McCarthy et al 2016
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EffectofatopicalanaestheticformulationonthecortisolresponsetosurgicalcastrationofunweanedbeefcalvesD.McCarthy†,S.Lomax,P.A.WindsorandP.J.WhiteFacultyofVeterinaryScience,TheUniversityofSydney,NSW2006,Australia(Received21October2014;Accepted1July2015)Impracticalityandcostofexistingpainmanagementstrategiesduringsurgicalcastrationofbeefcattlehavelimitedtheirwidespreadimplementationon-farm.Afarmer-appliedtopicalanaestheticformulation,originallydevelopedandusedcommerciallytomitigatethepainofmulesinginlambs,wasinvestigatedforitspotentialuseformanagingpaininsurgicallycastratedcalves.Thisformulationcontainedlidocaine,bupivacaine,adrenalinandcetrimide.Inthisstudy,24Angusbullcalveswererandomlyallocatedto(1)surgicalcastration(C,n=8),(2)surgicalcastrationwiththepost-operativeapplicationoftopicalanaesthetic(CTA,n=8)and(3)shamcastration/control(CON,n=8).Theexperimentwasconductedover2days,withtreatmentgroupsevenlyrepresentedacrosseachday.Calveswerehabituatedtohandlingbeforetheexperimentandbloodsampleswerecollectedforplasmacortisolmeasurementatde nedtimeperiodsbefore,atandposttreatment,(at−0.5,0h,then+0.5,1,1.5,2,4and6h).Therewasasigni canteffectoftimeoncortisolconcentrationsacrossalltreatmentgroups(P<0.01),withlowestconcentrationsat−0.5and6handpeakconcentrationat0.5hbeingsigni cantlyhigherthanthecortisolresponseat0h.Theeffectoftreatmentwasnotsigni cant(P=0.077),however,therewasatrendforCONcalvestodisplaylowercortisolconcentrationsthanCandCTAcalvesandCTAcalvestodisplaylowercortisolconcentrationsthanCcalves.Themeanareaunderthecurve(AUC)ofCONcalveswassigni cantlylowerthanthoseofCandCTAcalves(P=0.04),however,therewasnosigni cantdifferencebetweentheAUCsofCTAandCcalves.Immediateapplicationoftopicalanaestheticaftersurgicalcastrationdidnotsigni cantlyreduceplasmacortisolconcentrations.However,thetrendforCTAcalvestodisplaylowercortisolconcentrationsthanCcalveswarrantsfurtherinvestigationintotheuseofTAforpainreliefofsurgicallycastratedbeefcalves.Keywords:castration,cattle,cortisol,topicalanaesthetic,painmanagementImplicationsThisstudyinvestigatedtheuseofatopicalanaesthetic(TA)formulationforpost-operativepainreliefofcastratedcalves,
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icalanaesthetic,painmanagementImplicationsThisstudyinvestigatedtheuseofatopicalanaesthetic(TA)formulationforpost-operativepainreliefofcastratedcalves, whichoffersapracticaloptionforproducerstoprovidepainreliefon-farm.Inthisstudy,TAhadnosigni canteffectonthecortisolresponsetosurgicalcastrationofunweaned Anguscalves.However,therewasatrendforcalvestreated withTAtohavelowercortisolconcentrationsthanuntreated castratedcalvesatsometimepointsaftercastration.No conclusionscanbedrawnfromthecurrentstudyregarding theeffectivenessofTAtoamelioratepainduringcastration andfurtherresearchisrequired.IntroductionCastrationofmalecalvesisanimportantmanagementpracticeroutinelyperformedinbeefcattle(EarleyandCrowe,2002;Coetzee,2011)topreventunwantedbreeding,facilitatefattening(Puigetal.,2011)andimprovemeatquality(Coetzee,2013).Castrationalsoreducesaggressionandmountingbehavioursthatcauseinjuryandstressto othercattle(EarleyandCrowe,2002).Painandsufferinginanimalsusedinagricultureisofincreasingconcerntoconsumersoflivestockproducts(Earley andCrowe,2002).Althoughthepainofcastrationincattle hasbeenwelldocumented(Fisheretal.,1996;Coetzee,2011),theprocedureiscommonlyperformedwithoutanalgesicoranaestheticintervention.Considerableresearchonpainalleviationincastratedcalveshasbeenpublished (Fisheretal.,1996;EarleyandCrowe,2002).However,thepracticalityandcost-effectivenessofthesepainmanagement strategiesareamajorlimitationtotheirimplementation (Petherick,2005).Toaddresstheseissues,afarmer-applied ‘spray-on’topicalanaesthetic(TA)wasrecentlystudiedincalvesundergoingcastration(LomaxandWindsor,2013).ThisTAwasshowntoreducepain-relatedbehavioursandsensitivityofwoundsandsurroundingtissueforatleast24h
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incalvesundergoingcastration(LomaxandWindsor,2013).ThisTAwasshowntoreducepain-relatedbehavioursandsensitivityofwoundsandsurroundingtissueforatleast24h †E-mail:dominique.mccarthy@sydney.edu.auAnimal,page1of7©TheAnimalConsortium2015doi:10.1017/S1751731115001421 animal 1
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post-procedure(LomaxandWindsor,2013).Thisfollowedpreviousstudiesdemonstratingsuccessfulpainmanagement duringmulesingandcastrationoflambs(Lomaxetal.,2008;2010and2013).TheTA(Tri-Solfen®;BayerAnimalHealth,Pymble,NSW,Australia)usedinthesestudiesandthe currentstudyconsistsoflidocaine(40.6g/l),bupivacaine (4.2g/l),adrenalin(24.8mg/l)andcetrimide(5.0g/l).This productiscurrentlyonlyregisteredforuseinlambsunder- goingmulesing.Experimentaluseoftheproductincattleis conductedunderaresearchpermitissuedbytheAustralian pesticidesandveterinarymedicinesauthority.Assessmentofcortisolconcentrationhasbeenwidelyusedasameasureofacutedistressinanimals.Cortisolcon- centrationasameasureofpain-induceddistressisused extensivelybecausetheresponsemagnitudeandduration, asmeasuredbypeakheightandintegratedcortisolresponse, usuallyaccordwiththepredictednoxiousnessofcertain procedures(Melloretal.,2000).Measurementofcortisolhasbeenusedincattletoquantifytheeffectsofdifferentpainfulproceduressuchasdehorning(Sylvesteretal.,1998),branding(Layetal.,1992)andcastration(Fisheretal.,1996).TheaimofthisstudywastoinvestigatetheeffectofTAonthecortisolresponsetosurgicalcastrationofbeefcalvesand evaluatetheeffectivenessofTAasanoptionforpainrelief.It washypothesisedthatprovisionofTAwouldreducethe post-operativecortisolresponseofcalvesfollowingsurgicalcastration.MaterialandmethodsAnimalsandhousingAtotalof24unweaned,Angusbullcalves(3monthsold)weresourcedfromacommercialherdattheUniversityof Sydneyproperty‘Arthursleigh’(Marulan,NSW,Australia)inNovember2013.Theexperimentalprotocolwasapprovedbytheinstitutionalanimalethicscommittee(ApprovalNo. 5832).Calveshadnotpreviouslyundergoneanyhusbandry procedures.Calveswereheldwiththeirmothersfor5days beforetheexperimentina4hapaddock,adjacenttothe
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No. 5832).Calveshadnotpreviouslyundergoneanyhusbandry procedures.Calveswereheldwiththeirmothersfor5days beforetheexperimentina4hapaddock,adjacenttothe cattlehandlingfacilities.Duringthistime,cowsandcalves hadadlibitumaccesstowaterandpasture.Cowsandcalvesweresupplementedwithlucernehaydailyduetolowpasturelevelsintheholdingpaddockandtoencourageapositiveassociationwiththeexperimentalenvironment. Calveswereear-tagged2daysbeforeexperimentationand weighedusingcattlescales(Weighscaleanddatarecorder W810;GallagherGroupLtd,Hamilton,NewZealand).Calf BWrangedfrom77to102kg.Beforeear-tagging,calveshad notbeenseparatedfromtheirmothersandhadminimal exposuretohumans.Calveswerehabituatedtomovementthroughhandlingfacilitiestwicedaily(at0930and1600h)for4daysbeforeexperimentation.Cowsandcalveswere musteredintoaholdingyardandquietlymovedthroughthe racewiththeirmothers;1daybeforeexperimentation,calves wererestrainedinthecattlecrush(‘Ultimate’Crush;RPMRuralProducts,QLD,Australia)for2minbeforeexitingtherace.Restraintinvolvedmanuallycatchingthecalfinthe headbaleinastandingposition,andapplyingthesqueezeonthechutetoreducemovement.Thisemulatedhowthecalveswouldbehandledduringthetrialfortreatmentand bloodcollection.Cowsandcalveswerereleasedintothe 4hapaddockbetweenhabituationperiods.ExperimentaldesignandtreatmentsTheexperimentwasconductedover2days,withtreatment groupsevenlyrepresentedacrosseachday.Maximumdaily temperaturesforthesedayswere26.4and31.0°C.Oneachday,cowsandcalvesweremovedfromthepaddockintotheholdingyardadjacenttothecattlerace.Calveswere separatedfromtheirmothersintoaseparateholdingpen, andthecowswerereleasedbackintothepaddock.Calves weremovedthroughtherace,restrainedintheheadbale andreleasedaftertreatmentandbloodsamplingforevery timepoint.Calvesgenerallymovedthroughtheracewell.
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es weremovedthroughtherace,restrainedintheheadbale andreleasedaftertreatmentandbloodsamplingforevery timepoint.Calvesgenerallymovedthroughtheracewell. Ifrequired,calvesweregentlytouchedonthebacktoencouragemovement.Incorporatedwithintheraceweremanualslidegates,whichwereusedtoseparatecalves. Thisavoidedover- llingofracesandfacilitatedwithorderingofanimals.Calveswererandomlyassignedtooneof threetreatmentgroups:(1)shamcastration/control(CON,n=8);(2)surgicalcastration(C,n=8);and(3)surgicalcastrationwithpost-operativeapplicationofTA(CTA,n=8).Fourcalvesfromeachtreatmentgroupweretreatedeachday.Therandomorderoftreatmentswaspre-determinedbeforecalvesenteringtheraceusingtheanimals’identi cationnumbers.Allcalvesweretreatedwithina0.5htimeperiod,between1000and1030honeachofthe2experimental days.Forcastration,thesidegateofthecrushwasopened afterthecalveswererestrainedintheheadbale.Asingle, experiencedoperatormanuallyrestrainedthecalvesinastandingpositionwhileperformingtheprocedure.Calveswerecastratedstandingup,insteadofemployingtheuseof acalfcradle,toeliminateanypotentialstressassociatedwith lateralrecumbency(Tagawaetal.,1994;Pesenhoferetal.,2006).Castrationwasperformedusingatechniquethat requiredinitialtransverseexcisionofthedistalthirdofthe scrotalskinwithasterilisedknife.Eachtestiswasmanually exteriorisedbypullingfromthetunicavaginalis,andthespermaticcordcut~12cmproximaltotheheadoftheepididymis.Thismethodensuredthatalltissuethathadbeen handledorcontaminatedwasexteriorisedandremoved fromthecalf,reducingthechanceofinfectionofretracted material.ForCTAcalves,beforeremovalofeachtestis,the exposedtesticulartissuewascoatedwithTri-Solfen®byinsertingtheapplicatornozzlealongthespermaticcord
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material.ForCTAcalves,beforeremovalofeachtestis,the exposedtesticulartissuewascoatedwithTri-Solfen®byinsertingtheapplicatornozzlealongthespermaticcord insidethetunicavaginalis,intotheinguinalcavityandapplying3mlofTA.Aquantity(2to3ml)ofTAwasalsoappliedtothecutskinedgeofthescrotum.Thisapplication ofTAaimedtoprovidemaximumcoverageofexposedcut tissueandensuredtheretractedspermaticcordwascovered inapoolofanaestheticwithintheinguinalcanal.McCarthy,Lomax,WindsorandWhite2
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BloodsamplecollectionCalveswerenumbered(1to24)oneach ankwithwhiteroadmarkingspraypaintatthe rstbloodsamplecollectiontofacilitateorderingofthecalvesforeachsamplingtimepoint.Calveswerealwayssampledinthesameorder. Bloodsamples(~4to9ml)werecollectedinto9mlEDTA vacutainers(Vacuette®,WestHeidelberg,VIC,Australia)viajugularvenipuncturewithin2minofsecuringthecalvesin theheadbaleandmanuallyrestrainingtheirheads.Samples forbaselinecortisolweredrawn0.5handimmediately(0h) beforetreatment.Thereafter,samplesweredrawnat0.5,1,1.5,2,4and6hpost-treatment.The rstandlastbloodsampleswerecollectedat0930and1600h,respectively,on eachday.Calveswerekeptasagroupintheholdingyard adjacenttothecattleracebetweensamplingtimepointswheretheyhadadlibitumaccesstowaterandlucernehayandwheretheycouldseeandheartheirmothersthroughafence.Blood sampleswereplacedoniceimmediatelyaftercollectionandstoreduntilcentrifugation.Bloodsampleswerecentrifugedwithin4hofcollectionat3000r.p.m.for15min.Theplasmacomponentofthesampleswasseparatedusing1mlsterile pipettesandstoredin2mlcollectionvialsat−20°C.PlasmacortisoldeterminationPlasmacortisolconcentrationsweredeterminedusingacommerciallyavailableradio-immunoassaykit(Coat-A-Count CortisolRIA;SiemensPtyLtd,LosAngeles,CA,USA).Theinter-assayandintra-assaycoef cientsofvariationwere5.05%and5.15%,respectively.StatisticalanalysisTheprogramGenStat®(VSNInternationalLtd,HemelHempstead,UK)wasusedtoconductallstatisticalanalyses andgenerateLSDvalues.Dataoncortisolconcentrations weresubjectedtoresidualmaximumlikelihoodforrepeatedmeasures.The xedeffectsofthemodelweretreatment(CON,C,CTA),time(−0.5,0,0.5,1,1.5,2,4,6h),day(1,2)andBW(covariate).Therandomeffectofthemodelwascalf. Theintegratedcortisolresponse,orareaunderthecurve
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(CON,C,CTA),time(−0.5,0,0.5,1,1.5,2,4,6h),day(1,2)andBW(covariate).Therandomeffectofthemodelwascalf. Theintegratedcortisolresponse,orareaunderthecurve (AUC−0.5to6),wascalculatedforeachcalfandthenanalysedusingaone-wayANOVA.ThesuitabilityoftheAUC dataforparametricANOVAwastestedusingaprobability plotoftheresidualstodeterminethenormalityofthedata,andaplotofresidualsagainst ttedvaluestodeterminethehomogeneityofvariance.Forallstatistical calculations,P 0.05wasconsideredstatisticallysigni cantandP 0.05 0.1wereconsideredstatisticaltendencies.Differencesbetweenmeanswereconsideredstatistically signi cantiftheyweregreaterthanthegeneratedLSDs.ResultsTherewasnosigni cantinteractionbetweentimeandtreatment(F=0.99;d.f.=14,147;P=0.463,Table1).Therewasasigni canteffectoftimeoncortisolresponseacrossalltreatmentgroups(F=25.49;d.f.=7,161;P<0.001,Table2).Cortisolconcentrationsincreasedbetween−0.5and0.5hrelativetocastration,anddecreasedbetween1and6haftercastration.Lowestconcentrations wereat−0.5h(29.96nmol/l)and6h(23.39nmol/l)andpeakconcentrationat0.5h(77.98nmol/l)wassigni cantlyhigherthanthecortisolresponseat0h(62.77nmol/l). Thecortisolresponseat0hwassigni cantlyhigherthanthecortisolresponseat−0.5h.Therewasastatisticaltendencyfortreatmenttobesigni cant(F=2.95;d.f.=2,19;P=0.077).CON,CandCTAcalveshadmeancortisolconcentrationsof44.11±10.05,63.02±11.5and 59.03±10.68nmol/l,respectively.Therewasasigni canteffectoftreatmentonintegratedcortisolresponse (F=3.78;d.f.=2,21;P=0.04).ThemeanAUCforCONcalves(253±40.49nmol/lperh)wassigni cantlylowerthanthemeanAUCsofC(394±38.22nmol/lperh)andCTA(372±31.39nmol/lperh)calves.DiscussionTheresultsofthisstudydidnotsupportourhypothesisthatprovisionofTAwouldreducethepost-operativecortisol
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lperh)andCTA(372±31.39nmol/lperh)calves.DiscussionTheresultsofthisstudydidnotsupportourhypothesisthatprovisionofTAwouldreducethepost-operativecortisol Table1Meancortisolconcentration(nmol/l±s.e.m.)ofcalvesineachtreatmentgroupovertimeCortisolconcentration(nmol/l)CONCCTATime(h)Means.e.m.Means.e.m.Means.e.m.−0.528.57.1529.137.7332.277.56063.6712.2463.435.9961.227.00.563.6911.5784.898.0185.348.92 1589.1779.77.2078.3310.121.550.076.1780.210.1370.1110.42248.766.8779.6911.8970.028.70 428.46.1759.227.7844.438.13 611.761.5027.9211.1830.497.22CON=shamcastrated;C=castrated;CTA=castrated+topicalanaesthetic.Descriptivestatisticsarebasedonpredictedmeans(±s.e.m.).Nosigni cantinteractionwasfound(P=0.463). Table2Meancortisolconcentration(nmol/l±s.e.m.)ofallcalvesovertimeTime(h)Cortisolconcentration(nmol/l)s.e.m.−0.529.96a7.17062.77b8.470.577.98c9.91172.01bc9.271.566.79bc9.87266.16b10.42444.02d8.49623.39a7.99a,b,c,dValueswithinacolumnwithdifferentsuperscriptsdiffersigni cantlyatP<0.05.Descriptivestatisticsarebasedonpredictedmeans(±s.e.m.).Asigni canteffectwasfound(P<0.001).Topicalanaestheticforcastrationofcattle3
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responseofcalvesfollowingsurgicalcastration.Themain ndingwasthatTAhadnosigni canteffectoncortisolconcentrationsofsurgicallycastratedcalves.Thiswasthe rsttimethatthecortisolresponseofcastratedcalvestreatedwithTAhadbeenstudied.Inthisstudyweelectedtousecortisolasanindirectmeasureofpainassociatedwithcastrationasthecortisol responsetocastrationofcattle,andtheameliorationofthis responsebyuseofanaestheticsandanalgesics,hasbeen welldocumented(Coetzee,2011).However,despitethis,itis stillwidelyacceptedthatcortisolsecretionoccursinresponsetoavarietyofstressorsotherthanpain(MolonyandKent,1997).Thesestressorsincludeweaning,socialisolation, transport,socialmixing,novelty,restraintandhandling (Stilwelletal.,2010).Inaddition,thereareothervariables,suchasdiurnalchangesandindividualvariationthatin u-encecortisolconcentration.Thiscanimplicateinterpretation ofexperimentalresults(MolonyandKent,1997).Theresults ofthecurrentstudyhighlighttheresponsivenessofcortisoltofactorsotherthanpain.Whileitisidealtocombinemultiplephysiological,neuroendocrineandbehavioural measurestoreducetheimpactofnon-painfulfactorson results,thisusuallyrequiresusingseparategroupsofanimals foreachmeasure.Asweonlyutilisedasinglegroupofcalves forthisstudy,ouroptionsforobtainingotherdatainaddition tocortisolconcentrationswerelimitedduetotheconstant movementandrepeatedhandlingofthecalvesforbloodsampling.Inaddition,othermeasurementscouldhavecauseddistresswhichwouldhaveaffectedourcortisol ndings.However,apreviousstudyconductedbythesameresearch group,onthesameproperty,providesinformationonthe behaviouralresponseandwoundsensitivityofcalvessub- jectedtothesametreatmentsasthoseinthecurrentstudy (LomaxandWindsor,2013).Thestudyfoundthatcalves
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he behaviouralresponseandwoundsensitivityofcalvessub- jectedtothesametreatmentsasthoseinthecurrentstudy (LomaxandWindsor,2013).Thestudyfoundthatcalves treatedwithTAexpressedsigni cantlylesspain-relatedbehaviourthanuntreatedcalvesandwithstoodgreaterpres-sureappliedtothewoundandsurroundingskinasmeasured byanelectricvonFreyanaesthesiometer(0to1000g;IITCLife Sciences,WoodlandHills,CA,USA).Therewerenosigni canttreatmentdifferenceswhenwoundsensitivitywasmeasured withavonFreymono lament(300g;BaileyInstrumentsLtd,Manchester,UK)(LomaxandWindsor,2013).Inthisstudy,anincreaseinplasmacortisolfrom−0.5to0hwasapparentforalltreatmentgroups(Tables1and2).Thismayre ectthestressofseparationfrommothers(Lobergetal.,2008),handlingthrougharace,andrestraintinaheadbale(Cookeetal.,2009)beforetreatment.Cortisolconcentrationfurtherincreasedfrom0to0.5htoreacha peak,withtherisemoreapparentinCandCTAcalvesthan CONcalves(Table1).Inaddition,theintegratedcortisol responseofCONcalveswassigni cantlylowerthanthoseofCandCTAcalves.Thesigni cantlygreaterAUCsofCandCTAcalves,alongwiththetendencyforthesecalvesto displayhigherpeakcortisolconcentrations,isindicative ofaresponsetocastrationwhichinvolvestissuedamage, stimulationofnociceptors(EarleyandCrowe,2002)and haemorrhage(GannandEgdahl,1965).Thisstudyisnotthe rstto ndanon-signi canteffectoflocallyadministeredanaestheticonthecortisolresponseof castratedcalves(Fisheretal.,1996;Websteretal.,2013).TheeffectoflidocaineHCl,acomponentofTA,hasbeenwidelyinvestigatedforitseffectsonthecortisolresponseto castrationofcalves(Coetzee,2011).Onestudyfoundthat 2%lidocaineHCl,injectedintothetestesandscrotum 15minbeforecastration,didnotreducetheintegrated cortisolresponsetosurgicalorburdizzocastrationofFriesian
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%lidocaineHCl,injectedintothetestesandscrotum 15minbeforecastration,didnotreducetheintegrated cortisolresponsetosurgicalorburdizzocastrationofFriesian calves(Fisheretal.,1996),despitesigni cantlyreducingcortisolconcentrationsfrom0.25to1h.Fisheretal.(1996)suggestedthatthiswaslikelyattributabletotheshortdurationofaction(~1h)(ReichlandQuinton,1987)of lidocaineHCl.Thissuggestionisnotsuitabletoexplainthe lackofdifferencebetweentheintegratedcortisolresponseof CTAandCcalvesinthecurrentstudy.TheTAinthecurrent studyconsistsoftheanaestheticagentbupivacaineHClin additiontolidocaineHCl.Bupivacaineisalongactinglocal anaestheticwithadurationofactionof~5to8h(Coetzee,2011).Inaddition,theadrenalinecomponentofTAhasbeensuggestedtoslowtherateofsystemicabsorptionoflido- caineandbupivacaine,therebyprolongingtheirdurationof action(Lomaxetal.,2013).Anotherstudythatmeasuredcortisolconcentrationsofsurgicallycastrateddairycalves foundthat20mlof2%lidocaineHCladministeredina subcutaneousringblockattheneckofthescrotum,just abovethetestes,didnotreducethecortisolresponsetocastration(Websteretal.,2013).Itislikelythatadministra-tionoflidocainealoneasasubcutaneousringblockwas ineffectiveatmitigatingthepainofcastration.Itwassug- gestedthattherelativelyhighdoserateandtheinjection intothetestesratherthanthespermaticcordsmayhave causedtissueirritationorin ammation.Itwasalsoproposedthatthetwistingandseveringofspermaticcordsbythe Hendersontoolmayhavestimulatednociceptorsproximaltothesiteoflidocaineinjection(Websteretal.,2013).Inthecurrentstudy,thecastrationprocedureandthemodeof anaestheticapplicationdifferstothestudybyWebsteretal.(2013).Thespermaticcordswereseveredusingaknifeafter thedistalthirdofthescrotumwasexcisedandthetestes wereexposed.TAwasappliedpostoperatively,directlyonto
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13).Thespermaticcordswereseveredusingaknifeafter thedistalthirdofthescrotumwasexcisedandthetestes wereexposed.TAwasappliedpostoperatively,directlyonto exposed,injuredtissue.Therefore,amorelikelyexplanation forthelackofdifferencebetweenCandCTAcalvesinthecurrentstudyisthatthecastrationprocedureitselfcausedtissuedamage,in ammation,stimulationofnociceptors,andhaemmorhage,allofwhichcaninduceariseincortisol (GannandEgdahl,1965;EarleyandCrowe,2002).This explanationisalsoapplicablewhencomparingtheresultsof thecurrentstudytocontrastingresultsfrompreviouslitera- ture.Insomestudies,localadministrationof2%lidocaine HClhasbeenshowntosigni cantlyreducetheacutecortisolresponsetocastrationofFriesiancalves,thoughnotcom-pletelyeliminatingit(Tingetal.,2003;Stewartetal.,2010).Inthesestudies,lidocaineHClwasinjected10(Stewartetal.,2010)or20min(Tingetal.,2003)beforecastration.Pre-operativeadministrationoflidocaineHClwould haveensuredameliorationofbothperi-operativeandacuteMcCarthy,Lomax,WindsorandWhite4
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post-operativepain.TA,beingappliedpostoperatively,hadnoeffectonperi-operativepain,whichmayinducearisein cortisol(Melloretal.,2000).Inaddition,thestudybyTingetal.(2003)employedtheburdizzomethodforcastration,whichcausesarestrictioninblood owtothetestesbeforeremoval.Thisreduceshaemorrhage(StaffordandMellor, 2005),ofwhichcortisolsecretionisaconcomitant(Gannand Egdahl,1965).Itisimportanttonotethatinthepreviouslymentionedstudies(Staffordetal.,2002;Websteretal.,2013),cortisolconcentrationsofuncastratedcalvesweresigni cantlylowerthanthoseofuntreatedcastratedcalves.Inthecurrentstudy,althoughtheintegratedcortisolresponseofCON calveswassigni cantlylowerthanthatofCandCTAcalves,therewasnosigni cantdifferencebetweenthemeancorti-solconcentrationsofanytreatmentgroup.These ndingshavebeendemonstratedpreviouslyinastudycomparing plasmaconcentrationsofsubstancePandcortisolinbeef calvesaftercastrationorsimulatedcastration(Coetzeeetal.,2008).Inthisstudy,meancortisolconcentrationsofcastra-tedanduncastratedcalveswerenotsigni cantlydifferentatanypointupto4hfollowingtheprocedure.Inaddition,the meancortisolresponseofcastratedanduncastratedcalves wassimilarregardlessofwhethercastratedcalvesvocalised ordisplayedaversivebehaviourduringtheprocedure.Similar tothecurrentstudy,Coetzeeetal.(2008)usedAnguscrossbredcalvesandhabituationfortheexperimentcon-sistedofrestraintinaheadbaleandaropehalterfor15to30mindailyfor5days.Itwasproposedthatnon-painful stressors,suchashandling,hadaneffectoncortisolthat wasdisproportionaltothatofthenociceptivestimulusof castration(Coetzeeetal.,2008).Non-painfulstressorsexperiencedbythecalvesinthecurrentstudyinclude separationfromtheirmothers,novelexposuretohuman
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lusof castration(Coetzeeetal.,2008).Non-painfulstressorsexperiencedbythecalvesinthecurrentstudyinclude separationfromtheirmothers,novelexposuretohuman handling,andrestraint.Otherstudieshavehabituatedcalvestointensivehandlingandholdingfacilitiesfor3weeksbeforeexperimentationcommenced(Tingetal.,2003;Stewartetal.,2010).Thisextensivehabituationreducedtheeffectofhandlingontreatmentoutcomes,resultingina signi canteffectofcastrationoncortisolresponse(Tingetal.,2003;Stewartetal.,2010).Thecalvesusedinthecurrentstudyunderwentalessintensive,shorterhabituation process.Hence,theintensityanddurationofhabituationmaynothavebeensuf cienttoeliminatethestresscausedbyhandlingandrestraintinaheadbale.Furthermore,pre- viousstudiesalsoinsertedindwellingjugularcatheters1day beforeexperimentationtofacilitateintensivebloodsampling andminimiseanimalhandling(Tingetal.,2003;Stewartetal.,2010).Calvesinoneofthesestudieswereheldinindividualpensforthedurationofthetrial.Forthatreason, manualrestraintforeachbloodsamplewaspossible(Tingetal.,2003).Intheotherstudy,bloodsampleswereonlytaken−20,−10,15and20minrelativetocastration,whichmeantthateachcalfcouldberestrained oneatatimeinasqueezechuteforthedurationofsample collection(Stewartetal.,2010).Therefore,inbothofthesestudies,accesstothecatheterdidnotrequiremovementorheadrestraintofcalves.Duetothedesignofthecurrentstudy,calvesneededtobemovedthroughtheraceandinto thecrushandrestrainedinaheadbaleinordertocollectbloodsamples,regardlessofindwellingcatheterorjugularvenipuncture.Theriskofthecathetersbeingdamaged orpulledoutbythisformofrestraintmeantthatjugular venipuncturewasamorepracticaloption.Furthermore, therearecontradictoryresultsintheliteratureontheeffects ofvenipunctureoncortisol,withsomesuggestionsthatit hasnoeffect(AlamandDobson,1986)andsomesugges-
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erearecontradictoryresultsintheliteratureontheeffects ofvenipunctureoncortisol,withsomesuggestionsthatit hasnoeffect(AlamandDobson,1986)andsomesugges- tionsthatitcausesanincreaseincortisol(VeissierandLeNeindre,1988).Hopsteretal.(1999)suggestthatjugularvenipuncturemayinduceanincreaseincortisolconcentra- tion,butitseeminglyrelatestothehandlingexperienceof cattle.Inthecurrentstudy,manualrestraintforsampling, andjugularvenipuncture,mayhaveincreasedcortisolcon- centrations.Further,inthecurrentstudy,calveshadnever experiencedseparationfromtheirmothersbeforethe experimentaldays,wheretheywereseparatedforaperiodof7to8h.Studiesinvestigatingthestressofweaninghavefoundthatseparatingcalvesfromtheirmothers(Layetal.,1998;Lobergetal.,2008;O’Loughlinetal.,2014),andadditionally,alteringnormalmilkintake(Layetal.,1998),resultsinanelevatedcortisolresponse(Layetal.,1998;Lobergetal.,2008;O’Loughlinetal.,2014).Thecalvesusedinthecurrentstudywereunweanedbeefcalvesthatbefore experimentationhadminimalexposuretohumans.Studiesreportinganeffectofcastrationoncortisol(Tingetal.,2003;Stewartetal.,2010)useddairycalves,whichundercom-mercialconditionsarepermanentlyseparatedfromtheir mothersandarti ciallyrearedbyhumanswithinhoursoftheirbirth(BudzynskaandWeary,2008).Commercialbeef productionsystemstypicallyweancalvesat~6monthsof age,hencetheperiodofseparationfrommothersin thecurrentstudylikelycauseddistressandhenceamajorcortisolresponse.TheeffectofTAonthecortisolresponsetopainfulhusbandryprocedureshasbeenexploredinotherproduction animalspecies.AstudyinvestigatingashortactingTA, andalongactingTAfoundthatbothformulationswere unsuccessfulatreducingthecortisolresponsetocastrationin piglets.TheshortactingTAcontained14%Benzaine,2%
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andalongactingTAfoundthatbothformulationswere unsuccessfulatreducingthecortisolresponsetocastrationin piglets.TheshortactingTAcontained14%Benzaine,2% Butambenand2%TetracainehydrochlorideandthelongactingTAwasthesameproductasthatusedinthecurrentstudy(Sutherlandetal.,2010).Sutherlandetal.(2010)suggeststhatasTAisappliedpostoperatively,thepain ofthecastrationprocedureitselfmayhaveovershadowed anyeffectofTAoncortisol.Itwasalsosuggestedthat theanaestheticorapplicationmethodwasinadequate (Sutherlandetal.,2010).Theselimitationscanbeappliedtothecurrentstudy.AstudyinvestigatingthepainrelievingeffectsofthesameTAformulesingandtaildockinginlambsfoundthatTAsigni cantly,yetonlymoderately,reducedthepeakcortisolresponsetotheprocedureandithadnoeffect ontheAUC(Paulletal.,2007).Paulletal.(2007)foundthatcombiningthisTAwiththenon-steroidalanti-in ammatorydrugs(NSAIDs)carprofenand unixin,resultedinagreaterTopicalanaestheticforcastrationofcattle5
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decreaseinpeakcortisolthanTAalone,aswellasasigni cantreductioninAUC.Therefore,theeffectofTAincombinationwithanNSAIDonthecortisolresponseofcalvestocastrationisworthfutureinvestigation.ConclusionInthisstudytherewasnosigni canteffectoftreatmentonthecortisolresponseofunweanedbeefcalves.Itislikelythat aninsuf cienthabituationperiod,inadditiontoseparationofcalvesfromtheirmothers,mayhavecausedanincreasein calfcortisolconcentrationsindependentofpain.Thismay havemaskedanyeffectsofTAonthepainofcastration.The tendencyforcastratedcalvestreatedwithTAtohave reducedcortisolconcentrationsatsometimepointsaftercastrationandareducedintegratedcortisolresponsecom-paredwithuntreatedcastratedcalveswarrantsfurther investigation.Futurestudiesshouldincorporatemore extensivehabituationofcalvestoreducetheimpactofstress onresults.AcknowledgementsTheauthorsgratefullyacknowledgethe nancialsupportofMeatandLivestockAustraliaandBayerAnimalHealthAustralia.Theauthorsaregratefulforthetechnicalassistance ofSteveBurgunandhisstaffatArthursleigh,Marulan,NSW, andhonoursstudentsCharissaHarrisandSamanthaFaberfrom theUniversityofSydney.TheauthorsthankKimHeasman fromtheUniversityofSydneyforassistancewithlaboratory work.StatisticaladviceprovidedbyPeterThomsonfromthe UniversityofSydneyisgreatlyappreciated.ReferencesAlamMGSandDobsonH1986.Effectofvariousveterinaryproceduresonplasmaconcentrationsofcortisol,luteinisinghormoneandprostaglandinF2alphametaboliteinthecow.VeterinaryRecord118,7–10.BudzynskaMandWearyDM2008.Weaningdistressindairycalves:effectsofalternativeweaningprocedures.AppliedAnimalBehaviourScience112,33–39.CoetzeeJF2011.Areviewofpainassessmenttechniquesandpharmacologicalapproachestopainreliefafterbovinecastration:practicalimplicationsforcattle
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