 66.doi: 10.1016/0034-5288(95) 90090-X H AnimalProductionScience C.A.Espinoza etal . www.publish.csiro.au/journals/an