McCarthy et al. - 2016 - Effect of Topically Applied Anaesthetic Formulatio
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RESEARCHARTICLE EffectofTopicallyAppliedAnaestheticFormulationontheSensitivityofScoopDehorningWoundsinCalves DominiqueMcCarthy*,PeterAndrewWindsor,CharissaHarris,SabrinaLomax,PeterJohnWhite SchoolofLifeandEnvironmentalSciences,FacultyofVeterinaryScience,UniversityofSydney,Sydney,NSW,Australia*dominique.mccarthy@sydney.edu.au Abstract
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hite SchoolofLifeandEnvironmentalSciences,FacultyofVeterinaryScience,UniversityofSydney,Sydney,NSW,Australia*dominique.mccarthy@sydney.edu.au Abstract Thepost-operativeeffectsofthreeformulationsoftopicalanaestheticandacornualnerveblockonthesensitivityofscoopdehorningwoundsincalveswerecomparedintwotrials.InTrial1,21femaleHolsteindairycalvesaged8to24weekswererandomlyallocatedtotwogroups:(1)scoopdehorningwithapost-operativeapplicationofanoveltopicalanaestheticpowder(DTAP,n=10);and(2)scoopdehorningwithapost-operativeapplicationofanoveltopicalanaestheticethanolliquid(DTAE,n=11).InTrial2,18castratedmaleand18femaleHerefordbeefcalvesaged16to20weekswererandomlyallocatedtofourgroups:(1)scoopdehorningwithapre-operativecornualnerveblockoflignocaine(DCB,n=9);(2)scoopdehorningwithapost-operativeapplicationofthenoveltopicalanaestheticethanolliquidfromTrial1(DTAE,n=9);(3)scoopdehorningwithapost-operativeapplicationofatopicalanaestheticgel(DTAG,n=9);and(4)shamdehorning(CON,n=9).Sensitivitywasassessedbyscoringthebehaviouralresponseofcalvestostimulationofthewoundorskinattimepointsbeforeandaftertreatment.InTrial1,DTAPcalveshadagreaterprobabilityofdisplayingmoresevereresponsesthanDTAEcalvesat90and180min(P<0.001).InTrial2,at1h,DTAGcalveshadagreaterprobabilityofdisplayingmoresevereresponsesthanCONcalves.At2honwards,alldehornedcalveshadagreaterprobabilityofdisplayingmoresevereresponsesthanCONcalves(P<0.001).TherewerenodifferencesbetweentheresponsesofDCB,DTAGandDTAEcalvesatanytimepoint.Topicalanaestheticfor-mulationsresultinalmostimmediatebuttemporaryanaesthesiaofthewoundfollowingscoopdehorningincalvesandmayprovideapracticaloptionforpainreliefon-farm. Introduction
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resultinalmostimmediatebuttemporaryanaesthesiaofthewoundfollowingscoopdehorningincalvesandmayprovideapracticaloptionforpainreliefon-farm. Introduction Dehorningofcattleispainful,yetremainsacommonlyperformedprocedureinhornedbreeds[1]asitreducesinjuriesfromsocialcontact,improvessafetyforstock-persons[2],dehornedcattlerequirelessspaceduringfeedingandtransport[3]anddehorningreducesbruisetrimatslaughterbyapproximately50%[4].AsbruisingofcattlecoststheAustralian PLOSONE|DOI:10.1371/journal.pone.0163181September20,20161/13 a11111 OPENACCESSCitation:McCarthyD,WindsorPA,HarrisC,LomaxS,WhitePJ(2016)EffectofTopicallyAppliedAnaestheticFormulationontheSensitivityofScoopDehorningWoundsinCalves.PLoSONE11(9):e0163181.doi:10.1371/journal.pone.0163181Editor:FrancescoStaffieri,UniversityofBari,ITALYReceived:June7,2016Accepted:September2,2016Published:September20,2016Copyright:©2016McCarthyetal.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.DataAvailabilityStatement:AllrelevantdataarewithinthepaperanditsSupportingInformationfiles.Funding:ThisworkwasfundedbyMeatandLivestockAustralia(http://www.mla.com.au/Home).ThegrantnumberforthisfundingisP.PSH.0654.TopicalanaestheticproductsweresuppliedbyBayerAnimalHealthAustralia(https://www.bayer.com.au/en/about/profile-and-organisation/animal-health/).MeatandLivestockAustraliaandBayerAnimalHealthAustraliahadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.MeatandLivestockAustraliaapprovesthepublicationofthismanuscri pt.
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beefindustryapproximately$30millionannually[5],retentionofhornshasmajoreconomicimplications.ThenorthernbeefindustryinAustraliaisveryextensive,withaveragepropertysizereportedat23,436hafor2014–2015[6].ThemajorityofcattlebreedsinthisregionareBosIndicusorB.indicuscrossesandmostcalvesarebornwithhorns.AnimalmanagementonthesepropertiesdiffersgreatlyfromthesmallersouthernAustralianpropertiesandfromthatofmostotherbeefproducingnationsinthatcattlearehandledinfrequentlyduringtheyear.Theageatwhichbeefcalvesare‘marked’(thatis,animalsaremusteredandsubjectedtohus-bandrymanagementproceduresincludingeartagging,branding,vaccinationanddehorning)varies,butisusuallyinexcessof3.5monthsofage[1].Thehorntissueatthisstageismoredevelopedthanindairycalvesthatareusuallydehornedwithin1to3monthsofbirth.InnorthernAustralia,mostbeefcalvesaredehornedbyhornamputation,usingscoop,cuporknifedehorningtools[7].Thismethodofdehorninginvolvesremovalofthehornaswellasasmallareaofsurroundingskin[8]andhasbeenfoundtocausesignificantpainanddistress[9–11].Numerousstudieshaveshownthatacornualnerveblockusinglocalanaesthetics(LA)suchaslignocaineiseffectiveatalleviatingtheintra-operativeandacutepost-operativepaincausedbydehorning[10–13].However,theimpracticalityofinjectabledrugadministrationinthisenvironmenthaspreventedwidespreadadoptionofthistechniquebyAustralianproducers[1].TopicallyappliedLAmayprovideanalternativeoptiontoaddressthepost-operativepainofdehorningwoundsasadministrationdoesnotinvolveextrahandlingtimeorahighlevelofskill.Previousstudieshavedemonstratedtheefficacyofatopicalanaestheticformulation(Tri-Solfen1,BayerAnimalHealth,PymbleNSWAustralia)foruseduringmulesing[14],taildockingandsurgicalcastration[15]inlambsandsurgicalcastrationinbeefcalves[16].Pre-liminarystudieshaveshownthatmodifiedformulationsofTri-Solfen1appliedtodairycalvesduringscoopdehorningsignificantlyreducedthesensitivityofwoundsupto1.5h[17]and5h[18]followingtheprocedure.Theoverallaimofthepreviousstudies[17,18]andthecurrentstudy,aretoinvestigateoptionsforprovidingpainrelieftocalvesundergoingscoopdehorningthatarebotheffectiveandpracticaltouseon-farm.
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es[17,18]andthecurrentstudy,aretoinvestigateoptionsforprovidingpainrelieftocalvesundergoingscoopdehorningthatarebotheffectiveandpracticaltouseon-farm. MaterialsandMethodsAnimalsandHousing ExperimentalprotocolswereapprovedbytheAnimalEthicsCommitteeofTheUniversityofSydney(ApprovalNo.5832).Thestudyinvolvedtwotrialsonseparategroupsofcalvestoinvestigatetheeffectofthreedifferenttopicalanaestheticproductsonwoundsensitivitypostdehorning.Intrial1,calvesweresourcedfromacommercialdairy,“Schofields”,intheSouth-ernHighlands,NSW,Australia.Intrial2,calvesweresourcedfromacommercialbeefherd,“Ayrston”,intheCentralTablelandsofNSW,Australia.Attheconclusionofthetrials,calvesremainedonthepropertiesandcontinuedtobeusedascommerciallivestock.Trial1.Twenty-onefemalehornedHolstein-Friesiandairycalvesaged8to24weeksundergoingroutinedehorningwereusedinthistrial.Priortoexperimentation,calvesweremovedfromgrouppensintoaholdingpenadjacenttothecattlehandlingfacilities,wheretheyremainedforthedurationofthetrial.Thecalvesinthistrialhadbeenseparatedfromtheirmothersatbirthandhandrearedasperroutinedairyfarmpractice.Trial2.Eighteencastratedmaleand18femaleunweanedhornedHerefordbeefcalvesaged16to20weeksundergoingroutinedehorningwereusedinthistrial.Calvesweremoved TopicalAnaestheticforDehorningofCalves PLOSONE|DOI:10.1371/journal.pone.0163181September20,20162/13 CompetingInterests:TopicalanaestheticproductsweresuppliedbyBayerAnimalHealthAustralia(https://www.bayer.com.au/en/about/profile-and-organisation/animal-health/).BayerAnimalHealthAustraliaisacommercialfunderandthereforepotentiallyhascompetinginterests.ThisdoesnotalterouradherencetoPLOSONEpoliciesonsharingdataand materials.
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fromthepaddockintoaholdingpenadjacenttothecattlehandlingfacilities2daysbeforethetrial,wheretheywerehabituatedtomovementthroughhandlingfacilitiestwicedailyfor2daysbeforeexperimentation.Otherthanduringtimewhenmovedthroughthehandlingfacili-ties,thecalveshadaccesstotheirdams.Topicalanaestheticproducts.Tri-Solfen1isaregisteredandcommerciallyavailablelocalanaestheticandantisepticgelfortopicalapplicationimmediatelypostmulesingofmerinolambs.Itcontains40.6g/Llignocaine,4.2g/Lbupivacaine,5g/Lcetrimideand24.8mg/Ladrenalin.Wehavebeenstudyingtheefficacyofthisproductforpainreliefduringanumberofsurgicalinterventionsinfarmanimals[19],includingvariousmodificationsoftheoriginalformulatoenhanceadherenceandefficacyduringscoopdehorning[17,18].Trial1comparedthepracticalityandefficacyoftwonoveltopicalanaestheticagentsdevel-opedfordehorning(BayerAnimalHealth,PymbleNSWAustralia).Bothformulationscon-tained20%w/vlignocaineand4%w/vbupivacaine.Thesewerespecificallydesignedforapplicationtoamputationdehorningwoundswherehaemorrhagemayaffectabsorptionofanaestheticagents.Higherconcentrationsoflignocaineandbupivacainewereincludedinthesenovelformulationswiththeintentionofincreasingtheamountofactiveingredientscomingintocontactwiththetissuesurfaceimmediatelyuponapplication.Thefirstformula-tionusedaninertpowderbaseasacarrierinanattempttoimproveadherencetothewound.Thesecondformulationusedanethanol/waterbaseasacarrierdesignedtoevaporatefollow-ingapplication.Themosteffectiveformulationfromthistrialwasusedinthesecondtrialcom-paringthepracticalityandefficacywiththatofTri-Solfen1andacornualnerveblock(2%w/vlignocaine).Inbothtrials,practicalitywasassessedthroughobservationsofproductapplicationandeffi-cacywasevaluatedthroughassessmentofskinandwoundsensitivity. ExperimentalDesignandTreatments
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ywasassessedthroughobservationsofproductapplicationandeffi-cacywasevaluatedthroughassessmentofskinandwoundsensitivity. ExperimentalDesignandTreatments Trial1.Thetrialwasconductedover1day.Onthedayofthetrial,calvesweremovedoneatatimethroughtheraceandrestrainedinaheadbale(AustralianStockyardCo,Goul-burnNSWAustralia)fortreatmentanddatacollection.Calveswereblockedbyageandran-domlyallocatedtooneoftwotreatmentsbyuseofcomputergeneratedrandomnumbers(MicrosoftExcel2007,MicrosoftCorporation):(1)scoopdehorningwithapost-operativeapplicationofanoveltopicalanaestheticpowder(BayerAnimalHealth,PymbleNSWAus-tralia)(DTAP,n=10);and(2)scoopdehorningwithapost-operativeapplicationofanoveltopicalanaestheticethanolliquid(BayerAnimalHealth,PymbleNSWAustralia)(DTAE,n=11).Approximately5to10gofpowderwasappliedtoDTAPcalfwoundswithameasur-ingspoonandapproximately4mLoftheethanolliquidwasappliedtoDTAEcalfwoundsusingahouseholdspraybottle.Theamountofethanolliquidsprayedfromthebottlewascal-ibratedusinga3mLsyringe.Theliquidwassprayedintoacupandthesyringeusedtomea-suretheliquidanditwasdeterminedtherewas2mLofproductreleasedperspray.Theproductswereappliedimmediatelyafterdehorningsoastocompletelycoverthewoundandcutskinedge.Dehorningwasperformedbyasingle,experiencedtechnicianusingamediumsizescoopdehorningdevice(BainbridgeBarnesDehorner,TheFarmStore,Melbourne,VIC,Australia).Dehorningwasperformedbyplacingthedehorneroverthehornandpullingapartthehandlestoexcisethehornandsurroundingskin.Theprocedureofdehorningandapplyingtheethanolsprayorthepowdertookapproximately15sor30speranimal,foreachoftheseproductsrespectively.Datawascollectedimmediatelyprior(0h)totreatment,then1min,90minand180minposttreatment.Betweendatacollections,calveswerereleasedintoaholdingyard. TopicalAnaestheticforDehorningofCalves
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(0h)totreatment,then1min,90minand180minposttreatment.Betweendatacollections,calveswerereleasedintoaholdingyard. TopicalAnaestheticforDehorningofCalves PLOSONE|DOI:10.1371/journal.pone.0163181September20,20163/ 13
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Trial2.Thetrialwasconductedover2days.Oneachdayofthetrial,18calvesweremovedoneatatimethroughtheraceandrestrainedinaheadbale(AustralianStockyardCo,GoulburnNSWAustralia)fortreatmentanddatacollection.Calveswereblockedbyage,sexanddayoftrialandrandomlyallocatedtooneoffourtreatmentsbyuseofcomputergeneratedrandomnumbers(MicrosoftExcel2007,MicrosoftCorporation):(1)scoopdehorningwithapre-operativecornualnerveblockoflignocaine(IliumLignocaine201,TroyLaboratories,GlendenningNSWAustralia)(DCB,n=9);(2)scoopdehorningwithapost-operativeapplica-tionoftheethanolliquidfromTrial1(BayerAnimalHealth,PymbleNSWAustralia)(DTAE,n=9);(3)scoopdehorningwithapost-operativeapplicationofatopicalanaestheticgel(Tri-Solfen1,BayerAnimalHealth,PymbleNSWAustralia)(DTAG,n=9)and(4)shamdehorn-ing(CON,n=9).CalvesintheDCBgroupwereadministeredacornualnerveblock15minpriortodehorning.An18Gneedlewasinsertedtoadepthof1cmimmediatelybehindthetemporalridgeatapointmidwaybetweenthelateralcanthusoftheeyeandthebaseofthehorn[20,21].Lignocaine(5mL)wasinjectedintothetissueinthevicinityofthecornualnerveofeachhornandanaesthesiaofthehornareawasconfirmedbythepinpricktestwithan18Gneedleimmediatelypriortodehorning.ForcalvesintheDTAEandDTAGgroups,approxi-mately4mLofproductwasappliedtoeachwoundimmediatelyafterdehorningsoastocompletelycoverthewoundandcutskinedge.DehorningwasperformedasdescribedforTrial1.TheethanolsprayandthegelwereappliedusinghouseholdspraybottlesandtheamountappliedperspraywascalibratedasdescribedforTrial1.Theprocedureofdehorningandapplyingtheethanolliquidorthegeltookapproximately15speranimal.Shamdehorningwasperformedbyplacingthedehorneroverthehornbudandapplyinglightpressuretothesurroundingskin,withoutexcisinganytissue.Datawascollectedimmediatelyprior(0h)totreatment,then1h,2h,4hand6hposttreatment.Betweendatacollections,calveswerereleasedintoaholdingyard.
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inganytissue.Datawascollectedimmediatelyprior(0h)totreatment,then1h,2h,4hand6hposttreatment.Betweendatacollections,calveswerereleasedintoaholdingyard. AssessmentofSkinandWoundSensitivity Nociceptiveandanti-nociceptiveresponseswerenotedinbothtrials.MechanicalstimulationofthehornorwoundwasperformedusingvonFreymonofilaments(Touch-Test1SensoryEvaluators,NorthCoastMedicalandRehabilitationProducts,CAUSA).Thiswasperformedattwositesontheimmediateedgeofthehornbaseorwound(Area1)andtwositesontheskinsurroundingthehornorwound(Area2)(Fig1).Area2siteswere2cmfromtheedgeofthehornbaseorwound.VonFreymonofilamentsarecalibratedtobendatapre-determinedpressure.A75g/f(lighttouch)and300g/f(pain)monofilamentwereusedtodetermineallo-dyniaandhyperalgesia,respectively.Side(leftorrighthorn)andsitewererandomisedforeachrepeatedmeasure.Calveswereblindfoldedduringmeasurementtoeliminatevisualstimuliandreducestressandconsequentstrugglingbehaviours.Sensitivitywasassessedbyscoringthebehaviouralresponsesofthecalvestomechanicalstimulationonanumericalratingscaleof0to3adaptedfromEspinozaetal.[17]whereby:0=noresponse;1=mildresponseincludingminorwithdrawalreflexsuchasaslightheadmovementoranearflick;2=moderateresponseincludingpartialwithdrawalreflexsuchaspartialheadrotation;and3=severeresponseincludingfullwithdrawalreflexsuchasfullheadjerkorrotation. StatisticalAnalysis Samplesizecalculationwasbasedonthefollowingassumptions:thecornualnerveblockandshamtreatmentswillhaveresponsescoresof0or1,wherethetopicalanaestheticgroupswillhavemedianscores1or2,withanassumedstandarddeviation(SD)of1.8.Usingtheequation(a+b) 2 x2(SD) 2 /(mean1-mean2) 2 andassumingaType1error(a)of5%andaType2error TopicalAnaestheticforDehorningofCalves PLOSONE|DOI:10.1371/journal.pone.0163181September20,20164/ 13
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(b)of80%,8.13animalspergroupwouldberequired.Thereforeaminimumof9animalspergroupwereincludedtoallowforerrorinestimatesofthemeansandSD.Alldatawasanalysedusingordinallogisticregression(OLR)inASReml13.0statisticalsoftware(VSNInternationalLtd,HemelHempsteadUK).ForTrials1and2,thefixedeffectsoftheOLRmodelwereTreat-mentxTime,AreaandvonFrey.InTrial1,Calfwasincludedasarandomeffect.InTrial2,Calf,Dayoftrial,AgeandSexwereincludedasrandomeffects.DataispresentedascumulativeoddsratioswiththestatisticalprobabilitiesofcalvesineachtreatmentgroupdisplayingresponsescoreY=0,1,2and3.Forallstatisticalcalculations,Pvalues 0.05wereconsideredstatisticallysignificant. Results Therewerenoadverseclinicaleffectsregisteredforanyoftheanimalsfollowingtreatment. Trial1 TherewasasignificantTimexTreatmentinteraction(P<0.001)(Fig2).Allcalveshadagreaterprobabilityofdisplayingamoresevereresponseat90minthanat1min.DTAPcalvesalsohadagreaterprobabilityofdisplayingamoresevereresponseat180minthanat90min.DTAPcalvesweremorelikelytodisplaymoresevereresponsesthanDTAEcalvesat90and180min. Fig1.SitesSubjectedtoSensoryTesting.Greystarsrepresentsitesontheedgeofthehornbaseorwound(Area1)andblackstarsrepresentsitesontheskinsurroundingthehornorwound(Area2).doi:10.1371/journal.pone.0163181.g001 TopicalAnaestheticforDehorningofCalves PLOSONE|DOI:10.1371/journal.pone.0163181September20,20165/ 13
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TherewasasignificanteffectofArea(P<0.001)(Fig3).CalveshadagreaterprobabilityofdisplayingmoresevereresponsesforArea1thanforArea2.TherewasnosignificanteffectofvonFrey(P=0.916).Applicationoftheethanolliquidviaaspraybottlewaseasierandquickerthanapplicationofthepowderviaaspoon,withlesswastageofproduct.Thepowderwasdifficulttoapplydirectlytothedehornedareaanditwasnotedthatwastagewasanissue,particularlyinwindyconditions,raisinghealthandsafetyconcernsfortheoperatorwithpotentialinhalationofthepowderduringapplication. Trial2 TherewasasignificantTimexTreatmentinteraction(P<0.001)(Fig4).DCBcalveshadanincreasingprobabilityofdisplayingmoresevereresponsesateachtimepointfrom1honwards.DTAGcalveshadanincreasingprobabilityofdisplayingmoresevereresponsesateachtimepointfrom0to2h.DTAEcalveshadanincreasingprobabilityofdisplayingmoresevereresponsesateachtimepointfrom0to4h.Priortotreatment(0h),therewerenodiffer-encesbetweenanytreatmentgroups.At1h,DTAGcalvesweremorelikelytodisplayamoresevereresponsethanCONcalves.From2honwards,alldehornedcalvesweremorelikelytodisplaymoresevereresponsesthanCONcalves.TherewasasignificanteffectofArea(P<0.001)(Fig5).CalveshadagreaterprobabilityofdisplayingmoresevereresponsesforArea1thanforArea2. Fig2.ProbabilityofcalvesfromTrial1ineachtreatmentgroupdisplayingresponses(Y;0=noresponse,1=mild,2=moderate,3=severe)atdifferenttimepoints.ResultscombinetheeffectofbothvonFreymonofilamentsandallsitestested.(DTAE=scoopdehornedandtreatmentwithtopicalanaestheticethanolspray;andDTAP=scoopdehornedandtreatmentwithtopicalanaestheticpowder).a-bWithineachtimepoint,treatmentgroupsnotsharingacommonletteraresignificantlydifferent(P<0.05).A-CWithineachtreatment,timepointsnotsharingacommonletteraresignificantlydifferent(P<0.05).doi:10.1371/journal.pone.0163181.g002
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antlydifferent(P<0.05).A-CWithineachtreatment,timepointsnotsharingacommonletteraresignificantlydifferent(P<0.05).doi:10.1371/journal.pone.0163181.g002 TopicalAnaestheticforDehorningofCalves PLOSONE|DOI:10.1371/journal.pone.0163181September20,20166/ 13
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TherewasasignificanteffectofvonFrey(P=0.007)(Fig6).Calveshadagreaterprobabilityofdisplayingmoresevereresponsestothe300g/fvonFreythantothe75g/fvonFrey.Administrationofthecornualnerveblocktookmoretimeandrequiredmoreskillthanapplicationoftheethanolliquidandthegelviaaspraybottle. Discussion AmputationdehorningiswidelyusedintheAustralianbeefindustry,causinganopenwoundandhaemorrhage,pluspainanddistresstocalves[1,8].Currently,therearenocommerciallyavailable,farmer-appliedanaestheticoranalgesicoptionsforpainmanagementfordehorninginAustralia.Whileacornualnerveblockhasbeenshowntoeffectivelyminimiseacutepainassociatedwiththeprocedure[9,10],ithaslimitationsforuseinanextensivesetting[1].ThecurrentstudyextendedpreviousstudiesontheefficacyofmodifiedformulationsofTri-Sol-fen1fordehorningincalves[17,18],comparingtheeffectsofthreeformulationsoftopicalanaestheticandacornualnerveblockonwoundsensitivityinscoopdehornedcalves.Firstly,theeffectsoftwonoveltopicalanaestheticsdesignedforuseonscoopdehorningwoundswerecompared,toassesswhichformulationwasmoreeffectiveatprovidingwoundanaesthesia.Theeaseofapplicationwasalsoobservedtoevaluatethepracticalityoftheseprod-uctsforuseinafarmsetting.Thesenovelformulationsweredesignedtoimproveabsorptionofanaestheticagentsinthepresenceofarterialhaemorrhageresultingfromthescoopdehorn-ingprocedure[8].Thisissuewasidentifiedinapreviousstudyinvestigatingtheuseofthe Fig3.ProbabilityofcalvesfromTrial1displayingresponses(Y;0=noresponse,1=mild,2=moderate,3=severe)tostimulationofdifferentAreas.ResultscombinetheeffectofbothvonFreymonofilaments,bothtreatmentgroupsandalltimepoints.doi:10.1371/journal.pone.0163181.g003 TopicalAnaestheticforDehorningofCalves PLOSONE|DOI:10.1371/journal.pone.0163181September20,20167/ 13
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topicalanaestheticgelforsurgicaltail-dockingwoundsinlambs[15].Inthisstudyitwasnotedthatarterialbleedingassociatedwithsurgicaltailremovalmayhavepreventedeffectiveadher-enceoftheproducttothewound,resultinginreducedefficacy[15].Althoughmodifiedformu-lationsofTri-Solfen1haveshownsomeefficacyforscoopdehorningofcattle[17,18],compromisedadherenceofagelproducttothewoundmaystillbeanissue[17].Hencetheinvestigationofthepowderandethanol/waterbasecarriersoftopicalanaestheticinthisstudy.Trial1demonstratedthattheethanolspraywasmoreeffectivethanthepowder,asshownbygreateranti-nociceptiveresponsestowoundstimulationat90and180minposttreatment.Responsesofallcalvesincreasedinseverityfrom1to90minafterdehorning,indicatingheightenedsensitivityofthewound.WoundsensitivitycontinuedtoincreaseinDTAPcalvesfrom90to180min,suggestingawaningeffectofthepowderafter90mincomparedtotheeth-anolspray.Theefficacyandpracticalityoftheethanolspray,atopicalanaestheticgelandacornualnerveblockwerethencomparedinTrial2.Shamdehornedcalveshadthegreatestprobabilityofanti-nociceptiveresponses(score0)tostimulationatalltimepoints,indicatinganabsenceofpainorhypersensitivityintheintacttissue.Theincreaseinresponseseverityseeninalldehornedcalvesovertimedemonstratedahyperalgesicprogressionassociatedwiththepainescalationresponseofskinincisionsoropenwounds[14,22].Therewasnochangein
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dehornedcalvesovertimedemonstratedahyperalgesicprogressionassociatedwiththepainescalationresponseofskinincisionsoropenwounds[14,22].Therewasnochangein Fig4.ProbabilityofcalvesfromTrial2ineachtreatmentgroupdisplayingresponses(Y;0=noresponse,1=mild,2=moderate,3=severe)atdifferenttimepoints.ResultscombinetheeffectofbothvonFreymonofilamentsandallsitestested.(DCB=scoopdehornedandtreatmentwithcornualnerveblock;DTAG=scoopdehornedandtreatmentwithatopicalanaestheticgel;DTAE=scoopdehornedandtreatmentwithatopicalanestheticethanolspray;andCON=shamdehorned).a–cWithineachtimepoint,treatmentgroupsnotsharingacommonletteraresignificantlydifferent(P<0.05).A-DWithineachtreatment,timepointsnotsharingacommonletteraresignificantlydifferent(P<0.05).doi:10.1371/journal.pone.0163181.g004 TopicalAnaestheticforDehorningofCalves PLOSONE|DOI:10.1371/journal.pone.0163181September20,20168/ 13
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sensitivityfrombeforetreatmentto1hposttreatmentinDCBcalves,suggestingeffectivelocalanaesthesia.From2honwards,responseseverityofDCBcalvesincreasedateachtimepoint,indicatingdiminishingefficacy.Similarresponseshavebeenreportedinpreviousworkinvesti-gatingtheefficacyofalignocainecornualnerveblockoncortisol[10]andbehaviouralresponses[9]ofdehornedcalves.TheresponsescoreseverityofDTAGandDTAEcalvesincreasedupto2and4h,respectively,howeverdidnotchangeupto6h.Thissuggestsadelayedanaestheticeffectforthespray-onformulationswhichpersistedlongerthanthecor-nualnerveblock.Theethanolspraycontainedamuchhigherconcentrationofanaestheticagentscomparedtotheothertreatments.Thiscouldexplaintheextendeddurationofanaes-thesiacomparedtothecornualnerveblock.Inaddition,topicalapplicationoftheanaestheticagentsmayhaveimpactedtherateofabsorption,assuggestedinpreviousstudies[16,23,24].Extendeddurationoftopicallignocaineappliedtoburnwoundsinhumanshasbeenreported[23].Itwassuggestedthatthegradualabsorptionofthelignocainefromacreambaseresultedinextendedanaesthesia.Prolongedefficacyofthetopicalanaestheticgelformulationupto24hposttreatmenthasbeenobservedinmulesedsheep[24]andcastratedcalves[16].Thevaso-constrictivepropertiesofadrenalineintheformulationmaycontributetoslowingtherateofsystemicabsorptionoftheanaestheticagents,thereforeconcentrationatthewoundsiteispro-tracted.Additionally,theeffectofawoundbarriercreatedbythegelbaseissuggestedtoatten-uatepain,possiblybycoveringdamagednerveendingsandprotectingthewoundfromexposuretotheenvironmentandstimulation[24].Extensionoftheobservationperiodbeyond6hshouldbeconsideredinfuturestudiesonthedurationofefficacyoftopicalanaestheticfordehorningwounds.
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stimulation[24].Extensionoftheobservationperiodbeyond6hshouldbeconsideredinfuturestudiesonthedurationofefficacyoftopicalanaestheticfordehorningwounds. Fig5.ProbabilityofcalvesfromTrial2displayingresponses(Y;0=noresponse,1=mild,2=moderate,3=severe)tostimulationofdifferentAreas.ResultscombinetheeffectofbothvonFreymonofilaments,alltreatmentgroupsandalltimepoints.doi:10.1371/journal.pone.0163181.g005 TopicalAnaestheticforDehorningofCalves PLOSONE|DOI:10.1371/journal.pone.0163181September20,20169/ 13
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TherewerenotreatmentdifferencesinresponsesofDCB,DTAGorDTAEcalvesatanytimepoint,suggestingthatthepost-operativeefficacyofallanaesthetictreatmentswassimilarupto6h.DCBandDTAEcalvesrespondedsimilarlytoCONcalvesat1hposttreatment,indicatingeffectivelocalanaesthesiaatthistime.DTAGcalvestendedtohavemoremildandmoderateresponsescomparedtoCONcalvesatthistimepointwhichagaincouldbeattributedtoaslowerrateofabsorption,asmentionedpreviously[16,24].Therewereveryfewsevereresponsescoresdisplayedbyanydehornedcalvesatalltimepoints,particularlyat1hand2hposttreatment(Fig4),suggestingananaestheticeffect.Alter-nativelythiscouldindicatethatthe300g/fwasnotelicitinganoxiouspainstimulus,resultinginlesssevereresponses.InTrial2,althoughtherewasagreaterprobabilityofcalveshavingmoresevereresponsestostimulationwiththe300g/fvonFreythanthe75g/f,thiswasonlymarginal(Fig6).Conclusionsfromthisstudyarelimitedbythelackofacomparisontoanuntreateddehornedgroupofcalves,omittedduetowelfareconcernsforsuchanimals.However,thecomparableresultsofthepost-operativespray-onformulationstotheinjectedLAinthecor-nualnerveblockdemonstratesefficacyoftheseproducts[9,10,12].VonFreystimulationofbothArea1and2producednociceptiveresponsesfromcalvesinbothTrials1and2.TheeffectofAreaisareflectionofprimaryandsecondaryhyperalgesia, Fig6.ProbabilityofcalvesfromTrial2displayingresponses(Y;0=noresponse,1=mild,2=moderate,3=severe)tostimulationwithdifferentvonFreymonofilaments.ResultscombinetheeffectofbothAreas,alltreatmentgroupsandalltimepoints.doi:10.1371/journal.pone.0163181.g006 TopicalAnaestheticforDehorningofCalves PLOSONE|DOI:10.1371/journal.pone.0163181September20,201610/ 13
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withstimulationofArea1(woundsite)elicitinggreaterseverityofresponsefromcalvesthanthatofArea2(uncutsurroundingtissue)withinthe180min(Trial1)and6h(Trial2)obser-vationperiods.Primaryhyperalgesiadevelopsatthesiteofinjuryduetosensitisednociceptors.Secondaryhyperalgesiadevelopsinthetissuesurroundingthesiteofinjuryandisduetocen-tralsensitisation[25].Secondaryhyperalgesiaisaconsequenceofprimaryhyperalgesiaandthereforetendstodevelopataslowerrate[16]whenaccumulationofinflammatorymediators,initiatedfromwoundinjury,resultsindepolarisationofterminalnerveendingsandexcitationofnociceptors.Theinflammatorymediatorstaketimetoaccumulateandapainresponseisonlyinitiatedwhenanexcitationthresholdisreached,resultingingradualordelayeddevelop-mentofsecondaryhyperalgesia[26].Thisisthefirsttimethatthesecurrentformulationsoftopicalanaesthetichavebeeninvesti-gatedforuseonwoundsofdehornedcalvesandcomparedtoacornualnerveblockforpost-operativepainrelief.Theresultsofthisstudywarrantfurtherinvestigationintothepainreliev-ingeffectsoftopicalanaestheticforcalvesundergoingscoopdehorningasitspost-operativeeaseofadministrationwassuperiortoacornualnerveblockandapparentefficacyindesensi-tisingdehorningwoundswascomparabletothatofalignocainecornualnerveblock. SupportingInformation S1File.Experimentaldataset.(XLSX) Acknowledgments TheauthorsaregratefulforthetechnicalassistanceofGregSchofieldandChrisCrokerandtheirstaffat“Schofields”,Avoca,NSWand“Ayrston”,Golspie,NSW.Statisticaladvicepro-videdbyPeterThomsonandEvelynHallfromtheUniversityofSydneyisgreatlyappreciated. AuthorContributions
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ca,NSWand“Ayrston”,Golspie,NSW.Statisticaladvicepro-videdbyPeterThomsonandEvelynHallfromtheUniversityofSydneyisgreatlyappreciated. AuthorContributions Conceptualization:DMPAWSLPJW.Formalanalysis:DMCHSL.Fundingacquisition:PAWPJW.Investigation:DMCHSLPJW.Methodology:DMSLPJW.Projectadministration:DMSLPJW.Supervision:DMSLPJW.Validation:DMSLPJW.Visualization:DMCHSL.Writing–originaldraft:DMCH.Writing–review&editing:DMPAWCHSLPJW. References 1.PetherickJC.Animalwelfareissuesassociatedwithextensivelivestockproduction:Thenorthernaus-tralianbeefcattleindustry.ApplAnimBehavSci.2005;92:211–234. TopicalAnaestheticforDehorningofCalves PLOSONE|DOI:10.1371/journal.pone.0163181September20,201611/ 13
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dsorPA.Durationofactionofatopicalanaestheticformulationforpainmanage-mentofmulesinginsheep.AustVetJ.2013;91:160–167.doi:10.1111/avj.12031PMID:23521101 TopicalAnaestheticforDehorningofCalves PLOSONE|DOI:10.1371/journal.pone.0163181September20,201612/ 13
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25.MeyerRA,RingcampM,CampbellJN,RajaSN.NeuralMechanismsofhyperalgesiaaftertissueinjury.JohnsHopkinsAPLTechDig.2005;26:56–66.26.GregoryNG.Pain.Physiologyandbehaviourofanimalsuffering.Oxford,UK:BlackwellScience;2004. TopicalAnaestheticforDehorningofCalves PLOSONE|DOI:10.1371/journal.pone.0163181September20,201613/ 13