Ferrer et al_2020_Animals

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ArticleImpactofaTopicalAnaesthesiaWoundManagementFormulationonPain,InflammationandReductionofSecondaryInfectionsafterTailDockinginLambsLuisMiguelFerrer1,DeliaLacasta1,* ,AuroraOrtín1 ,JuanJoséRamos1,MaríaTeresaTejedor2 ,MartaBorobia1,MaríaPérez1,EnriqueCastells3,MartaRuizdeArcaute1,HéctorRuiz1andPeterAndrewWindsor4 1AnimalPathologyDepartment,InstitutoAgroalimentariodeAragón-IA2,VeterinaryFacultyofZaragoza,UniversidaddeZaragoza-CITA,C/MiguelServet177,50013Zaragoza,Spain;lmferrer@unizar.es(L.M.F.);aortin@unizar.es(A.O.);jjramos@unizar.es(J.J.R.);mborobia@unizar.es(M.B.);mariaperezb3@hotmail.com(M.P.);martarda@unizar.es(M.R.d.A.);hectorruiz353@gmail.com(H.R.)2Anatomy,EmbryologyandAnimalGeneticsDepartment,VeterinaryFacultyofZaragoza,CIBERCV,UniversidaddeZaragoza-IIS,C/MiguelServet177,50013Zaragoza,Spain;ttejedor@unizar.es3CentroClínicoVeterinario,C/MadreGenovevaTorresMorales8,50006Zaragoza,Spain;kikevet39@gmail.com4SydneySchoolofVeterinaryScience,TheUniversityofSydney,Camden,NSW2570,Australia;peter.windsor@sydney.edu.au*Correspondence:dlacasta@unizar.es;Tel.:+34-609676727Received:29June2020;Accepted:22July2020;Published:24July2020

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Australia;peter.windsor@sydney.edu.au*Correspondence:dlacasta@unizar.es;Tel.:+34-609676727Received:29June2020;Accepted:22July2020;Published:24July2020 SimpleSummary:Taildockingisroutinelyconductedinsheephusbandryaroundtheworld,oftenwithoutanaesthesiaoranalgesiadespiterecognitionthatitisapainfulprocess.Severalmethodsareused,althoughtheCouncilofEuropestatesthatsurgicalmethodswithanaesthesiashouldbeusedtodockthetail,withtheserecommendationscopiedintonationallegislationbymostEuropeancountries.However,nostudieshavebeendocumentedcomparingtheuseofgeneralanaesthesiaandstitching.Thisstudycompareddi erentsurgicalapproacheswithandwithoutgeneralanaesthesia,andwithandwithoutsurgicalstitching,plusexaminedthee cacyofapplyingacommerciallyavailablewoundgelspraycontainingtopicalanaestheticsandanantiseptic,immediatelyontailamputation.Resultsdemonstratedthatlambsdisplaymorepainwhenthesurgeryincludedstitchingandthesewoundsbecamemorereadilyinfected,potentiallyrequiringantibiotictherapy.Asthegenerationofpotentialantimicrobialresistanceanddrugresiduesissuesinfood-producinganimalsfromover-useofantibioticsisanemergingonehealthconsideration,theavailabilityofatopicalanaestheticformulationthatprovidespainreliefandcontainsanantisepticthatreducessecondaryinfectionsandhastenwoundhealing,isofpotentialimportanceforimprovinghusbandryproceduressuchastaildocking.Abstract:Weexaminedseveralproceduresforsurgicaltaildocking;withandwithoutgeneralanaesthesia(GA),includingtheuseofatopicalwoundgelformulationtoprovidepainrelief(PR)andimprovehealingaftersurgery,containinglocalanaestheticslignocaineandbupivacaine,withcetrimideandadrenalin.Forty-fourlambswererecruitedintofourequalcohorts:GroupsAandC,thetailwasexcisedwithascalpelwithoutanaesthesiaorstitches;GroupsBandD,thetailwassurgicallyexcisedandstitchedunderGA;GroupsCandDwoundswereimmediatelysprayedwithPR.BehaviouralobservationsidentifiedthatGroupsAandCdisplayedsignificantlylesspain-relatedbehavioursthanGroupsBandDshortlyaftertheprocedure,especiallyiftreatedwithPR.Similarly,themeanofdayswhenanimalsshowednosignsofwoundinfectionwaslongerinthegroupsnotundergoingstitching.Finally,treatmentwithPRappearedtoreducethecortisolresponseandavoidedtheelevationofserumamyloidAinlambswherethetailwasexcisedwithoutgeneralAnimals2020,10,1255;doi:10.3390/ani10081255www.mdpi.com/journal/animals

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Animals2020,10,12552of14anaesthesia.Inconclusion,surgicaltail-dockingwithoutGAbutwherewoundsareimmediatelysprayedwithPR,appearsasana ordableandmorewelfare-appropriatemethodforconductingtaildockinginlambs.Keywords:lambs;taildocking;painresponse;woundinfection;cortisol;serumamyloidA

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a ordableandmorewelfare-appropriatemethodforconductingtaildockinginlambs.Keywords:lambs;taildocking;painresponse;woundinfection;cortisol;serumamyloidA 1.IntroductionAmputationofthetailinlivestockortaildocking,isaroutinehusbandryprocedureinsheepproductionworldwideforpreventativemanagementofmyiasis[1–3]andimprovedfertility[4].Althoughnumerousstudieshavedemonstratedthattaildockingcausesacutepaininlambs[5–7],itremainsacommonprocedure,oftenperformedwithoutpainrelief.Despitejustificationforsuchaversivehusbandryprocedurestomanageanimalhealthandproductionissues,thereisagrowingconcernforthewelfareofanimalsundergoingtheseprocedures,withdemandstheyceaseoratleastincludepainrelief[8].Taildockingpracticesvarybetweenregionsandcountries,mostcommonlyinvolvingeitherrapidamputationbysurgicalexcision,ordelayedischaemicremovalbyapplicationofarubberring,despiteclearevidencethatitcausesextremepain.IntheUK,itwasestimatedthat90%offarmersremovetails,withuseofringsmostcommon(86%),followedbysurgical(3%)andothermethods(2%)[7,9].Theacuteischaemiainthetaildistaltotherubberringleadstolocalisedtissuenecrosisthat,afteranextendedperiod,eventuallycausessloughing.Taildockingbysurgeryinvolvesseveringthetailusingasharpknifeorscalpel,orahotknifeordockingirontocauterisethewound[10,11].Theseprocedureshavemostlybeenperformedbyfarmersortheircontractorswithoutanalgesiaoranaesthesiaastheuseofanaesthesiausuallyrequirescostlyparticipationorsupervisionofaveterinariananduntilrecently,therehavebeennopainreliefproductsavailableforusebyfarmers[8,10].TheCouncilofEurope(CoE),in1992[12],statedinArticle30thatmutilationsarenotpermitted,exceptfortaildocking(aslongassu cientlengthofthetailislefttocoveranusandvulva).Itissaidthatrubberringsshouldbeavoided,andsurgicalmethodswithanaesthesiashouldbeused.TheCoErecommendationsarecopiedintonationallegislationbymostEuropeancountries,andthereareonlythreecountriesthatbanlambdockinginEurope:Lithuania,Finland,andSweden.Withthegrowingdemandforhumanemanagementofproductionanimals,thereisasocietalexpectationthatanysu eringfromsuchaversiveproceduresisminimised.Thereisanurgentneedforpracticalanda ordableapproachestotaildockingwithpainreliefsuitableforusebyfarmers,thatreducesanimalsu ering.Astheevaluationofpainresponsesisdi cult,especiallyinlambs,mainlysincebehavioursthatreflectpainarenotdisplayedasintensivelyasinotherspecies[13],behaviouralandphysiologicalmethodsmaybeusedtoevaluatepost-proceduralresponsesfollowingtaildocking.Globalratingsofpainhavebeenusedtoassessanimalpaininclinicalsituations[14,15],withseveralratingmethodsdevelopedtodirectlyassesspain-relatedbehaviour[16,17].Theactivityofthehypothalamic-pituitary-adrenocortical(HPA)systemhasbeenextensivelymeasuredtoindirectlygaugethedistressoftaildockingandthee ectivenessofanalgesics[18–20].PlasmaorserumcortisolconcentrationsprovideanindicationofHPAactivityandareusefulindicesas,withincertainlimits,secretionoccursinagradedwayinresponsethatmayreflectthepresumednoxiousnessofdi erentexperiences.Theconcentrationofacutephaseproteins(APPs)changesinresponsetoinflammation,infection,trauma,andstress[21,22].Althoughknowledgeoftheacutephaseresponseislessinsmallruminantsthaninotherspecies,serumamyloidA(SAA)andhaptoglobin(HP)areconsideredthemainAPPsinthesespecies.Theycanincreaseupto10–100timesinresponsetostimulation[23],andelevatedconcentrationsinsmallruminantshavebeenreportedundercertaininfectiousconditions,experimentalinflammatorystimulus[23,24],andinresponsetotransportationstress[25].TheSAA

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Animals2020,10,12553of14responseisgenerallystrongerthanthatofHP,anditsconcentrationtendstoincreasesrapidly,whileHPincreasesataslowerrate[21].ThestudyoftheevolutionofSAAlevelsfollowingtaildockingcouldenablemeasurementofthetissuetraumaandtherecoveryoftheanimalsfollowingthedi erentproceduresused.Inthisstudy,twodi erentproceduresofsurgicaltaildocking,withandwithoutgeneralanaesthesia(GA)andwithandwithoutsurgicalstitching,wereassessed.Inaddition,theimmediateapplicationofatopical‘spray-on’woundmanagementformulationafterbothtechniqueswasassessed.Thistherapycontainsthelocalanaestheticslignocaineandbupivacaine,pluscetrimideandadrenalin,inagelmatrix(Tri-Solfen®,BayerAnimalHealth,Gordon,NSW,Australia),thatwasdevelopedinAustraliatoprovidepainrelief(PR)duringaversivehusbandryprocedures.TherapiduptakeandwidespreadadoptionofthisproductinAustralianlivestockhusbandryusehasbeendescribedasa‘painmanagementrevolution’,withincreasedtractioninotherjurisdictionsleadingtostudiesforuseinmanagingwoundsinhumans[26].Thecurrentstudyinvolvedassessmentofpain-relatedbehaviourbyanumericalratingscale(NRS),developedfromacombinationofpreviousfieldobservationsandreportsofbehaviouralchangesinresponsetomulesingand/orcastration[17].Therateofwoundhealingandtheonsetofsecondarybacterialinfectionwasassessed.Further,thestudyincludedmeasurementoftheconcentrationofserumcortisol(SC),andtheacutephaseproteinserumamyloidA.2.MaterialsandMethodsAllprocedureswerecarriedoutunderProjectLicencePI37/19approvedbytheEthicsCommitteeforAnimalExperimentsfromtheUniversityofZaragoza.ThecareanduseofanimalswereperformedaccordinglywiththeSpanishPolicyforAnimalProtectionRD53/2013,whichmeetstheEuropeanUnionDirective2010/63ontheprotectionofanimalsusedforexperimentalandotherscientificpurposes.2.1.SelectionofFarm,Animals,andTreatmentGroupsTheselectedfarmwaslocatednearZaragoza(Spain)andfollowstheprototypeoftheproductionsysteminthearea.Thisinvolvesasemi-intensivesystemforternasco-typelambproduction,wheresheeparehousedattheendofgestationandduringlactation,withlambskeptindoorsuntilslaughteratapproximatelythreemonthsofage.Forty-four25to35-day-oldRasaAragonesafemalelambswithsimilarweights(8–10kg)wererecruitedforthisstudyandrandomlydividedintofourequalcohorts(n=11).Thefourequalcohortsoflambswere:GroupA,thetailwasexcisedwithascalpelwithoutanaesthesiaorstitches;GroupB,thetailwassurgicallyexcisedandstitchedunderGA;GroupC,thetailwasremovedwithascalpelwithoutprioranaesthesiaandtreatedimmediatelywithPR;GroupD,thetailwassurgicallyexcisedandstitchedunderGAandtreatedwithPRappliedbeforesuturingofperi-woundskintoclosethewound.AsingleapplicationofthepainreliefproductTri-Solfen®wassprayedonthewounds,usingaproductapplicator(gun)providingatotaldoseof1.5mLperanimalupto10kg.Noadditionaltreatments,includingantibiotics,wereappliedtothelambsthroughouttheentirestudy.Allsurgerywasperformedonthesamedayandfollowingtheprocedure,theanimalswerereintroducedintotheirproductionlot,eachconsistingof96sheepand150lambs.Thefacilitieswerecleanedanddisinfectedbeforeeachlambingperiodandbeddingstrawwasused.2.2.TailDockingSurgicalProceduresForsurgeryundergeneralanaesthesiainGroupsBandD(n=22),dexmedetomidinewasusedaspre-anaestheticatadoseof2.5–5mg/kgofbodyweightintravenously(iv),andbuprenorphineatadoseof0.01mg/Kgiv,inoculatingpropofolatadoseof1mg/Kgivwhentheanimalshowedsignsofsedation.Thisregimenprovidedbetween5to10minofanaesthetictoconductthesurgicalamputation,performed,afterdisinfectingtheskinwithiodinesolution,byincisingwithascalpelbetweenthe68thcoccygealvertebrae,thensuturingofperi-woundskintoclosethewoundswithloosestitches.InlambsbelongingtoGroupD,PRwassprayedonthewoundswithaspraygunthatapplied

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Animals2020,10,12554of141.5mLineachapplication,justpriortosuturing.Thetimerequiredtoperformtheentireprocedurewasrecorded.Forsurgerywithoutgeneralanaesthesia,inGroupsAandC(n=22),thesurgicalamputationofthetailwasperformedusingthetraditionalmethod,consistingofproducinghaemostasiswiththepressureofthefingersproximaltothesixthcoccygealvertebrae,thendockingthetaildistaltothatlevelwithascalpel.Fingerpressurewasmaintaineduntiltherewasniloronlyminimalhaemorrhage,thewoundswerenotsutured,plusPRwassprayedonthewoundsofGroupCwithaspraygunthatapplied1.5mLineachapplication.Thetraditionalprocedurewasconductedbythefarmerwhousuallydiditonthefarm,withanadditionalpersonapplyingthetopicalPRtreatmentonthelambsthatbelongedtoGroupC.Thelambswererestrainedbythefarmerbetweenhislegsduringtheintervention.Thetimerequiredtoperformthewholeprocedurewasrecorded.2.3.AssessmentofPain-RelatedBehaviourPain-relatedbehaviouraftertaildockingwasassessedusingtheNRSdevelopedpreviously[8].AtrainedscientistblindedtotreatmentwithPR,observedthelambswithin5minfollowingtheirreleaseintothelotwiththeirmothers,whentheycouldfreelyexpresstheirnaturalbehaviour(R1),2.5h(R2)and5h(R3)aftertaildocking.ForlambsrecoveringfromGAreleaseoccurredwithin10minofcompletionofthesurgery,whereasforlambswithoutGA,releaseoccurredwithinaminutefollowingthesurgery.IndividuallambswereascribedanNRSscorebetween0and3,where:0=nopain-relatedbehaviour;1=mildlyabnormalposture,gaitorbehaviour,includingmildkyphosiswithouthyperextensionofhindlegs,ventralrecumbencywithhindlegspartiallyextendedormildsti eningofgaitwithoutovertlimpingorlegdragging;2=moderatelyabnormalposture,gaitorbehaviour,including‘statuestanding’withheaddownandprominentkyphosis,moderatesti eningorslowingofgaitorhyperextensionand/orabductionofhindlegs,ventralrecumbencywithhindlegsfullyextended;3=displayingseverelyabnormalposture,gaitorbehaviour,includingmarkedagitationwithtwistingorwrithing,highfrequencyofposturalchangefromlyingtokneelingorstanding,distressedvocalisation,lateralorprostratelying,kneeling,dogsittingortremors,shakingorlipcurling.Alltheproceduresandthesubsequentbehavioursofthelambswererecorded.2.4.WoundManagementFollowingthetaildockingprocedure,theanimalswereexamineddailyfor23days,withlesionsphotographed.Thisenabledanalysisofwoundhealingaftertaildockingandthedetectionofsecondaryinfections.Woundmanagementwasevaluatedasdaysinwhichtheanimalsshowednosignsofwoundinfection.Itwasconsideredthatawoundwasinfectedwhenyelloworgreenish-colouredsurfaceordrainingexudatewasobserved.2.5.HaematologySamplesof3mLofwholebloodweretakenfromthejugularveinthroughavacutainersystemtoperformhaematologyinalltheanimalspriortotaildocking(T0)andsevendayslater(T1).HaematologywasperformedusinganautomatichaematologicalcounterVet-ABC(DIVASA-FARMAVICS.A.,Barcelona,Spain).Measuredparameterswereleucocytes(109cells/L),erythrocytes(1012cells/L),haemoglobin(g/L),PCV(packedcellvolume;%),platelets(109cells/L),MCV(MeanCorpuscularVolume;fl),MCH(meancorpuscularhaemoglobin;pg)andMCHC(meancorpuscularhaemoglobinconcentration;g/L).2.6.SerumCortisolConcentration(SC)ForassessmentofSCfollowingthedi erenttaildockingprocedures,3mLbloodsampleswithoutanticoagulantsfromthejugularveinwerecollectedfromeachlambintoavacutainerwithoutanticoagulants,priortotaildocking(C0),andat30min(C1),5h(C2)and48h(C3)intervalspost-tailexcision.Serawereremovedandstoredat 20 CuntilSCconcentration(ng/mL)wasanalysedusing

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Animals2020,10,12555of14acompetitiveELISAassay(SalivaryCortisolELISASLV-2930,DRGDiagnostics,Marburg,Germany).Theintra-andinter-assayCVwererespectively2.6%and4.3%andthesensitivitywas0.09ng/mL.2.7.SerumAmyloidAConcentration(SAA)Inthesamplesthatcoincidedintime,thesameextractedserumwasusedtocarryoutthequantificationofSCandSAA.ForSAA,the3mLbloodsampleswithoutanticoagulantsweredesignatedas:priortotaildocking(S0);andat5h(S1);48h(S2)intervals;plus,sevendayslater(S3).Serawereobtainedandstoredat 20 CuntilanalysedtodetermineSAAconcentrations(ng/mL),usingasolid-phasesandwichELISAkit(PHASETMSerumAmyloidAAssay,TrideltaDevelopmentLtd.,Maynooth,Ireland).Theintra-andinter-assayCVwererespectively,5.0%and11.4%andthesensitivitywas0.3 g/mL.2.8.StatisticalAnalysisofResultsStatisticalanalyseswereperformedusingIBMSPSSstatisticsversion26(2019)software(IBM,Armonk,NY,USA).Shapiro-Wilk’stestwasusedtoassessnormalityofconsideredvariables.Leucocytecountandpain-relatedbehaviourwereanalysedusinganon-parametrictest(theMann–WhitneyUtestandWilcoxontest,wherenecessary).Theproportionofindividualswithleucocytosiswerecomparedamonggroupsbythechi-squaredtest.Alogarithmictransformation(log10)wasusedtonormaliseSCandSAAconcentrationsbeforestatisticalanalysis.Foreachprocedure(withorwithouttheuseofgeneralanaesthesia),atwo-waymixedANOVAwasruntounderstandthee ectsoftreatment(withorwithoutPR)andtimeonerythrocytecount,log10SCconcentration(C0toC3),andlog10SAAconcentration(S1toS4).Levene’stestandBox’sMtestwereappliedtoassessthehomogeneityofvariancesandcovariances,respectively.Whenadequate,Mauchly’stestwasappliedtotesttheassumptionofsphericityfortwo-wayinteraction.Finally,woundmanagementdatawereanalysedusingproportioncomparisonbyFisher’sexacttestandsurvivalanalysis(theKaplan–MeyermethodandBreslowtest),evaluatingthedaysuntilthewoundwasfoundinfected.ThepossibleassociationbetweenthelevelofSAAatthedi erentsamplingtimesandthewoundhealingwasinvestigatedbydeterminingthePearsoncorrelation.Ineverytest,pvalues<0.05wereconsideredstatisticallysignificant.Whensignificantdi erencesamongmorethantwogroupswerefound,pairwisecomparisonwithBonferronicorrectionwascarriedout.3.ResultsThetimerequiredtoperformthesurgeryundergeneralanaesthesiaandsubsequentstitchingofeachlambexceeded10minandthetraditionalmethodwithoutGAtooklessthanaminute.Theestimatedcostofanaestheticandthematerialnecessaryforthesurgerywas7.90euros/lambandtheeconomiccostinmaterialinthetraditionalmethodwas0.50euros/lamb.ThecostofPRperanimaltreatedwas0.50euros.Nolambmortalitywasrecordedthroughoutthestudy.3.1.AssessmentofPain-RelatedBehaviourAsdisplayedinTable1andFigure1,thegroupstaildockedbythetraditionalmethod(AandC)displayedsignificantlylesspain(p=0.004)thantheothers(BandD)shortlyafter(R1)theprocedure(1.27and0.68vs.2.36and2.04)(Table1).Inaddition,lambstreatedwithPRinthegrouptail-dockedwithoutgeneralanaesthesia(C)displayedalowerpainscorethanthoseuntreated(A)(0.68vs.1.27)(p=0.013).By2.5haftertaildocking(R2),theNRSscorestendedtodeclineandequalize.IngroupswithoutPR(AandB),themeanofthepainresponsewas1.14and1.09,whereas,ingroupstreatedwithPR(CandD),themeanwas0.86and0.81,respectively,althoughthesedi erenceswerenotfoundsignificant(p>0.05).Finally,at5h(R3)mostoftheanimals,independentofthegrouptheybelongedto,werenolongerdisplayingpainbehaviours(Table1).

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Animals2020,10,12556of14Table1.MeanandSDofpain-relatedbehaviourscore(NRS)followingtaildockingby“Surgery”(conductedundergeneralanaesthesia(GA)withstitching;GroupsBandD)or“Traditional”(conductedwithoutgeneralanaesthesiaorstitching;GroupsAandC),withorwithouttreatmentwithpainrelief(PR)(yesorno,respectively).Onlysignificantdi erenceswerefoundforR1:a,b:di erentlettersmeansignificantdi erencesbetweengroupswithinprocedure(p<0.05),A,B:di erentlettersmeansignificantdi erencesbetweenprocedures(p<0.05). TimeProcedurePainReliefTreatment(Group)NMean SD R1TraditionalNo(GroupA)111.27 0.754a,AYes(GroupC)110.68 0.462b,A SurgeryNo(GroupB)112.36 0.674a,BYes(GroupD)112.04 0.650a,B R2TraditionalNo(GroupA)111.14 0.504Yes(GroupC)110.86 0.552 SurgeryNo(GroupB)111.09 0.584Yes(GroupD)110.82 0.560 R3TraditionalNo(GroupA)110.50 0.316Yes(GroupC)110.54 0.472 SurgeryNo(GroupB)110.54 0.610Yes(GroupD)110.68 0.751 Figure1.Meanofpain-relatedbehaviourscore(NRS)inthefourGroupsA,B,C,andD,shortly(R1),2.5h(R2)and5h(R3)followingtaildocking(Errorbar:95%CI).GroupsAandC,thetailwasexcisedwithascalpelwithoutanaesthesiaorstitches;GroupsBandD,thetailwassurgicallyexcisedandstitchedunderGA;withGroupsCandDimmediatelysprayedwithPR.3.2.WoundManagementIntheperiodstudied(23days),73%(16/22)oflambsinthetwogroupsnotundergoingsurgicalstitching(AandC),and91%(20/22)oflambsinthetwogroupsundergoingsurgicalstitching(BandD),developedsignsofwoundinfection,althoughtheseproportionsdidnotdi ersignificantly(p=0.240).Themeanofdaysinwhichtheanimalsshowednosignsofwoundinfectionaftertaildockingwashigherintraditionalgroups(Table2;A:7.54andC:8.73vs.B:4.27andD:3.64).Significantdi erences

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Animals2020,10,12557of14werefoundwhencomparingthetwomethods,withandwithoutstitchingamongthosetreatedwithPR(Cvs.D;p=0.015),anditwasobservedthatgroupC,beingthoselambswhosetailwasexcisedwithoutstitchingandthewoundsweretreatedwithPR,hadthehighernumberofdayswithoutsignsofwoundinfection.Table2.MeanandSEofdaysfreeofsignsofinfection,followingtaildockingby“Surgery”(conductedundergeneralanaesthesiawithstitching;GroupsBandD)or“Traditional”(conductedwithoutgeneralanaesthesiaorstitching;GroupsAandC),withorwithouttreatmentwithPR(yesorno,respectively).A,B:di erentlettermeansignificantdi erencesbetweenprocedures(p<0.05). ProcedurePainReliefTreatment(Group)Mean SE TraditionalNo(GroupA)7.54 2.867AYes(GroupC)8.73 2.666A SurgeryNo(GroupB)4.27 1.805BYes(GroupD)3.64 1.869B

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. ProcedurePainReliefTreatment(Group)Mean SE TraditionalNo(GroupA)7.54 2.867AYes(GroupC)8.73 2.666A SurgeryNo(GroupB)4.27 1.805BYes(GroupD)3.64 1.869B 3.3.HaematologyForleucocytecounts,nosignificantdi erenceswerefoundatT0andT1betweengroups.IngroupB,onlytheleucocytecountsignificantlyincreasedinthesecondsampling(T1),sevendaysaftertheprocedure(p=0.041)(Table3).Althoughnosignificantdi erenceswerefound(p=0.553),thenumberoflambswithleukocytosisinthesecondsamplingwashigheringroupsnottreatedwithPR,AandB(A:36.4%andB:45.5%vs.C:18.2%andD:27.3%).Fortheerythrocytecount,thevaluesweresignificantlyhigherinT0thaninT1inallthecases(p<0.01)andtheerythrocytecountwaslowerwhentaildockingwascarriedoutwithoutanaesthesia(p=0.009)(Table3).Nodi erenceswerefoundinanyoftheotherhaematologicalparametersexamined.Table3.MeanandSDoftheleucocyte(WBC,109cells/L)anderythrocytecounts(RBC,1012cells/L)atT0(immediatelypriortotaildocking)andT1(sevendaysafterprocedure),where“Surgery”wastaildockingperformedundergeneralanaesthesia(GroupsBandD),“Traditional”wastaildockingconductedwithoutgeneralanaesthesia(GroupsAandC),and‘yes’or‘no’waswithorwithoutPR,treatmentapplied,respectively.a,b;di erentlettersmeansignificantdi erencesbetweenT0andT1(p<0.05).A,B;di erentlettersmeansignificantdi erencesbetweenTraditionalandSurgerygroupsforRBCinbothT0andT1(p<0.05). VariableGroupPainReliefTreatment(Group)NT0T1 Mean SDMean SD Leucocytecount(WBC,109cells/L)TraditionalNo(GroupA)119.85 2.799a10.40 2.999a Yes(GroupC)1110.14 5.358a11.18 8.253a SurgeryNo(GroupB)1110.27 3.604a12.34 3.803b Yes(GroupD)118.80 3.133a10.34 2.202a Erythrocytecount(RBC,1012cells/L)TraditionalNo(GroupA)1110.78 1.300a,A10.20 1.087b,A Yes(GroupC)1110.51 0.868a,A9.14 1.280b,A

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18.80 3.133a10.34 2.202a Erythrocytecount(RBC,1012cells/L)TraditionalNo(GroupA)1110.78 1.300a,A10.20 1.087b,A Yes(GroupC)1110.51 0.868a,A9.14 1.280b,A SurgeryNo(GroupB)1111.30 0.819a,B10.33 0.850b,B Yes(GroupD)1110.99 0.591a,B10.64 0.793b,B 3.4.SerumCortisolConcentration(SC)SCresultsaredisplayed(Figure2andTableS1).Ingroupstail-dockedundergeneralanaesthesia(BandD),SCconcentrationdidnotdi ersignificantlybetweenthegrouptreated(D)andnottreatedwithPR(B)(p=0.919),andSCconcentrationdidnotchangesignificantlyovertime(p=0.205).Ingroupstail-dockedwithoutgeneralanaesthesia(AandC),althoughnosignificantdi erenceswere

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Animals2020,10,12558of14foundforSCconcentrationbetweenthem(p=0.162),inthesetwogroups,SCpeakedat30minpost-tailremoval(C1),anddecreasedthereafter,withC1valuessignificantlyhigherthantheremainingvalues(p<0.01).Althoughstatisticallysignificantdi erenceswerenotdetectedbetweenthegroups(p=0.162),SCatC1andC2forGroupCwerelowerthanforGroupA(Figure2). Figure2.Meanoflog10cortisolinthefourGroupsA,B,C,andD,priortotaildocking(C0),andat30min(C1),5h(C2)and48h(C3)intervalspost-tailexcision(Errorbar:95%CI).GroupsAandC,thetailwasexcisedwithascalpelwithoutanaesthesiaorstitches;GroupsBandD,thetailwassurgicallyexcisedandstitchedunderGA;withGroupsCandDimmediatelysprayedwithPR.3.5.SerumAmyloidAConcentration(SAA)SAAresultsaredisplayed(Figure3andTableS2).Ingroupstail-dockedundergeneralanaesthesia(BandD),SAAincreasedfromS0toS2anddecreasedinS3,withtheS2valuesignificantlyhigherthantherestofthevalues(p<0.01).Thegrouptail-dockedwithPR(D)hadconsistentlylowerSAAlevelsthanthegroupwithoutthiswoundtreatment(B),althoughstatisticallysignificantdi erenceswerenotdeterminedbetweenthesegroups(p=0.604).Ingroupstail-dockedwithoutgeneralanaesthesia(AandC),SAAevolvedsimilarlyasafunctionoftime,withonlytheS2valuedi eringsignificantlyfromS3(p=0.003).AdetailedexaminationofdatafromGroupsAandCfoundthatSAAconcentrationinGroupAclearlyshowedamaximuminS2.TheSAAinthePRtreatedgroup(C)failedtoachievethemaximumconcentrationsatS2andweresimilartoS0andS1,decreasingbyS3,althoughstatisticallysignificantdi erenceswerenotdetectedbetweenlambstreatedandnon-treatedwithPR(p=0.913).Finally,asignificantbilateral(0.014)negativecorrelation( 0.368)wasfoundbetweenthedaysuntilthewoundwasfoundinfectedandtheSAAconcentrationatS2(48hpost-taildocking),thetimeatwhichsignificantelevationsofSAAareexpectedafteranoxiousstimulus.

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Animals2020,10,12559of14 Figure3.Meanoflog10SAAinthefourGroupsA,B,C,andD,priortotaildocking(S0),andat5h(S1)and48h(S2)intervals,plussevendayslater(S3)(Errorbar:95%CI).GroupsAandC,thetailwasexcisedwithascalpelwithoutanaesthesiaorstitches;GroupsBandD,thetailwassurgicallyexcisedandstitchedunderGA;withGroupsCandDimmediatelysprayedwithPR.4.DiscussionTaildockingisroutinelyconductedinsheephusbandryaroundtheworld,oftenwithoutanaesthesiaoranalgesiadespiterecognitionthatitisapainfulprocess[5–7].Severalmethodscanbeusedtoperformtaildocking,mostcommonlytheuseofasharpknife,scalpel,hotknifeorrubberrings[11].Allthesetechniqueshavebeendemonstratedtobepainfulforlambs.TheCouncilofEuropestatesthatsurgicalmethodswithanaesthesiashouldbeusedtodockthetail[12].Totheauthors’knowledge,nostudieshavebeendocumentedcomparingtheuseofgeneralandlocalanaesthesiaintaildockinginlambs.Thepresentstudywasdevelopedtocomparetwosurgicalapproachesappliedtotaildockingandexamineiftheuseofacommerciallyavailable‘spray-on’woundformulationcontainingtwotopicalanaesthetics(PR)canhelpalleviatepainandassistrecoveryfollowingtailamputation.Thestudycomparedsurgicalexcisionofthetailundergeneralanaesthesiaandsubsequentstitching,withthetraditionalmethod,wherethetailisexcisedwithascalpelwithoutanaesthesiaorstitching.Theresultsdemonstratedthatthelambsdisplayedmorepainwhenthesurgeryincludedstitchingandthewoundsbecomemorereadilyinfected.Thisapproachalsoinvolvescostsintimeandmoneythatdonotappeartobecompensatedbyimprovedanimalwelfare.Thetimerequiredtoperformthesurgerywiththestitchingofeachlambexceeded10min,withthelambshavingtobemanagedtwice;oncetocarryoutthesedationandlaterthesurgery.Thiscomparedtothefewsecondsrequiredtoconductthetraditionalmethod.Further,iftheprocedureisperformedinveryyounglambsandthee ectsofanaesthesiaareprolonged,maternal-filialrecognition(referredtoas‘motheringup’insomecountries)maybecompromised.Inourstudy,itwasobservedthattheanimalstaildockedundergeneralanaesthesiasu eredsignificantlymorepainduringrecoveryfromanaesthesiathanthosethatdidnothaveGA.Thislikelyreflectsthatsurgerywithstitchinginflictsseveralpainfulprocedures,including:injectionoftheintravenouspre-anaesthetic;injectionoftheintravenousanaesthetic;thetaildocking;and,finally,theapplicationofstitchesfollowingexcisionthatappearstoincreasepainduringrecovery.Further,theincreasedriskofdeathoflambsundergeneralanaesthesiaisaconsideration,

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Animals2020,10,125510of14asisthegenerationofpotentialdrugresiduesinfood-producinganimals.Incompanionanimals,generalanaesthesiaisdoneundermuchsaferconditionsthaninproductionanimals,andamortalityrateofbetween0.1to0.24%isreported[27].Inhorsesattendedinaveterinaryhospital,themortalityratefollowinggeneralanaesthesiaincreasedto1.4%[28].Althoughnomortalitieswereobservedinourstudy,thepercentagefollowinggeneralanaesthesiainlambsunderfarmconditionsislikelytoexceedthoseincompanionanimalsandhorses.Itisconsideredcriticallyimportantforthesustainabilityoflivestockproductionthatimprovedanimalwelfareoccursonfarms.Theidentificationofe caciousandeconomicallya ordableprotocolsforusebyfarmersconductingaversivelivestockhusbandryproceduresisincreasinglynecessary.Theseprotocolsshouldbesafeandsu cientlymitigatepost-proceduralpainaftertaildockingtoensurethattheimprovedanimalwelfareisclearlyvisibletofarmers,motivatingtheircontinuedinvestmentinproductsused,preferablyavoidingthecostlyneedforaveterinariantobepresent.Inourstudy,wefoundthefarmer-applied,spray-ontopicalanaestheticformulation(Tri-Solfen®)successfullymitigatedthepaindisplayedbylambsaftertaildocking.ThisproductwasdesignedinAustraliaforwoundpainmanagementinsheep,providingmaximumadhesionanddurationofe ect,actingdirectlyonexposedperipheralnervoustissue,elicitingareversibleblockoftheconductionofthenociceptivesignalsresponsibleforthesensationofpain.Theproducthasbeenshownpreviouslytobee ectivefortaildockingwithahotknife[10].Whenappliedtoopenwounds,PRe ectsrapidandprofoundlocalanaesthesia,withourresultsinagreementwithothersthathavetestedtheuseoftopicalanaestheticstomitigatepaininlambsaftertraumaticprocedures[8,10,17,18,29].Althoughtheimmediateuseoftopicalanaesthesiaafteratraumaticpracticecouldseemcontradictory,postoperativepainmaybemoree ectivelyaddressedastheactivesaredelivereddirectlytothetraumatisednervefibresandtissuesthatarethesourceofpain.Inaddition,theapplicationoftopicalanaesthesiapreventsthesubsequentpainescalationresponsepathway,consequentlyimprovingthegeneralwelfareoflambs[10].Asitcanbedi culttoquantifypaininlambs[13],moststudiesreportacombinationofapproaches.Weusedseveralmethodstoassessbothbehaviouralandphysiologicalchangesrelatedtopain.Thechosenmethodtoassessthepain-relatedbehaviourusedanumericalratingscale(NRS)thatwasdevelopedinapreviousstudy[17]andhasbeenusedinseveralsubsequentstudies[10,29].Themostaccuratepain-relatedbehaviourmeasurementsareobtainedwithdatacollectedfromthefirst60minaftertreatment[16],althoughasourstudyusedameasureat30min,acceptableaccuracymayalsobeobtainedwithshorterperiods.Further,behavioursrecordedaseventsratherthantimespentcarryingouttheactivity,areconsideredaspreferredindicatorsofpain,duetothebroadvariationinresponsetothesamestressor[30].Inourstudy,combiningdi erentactivitiesintoasingleindexcompensatestosomeextentforthisindividualvariation[16].Anadditionalobjectiveofthepresentworkwastoevaluatetheprogressionoftaildockingwoundsfollowingeitherofthetwoproceduresapplied,assessingimpactsofusingthetopicalanaestheticwoundsprayPR.Almost82%ofthestudiedlambsdevelopedsignsofwoundinfectionaftertaildocking.Thishighpercentageofinfectionslikelyreflectsthatthelambsremainedstabledthroughouttheperiodofstudy,andantibiotictreatmentwasnotappliedduringtheprocedure,despitethisbeingausualpractice.However,themeanofdaysonwhichtheanimalsshowednosignsofwoundinfectionwerehigherinthetwogroupsnotundergoingsurgicalstitching.Thissuggeststhatclosingthewoundwiththesurgicalstitchesandnottreatingtheanimalwithantibioticsinapotentiallycontaminatedenvironment,increasesinfectionrisk,delayingwoundhealing.Althoughtheuseofantibioticsisacommonpracticeaftersurgicalproceduresinanimals,thisincreasesboththecostoftheprocedureandrisksofantimicrobialresidueandresistance.Further,thepracticeisinviolationoftheantibioticusereductionpolicyaspromulgatedbytheEU.Importantly,itwasobservedthatbestresultswereobtainedinthegroupoflambswherethetailwasexcisedwithoutstitching,andthewoundsweretreatedwithPR.ThisaccordswithreportsthatPRimproveswoundhealinginaversivehusbandryproceduresconductedinAustralia,includingmulesing[17]andtaildocking[10]oflambs

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Animals2020,10,125511of14andcastrationofcalves[31].Ofnoteisthatthesestudieswereconductedinextensively-raisedanimals,andtherewasnoevidenceofdelayedwoundhealingduetohighratesofinfection.Plasmaorserumcortisolconcentrationhavebeenwidelyusedtoassesstheresponsestopainfulexperiencesinlambs[18–20].However,thereissomecontroversyontheapplicationofcortisolinstudiesofpain,consideredasanindirectmeasureofstress,notpain.Thehandlingandshort-termremovalofthelambfromthedamtocarryouttaildockingisanimportantsourceofstresstothelamb[19],andstressorssuchasrestraintorsocialisolationincreasecortisollevelsinsheep[31].Further,asserumcortisolshowsa‘ceilinge ect’thislimitsitsuseforgradingseverelypainfulprocedures[16],pluscortisolwillbeelevatedifthereishaemorrhagepresent,limitingthecomparisonofsurgicalversusnon-surgicalprocedures[32].Inourstudy,ingroupstail-dockedwithoutgeneralanaesthesia,serumcortisolconcentrationspeakedat30minpost-tailremoval,decreasingthereafter,aswidelyreportedinpreviousstudies[19,33].However,ingroupssubjectedtogeneralanaesthesia,thiscortisolpeakwasabsent,despitetheselambsdisplayingsignificantlyincreasedpain-relatedbehavioursfollowingtaildocking.Thiswasmostlikelyduetothefactthattheanimals,whenanesthetized,didnotsu erthestressoftheprocedure,buttheconsequentpainthatisdisplayed,isnotreflectedinthecortisolpeak.Thisobservationcorroboratesotherevidence,particularlyincalves,thatserumcortisolismeasuringstressfulmanagement,suchashandling,restraintandpotentiallyhaemorrhage,ratherthanprimarilythepainaftertaildocking[30,31].Theabsenceofe ectofNSAIDsontheacutecortisolresponsehasbeenobservedinotherstudies[18,34],afurtherindicationthatserumcortisolresponsesaremeasuresofstressfulexperiences,ratherthanjustpain.Inourstudy,thePRtreatedgroupwithoutgeneralanaesthesiaandsubsequentstitchinghadSCconcentrationslowerthaninthePRuntreatedgroup,althoughthesedi erenceswerenotstatisticallysignificant.TheseresultscouldimplythattreatmentwithPRdidreducethecortisolresponseelicitedafterthesurgerywithoutgeneralanaesthesia,althoughthelowstatisticalpowerduetosmallsamplesizesused,couldwellhavepreventedthesedi erencesasbeingsignificant.Thisisinaccordancewithpreviousfindingsinwhichtopicalanaestheticappliedtothecastrationwoundreducedthecortisolpeakat30min[18],andthatsmallgroupsizesarelikelytocompromisestatisticalsignificanceoftrendsinlowercortisolresponsesbetweentreatedanduntreatedgroupsofcastrated[31]anddehornedcalves[32].Thequantificationoftheacutephaseproteinscanassisttheassessmentofinfection,inflammation,trauma,tissuedamageandstressinanimals,andareappliedinordertoimprovethedetection,diagnosis,prognosisandmonitoringdi erentdiseasesandpathologicalprocessesofdomesticanimals[21,22,24].ElevatedconcentrationsofHPandSAAhavebeenreportedinsmallruminantsundercertaininfectiousconditions,experimentalinflammatorystimuli[23,24],andinresponsetotransportationstress[25].However,althoughHPisane ectivemarkerformonitoringprocessessuchastaildockingandcastrationincattle[21,35],HPlevelsremainunchangedinlambsfollowingtheseprocesses[35].Inourstudy,asignificantincreaseinSAAconcentrationwasobservedinthegroupsoflambssubmittedtosurgerywithstitchingfollowingtaildocking.TheSAAresponseinlambsisgenerallystrongerthanthatofHP,anditsconcentrationtendstoincreasemorerapidly,reachingmaximumconcentrationswithin24to48hafterstimulation,witharapidnormalisationwithinfourtosevendays,ifnofurtherstimulusoccurs[21].OurresultsareinaccordancewiththisfindingsincethehighestconcentrationofSAAinthisstudywasmeasuredat48hpost-taildocking.AriseinSAAconcentrationatthistimepoint,althoughnotsignificant,wasalsoobservedinthegrouptail-dockedwithoutgeneralanaesthesiaandnottreatedwithPR,whereas,inthePRtreatedgroup,SAAconcentrationremainedunchanged.AlthoughelevatedconcentrationsofAPPsalsooccurwithsomeconditionsnotgenerallyassociatedwithinflammation,infectionortissuedamage,suchasstress,thee ectofstressonserumconcentrationofAPPsremainscontroversialsinceitisdi culttodistinguishthee ectofstressfromthee ectoftrauma[21].Inourstudy,theincreaseinSAAconcentrationobservedislikelyaconsequenceofpainassociatedwiththeprocedureandareflectionofthetissuedamage,inflammationandparticularly,woundinfection.Groupstail-dockedwithgeneralanaesthesia,inwhichSAArisewassignificant,

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Animals2020,10,125512of14showedsignificantlyhigherpain-relatedbehaviouraftertaildockingthantheothers,withthesurgicalstitchingintheselambslikelytoberesponsibleforgreatertissuedamage,inflammationandriskofinfection.Ofinterest,thegroupnotundergoingsurgicalstitchingthatwastreatedwithPRsu eredtheleastpainandshowedunchangedSAAconcentrationat48hpost-taildocking.Further,stressdoesnotseemtobeinvolvedintheacutephaseresponseobserved,asingroupssubmittedtosurgerywithstitching,serumcortisolconcentrationsdidnotpeakat30minpost-tailremoving.TherelationshipbetweenwoundhealingandSAAinourstudydeservesconsideration,asitislikelythatwoundinfectioncouldhavecontributedtotheelevationinSAAat48hpost-taildocking.Themeanofdaysonwhichtheanimalsshowednosignsofwoundinfectionwassignificantlyshorterinthetwogroupsundergoingsurgicalstitching,whereasignificantincreaseinSAAconcentrationwasalsomeasured.Further,inthegroupoflambswherethetailwasexcisedwithoutstitching,andthewoundsweretreatedwithPR,theSAAconcentrationdidnotchangewithin48hfollowingtheprocedure,andtheseanimalsdeveloplowersignsofwoundinfection.Asignificantbilateralnegativecorrelationwasfoundbetweenthedaysuntilthewoundwasfoundinfected,andtheSAAconcentrationat48hpost-taildocking.Althoughtheintensityofassociationbetweenbothparametersismedium,theseresultsappeartoindicatethathigherSAAconcentration48haftertaildockingcoulddetectaworseevolutionofthewoundrepairprocess,duetomorerapidonsetofinfection.ThesepromisingresultsindicatethatSAAcouldbeausefulbiomarkerformonitoringacutephaseresponsesaftertaildockinginlambsandpredicttheevolutionofwoundhealing,althoughfurtherresearchisneededtocorroboratethesefindings.Thisstudysuggeststhatthemethodtraditionallyperformedbyfarmersappearstodemonstrateclearadvantagescomparedtotaildockingundergeneralanaesthesia.Importantly,theestimatedcostofPRperanimaltreatedinthisstudywasEuro0.50,comparedtoEuro7.90fortheGAprotocol.5.ConclusionsTheresultsfromthisstudyandotherreportsareencouraging,suggestingthatsurgicaltaildockingwithoutgeneralanaesthesiaandsubsequentstitchingbutwherewoundsareimmediatelysprayedwithTri-Solfen®isana ordableande caciousmethodofconductingtheprocedurebyfarmersthatimprovesanimalwelfare.Thistopicalanaesthesiawoundtreatmentformulationalleviatespainandhastenshealing,reducingsecondaryinfectionsandavoidingtheunnecessaryuseofantibioticsthatmaycontributetotherisksofdevelopingantimicrobialresistanceandresidues.SupplementaryMaterials:Thefollowingareavailableonlineathttp://www.mdpi.com/2076-2615/10/8/1255/s1,TableS1.MeanandSDoflog10cortisolpriortotaildocking(C0),andat30min(C1),5h(C2),and48h(C3)intervalspost-tailexcision,where“Surgery”wastaildockingperformedundergeneralanaesthesia(GroupsBandD),“Traditional”wastaildockingconductedwithoutgeneralanaesthesia(GroupsAandC),and‘yes’or‘no’waswithorwithoutPR,treatmentapplied,respectively.Onlysignificantdi erenceswerefoundforGroupsAandCamongtime,beingvaluesforC1significantlyhigherthantherest(p<0.05):a,bdi erentlettersmeansignificantdi erencesforA,CGroupsamongtime(p<0.05).TableS2.MeanandSDoflog10SAAatS0priortotaildocking(S0),andat5h(S1)and48h(S2)intervals,plussevendayslater(S3),where“Surgery”wastaildockingperformedundergeneralanaesthesia(GroupsBandD),“Traditional”wastaildockingconductedwithoutgeneralanaesthesia(GroupsAandD),and‘yes’or‘no’waswithorwithoutPR,treatmentapplied,respectively.Onlysignificantdi erenceswerefoundamongtime:a,b:di erentlettersmeansignificantdi erencesamongtimefortraditionalprocedure(p<0.05);A,B:di erentlettermeansignificantdi erencesamongtimeforsurgeryprocedure(p<0.05).AuthorContributions:Conceptualization,D.L.andP.A.W.;datacuration,M.T.T.;formalanalysis,A.O.andM.T.T.;fundingacquisition,P.A.W.;investigation,L.M.F.,D.L.,M.B.,M.P.,M.R.d.A.,H.R.andP.A.W.;methodology,L.M.F.,J.J.R.andE.C.;projectadministration,D.L.;resources,J.J.R.;software,M.P.andH.R.;validation,L.M.F.,A.O.andJ.J.R.;visualization,M.T.T.;writing—originaldraft,D.L.;writing—reviewandediting,L.M.F.,A.O.,M.T.T.andP.A.W.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.Funding:ThisresearchwassupportedbyprovisionofproductfromtheAustraliancompanyAnimalEthicsPtyLtd.withfinancialsupportforthetravelofseniorco-author(PW)toattendthetrialandfinancialcompensationofsomeofthecostsincurred.TheworkwasalsosupportedbytheAragónGovernmentandtheEuropeanSocialFund(A15_17R,ConstruyendoAragón2016-20).

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Animals2020,10,125513of14Acknowledgments:AuthorswouldliketoacknowledgethefarmerTomásYagüeforlettingusperformthewholestudyathisfarm.ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.Thefundershadnoroleinthedesignofthestudy,inthecollection,analyses,orinterpretationofdata,orinthewritingofthemanuscriptanddecisiontopublishtheresults.References1.French,N.P.;Wall,R.;Morgan,K.L.Lambtaildocking:Acontrolledfieldstudyofthee ectsoftailamputationonhealthandproductivity.Vet.Rec.1994,134,463–467.[CrossRef][PubMed]2.Webb-Ware,J.K.;Vizard,A.;Lean,G.R.E ectsoftailamputationandtreatmentwithandalbendazolecontrolled-releasecapsuleonthehealthandproductivityofprimelambs.Aus.Vet.J.2000,78,838–842.[CrossRef][PubMed]3.Fisher,N.W.;Gregory,N.G.Reconcilingthedi erencesbetweenthelengthatwhichlambs´tailsarecommonlydockedandanimalwelfarerecommendations.NZSAP2007,67,32–38.[CrossRef]4.Marai,I.F.M.;Bahgat,L.B.Fat-tailedsheeptraitsasa ectedbydocking.Trop.Anim.HealthProd.2003,35,351–363.[CrossRef][PubMed]5.Molony,V.;Kent,J.E.Assessmentofacutepaininfarmanimalsusingbehaviouralandphysiologicalmeasurements.J.Anim.Sci.1997,75,266–272.[CrossRef][PubMed]6.Kent,J.E.;Molony,V.;Graham,M.J.Comparisonofmethodsforthereductionofacutepainproducedbyrubberringcastrationortaildockingofweek-oldlambs.Vet.J.1998,155,39–51.[CrossRef]7.Sutherland,M.A.;Tucker,C.Thelongandshortofit:Areviewoftaildockinginfarmanimals.Appl.Anim.Behav.Sci.2011,135,179–191.[CrossRef]8.Windsor,P.A.;Lomax,S.AddressingwelfareconcernsregardingcontrolofcutaneousmyiosisinAustralia.SmallRum.Res.2013,110,165–169.[CrossRef]9.French,N.P.;Morgan,K.L.Neuromasindockedlambs’tails.Res.Vet.Sci.1992,52,389–390.[CrossRef]10.Lomax,S.;Dickson,H.;Sheil,M.;Windsor,P.A.Topicalanaesthesiaalleviatesshort-termpainofcastrationandtaildockinginlambs.Aus.Vet.J.2010,88,67–74.[CrossRef]11.Orihuela,A.;Ungerfeld,R.Taildockinginsheep(Ovisaries):Areviewontheargumentsforandagainsttheprocedure,advantages/disadvantages,methods,andnewevidencetorevisitthetopic.Livest.Sci.2019,228,136–143.[CrossRef]12.CouncilofEuropeRecommendationsConcerningSheepandtheCouncilofEuropeRecommendationConcerningGoatsBothfrom1992AdoptedbytheStandingCommitteeoftheEuropeanConventionfortheProtectionofAnimalsKeptforFarmingPurposes.1992.Availableonline:https://www.coe.int/t/e/legal_a airs/legal_co-operation/biological_safety_and_use_of_animals/farming/Rec%20sheep%20E.asp(accessedon23July2020).13.Manteca,X.;Temple,D.;Mainau,E.;Llonch,P.;FarmAnimalWelfareEducationCenter(FAWEC).Evaluacióndeldolorenelganadoovino.FarmAnimalWelfareEducationCentre(FAWEC)PainAssessmentinSheep.2017.Availableonline:https://www.fawec.org/es/fichas-tecnicas/49-ganado-ovino/237-evaluacion-dolor-ovino(accessedon15April2020).(InSpanish).14.Holton,L.L.;Scott,E.M.;Nolan,A.;Reid,J.;Welsh,E.;Flaherty,D.Comparisonofthreemethodsusedforassessmentofpainindogs.J.Am.Vet.Med.Assoc.1998,212,61–66.[PubMed]15.Thornton,P.D.;Waterman-Pearson,A.E.Quantificationofthepainanddistressresponsestocastrationinyounglambs.Res.Vet.Sci.1999,66,107–118.[CrossRef][PubMed]16.Molony,V.;Kent,J.E.;McKendrick,I.J.Validationofamethodforassessmentofanacutepaininlambs.Appl.Anim.Behav.Sci.2002,76,215–238.[CrossRef]17.Lomax,S.;Sheil,M.;Windsor,P.A.Impactoftopicalanaesthesiaonpainalleviationandwoundhealinginlambsaftermulesing.Aus.Vet.J.2008,86,159–168.[CrossRef]18.Paull,D.R.;Lee,C.;Colditz,I.G.;Atkinson,S.J.;Fisher,A.D.Thee ectofatopicalanaestheticformulation,systemicflunixinandcarprofen,singlyorincombination,oncortisolandbehaviouralresponsesofMerinolambstomulesing.Aus.Vet.J.2009,85,98–106.[CrossRef]

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Animals2020,10,125514of1419.Clark,C.;Mendl,M.;Jamieson,J.;Arnone,A.;Waterman-Pearson,A.;Murrell,J.Dopsychologicalandphysiologicalstressorsaltertheacutepainresponsetocastrationandtaildockinginlambs?Vet.Anaesth.Analg.2011,38,134–145.[CrossRef]20.Small,A.H.;Marini,D.;LeFloch,M.;Paull,D.;Lee,C.Apenstudyevaluationofbuccalmeloxicamandtopicalanaestheticatimprovingwelfareoflambsundergoingsurgicalmulesingandhotknifetaildocking.Res.Vet.Sci.2018,118,270–277.[CrossRef]21.Petersen,H.H.;Nielsen,J.P.;Heegaard,P.M.H.Applicationofacutephaseproteinmeasurementsinveterinaryclinicalchemistry.Vet.Res.2004,35,163–187.[CrossRef]22.Eckersall,P.D.Proteins,proteomics,andthedysproteinemias.InClinicaBiochemistryofDomesticAnimals,6thed.;Kaneko,J.J.,Harvey,J.W.,Bruss,M.L.,Eds.;ElsevierAcademicPress:Davis,CA,USA,2008;pp.117–155.ISBN9780123704917.23.Ceciliani,F.;Ceron,J.J.;Eckersall,P.D.;Sauerwein,H.Acutephaseproteinsinruminants.J.Proteom.2012,75,4207–4231.[CrossRef]24.Tothova,C.;Nagy,O.;Kovac,G.Acutephaseproteinsandtheiruseinthediagnosisofdiseasesinruminants:Areview.Vet.Med.2014,59,163–168.[CrossRef]25.Piccione,G.;Casella,S.;Giannetto,C.;Giudice,E.;Fazio,F.Utilityofacutephaseproteinsasbiomarkersoftransportstressinewes.SmallRum.Res.2012,107,167–171.[CrossRef]26.Roberts,C.D.;Windsor,P.A.Innovativepainmanagementsolutionsinanimalsmayprovideimprovedwoundpainreductionduringdebridementinhumans:Anopinioninformedbyveterinaryliterature.Int.WoundJ.2019,16.[CrossRef][PubMed]27.Brodbelt,D.C.;Pfei er,D.U.;Young,L.E.;Wood,J.L.N.L.Resultsoftheconfidentialenquiryintoperioperativesmallanimalfatalitiesregardingriskfactorsforanesthetic-relateddeathindogs.J.Am.Vet.Med.Assoc.2008,233,1096–1104.[CrossRef]28.Laurenza,C.;Ansart,L.;Portier,K.Riskfactorsofanesthesia-relatedmortalityandmorbidityinoneequinehospital:Aretrospectivestudyon1,161casesundergoingelectiveoremergencysurgeries.Front.Vet.Sci.2020,22,6–514.[CrossRef][PubMed]29.Lomax,S.;Sheil,M.;Windsor,P.A.Durationofactionofatopicalanaestheticformulationforpainmanagementofmulesinginsheep.Aus.Vet.J.2013,91,160–167.[CrossRef]30.Wiepkema,P.R.Developmentalaspectsofmotivatedbehaviourindomesticatedanimals.J.Anim.Sci.1987,65,1220–1227.[CrossRef]31.McCarthy,D.;Lomax,S.;Windsor,P.A.;White,P.J.E ectofatopicalanaestheticformulationonthecortisolresponsetosurgicalcastrationofunweanedbeefcalves.Animal2016,10,150–156.[CrossRef]32.Espinoza,C.;Lomax,S.;Windsor,P.A.TheE ectofTopicalAnaesthesiaontheCortisolResponsesofCalvesUndergoingDehorning.Animals2020,10,312.[CrossRef]33.Marini,D.;Colditz,I.G.;Hinch,G.;Petherick,J.C.;Lee,C.Self-administrationbyconsumptionofflunixininfeedalleviatesthepainandinflammationassociatedwithcastrationandtaildockingoflambs.Appl.Anim.Behav.Sci.2017,188,26–33.[CrossRef]34.Turner,A.;Canny,B.;Hobbs,R.J.;Bond,J.D.;Clarke,I.J.;Tilbrook,A.J.InfluenceofsexandgonadalstatusofsheeponcortisolsecretioninresponsetoACTHandoncortisolandLHsecretioninresponsetostress:Importanceofdi erentstressors.J.Endocrinol.2002,173,113–122.[CrossRef][PubMed]35.Murata,H.;Shimada,N.;Yoshioka,M.Currentresearchonacutephaseproteinsinveterinarydiagnosis:Anoverview.Vet.J.2004,168,28–40.[CrossRef]

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rossRef][PubMed]35.Murata,H.;Shimada,N.;Yoshioka,M.Currentresearchonacutephaseproteinsinveterinarydiagnosis:Anoverview.Vet.J.2004,168,28–40.[CrossRef] ©2020bytheauthors.LicenseeMDPI,Basel,Switzerland.ThisarticleisanopenaccessarticledistributedunderthetermsandconditionsoftheCreativeCommonsAttribution(CCBY)license(http://creativecommons.org/licenses/by/4.0/).