QualitativeBehaviouralAssessmentofBosindicuscattle

followingsurgicalcastration

ThisthesisissubmittedtoMurdochUniversity

forthedegreeofMasterofPhilosophyby

ThinzaVindevoghel

B.AnimalScience(Hons)(MurdochUniversity)

SchoolofVeterinaryandLifeSciences

MurdochUniversity

2016

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Declaration

Ideclarethisthesisismyownaccountofmyresearch,andcontainsasitsmaincontentwork

thathasnotpreviouslysubmittedforadegreeatanyuniversity.

ThinzaVindevoghel

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Abstract

SurgicalcastrationofcattleiscommonhoweverprovidinganalgesiaintheAustraliancattle

industry is rare. The aimsof this studywere to: 1) describe thebehaviour of cattle post-

castrationusingquantitativeandqualitativemethodologyand2)determinetheeffectiveness

ofanalgesiaforsurgicalcastration.Forty-eightBosindicus(Brahman)calvesweredividedinto

sixgroups(n=8):1)castratedwithoutanalgesia(C),2)non-castrated(NC),3)castratedwith

Meloxicampre-castration (CMpre),4)castratedwithMeloxicampost-castration (CMpost),5)

castrated with Lignocaine (CLA) and 6) castrated with Lignocaine and Meloxicam post-

castration(CLMpost).Cattlewerefilmedinthepaddockandfeederyardondays-1to13,and

wereassessedinthecrushatsurgeryandastheyexitedthecrush.Videofootagewasshown

to volunteered observers using Qualitative Behavioural Assessment (QBA). In addition,

quantitative behaviour scoring by one observer was conducted. There was good inter-

observer reliability for the behavioural expression of cattle. Results showed significant

TreatmentgroupsxDayinteractioneffects(P<0.05)forthepaddockandfeederyardcontext.

Correlations between qualitative and quantitative measures of behaviour were also

identified.

The behavioural responses of cattle varied; in Generalised Procrustes Analysis 1 (GPA 1),

cattleincastrated(C)groupinthefeederyardweredescribedasrelativelymore‘agitated’

and‘anxious’onday-1andmore‘calm’and‘relaxed’onday+1.Someofthebehaviourwas

unexpected regarding the treatment;however, thebehaviourcanbeexplainedby factors

suchastheenvironmentortheinnatecharacteristicsoflivestock.Thisstudydemonstrated

thatBosindicusbullsarelikelytobenefitfromtheadministrationofanalgesiaatcastration,

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however we recommend the importance of careful interpretation of behaviour when

assessingtheeffectsofanalgesia.

.

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TableofContents

Declaration...............................................................................................................................2

Abstract....................................................................................................................................3

TableofContents.....................................................................................................................5

ListofTables...........................................................................................................................10

TableofFigures......................................................................................................................11

Acknowledgements................................................................................................................12

1 GeneralIntroduction.......................................................................................................13

1.1 Animalwelfareandhusbandry................................................................................14

1.2 Pain..........................................................................................................................15

1.3 Analgesia..................................................................................................................17

1.4 AnimalWelfareAssessment....................................................................................17

1.4.1 Physiologicalmeasures.....................................................................................18

1.4.2 Behaviouralmeasures......................................................................................19

1.5 QualitativeBehaviouralAssessment(QBA).............................................................20

1.6 Generalaims............................................................................................................22

1.7 Thesisoutline...........................................................................................................23

2 GeneralMethodsandMaterials.....................................................................................24

2.1 Animals....................................................................................................................24

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2.2 AnalgesiaandSurgicalProcedures..........................................................................25

2.3 Painassessment.......................................................................................................26

2.4 Behaviouralmeasurements.....................................................................................27

2.4.1 CompositeBehaviourScoring(CBS).................................................................27

2.4.2 QualitativeBehaviouralAnalysis(QBA)............................................................28

2.4.3 Termgenerationsession..................................................................................29

2.4.4 Viewingsessions...............................................................................................30

2.4.5 StatisticalanalysisforQualitativeBehaviouralAnalysis...................................30

3 Applyingcompositebehaviourscoring(CBS)todeterminecastrationpainincattle.....32

3.1 Abstract...................................................................................................................32

3.2 Introduction.............................................................................................................34

3.3 Method....................................................................................................................35

3.3.1 AnimalsandQuantitativeBehaviourAnalysis..................................................35

3.4 Results.....................................................................................................................37

3.5 Discussion................................................................................................................38

3.6 Conclusion...............................................................................................................41

3.7 Appendix..................................................................................................................42

4 UsingQualitativeandQuantitativebehaviouralassessmentoncattleundergoingsurgical

castrationwithdifferentanalgesicprotocols.........................................................................48

4.1 Abstract...................................................................................................................48

4.2 Introduction.............................................................................................................50

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4.3 Methods..................................................................................................................52

4.3.1 Animalsandtreatmentgroups.........................................................................52

4.4 QualitativeBehaviouralAssessment(QBA).............................................................53

4.5 QuantitativeAnalysis...............................................................................................54

4.6 Results.....................................................................................................................55

4.6.1 QualitativeBehaviouralAssessment(QBA)......................................................55

4.7 Discussion................................................................................................................57

4.8 Conclusion...............................................................................................................61

4.9 Appendix..................................................................................................................63

5 QualitativeBehaviouralAssessmenttodistinguishsurgicallycastratedcattlefromnon-

castratedandanalgesiacontrols............................................................................................67

5.1 Abstract...................................................................................................................67

5.2 Introduction.............................................................................................................69

5.3 Methods..................................................................................................................71

5.3.1 Animalsandvideorecording............................................................................72

5.4 QualitativeBehaviouralAssessment(QBA).............................................................73

5.4.1 Observers.........................................................................................................73

5.4.2 QuantitativeAnalysis........................................................................................75

5.5 Results.....................................................................................................................75

5.5.1 QualitativeBehaviouralAssessment(QBA)......................................................75

5.5.2 Terms................................................................................................................76

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5.5.3 Treatmenteffects.............................................................................................77

5.5.4 Correlationsbetweenquantitativeandqualitativebehaviour........................78

5.6 Discussion................................................................................................................78

5.7 Conclusion...............................................................................................................82

5.8 Appendix..................................................................................................................84

6 GeneralDiscussion..........................................................................................................90

6.1 GeneralConclusion..................................................................................................94

7 Appendices......................................................................................................................96

7.1Behaviourscoringinpaddock(CompositeBehaviouralScoringchart)........................96

7.1 Post-graduatePosterDayNovember2013.............................................................97

7.2 BehaviourConference-CairnsAugust2015...........................................................99

7.3 QBAFlyer...............................................................................................................100

7.4 ParticipantInformationLetter...............................................................................101

7.5 InstructionSheet–ForQBAtermgenerationsession...........................................102

7.6 InstructionSheet–ForQBAviewingsessions.......................................................103

7.7 QBAobserverssurveypart1.................................................................................105

7.8 QBAobserversurveypart2...................................................................................113

7.9 QBAobserversurveypart3Aand3B.....................................................................116

8 References....................................................................................................................122

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ListofTables

Table2.1Samplingprotocolperday......................................................................................26

Table3.1Quantificationofbehaviouracrossthreecontexts................................................42

Table4.1Quantificationofbehaviour(a)duringcastrationand(b)postcastration.............63

Table 4.2 Termsused by observers to describe qualitative behaviour expression of calves

duringandimmediatelyaftersurgicalcastration..................................................................64

Table5.1Treatmentgroupsandlocationsusedinthestudy................................................73

Table5.2Termsusedbyobserverstodescribequalitativebehaviourexpressionofcalvesin

paddockpre-andpost-castration(SessionA)........................................................................84

Table5.3Termsusedbyobserverstodescribequalitativebehaviourexpressionofcalvesin

feederpre-andpost-castration(SessionB)............................................................................86

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TableofFigures

Figure3.1.CompositeBehaviourScoring(CBS)ofcattleinthepaddockshowing5behavioural

displaywhichhadsignificantdifferences(P=0.004).............................................................44

Figure 3.2. Castrated (C) vs.Non-castrated (NC) cattle on pre-and post-surgery (Day-1 vs.

Day+1)....................................................................................................................................45

Figure3.3.Castrated (C) vs.Castratedwith Local anaestheticandpost-surgeryMeloxicam

(CLMpost)cattleonpre-andpost-surgery(Day-1vs.Day+1)...................................................45

Figure 3.4. Castrated with pre-surgery Meloxicam (CMpre) vs. Castrated cattle with post-

surgeryMeloxicam(CMpost)onpre-andpost-surgery(Day-1vs.Day+1)...............................45

Figure3.5.Treatmentgroupcomparisonofbehaviouralstatesobservedascalvesexitedthe

crushaftercastration.............................................................................................................46

Figure3.6Treatmentgroupcomparisonofbehaviouraleventsobservedascalvesexitedthe

crushaftercastration.............................................................................................................47

Figures4.1(a-f).ComparisonofQualitativeBehaviouralExpressionduringcastration(a,b,c)

andpostcastration(d,e,f)....................................................................................................66

Figure5.1QBASessionA(paddock)–CvsNCcattleforeachGPAdimensions....................88

Figure5.2QBASessionA(paddock)–CvsCLMpostcattleforallGPAdimensions................88

Figure5.3QBASessionA(feederyard)–CvsNCcattleforallGPAdimensions...................89

Figure5.4QBASessionB(feeder)–CvsCLMpostcattleforallGPAdimensions....................89

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Acknowledgements

I am very grateful for the opportunity to undertake this study and learn from my very

supportiveandknowledgeablesupervisors,DrTeresaCollinsandA/ProfTrishFleming.My

sincerethankstoboth.ThankyoutoMeatandLivestockAustraliaforfundingthisprojectand

theteamatMurdochUniversity,SchoolofVeterinaryandLifeScienceswhoparticipatedand

assistedinthisstudy.Tothevolunteerswhoparticipatedinthestudy,thankyoutoeachof

you.TomyfamilytravellingfromabroadtolookaftermydaughterAnyasoIcouldwritethis

thesisandtomyveryunderstandinghusband,Cole,myITsupport–thankyousomuch!!I

amforevergratefulforallofyouforgivingmeyourtime.Iwouldnothavebeenabletodo

thisalone.Ithastaken3.5yearstocompletethisproject,butthisisonlythebeginningandI

Iookforwardtothefuture.

“ThankyoutotheBrahmancattleforlettingusstudythem!”

“You’renotjustsittinginthepaddockwatchingcows,you’relearningfromthemand

formingbonds”

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1 GeneralIntroduction

Castrationofmalecalves isa routinehusbandryprocedurecommonlyperformedwithout

anaesthesia or analgesia on cattle herds around the world (Lomax and Windsor, 2013).

Castrationhasbeenwell-documentedaspainful(Molonyetal.,1995;Fisheretal.,1996),but

it is considered necessary for economic, safety and management reasons (Lomax and

Windsor, 2013). There is controversy about whichmethod of castration causes the least

distressandseveralfactorscontributetoit,including,ageofanimals,andmethodsofrearing

(Staffordetal.,2000).Surgicalcastrationhasproventocausemoredistressthancastration

usingrubberringorclamporchemicalcastration(Felletal.,1986;Cohenetal.,1990;Kinget

al.,1991).However,otherauthorsfoundthatbandcastrationelicitedthemaximumcortisol

response(Staffordetal.,2002)and“surgicalcut”castrationwasnotsignificantlygreaterthan

thatofcontrolgroup(Fisheretal.,1996).Growingpublicconcernaboutfarmanimalwelfare

makesitincreasinglyimportanttofindamoreethicalandwelfareappropriatemethodsof

conductingroutinehusbandryproceduresbyincorporatingpracticalandaffordablemethods

ofpainreliefintoroutinesurgicalprocedures(LomaxandWindsor,2013).

Theaimofthisresearchwastoidentifywhethertherewerebehaviouralindicatorsofpainin

cattle during castration. We used composite behaviour scoring for quantitative

measurements,aswellasapplyingQualitativeBehaviouralAssessment(QBA).Quantitative

analysisusingdifferenttypesofbehaviourscoringoncattlewhilstincrush/outofcrushon

thedayofsurgeryandinthepaddock.Aspecificlistofbehaviourwereobservedwhilstcattle

wereinthepaddockcalledCompositebehaviourscoring(CBS).QBAisatechniquethatallows

observerstousedescriptivewordstodefinehowtheanimalisbehavingratherthanwhatthe

animal is doing. This form of assessment has proved to be as effective in measuring

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behaviouralexpressionofvariousspeciesof livestock.This isthefirststudytouseQBAto

assess painful behaviour in cattle. This literature review will cover concepts of cattle

behaviour, theneed tomitigatepainfulhusbandryprocedures suchas surgical castration,

varioustypesofwelfareassessmentincludingQBAmethodologyasatoolinassessingpainin

cattle.

1.1 Animalwelfareandhusbandry

Thedefinitionofanimalwelfareisnotstraightforward.AsWebster(2005)stated,itisupto

thosewhostudyanimalwelfaretocreatetheirowndefinitions.Dawkins(2004)arguedthat

animal welfare should be directed at answering two key questions: 1) Are the animals

healthy?2)Dotheyhavewhattheywant?Theconceptofanimalwelfarehasevolvedfrom

focusing primarily on an animal’s physical health and their ability to cope with their

environmenttorecognisingthatanimalsaresentientbeingscapableofexperiencingpositive

and negative emotions (Wemelsfelder and Mullan, 2014). It is those who are directly

concernedwith themanagementofanimals thathave the responsibility topromote their

welfarethroughthepracticeofgoodhusbandry(Webster,2005).

Australiaisamongtheworld'slargestandmostsuccessfulproducersofcommerciallivestock

andaleaderintheexportofredmeatandlivestockwithanestimatedvalueofA$16billion

(MLA,2011).Asanationthathasasignificanteconomicrelianceonthelivestockindustry,it

isparamounttoensureanimalwelfareguidelinesareadheredto.Inadditiontotheethical

issues regarding animalwelfare, livestockproduction canbenegatively impactedbypoor

animalwelfare(DobsonandSmith,2000)asitcanresultinincreasedmortality,reducedmeat

quality, reducedmilkproduction, reducegrowth rate and increased incidenceofdiseases

(Broom, 1986). For example, Kauppinen et al. (2012) found that providing piglets with

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favourableenvironmentsincreasedthenumberofweanedpiglets,andfarmerswithpositive

attitudes to animals gainedmorepiglets per litter. Similarly, lambsplaced in an enriched

environmentshowedhigheraveragedailygain,heaviercarcassweightandhigherfattening

scoresthanthoseplacedinaconventionalpen(Aguayo-Ulloaetal.,2014).Correctstocking

densitiesofcattle,sheepandpigsarealsocrucialtoensureanimalshavesufficientspaceto

stand,liedown,turnandaccesstofoodandwatertopreventunnecessaryinjuriesandstress

thatcancompromisetheirwelfare(Weeks,2008).

Inadditiontogoodhusbandrypractices,theattitudesofstockmendirectlyinvolvedwiththe

animalscanalsoaffectproductionrates.Forexample,highermilkyieldwasreportedinfarms

with positive indicators of human-animal interaction (Breuer et al., 2000).Weeks (2008)

statedthatdairycattleexposedto loudnoisesmadebyhumansnotonlyshowedgreater

reactivityandincreasedheartrates,butalsoreducedmilkproduction.

Certainhusbandryprocedure suchas surgical castrationanddehorning can create severe

negativeeffectsonwelfareaspainisinflictedupontheanimals(Petherick,2005).

1.2 Pain

Painisdefinedas“anunpleasantsensoryandemotionalexperienceassociatedwithactualor

potential tissue damage, or described in terms of such damage” (IASP, 1979). Painful

proceduresperformedon animals are among themost emotiveof public concerns about

animalwelfare (Wearyet al., 2006).Assessingpain in animals canbedifficult but it is an

importantpartofanimalwelfareresearch(Rutherford,2002).Routinehusbandryprocedures

suchassurgicalcastrationofmalebeefcattleispartofnormalmanagementoncattlestations

inAustralia, yet there is ampleevidence that castration causespain (StaffordandMellor,

2005a).Castrationisperformedonbullcalvestoreduceaggression,preventphysicaldanger

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to both handlers and other animals, prevent unwanted breeding, and to improve meat

tenderness(delCampoetal.,2014).InAustralia,thereisnolegalrequirementformanaging

painforcattleunder6monthsofageandtherearefewanalgesicdrugsapprovedforpain

relief in cattle and these must be administered under veterinary supervision. Castration

withoutlocalorgeneralanaestheticshouldbeconfinedtocalvesundersixmonthsold,orat

theirfirstmusterpriortoweaning(PISC,2004).Althoughtheuseofanalgesiaiswidespread

incompanionanimalmedicine(Lascellesetal.,1999),studieshaveshownthatfarmanimals

oftenreceivenoanalgesiaforpainfulprocedures(Hewsonetal.,2007a).SimilartoAustralia,

proceduresperformedonpigletsarepainful,howeverpainmitigationisnotprovidedtothe

animal reared in swine production in the USA due to economic barriers, impracticality,

historical precedent, uncertainty about need, legality the use of analgesic drugs and its

efficacy (O'Connor et al., 2014).Millman (2013)quotedanAustralian study that livestock

producers, transporters, veterinarians, animal welfare scientists, and animal welfare

advocates generally agreed that provision of pain relief for invasive procedures, such as

castration,dehorning,andtaildocking,wasofgreaterimportancethanthetechniquesused

toperformtheseprocedures.However,providinganalgesiatocattleonextensiverangeland

propertiespresentschallengesascattlearecommonlymusteredbyhelicopteronceannually,

nothabituatedtopeople,andinfrequentlyhandledduetolargedistancesandinadequate

yardsformonitoring.

Todetermineiftheuseofanalgesiaisrequiredforsurgicalcastration,researchersneedto

showthatthewelfareoftheseanimalsisimprovedbyprovisionofanalgesia.Thiscanbedone

viavarious typesofanimalwelfareassessments.Researchtodateonpainassessments in

animalshastendedtouseoneofthreeapproaches:measuresofbodyfunctioning(suchas

foodandwaterintakeorweightgain),measuresofphysiologicalresponses(suchasplasma

17

cortisol concentrations), andmeasures of behaviour (such as vocalisation) (Weary et al.,

2006). When assessing behavioural changes induced by pain, as pain is a complex

multidimensional phenomenon and the responses of animals to it are also complex,

qualitativeobservationbyanexperiencedobserver isargued tobe theonlymethod truly

capableofcapturingthiscomplexity(Rutherford,2002).Studieshaveshownthatsurgically

castrated calves struggled and kicked during the procedure (Fell et al., 1986) and escape

behaviourswereseenatcastrationbutnotafterwards(Melloretal.,2000).Researchershave

usedacombinationofbehaviouralandphysiologicalmeasurestoassesspainasitisgenerally

thoughttobemorecomprehensivethaneitheralone.

1.3 Analgesia

Localanaesthetics(LA)suchasLignocainearethemostcommonlyprescribedpre-emptive

drugs used in food animal practice along with systemic analgesic non-steroidal anti-

inflammatory (NSAID) such as Meloxicam or Ketoprofen (Stafford et al., 2002; Coetzee,

2013b). Both LAs and NSAIDs have been proven to reduce cortisol responses in cattle

undergoingcastrationandalleviatetheassociatedstressresponse(EarleyandCrowe,2002;

Stafford et al., 2002). However, various factors serve as a disincentive for producers to

provideroutinepre-emptiveanalgesiatocattle.InAustralia,thereisnolegalrequirementfor

managing pain for cattle under 6 months, however the cattle model code of practice

stipulatesthatcastrationshouldbeconductedbyaveterinarian(PISC,2004).

1.4 AnimalWelfareAssessment

Conditionsthatcompromiseanimalhealthand/orputthemathighriskofdyingarebadfor

welfare.However,goodhealthisanecessaryrequirementforgoodwelfare(Dawkins,2003).

Thereisagrowingneedtomonitorthehealthandwelfareoflivestockefficientlyandreliably

(Wemelsfelderet al., 2001). It is generally accepted thatwelfareassessment requires the

18

combinationof several indicatorsbasedonhealth, longevity,productiveandreproductive

success, changes to behaviour and physiology, and measures of animal preferences or

motivations (Broom and Johnson, 1993; Duncan and Fraser, 1997). Various types of

physiologicalmeasurementswillbediscussed,followedbybehaviourmeasurementswitha

focusonbothquantitativemethodsandqualitativebehaviourassessment(QBA).

1.4.1 Physiologicalmeasures

Physiologicalmeasurescommonly includeshort termresponsessuchas increases inheart

rates,bodytemperature, respiratoryrates,circulatingcorticosteroids,glucose, lactateand

freefattyacids(BroomandJohnson,1993).Longertermresponsesincludeenlargementof

adrenalglandsanddepressionofimmunefunction(Moberg,1985).

Physiologicalmeasurementsalonecancloudthedifferencebetweenbadandgoodwelfare

withoutknowingthetypeofenvironmentalconditiontheanimalisin.Forexample,hunted

deer have higher serum levels of creatine kinase, aspartate aminotransferase, lactate

dehydrogenase, plasma glucose, lactate, sodium, cortisol and endorphins than those that

werenotbeinghunted(BatesonandBradshaw,1997),whichcouldsimplyreflectdifferences

between deer that have been running and those that were not (Dawkins, 2003). Other

physiological indicators, suchasheart rateand respiratory ratecanbecausedbyphysical

activitiesorarousalsuchasduringcoitus,exerciseandanticipationforfood(Dawkins,2003).

Physiologicalindicatorsalonetoassessanimalwelfarecanthereforeproducediscrepancies

and misinterpretation of what the animals are experiencing, especially regarding their

emotionalstate.

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1.4.2 Behaviouralmeasures

Dawkins(2004)pointedoutthatbehaviouriscrucialingaugingwhatanimalswant,intheir

choicesandpreferencesandemphasizedthatbehaviourwillbecomeevenmoreimportant

thatithasbeenasthereisagrowingneedtofindamorereliable,lessinvasivemethodsof

welfareassessment.Usingbehaviourintheassessmentofanimalwelfarehasseveralmajor

advantagesasitcanbelessinvasiveorintrusive(Dawkins,2004).Behaviourisalreadyused

widelyintheclinicalassessmentofanimalhealthandintheassessmentofpain(Rutherford,

2002). Veterinarians traditionally use behaviour as components of assessment performed

during clinical examination (Millman, 2013) for example, the use of gait scores to assess

lameness in dairy cattle (Kestin et al., 1992) and repetitive leg and tailmovementswere

reportedtobeassociatedwithpainfromcastration(Molonyetal.,1995;Fisheretal.,2001).

Theadvantageofusingbehaviour toscore theabilityofa farmedchickenwalkinggivesa

quick,on-farmmethodofassessmenteventhoughtheunderlyingpathologycanvaryfrom

cartilageabnormalitiestojointinfectionordistortedbonegrowth(Thorp,1994;Bradshawet

al., 2002). Behavioural choices can also indicate what animals want. For example, a link

betweenchickenwithpoorgait scoresand those that choose to self-administeranalgesic

drugsshowedthatthebirdswantedareductioninpain(Weeksetal.,2000).

Differentmethodsofon-farmmonitoringhavebeendevelopedandtested,somefocuson

physical health, better management of animals and others on assessing animal welfare

(Wemelsfelder et al., 2001). Quantifying behaviour has been performed in various cattle

studiessuchaslamenessindairycattleandpennedandfreerangeyoungcattle(Ishiwataet

al.,2008;Walkeretal.,2008;GomezandCook,2010).Itisatraditionalmethodofstudying

animalbehaviourrangingfromlargecarnivorepredatorybehaviour(MacNultyetal.,2007)

20

toemotionalityingrowingpigs(Rutherfordetal.,2012).Inthisstudy,alistofbehavioural

patternswerecollatedfromthepublishedliterature(Sylvesteretal.,2004;Thüeretal.,2007;

Napolitanoetal.,2012;Sutherlandetal.,2013;Pethericketal.,2014)todevelopasuitable

listofbehavioursforscoringcattlebehaviourbefore,duringandaftercastration.Dawkins

(2004)emphasizedthatbehaviourwillbecomeevenmoreimportantthatithasbeen,asthere

isagrowingneedtofindamorereliable,lessinvasivemethodsofwelfareassessment.

1.5 QualitativeBehaviouralAssessment(QBA)

Onemethodofmonitoringbehaviourthathasthepotentialtoimproveanimalhealthand,

welfare is assessing the ‘whole animal’, using called Qualitative Behavioural Assessment

(QBA). This qualitative assessment of behaviour integrates and summarises the different

aspectsofananimal’sdynamicstyleofinteractionwiththeenvironment,whichreflectson

theanimal’sexperienceofasituation.Thisexperienceisdirectlyrelevanttoevaluationsof

welfare (Wemelsfelder, 1997). Observers were asked to score animals against a set of

qualitativedescriptorssuchas‘calm’,‘anxious’,‘excited’,‘annoyed’.Thisapproachcanuse

eitherafixedlistsofdescriptiveterms,orhaveobserversgeneratetheirownlistofterms

under ‘Free-Choice Profiling’ (FCP), a method developed and tested in food sciences

(Oreskovichetal.,1991).Observersusingthismethodwerelessbiasedastheygeneratetheir

owndescriptivewordsaccordingtotheirjudgmentofwhattheanimalsmightbeexperiencing

(Wemelsfelderetal.,2001).Clarkeetal.(2016a)demonstratedthatregardlessofwhether

observersweregivenfixedtermsorallowedtogeneratetheirownterms,observersscore

sow body language in a similar way. QBA has previously been applied to cattle during

transport(Stockmanetal.,2011),assessingcattlebehaviourbeforeslaughter(Stockmanet

al.,2012)aswellasdairycowundervarioussocialconditions(RousingandWemelsfelder,

21

2006);whichallrevealedhighagreementinhowobserversassessedtheanimalsregardless

ofcontext.

Eachofthesemethodshasbenefitsandlimitationsintermsofhowtheycanbeappliedto

welfareassessments,andcarefulreviewofthemethodswillhelpclarifytheiruse(Fleminget

al.,2016).

QBAhasbeenused inavarietyof studies includingsocialbehaviourofdairycows, sheep

transport,cattletemperamentandpigs’emotion,andassessinghorses’demeanourduring

endurance races.Animalscanexecuteabehaviour in severaldifferentways; forexample,

they canbewalking in a relaxedor tensedmanner (Fagenet al., 1997). Therefore,when

focusingonthewholeanimal,behaviourisnotjustaphysicalmovement,butitisevaluated

ina largercontextandacquiresanexpressive,psychologicalquality (BavidgeandGround,

1994;Wemelsfelder,1997;Segerdahletal.,2005).

SomescientistsinitiallywereapprehensivethatQBAmightbeanthropomorphicjudgements

ofuncertainvalidityassomecharacterizequalitativeassessmentas‘subjective’assessment

(Stevenson-Hindeetal.,1980;Kennedy,1992;Heyes,1993).Toanswertheaboveargument,

research with pigs has shown spontaneous qualitative behaviour assessments made by

untrained observers to be reliable and repeatable, irrespective of observer background

(Wemelsfelderetal.,2000;Wemelsfelderetal.,2001;WemelsfelderandLawrence,2001).

Observerscouldidentifydifferencesinbehaviouralexpressionbetweencattlethatwerenaïve

versus habituated to road transport (Stockman et al., 2011) and these differences were

supported by physiological measurements. Studies conducted on sheep and dairy cattle

indicated that the qualitative assessment procedure is a reliablemethod (Dungey, 2003;

RousingandWemelsfelder,2006).

22

Various professionalsworkingwith animals routinely use expressive terms to discuss and

managetheiranimal’shealthandwelfarestate.Therefore,QBAhasthepotentialofbecoming

anon-farmwelfareassessmenttool(Wemelsfelder,1997).

1.6 Generalaims

Theaimsofthisthesisaretodetermine:

1). To determine any variation in the behaviour of Bos indicus cattle following surgical

castration

2).TodetermineifthereisconsensusinhowobserversuseQualitativebehaviouralanalysis

(QBA)tointerpretcattlebehaviourfollowingsurgicalcastration

3).TodetermineifeffectiveanalgesiacouldbeachievedfollowingsurgicalcastrationinBos

indicuscattle

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1.7 Thesisoutline

Chapter1–containsaliteraturereviewonanimalwelfare,painandtypesofanalgesia

availableforcattle.ItalsocoversanimalwelfareassessmentandtheuseofQBAon

cattlebehaviour.

Chapter2–describeQBAmethodology,treatmentgroups,andcastrationmethod.

Chapter3-describeananalysisofcompositebehaviourscoring(CBS)developedfor

behaviourinthreedifferentcontexts(paddock,feederyardandcrush)oncattlewith

andwithoutanalgesia.

Chapter 4 – describes QBA on treatment groups during castration (in crush) and

immediately after castration (exit crush) to determine if therewas an effect with

analgesia.QuantificationofbehaviourwasperformedforcorrelationwithQBA.

Chapter 5 – applies QBA to distinguish castrated cattle from non-castrated and

analgesiacontrolsonpre-and-postcastrationdaysintwodifferentcontexts(paddock

andfeederyard).CBSwasperformedtocheckforcorrelationwithQBA.

Chapter6–includesafinaldiscussionabouttheresults,generalquestionsandfuture

research.

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2 GeneralMethodsandMaterials

Thischapterdescribesthegeneralmaterialsandmethodsusedinthisstudyhoweversome

additionalmethodsaredescribedintherelevantchapters.Allexperimentalprocedureswere

reviewed and approved by the animal ethics committee (Permit Number R2551/13) at

Murdoch University (Perth, Australia) and the survey procedures were approved by the

humanethicscommittee(PermitNumber2008/021)atMurdochUniversity(Perth,Australia).

2.1 Animals

Forty-eightBrahmanbullcalves(meanweight165±17.5kg)fromanextensivecattlestation

inthenorth-westregionofAustralia(Pilbararegion)weretransportedtoMurdochUniversity

vetfarmfortheprojectduration.Theyweretransportedfromthefarmoforigin8daysprior

tothesurgerydaytoallowforacclimatisationtotheMurdochUniversityfarm.Thecattlehad

notbeenhandledbythefarmerandwerenotusedtohumans.Accesstohayandwaterwas

allowedadlibandEasyBeefpelletswerefeddaily(MilneAgriGroupPtyLtd,Welshpool,WA,

Australia)atapproximately3%ofbodyweight.Thedayafterarrivalthecattlewerebrought

intotheraceforidentification.Aneartagwasplacedintherightpinnaandthesamenumber

waspaintedontoeachrumpforeasyidentificationfromadistance.Apedometerwasfitted

withastraptothelefthindlegjustabovethefetlockjointforanotherstudy(Laurenceetal.,

2016).Thefollowingdaythecattlewereonceagainbroughtintotheraceforprophylactic

parasitetreatment(Moxidectin5g/LCydectinPour-on,Virbac).Bloodsampleswerecollected

fromthetailveinforantigencaptureenzymelinkedimmunosorbentassaytestingforBovine

ViralDiarrhoeaVirus.

Cattlewererandomlydividedintosixequalgroups(n=8foreachgroup):

25

1) NC–non-castrated(negativecontrol).Calveswerepassedthroughtheraceandthen

heldinthecrush(restrainedviaaheadbail)fortheequivalentamountoftimeasthoseheld

forsurgery(5min)

2) CLA–castratedwithlocalanaestheticLignocaine(2mg/kg,Lignocaine20,Ilium,Troy

Laboratories,Glendenning,NSW,Australia)injectedintothetesticleandsubcutaneouslyon

theincisionsite5minpriortosurgery(whilstinthecrush)

3) CMpre – castrated with NSAIDMeloxicam (0.5mg/kg,Meloxicam 20) administered

subcutaneously30minpriortosurgery(whilstintherace)

4) CMpost – castratedwithNSAIDMeloxicam (0.5mg/kg,Meloxicam20)administered

subcutaneouslyimmediatelypost-surgery(whilstinthecrush)

5) CLMpost–castratedwithbothLignocaineadministeredsubcutaneously30minprior

tosurgery(whilstheldintherace)andMeloxicam(administeredsubcutaneouslyimmediately

post-surgerywhilstheldinthecrush)

6) C–castratedwithoutanalgesia(positivecontrol)inthecrush

Allanimalswerehandledinthesamemannerandheldintheraceandcrushforequivalent

periodsoftime.

2.2 AnalgesiaandSurgicalProcedures

AnalgesiawasprovidedbyeitherLignocaine(L)(2mg/kg,Lignocaine20)orMeloxicam(M)

(0.5mg/kg,Meloxicam20)oracombinationofboth.EachLignocaineinjectionwassplitinto

approximately6mlintra-testicularlyandtheremaining2mlsubcutaneouslyintothescrotal

skin.AllLignocaineinjectionswerecarriedoutinthesamemannerbytheveterinarianwho

26

castratedthecattle5minpriortosurgery.Meloxicamwasinjectedsubcutaneouslyintheright

lateralneckarea30minpriortosurgery.

Surgicalcastrationwasperformedbytwoveterinarianswithextensiveexperienceusingthe

opentechnique.Theanimalwasrestrainedinthecrushandheadbailandthescrotumwas

cleanedwithdilutechlorhexidinesolution.Onetesticlewasheldagainstthebottomofthe

scrotalskinandafirmincisionwasmadeusingascalpelbladealongthescrotumallowingthe

testicletobeexteriorised.Thespermductandfibroustissuewascut,andthetesticlepulled

away.Theprocedurewasrepeatedforthesecondtesticle.

2.3 Painassessment

Painwasassessedusingseveralmeasures,andthefocusofthisthesis,wasthebehavioural

responsestocastration,whichformedapartofalargerstudy.Physiologicalandbehavioural

painassessment strategieswereemployedat specific timepoints (Table2.1): liveweight,

serumcortisollevels,nociceptivethresholdtesting,pedometerreadings,balkscoreandcrush

scoreweretakenforanotherstudy(Laurenceetal.,2016;Musketal.,2016).

Table2.1Timelineofsamplingthroughouttheexperiment

Day QBA Quantitative(CBS)

Weight Cortisol Balkscore Crush

score

NTT

-6 √ √ √ √ √ √

-1 √ √ √ √ √ √ √

0(surgery)

√ √ √

1 √ √ √ √ √ √ √

2 √ √ √ √ √ √

6 √ √ √ √ √ √ √

10 √ √ √ √ √ √

13 √ √ √ √ √

*QBA–QualitativeBehaviouralAssessment*CBS–CompositeBehaviouralScoring

*NTT–NociceptiveThresholdTesting

27

2.4 Behaviouralmeasurements

2.4.1 CompositeBehaviourScoring(CBS)

Quantifyingbehaviourhasbeenperformedinvariouscattlestudiessuchaslamenessindairy

cattleandpennedandfree-rangebehaviourofyoungcattle(Ishiwataetal.,2008;Walkeret

al.,2008;GomezandCook,2010).Inthisstudy,alistofbehaviouralactivitieswerecollated

frompublishedliterature(Sylvesteretal.,2004;Thüeretal.,2007;Napolitanoetal.,2012;

Sutherland et al., 2013; Petherick et al., 2014) to develop a suitable list of behaviour for

quantifyingcattlebehaviourbefore,duringandaftercastration.Ourmethodofquantification

iscalledcompositebehaviourscoring (CBS),which isslightlydifferent fromthetraditional

ethogramandtime-budgetmethodologies.

Allobservationswereconductedbyoneobserverwithouttheknowledgeofindividualcalf

treatmentgroup.

Quantitativemeasurementsweretakenwhilstcattlewerebeingcastrated(incrush)andpost-

castration(exitcrush)basedontwo(2)listsofbehaviouraldisplay(Table3.1–Chapter3).

Observationsweredoneonthecattlefacialexpressionduringcastrationasmostoftheirbody

wascoveredbythecrushandonlytheheadwasvisible.Uponexitingthecrush,entirebody

demeanourwasobserved.

Adifferentlistofbehaviouraldisplaywasusedwhilecattlewereinthepaddock(Table7.1).

Compositebehaviourscoring(CBS)wasconductedeachmorningatsunriseforgoodvisibility

(approximatelyat7:30am)inthepaddockforamaximumof2minutesoneachanimalon

pre-and-postcastrationdays.Cattleweresampledfromlefttorightofthepaddock.Forthe

dayofcastration(Day0),scoringwasdoneintheafternoon,afterthecattlehadreturnedto

theirhomepaddock.Eightcommontypesofbehaviourincattlewerechosenforscoring.The

28

firstfourbehaviours:PositioninGroup,Grazing,RuminatingandSocialBehaviour(included

groomingeachother, sniffingor licking)were scoredas follow:0= isolated/nil displayof

behaviour, 1= semi-isolated/intermittentand2=positioned together/constantdisplayof

behaviour. The next four behaviours: Weigh Shifting, Hindleg Stomping, Scrotal Area

Grooming and Tail Swishing were scored as follow: 2 = nil display of behaviour, 1 =

intermittent and 0 = constant. The last four behaviourswere thought to be abnormal or

indicative of pain (Petherick et al., 2014a) andwere scored low (2) if behaviourwas not

present and high (0) if behaviour was constantly shown. Positioning in group, grazing,

ruminating and social behaviour (SB) were expected to be normal behaviour for grazing

animals.(Appendix7.1)

CBS data were analysed using Repeated Measures Analysis of Variance (RM-ANOVA)

(Statistica)tocheckforsignificantdifferencesbetweentreatmentgroupsandday.

2.4.2 QualitativeBehaviouralAnalysis(QBA)

CattlewerefilmedimmediatelypostCBS,scanninganimalslefttorightinthepaddock.The

videoclipscapturedtheirwhole-bodydemeanourandusingthezoomtoallowanimalviewing

atcloserange.Theywerealsofilmed inthefeederyardaftersurgery,and inthecrushat

timesofcastrationandastheyexitedthecrushimmediatelyaftercastration.Whileinthe

feederyard,videofootagewasrecordedthesamewayaspaddock.Duringcastration,only

the head and face of the cattle were shown to observers so not to reveal what type of

procedurewastakingplace.Ascattleexitedthecrush,observerswereagainabletoviewthe

wholebodyoftheanimal.Approximately3minofvideofootagewasrecordedforeachcalf

inthepaddockusingahandheldPanasonicdigitalvideocameraat7-7:30amonpre-and

post-surgerydays.Footagewaseditedusing,AdobePremiereProCS3andAdobeAfterEffects

29

CS3toproduce40-50secondsclipofeachcalfthatwasshowntoobserversforscoring.All

clipswereeditedinsimilarway,withoutknowingthecalves’treatmentgroupsandselecting

a similar startandend timepointoneach footage.Someclipsdurationwere longer than

othersascalvesweremovingconstantlyorhidingbehindoneanother.Henceeditingofthe

clipswerenecessarytocapturethewhole-bodyviewandofsimilartimeframeforeveryclip.

Volunteerobserversthatscoredthevideoswererecruitedviaflyers,postersadvertisements

andthroughtheMurdochUniversitysocialmediapage.Therewere20recruitedforthefirst

session and 30 for the following session. Observers included university staff members,

students,primaryproducersandthegeneralpublic.Eachobserverwasrequiredtocomplete

foursessionsviacorrespondenceoroncampus.Beforescoringcattle,observerswereasked

tocompleteaquestionnaireregardingtheirdemographicbackground,experiencewithcattle

andtheirattitudesandopinionstowardscattlebehaviourandanimalwelfare(Appendix7.4

to7.10).ObserversweregivendetailedinstructionsoncompletingthefourQBAsessionsbut

wereunawareofthetreatmentgroups.

2.4.3 Termgenerationsession

Observerswereshown15videoclipsofindividualorgroupsofcattleinthepaddock,holding

yardor feederareademonstratingarangeofbehaviourtoallowobservers todescribeas

manyaspectsofcattlebehaviouraspossible.Afterwatchingeachclip,observerswrotedown

termsthattheythoughtdescribedthatanimal’sbehaviouralexpression.Therewasnolimit

imposedtothenumberofdescriptivetermsanobservercouldgenerate,buttermsneeded

todescribenotwhattheanimalwasdoing(i.e.physicaldescriptionsoftheanimalsuchas

eatingorwalking),buthowtheanimalwasdoingit(i.e.emotionaldescriptionsoftheanimal

suchasrelaxedoranxious).Eachdescriptivetermwasattachedtoa100-mmvisualanalogue

30

scale(minimum=0tomaximum=100)inanexcelworksheet.Thelistoftermswasarranged

alphabetically,althoughensuringthattermswithasimilarmeaningwerenotlistedtogether.

2.4.4 Viewingsessions

Beforesessioncommencementobserversweregivendetailedinstructionsonhowtoscore

each animal’s expression using the visual analogue scale: they were told to think of the

distancebetweenthezero-pointandtheirmarkonthescaleasreflectingtheintensityofthe

animal’sexpression.Observersviewedandscoredvideoclipsofindividualanimalsusingtheir

ownuniquelistofdescriptiveterms.Insession1,observersviewed32clipsofindividualcattle

in the paddock on pre-and post-surgery days (Day-1 vs. Day +1) for treatment groups

castrated(C)vs.non-castrated(NC).Insession2,observersviewed32clipsofindividualcattle

in the paddock on pre- and post-surgery days (Day-1 vs. Day+1) for treatment groups

castrated (C)vs. castratedwith localanaestheticandpost-surgerymeloxicam(CLMpost). In

session3,observersviewed48clipsofindividualcattleinthecrushondayofsurgery(Day0)

forallsixtreatmentgroups.Thesamenumberofsessionswererepeatedforfeederyardand

exitcrushviewingsessions:Session1with32clips,Cvs.NC,Session2with32clips,Cvs

CLMpostand48clipsascattleexitedthecrushimmediatelyaftercastration.

2.4.5 StatisticalanalysisforQualitativeBehaviouralAnalysis

Thedistancefromthestartofthevisualanaloguescaletowheretheobserverhadmadea

markwasautomaticallymeasuredinmillimetresintoExcel(MicrosoftExcel2003,NorthRyde,

NSW, Australia) files. These data were submitted to statistical analysis with Generalised

Procrustes Analysis (GPA) as part of a specialised software packagewritten for Françoise

Wemelsfelder(Wemelsfelderetal.,2000;GenStat,2008).GPAcalculatesaconsensusor‘best

31

fit’profilebetweenobserverassessmentsthroughcomplexpatternmatching.GPAprovides

astatistic(theProcrustesStatistic)thatindicatesthelevelofconsensus(i.e.thepercentage

ofvariationexplainedbetweenobservers)thatwasachieved.Thisprocedurerearrangesat

randomeachobserver’sscoresandproducesnewpermutateddatamatrices.ByapplyingGPA

tothesepermutatedmatrices,a‘randomised’profileiscalculated.Thisprocedureisrepeated

100times,providingadistributionoftheProcrustesStatisticindicatinghowlikelyitistofind

anobserverconsensusbasedonchancealone.Subsequentlyaone-wayt-testwasusedto

determine whether the actual observer consensus profile falls significantly outside the

distributionofrandomisedprofiles.

ThroughPrincipleComponentsAnalysis(PCA),thenumberofdimensionsoftheconsensus

profileisreducedtoseveralmaindimensions(usually2or3)explainingthevariationbetween

animals.Eachanimalreceivesaquantitativescoreoneachofthesedimensions,sothatthe

animal’sposition intheconsensusprofilecanbegraphicallyrepresented intwo-orthree-

dimensionalplots.GPAdimensionsareinterpretedbycorrelatingtheanimals’scorestothe

observers’ individual scoring patterns, producing individual observer word charts that

describetheconsensusdimensionsthroughtheirassociationwitheachindividualobserver’s

terms.Thesewordchartscanthenbecomparedforlinguisticconsistency.Fromtheseword

charts,alistoftermsdescribingtheconsensusdimensionswasproduced,byselectingterms

foreachobserverthatcorrelatedstronglywiththosedimensions.Tocomparetreatments,

the GPA scores for each dimension were analysed using repeated-measures ANOVA for

session1and2,andone-wayANOVAforsession3.

32

3 Applying Composite behaviour scoring (CBS) to determine

castrationpainincattle

3.1 Abstract

Castrationofmalecattleistraditionallyperformedfordocility,toenhancecarcassquality,as

well as to prevent unwanted breeding. There have been various studies conducted to

investigatewaystominimisepain in livestockandto improvetheirwelfare.Weexamined

behaviourdisplayedinresponsetosurgicalcastrationincattletoidentifyindicatorsofpain.

Forty-eightBrahmanbullcalvesweredividedintosixtreatmentgroups,witheightanimals

ineachgroup(n=8).Thereweretwocontrolgroups:1.castratedwithoutanalgesia(C),and

2. non-castrated (NC) and four analgesic groups: 1. castrated with local anaesthesia -

Lidocaine (CLA), 2. castratedwith post-surgery non-steroidal anti-inflammatory (NSAID) –

Meloxicam (CMpost), 3. castrated with pre-surgery NSAID – Meloxicam (CMpre), and 4.

castratedwith combined Lidocaine and post-surgeryMeloxicam (CLMpost). Behaviourwas

recorded from footage collected under three contexts: in the paddock on pre-and post-

castrationdays,of theanimal’sheadas itwasheld in thecrushduringcastration,andas

animalswerereleasedfromthecrushintoapenimmediatelyaftercastration.Therewereno

significant differences amongst treatment groups evident for footage collected in the

paddockorduringcastration,butdifferencesinbehaviourwereevidentimmediatelyafter

castration.Thisstudyrevealedthatthetimingofbehaviouralobservationsandthecontext

under which they are filmed are important in determiningwhether pain associatedwith

castrationislikelytobedetectable.Thecontextunderwhichanimalsarefilmedinfluences

partsoftheanimalthatcanbevisualisedaswellasthedegreeofmovementandactivityor

body language.Controlling thesecontextual factorswouldallowclearercomparisonsover

33

timewithfuturestudies.Suchinformationwillbevaluableforfurtherinvestigationsonpain

reliefforcattle.

Keywords:Cattle,Surgicalcastration,Analgesia,Pain,Compositebehaviourscoring(CBS)

34

3.2 Introduction

Castratingyoungmalecattleisacommonpractice,performedonpropertiesusuallybythe

farmer. Reasons for doing so are for docility, enhanced carcass quality and to prevent

unwantedbreeding(NielsenandThamsborg,2005).Varioustechniqueshavebeenadopted

throughout theyearsanddevicesdeveloped tomake theprocessashumaneaspossible.

Physical castration is themost invasive, yetmost widely used compared to hormonal or

chemicalmethodsandwelfareconcernsaredrivingresearchintominimisingthesideeffects

(Stafford and Mellor, 2005b). Nielsen and Thamsborg (2005) and Bretschneider (2005)

believedthattherewerenomajorwelfarevariationsbetweenthemethods,becausetheyall

result in comparable situations involving pain. Behaviour has been used as ameasure of

welfarestateandapparentlycanalsobeanindicatorofpain(StaffordandMellor,2005b).

Surgicallycastratedcattlehavebeenobservedtostomptheirhind-legs,swishtheirtailsand

grazetheleastcomparedtonon-castratedcattle(StaffordandMellor,2005b).Rubber-ring

castrated calves assumed various stances and tried all means to reach their testicles to

removetheagentofirritationforweekspost-castration(StaffordandMellor,2005b)which

canbe interpretedasbeing lessquiet thansurgically castratedcattle.However, surgically

castratedcattlewereinterpretedasquieterpost-castration,asitcouldbepathologicalpain

thatwouldbeaggravatedbyexcessivelocomotion(StaffordandMellor,2005b).

Cattlehavequiteanextensiverepertoireofbehaviour(Kilgour,2012)anditisimportantto

be able to identify normal versus abnormal behaviour to assess pain and discomfort

associatedwithnegativestimuli.Forexample,grazing,ruminatingandrestingaretypicalof

normalcattlebehaviour,whichmakesup90-95%oftheirday(Kilgour,2012).Disturbances

totheirnormalrageofbehaviourmaythereforebeevidentasreducedtimespentgrazing,

35

ruminatingandresting,whileincreasesinagitationorlocomotionmaybecomeevidentfor

animalsexperiencingphysicalormentalpain.

The aim of this studywas to identify cattle behaviour in response to surgical castration,

comparingbetweendifferentanalgesiatreatmentsandrecordingtheirbehaviourunderthree

differentcontexts:undisturbedinthehomepaddock(paddock),lockedintheheadbailwhile

inthecrushduringsurgery(incrush)andexitingthecrushimmediatelyaftersurgery(exit

crush).Weexpectedtoseetreatmenteffectsforallthreecontexts,inparticularforCvs.NC

andCvsCLMposttreatmentgroups.

3.3 Method

ThisstudywasapprovedbyMurdochUniversityAnimalEthics (PermitNumberR2551/13)

andHumanEthics(PermitNumber2008/021)committees.

3.3.1 AnimalsandQuantitativeBehaviourAnalysis

Forty-eight(48)Bosindicus(Brahman)bullcalvesofsix(6)toeight(8)monthsofagewitha

meanbodymassof165.5(±17.5)kg.TheyweretransportedtoMurdochUniversityfarmfrom

anextensivecattlestationinthenorth-westregionofAustraliaduringthewintermonths.

Calveswereplaced inthesamepaddockeight (8)dayspriortocastrationforacclimation.

Calveswerehandleddailybythesamestaffmembers,withthecalvesmovedthrougharace

as a group, placed in a crush individually for weighing, blood sampling for baseline

measurementofbloodcortisol,nociceptive threshold testingandpedometer readings for

anotherstudy(Laurenceetal.,2016;Musketal.,2016).Theywererandomlydividedintosix

treatmentgroups(n=8):

36

1).NC–non-castrated

2).CLMpost–castratedwithMeloxicampost-castrationandLidocaine

3).CLA–castratedwithLidocaine

4).CMpre–castratedwithMeloxicampre-castration

5).CMpost–castratedwithMeloxicampost-castration

6).C–castrated

Quantificationofbehaviourwascarriedoutforcalvesunderthreecontexts;undisturbedin

thehomepaddock(paddock), lockedintheheadbailwhile inthecrushduringsurgery(in

crush)andexitingthecrush immediatelyaftersurgery (exitcrush).Behaviourscoreswere

counted by one observer each morning (07:00-08:00) from left to right direction of the

paddockoneachcalffortwo(2)minonpre-andpost-castrationdays(Day-6,-1,+1,+2,+3,

+6, +10, +13). Eight sets of cattle behaviours were put together for observation in the

paddock, called Composite Behaviour Scoring (CBS) Scoring chart – Appendix 7.1). Calves

wereobservedfromadistanceusingbinocularstoavoidanydisturbancetotheirbehaviour.

Scoresof0,1or2wereassignedtoeachbehaviourtodistinguishanyvariationinbehavioural

display.

In each context, two categories of behaviour were scored; i) states that were mutually

exclusiveandmeasuredasaproportionoftime,andii)eventsthatwereactivitiescarriedout

for less than five (<5) seconds each, andmeasured by counts (Table 3.1). On the day of

castration,inthecrush,videofootagewastakenbyastationarycameramountedonarailing

located approximately two (2) metres in front of the exit gate. Exit crush footage was

collectedwithahandheldcamerafromanobserverapproximatelythree(3)metresonthe

sideofthecrush.Thesameobserverviewedtherecordedfootageofcattleduring(incrush)

andimmediatelyafter(exitcrush)castrationforthescoringthebehaviour(Table3.1).The

37

CBS performed on cattle in crush, and exit crush, recorded 11 states and 11 events,

respectively.

DataanalysiswasperformedbyrepeatedmeasuresANOVA(RM-ANOVA),Statistica(StatSoft,

2007) and Paleontological Statistics (PAST) (Hammer, 2014) using One-way analysis of

similarities(ANOSIM)andsimilaritypercentage(SIMPER)tocheckforsignificantdifferences

betweentreatmentgroups.

3.4 Results

InthepaddockusingCBS,therewerenosignificantdifferencesbetweenbehaviourpreand

post-surgerydays(Day-6,-1,+1,+2,+3,+6,+10,+13)inall6treatmentgroups(P=0.866)

whenanalysedwithRM-ANOVA.Therefore,Day-1andDay+1wereselectedtodetermine

differencesbetweentreatments(Figure3.1).Thereweresignificantdifferencesbetweenthe

twodaysintermsofpositioningingroup,grazing,ruminating,socialbehaviourandweight

shifting (P = 0.004). Three behaviours (hind leg stomping, scrotal area grooming and tail

swishing)werenotdetectedinthepaddock.Furtheranalysiscomparingpre-andpost-surgery

days(Day-1vs.Day+1)onthreepairsoftreatmentgroups:Cvs.NC(Figure3.2),Cvs.CLMpost

(Figure3.2),andCMprevs.CMpost(Figure3.3)wereconducted.Weightshiftingwasdetected

inCtreatmentgrouponDay-1butinbothCandNCtreatmentgroupsonDay+1.However,

therewerenosignificantdifferencesintreatmenteffectbetweenCandNCgroupsonDay+1

(P=0.810).Novarianceinotherbehaviourwasseen.Comparisonofweightshiftingbetween

C and CLMpost, and between CMpre vs CMpost treatment groups showed no significant

differences (P= 0.810). One-way ANOSIM analysis found no significant differences found

betweentreatmentgroupsonallbehaviour(P=0.251)duringcastration(incrush).Onexiting

thecrushaftercastration,thereweresignificanttreatmenteffectfoundbetweentwopairs

38

usingone-wayANOSIMCvsNC(P=0.024)andCMpostvsNC(P=0.044).SIMPERanalysiswas

thenappliedonthetwopairsoftreatmentgroups(CvsNC)and(CMpostvsNC)tocheckfor

behaviourthatshowedsignificantdifferences.Inpercentageoftimeofcalvesrunningupon

releaseforNC(9%),CMpost(7%)andC(0%)(Figure3.5).Proportionsofcalvesthatwalked

(forwardandbackwards)were,NC(53%),CMpost(54%)andC(60%)andstoodwere,NC(38%),

CMpost(39%)andC(39%)(Figure3.6).Proportionsofcalves’weightshiftedwere,NC(100%),

CMpost(45%)andC(94%),tailswishingwasonlyseeninCMpost(45%).Urinate/defecatewas

identifiedinC(6%)andCMpost(9%)respectively.Therewerenosignificantdifferencesfound

betweengroups(P=0.251)forbehaviourrecordedduringcastration.

3.5 Discussion

Significant differenceswere evident between treatment groups as cattle exited the crush

after castration. However, there was little significant difference in time-budgets

measurementsbetweentreatmentgroupsforcattleobservedwhileinthepaddockorduring

castration. This study therefore reveals that the context in which cattle are observed is

important in showingevidenceofpain. Identifyingwhenpain ismost likely tobeevident

wouldaidourunderstandingoftheefficacyofanalgesia.

Previous studieshave shown that castration in cattle is painful. For instance, pain canbe

associatedwithdecreasesinactivity,eatingandruminating(AndersonandMuir,2005),as

wellasabnormalgaitandpostures(StaffordandMellor,2005a;Coetzee,2011).Inourstudy,

therewasnodifferenceingaitoractivitywhilstinthepaddock.Oneexplanationwouldbe

that thecattleweremaskingthepain.Livingston (2010),describedthatsuchmasking isa

result of the behavioural evolution of prey species to avoid overt displays of abnormal

behaviourthatmaymakethemvulnerabletopredators.Calvesmightbehavecautiouslyas

39

theywereexposedtovariousenvironmentalelementsbeingoutintheopenpaddockand

theymayalsohavebeenmimickingthebehaviourofoneanotherasbeingamongstother

cattle is crucial for their safety. Cohesive relationships are important inherd animals and

cattle often display social grazing behaviour as they follow each other independently

(ReinhardtandReinhardt,1981).

Fearwhile being held in the crush (head bail), can overpower other behavioural display,

especiallyascalveshadtobesoclosetohumans.Therefore,notreatmentdifferenceswere

detected during castration. Cattle, by nature, are fearful animals and aware of their

surroundings and the presence of people. Studies have suggested that cows alter their

vigilance according to their degree of fearfulness toward people and toward different

environment(Welpetal.,2004).Therefore,itisimportanttobeabletoseparatetheeffects

ofpainfromtheeffectsofhumanpresence.

Thereweresomedifferencesascattleexitedthecrushaftercastration.NoneoftheCcalves

ranoutofthecrush,whichsuggeststhatcastrationmaycausemorediscomfortthancontrols

(NCandCMpost).Walkingwas seenmore inC andCMpost group thanNC,whichmight be

explainedbycastratedgroupsexperiencingmostdiscomfortandthereforelesslikelytorun.

Theanalgesiceffectwasunlikelytohavebeeneffectiveforaleastthirty(30)minutespost-

administrationsinceMeloxicamtherapeuticplasmaconcentrationisfromthirty(30)minutes

toforty-eight(48)hourspost-administration(DumkaandSrivastava,2004).Hence,CMpostand

Cwouldbesimilarintheirsensitivitytopainastheyexitedthecrush.CMpostandCgroups

demonstrated this through their display of forward walking behaviour and less running

comparedtoNCcalves.

40

NCshowedthemostweightshiftinguponreleasefromthecrush,whichcouldbeduetothem

tryingtomoveawayfromhumansastheywerefearfuloftherecentrestraintinthecrush.

Some of the behavioural displays that indicate distress and fear are standing/escape or

avoidance movements, pacing and restlessness (Mellor et al., 2000). However, these

behaviouraltypescouldalsomeanthecattlewereinpain.LessweightshiftinginCandCMpost

wasseenastheywerereleasedfromthecrush,whichcouldbeasignofpainasmovement

inthehindlegswouldlikelyaggravatethewoundregion.TailswishingwasseenonlyinCMpost

groupbutnotinCastheyexitedthecrush.Itwastheexpectationthatbothcastratedgroups

havesimilarbehaviouraldisplays.However,bothCandCMpostshowedsignsofnervousness

astheyurinated/defecateduponexitingthecrushunliketheNCgroup.Theproceduremost

likelyaggravatedthealreadyfearfulcattle.Thismaybewhythesecattleweremorestoicand

minimaldisplayofexpectedbehaviourchanges.TheNCgroupwereplacedinthecrushand

blood sampled. Therefore, they would still be in a state of fear that may look like the

behaviourof castratedcattle.Prior to this study, thesecattlehad little tonoexposure to

humanstheirbehaviouraldisplay indicatedtheyremainedreactive tohumansthroughout

thestudy.Thereisevidencethatcattlecanbetrainedorhabituatedtohumansandbecome

lessfearful(BoissyandBouissou,1988;Pajoretal.,2000;HemsworthandColeman,2011).

Repeated exposure to aversive handling procedures can result in cattle becoming more

reactiveandfearfulofpeople(Grandin,1997;HemsworthandColeman,2011).

Timingofmonitoringofthesecattlemayhavebeenproblematicastheymightneedclose

monitoringduringthefirstforty-eight(48)hourspost-proceduretocaptureanytreatment

effects.Anotherissuemightbethenumberofcattleusedforeachtreatmentgroup.Sample

sizeiscrucialastoobtainthemostaccurateresults.

41

3.6 Conclusion

Behaviour is an apparent indicator of pain (Stafford andMellor, 2005a) and any type of

behaviourshouldbeconsideredwithinabroaderperspectiveoftheanimals’environmentas

somebehaviouraldisplaymightbeconflicting.Ourstudyshowedunexpectedfindings,aswe

didnotseeamarkeddifferenceincalfbehaviourinthecastratedvsnon-castratedgroups

regardlessofthecrushorpaddockcontext.Hence,wecanconcludeeitherthatsurgeryisnot

painfulforthisspeciesorthatinthecontextofourstudy,thecalvesdidnotexhibitovertpain

behaviour. Itwasexpectedthattherewouldbechanges inbehaviour incattlebeforeand

after castration (Day -1 vs Day +1) and treatment effects during and immediately after

castration.However,inthisstudy,theprovisionofpainreliefdidnotappeartosignificantly

alterthebehaviourofthecattleduringoraftersurgery.Webelievethelatterismorelikely

andoverriddenbythefear/stressofhandlingandbloodsamplingforthecattleinandaround

thecrush,maskinganypainresponse.Inthepaddock,wecanexplainthelackoftreatment

differenceascattlewerewiththeirherdmatesandmayhavedisplayedsimilarbehaviourto

oneanother.Withpreyspecies,suchascattle,strongfearfulinstinctsandsubtlebehavioural

displaysprovideonlyasmallwindowofopportunitytowitnesschangesinbehaviourdisplay

andtounderstandtheirexpression,whichrequiresadditionaltimeandpatience.Weknow

surgicalprocedurescausepainandthisstudyhasshowndifficultiesinidentifyingtheovert

expressionofpainincattlenothabituatedtohumans.Anoverwhelmingfearofbeingina

confinedcrushandclosetohumansmayhavesignificantlyshiftedtheirdemeanour.Hence,

fearmayreplaceanydisplayofpainfulbehaviour.

42

3.7 Appendix

Table3.1Thelistofbehaviourdevelopedforscoringacrossthreecontexts(paddock,incrush

andoutofcrush).

Category Behaviour Description Measure Paddock Incrush(duringcastration)

Existcrush(aftercastration)

States Stand(still) Stationary-

torsoupright

position

Proportion

oftime �

Stand

(hunched)

Stationary–

shoulderslightly

lowered

Proportion

oftime �

Stand

(struggling)

Notstationary–

torsomoving

vigorously

Proportion

oftime �

Walk(forward) Forward

locomotion

Proportion

oftime �

Walk

(backwards)

Backwards

locomotion

Proportion

oftime �

Stand Nomovementof

body,stationary

Proportion

oftime �

Run Continuous

motionof

forward

movement

Proportion

oftime

�

Positionin

group

Withanother

coworbeing

alone

Proportion

oftime �

Grazing Eatinggrass Proportion

oftime�

Ruminating Chewingcud Proportion

oftime�

SB*grooming

(licking/

sniffing)

Grooming

anothercow

Proportion

oftime �

Events Earsflick Movementof

oneorbothears

Counts �

Swallowing

saliva

Visible

swallowing

reflex

Counts

�

Drooling Producingclear

oralsecretion

Counts �

Head(shaking/

struggling)

Vigorously

movinghead

Counts �

43

Eyeswidening Wideningof

eyelids/sclera

visible

Counts

�

Head(lowering

toground)

Headbelow

brisket,almost

touchingground

whilestanding

Counts

�

Scrotalarea

grooming

Lickingscrotal

area

Counts� �

Hindleg

stomping

Stompingof

eitherhindfoot

onground

Counts

� �

Weightshifting Liftingeither

footwhile

maintaining

balance

Counts

� �

Tailswishing Movementof

tailfromsideto

side

Counts

� �

Urinate/

Defecate

Excretionof

bodilyfluid/

solid

Counts

�

44

Figure3.1.CompositeBehaviourScoring(CBS)ofcattleinthepaddockshowing5behavioural

display,whichhadsignificantdifferences(P=0.004).

45

Figure 3.2. Castrated (C) vs.Non-castrated (NC) cattle on pre-andpost-surgery (Day-1 vs.

Day+1)

Figure3.3.Castrated (C)vs.CastratedwithLocalanaestheticandpost-surgeryMeloxicam

(CLMpost)cattleonpre-andpost-surgery(Day-1vs.Day+1)

Figure 3.4. Castrated with pre-surgery Meloxicam (CMpre) vs. Castrated cattle with post-

surgeryMeloxicam(CMpost)onpre-andpost-surgery(Day-1vs.Day+1)

46

Figure3.5.Treatmentgroupcomparisonofbehaviouralstatesobservedascalvesexitedthe

crushaftercastration.

0%

20%

40%

60%

80%

100%

C CMpost NC

55% 53% 42%

5% 1% 11%

39% 39% 38%

0% 7% 9%

Proportionoftime

Run Stand Walk(Backwards) Walk(Forward)

47

Figure3.6Treatmentgroupcomparisonofbehaviouraleventsobservedascalvesexitedthe

crushaftercastration.

94.44%

45.45%

100.00%

0.00%

45.45%

0.00% 5.56% 9.09% 0.00%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

C CMpost NC

Prop

ortio

noftime

Wtshift TailSwish Urinate/Defecate

48

4 Using Qualitative and Quantitative behavioural assessment on

cattle undergoing surgical castration with different analgesic

protocols

4.1 Abstract

Castration isapainfulprocedure that is routinelyperformedon livestock. Identifyingbest

practice for castration requires objectivemeasures of pain assessment. In this study, the

behaviouralexpressionofBrahmancalvesduringandimmediatelyaftersurgicalcastration

wasexaminedusingqualitativeandquantitativemethods.Theobjectivewastocomparethe

behaviourofcontrolcattlewiththosesubjectedtodifferentanalgesicprotocolsusinganon-

steroidalanti-inflammatorydrug(Meloxicam)administeredpre-orpost-surgicalcastration

andalocalanaesthetic(Lignocaine)whiletheanimalwasheldinacrush.Forty-eightBrahman

bullcalvesweredividedintosixtreatmentgroups(n=8):1)nocastration(NC),2)castration

withnoanalgesia(C),3)castrationwithMeloxicamadministeredpre-castration(CMpre),4)

castrationwithMeloxicamadministeredpost-castration(CMpost),5)castrationwithLidocaine

(CLA)and6)castrationwithLidocaineandMeloxicampost-castration(CLMpost).Videofootage

recordedcattle(a)duringsurgicalcastration(facialfeaturesvisiblewhilstrestrainedinthe

crush)and(b)post-castration(wholeanimalvisibleuponreleasefromthecrush)andshown

to volunteer observers for analysis by Qualitative Behavioural Assessment (QBA) in two

separateviewingsessions.Theconsensusprofileexplained44.5%and35.36%ofthevariation

in observer’s scoring patterns for the two viewing sessions, respectively. There were

significant differences between treatment groups for both sessions (P < 0.001) where

castratedcattle(C)showedthedifferencesfromgroupsgivenanalgesia.Descriptiveterms

suchas‘agitated’,‘uneasy’and‘sore’usedbyobserverssuggestedthatcastratedcattle(C)

experienceddiscomfortfromtheprocedure.Cattlethatreceivedbothformsofpaincontrol

49

(CLMpost)weredescribedas‘calm’,relaxed’and‘contented’.Spearmancorrelationsbetween

QBAandquantitativeanalysissupportedtheinterpretationthattheobserversscoredcattle

behaviourconsistentlywiththeinterpretationthatthecattledemonstrateddifferingdegrees

ofpain.

Keywords: Castration, Cattle, Qualitative behavioural assessment (QBA), Pain, Analgesia,

Quantitativeanalysis

50

4.2 Introduction

Castration is a painful procedure that is routinely performedon livestock. There is ample

evidencethatcastrationcausespain(StaffordandMellor,2005a;Coetzee,2011).Themost

commonmethodsofcastrationcurrentlyusedarebandorrubberring,surgicalcastrationand

usingaBurdizzoclamp(Staffordetal.,2000).Surgicalcastrationisthemostcommonmethod

used,eventhoughitincreasesthecortisolresponse(EarleyandCrowe,2002;Sutherlandet

al.,2013).Tensionbandingcastrationhasgainedfavourasitisrelativelysimpletoperform

andpromotedbyretailersofthebandersasahumanemethodofcastration(Pethericketal.,

2014).Comparingthesemethodstoguidebestpracticerequiresobjectivemeasuresofpain,

anareaofgrowingresearch.

Given the growing concern for animal welfare amongst the public, it is appropriate to

determinewhethereffectiveanalgesiacanbeachieved (WearyandFraser,2004).Several

pain mitigating strategies have been proven to be effective in mitigating pain during

castration including the use of local anaesthetics and systemic non-steroidal anti-

inflammatory drugs (NSAID). Petherick et al. (2014) found that the NSAID, Ketoprofen

appeared effective inmoderating pain-related behaviour in the three (3) hours following

castration inmatureBrahmanbulls,butwas lesseffective inyoungweaners.Wearyetal.

(2006)indicatedthatNSAIDswereeffectiveatreducingpost-operativepaininthehoursthat

followed the procedure. However, otherNSAID studies have shown Ketoprofen, and oral

Meloxicam, tobe ineffective atmitigatingpain after surgical castration (Repenninget al.,

2013;Moyaetal.,2014).Whilesystemicanalgesia(e.g.Ketoprofen)maybemoreeffective

thanlocalanaesthesiaduringcastrationbecauseitservestoalleviatetheassociatedstress

response(EarleyandCrowe,2002),localanaesthesia(e.g.Lidocaine)mayprovideeffective

pain-relieffortwo(2)tothree(3)hourspost-surgery(McMeekanetal.,1998a;McMeekanet

51

al.,1998b).Indirectsymptomsthatcanserveasindicatorsofpaininanimals,includechanges

in both physiological and behavioural parameters (Molony and Kent, 1997). Clinical

assessment of animal health and assessment of pain widely uses behaviour (Rutherford,

2002; Petherick et al., 2014). Dawkins (2004) added that behaviour has several major

advantages inwelfare studies as it is not only non-invasive, it is also inmany cases non-

intrusive.MortonandGriffiths(1985)proposedthatthestudyofbehaviouralpatternsshould

constituteasubstantialpartofpainassessment.Applyingquantitativemethodssuchasusing

composite behaviour scoring for pain assessment is anotherway of studying behavioural

patternsasdescribedinChapter3.

Qualitative Behavioural Assessment (QBA) uses observers to score qualitative aspects of

behaviouralexpression(Flemingetal.,2016).Theobserversareaskedtoconsiderthewhole

animal and integratemanypiecesof information, including incidentalbehavioural events,

subtledetailsofmovementandposture,andaspectsofthecontextinwhichbehaviouroccurs

(Wemelsfelderetal.,2001).QBAcapturesnotsomuchwhatananimaldoes,buthowitdoes

soandtheanimal’sdynamicstyleofinteractionwiththeenvironment(Wemelsfelder,1997).

Thisstudyexaminedthebehaviouralexpressionofcattleallocatedtosixtreatmentgroups,

includingacontrolgroup(no-castration),acastratedgroup(noanalgesia),andfourdifferent

analgesia protocols given to castrated groups. The bulls were filmed as they were being

castratedandastheywalkedoutofthecrushimmediatelyaftercastration.Thefootagewas

showntovolunteerobserversforscoringusingQBA,andasingleobserverforCBSanalysis.

Theaimsofthisexperimentwereto:

1. Determine whether observers can distinguish between treatment groups using

quantitativeandqualitativebehaviouralassessment;and

52

2. Determinewhetheranalgesiareducesthepainassociatedwithcastration.

4.3 Methods

ThisstudywasapprovedbyMurdochUniversityAnimalEthics (PermitNumberR2551/13)

andHumanEthics(PermitNumber2008/021)committees.

4.3.1 Animalsandtreatmentgroups

Forty-eight (48)Brahmanbull calves (meanweight165±17.5kg) fromanextensivecattle

stationinthenorth-westregionofAustralia(Pilbararegion)weretransportedtoMurdoch

Universityvetfarmfortheprojectduration.Thecalveswereheldinacommunalpaddockfor

eight(8)daysforacclimationpost-arrival,duringwhichtimetheywerepassedthrougharace

and crush for habituation to handling andmeasurement of baseline data formechanical

nociceptivestudy(Musketal.,2016)andpedometermeasurement(Laurenceetal.,2016).

Theywererandomlydividedintosix(6)treatmentgroupswitheight(8)calvesineachgroup.

Treatment groups contained: castrated (C) without any analgesia, non-castrated (NC),

castratedwithMeloxicampost-castrationandLidocaine(CLMpost),castratedwithLidocaine

(CLA),castratedwithMeloxicampre-castration(CMpre)andcastratedwithMeloxicampost-

castration(CMpost).

Calvesweremoved througha race individually into a crush for either sham treatmentor

surgicalcastrationusingtheopentechnique.Theywererestrainedinthecrushusingthehead

bail,andthescrotumwasdisinfectedusing0.05%chlorhexidinesolution.Onetesticlewas

heldagainstthebaseofthescrotalskinandaquickfirmincisionwithascalpelbladewas

madeallowing the testicle tobeexteriorised. Thevasdeferens and tunica vaginaliswere

separated from the testicle. The testicle was then pulled away from the body such that

vasculaturetoreandretractedbackintotheabdomen.Thestepswererepeatedforsecond

53

testicle. Once complete surgical castration, they underwent routine blood sampling for

anotherstudy(includingNCcalves)andreleasedfromthecrushviatheforwardgate.

Eachindividualcalfwasfilmedcontinuouslyusingafixed-positiondigitalhandheldcamcorder

(PanasonicSDR-H250)asitwasheldinthecrush,andbyasecondhand-heldcamcorderasit

wasreleasedfromthecrushanditwalkedintotemporaryholdingyardsbeforebeingreleased

backintotheircommunalpaddock.Thecontinuousfootageinthecrushwaseditedtoisolate

onlyfootageatthetimeofthesurgicalincisionforeachanimal.

4.4 QualitativeBehaviouralAssessment(QBA)

Videofootagewasrecordedandedited(AdobePremierProCS3andAdobeAfterEffectsCS3,

Chatswood,NSW,Australia)intoforty(40)tofifty(50)secondclips.Observerswererecruited

onavolunteerbasis.Eachobserverwasshownthesameclipsofindividualcattle,without

beinginformedabouttheirtreatmentgroups.FreeChoiceProfiling(FCP)methodologywas

used for scoringcattle,which reliesonobserversgenerating theirownuniquedescriptive

termstodescribeanimalsforaseriesofvideoclipsshowntotheobserversaspartofaterm

generationsession(Wemelsfelderetal.,2001).

Two (2) qualitative viewing sessions were carried out, where (a) twenty (20) observers

watchedfootagecollectedatthetimeofsurgery,and(b)thirty(30)observerswatchedthe

footageascalveswerereleasedfromthecrush.Duringthesesessions,eachobserverused

thetermstheyhadgeneratedtoscorecattlebehaviouronavisualanaloguescaleranging

from0to100.Theobserverswereaskedtomarkonthescaletheintensityofthebehavioural

expression for each of their descriptive terms. These measurements were entered to

individualExcelfiles(MicrosoftExcel2013,NorthRyde,NSW,Australia)foreachobserver.

Thescoresobtained foreachclipwereanalysedbyGeneralisedProcrustesAnalysis (GPA)

54

usingaspecialisedsoftwareeditionwrittenforFrancoiseWemelsfelder(StatSoft,2007).A

detailed description of GPA procedures is outlined in (Wemelsfelder et al., 2000;

Wemelsfelderetal.,2001).

GPAdevelopsaconsensusprofiledescribingallobservers’scorescollatedwithinaviewing

sessionby transformingall thedata aswell as identifying complexpatterns.AProcrustes

statistic is calculated, quantifying the percentage of variation between observers that is

explainedbytheconsensus.Whetherthisconsensusisasignificantfeatureofthedataset,

or,alternatively,anartefactoftheProcrusteancalculationprocedures,isdeterminedthrough

a randomisation test (DijksterhuisandHeiser,1995).One-way t-test isused todetermine

whethertheactualobserverconsensusprofilefallssignificantlyoutsidethedistributionof

randomisedprofiles(Flemingetal.,2015).Subsequently,thenumbersofdimensionsofthe

consensusprofileisreducedthroughPrincipleComponentAnalysis(PCA).Thetermsusedby

eachobservertoscorecattlebehaviourarecorrelatedwitheachGPAdimensionstoidentify

thosetermsthatshowthestrongestcorrelations(r)witheachGPAdimension.Theseterms

wereidentifiedbycalculating>75%ofthehighestabsolutecorrelationcoefficientvaluefor

that dimension (Mardia et al., 1979). By using Mixed-model ANOVA, treatment groups

comparisonsweremadeforeachofthethree(3)GPAdimensionswithineachsession.

4.5 QuantitativeAnalysis

Alistofbehavioursuitableforquantifyingbehaviourwascollatedbasedonpublishedstudies

on cattle behaviour (Sylvester et al., 2004; Thüer et al., 2007; Napolitano et al., 2012;

Sutherland et al., 2013; Petherick et al., 2014). A total of ninety-six (96) clips from both

sessions(fourty-eight(48)videoclipseach)wereobservedforidentificationofninebehaviour

categories:fourmutuallyexclusivestates(durationofmorethanfive(5)seconds;measured

55

asproportionoftime)andfive(5)events(countsofbehaviourwithadurationoflessthan

five(5)seconds)(Table4.1).Spearmancorrelationwasusedtocheckforcorrelationsagainst

QBAresults.

4.6 Results

4.6.1 QualitativeBehaviouralAssessment(QBA)

Atotalofninety-seven(97)descriptivetermsweregeneratedbytwenty(20)observersfor

duringsurgery(incrush);with14±5(range7-23)termsperobserver.Aftersurgery(exit

crush),thirty(30)observersgeneratedonehundredthirty-seven(137)descriptiveterms;with

15±5(range7–28)termsperobserver(Table4.2).Foreachsession,therewerethreeGPA

dimensionsthatexplained81.8%and63.4%ofthevariationinthescoringpatternbetween

cattle (percentage breakdown for each dimension are shown in Table 4.2). There were

significant treatment group differences for all three (3) GPA dimensions for the footage

collectedduringcastration(Table4.2a).

Duringcastration (incrush),onGPAdimension1,observersdescribedcalves in theCand

CMpreasthemost‘agitated’,‘restless’andthoseintheNCandCLMposttreatmentgroupsas

beingthemost‘calm’,‘relaxed’(Figure4.1a).GPAdimension1explainedthemajority(66.2%)

ofthevariationinscoringbetweencattle,withtermssuchas‘calm’,‘relaxed’correlatedwith

thelowendoftheaxisand‘agitated’,‘restless’correlatedwiththehighendoftheaxis.There

werecorrelationswithquantitativebehaviouralscoresonthisaxis,withanimalsthatwere

attributed high on GPA dimension 1 scores (more ‘agitated’, ‘restless’) scored as stand

(struggling)(rs48=0.82,P<0.001),headshaking/struggling(rs48=0.84,P<0.001),andhead

lowering(rs48=0.37,P<0.05)tothegroundcomparedwithcattlethatwereattributedlow

GPAdimension1scores(more‘calm’,‘relaxed’).

56

OnGPAdimension2,CandCLMpostgroupsweredescribedasmore‘playful’,’restless’,while

theremainingtreatmentgroupsCLA,NC,CMpreandCMpostwerecategorisedasmore‘unsure’

byobservers (Figure4.1b).GPAdimension2 (10.5%ofvariability)wasdescribedbyterms

suchas ‘playful’, ‘uneasy’correlatedwith lowendoftheaxisand‘unsure’correlatedwith

highendoftheaxis.Quantitativescoresforstand(hunched)(rs48=-0.57,P<0.001)and

swallowingsaliva(rs48=0.33,P<0.05)correlatedwithlowandhighGPAdimension2values

respectively.

OnGPAdimension3,theCLMpostgroupweredescribedasmost‘sleepy’,‘depressed’andCLA

andCMpostthemost‘sore’,‘frustrated’(Figure4.1c).GPAdimension3describedonly5.1%of

thevariationbetweencattle,andwasdescribedbytermssuchas‘sore’,’frustrated’atthe

lowendand‘sleepy’,‘depressed’atthehighend.Head–lowering(rs48=0.36,P<0.05)to

groundwascorrelatedwiththehighvaluesonthisdimension(more‘sleepy’,’depressed’).

Quantitativebehaviourthatdidnotcorrelate(P>0.05)toanyoftheGPAdimensionsduring

castrationare:stand(still),ears(flick),producing(saliva)andeyes(widening).

For animals filmed after castration (exit crush), there were significant treatment group

differences for all three (3) GPA dimensions for the footage collected immediately after

castration(Table4.2b).OnGPAdimension1,observersdescribedcalvesintheCandCMpost

as the most ‘calm’, ‘relaxed’ compared with the other treatment groups, which were

describedasmore‘agitated’,‘anxious’(Figure4.1d).GPAdimension1explained50.1%ofthe

variationinscoringbetweencattle,withtermssuchas‘agitated’,‘anxious’associatedwith

thelowendoftheaxisand‘calm’,’relaxed’associatedwiththehighendoftheaxis.There

werecorrelationswithwalking(backwards)(rs48=-0.40,P<0.05)andrunning(rs48=-0.57,

P < 0.001) on this GPA dimension, with ‘agitated’, ‘anxious’ at the low end and walking

57

(forward)(rs48=0.57,P<0.001)atthehighendwith‘calm’,‘relaxed’.InGPAdimension2,

theCMpregroupweredescribedasmost‘watchful’,‘frightened’,whiletheothertreatment

groupsweredescribedasmore‘relaxed’,‘confused’(Figure4.1e).GPAdimension2(7.9%of

variabilitybetweencattle)wasdescribedbytermssuchas ‘relaxed’, ‘confused’associated

withthelowendoftheaxisand‘watchful’,‘frightened’associatedwiththehighendofthe

axis. There were no correlations between qualitative and quantitative analysis in GPA

dimension2.InGPAdimension3,CLAandCLMposttreatmentgroupsweredescribedasmost

‘affectionate’,‘comfortable’whiletheCandNCgroupsweremost‘angry’,‘annoyed’(Figure

4.1f).

GPA3describedonly5.4%ofthevariationbetweencattle,andwasdescribedbytermssuch

as‘angry’,‘annoyed’atthelowendand‘affectionate’,‘comfortable’atthehighend.Running

(rs48=0.38,P<0.005)wascorrelatedwithmore‘affectionate’,‘comfortable’termsatthe

highendwhileweight-shifting(rs48=-0.29,P<0.05)andurinate/defecate(rs48=-0.33,P<

0.05)werecorrelatedwith‘angry’,‘annoyed’atthelowend.Quantitativebehaviourthatdid

notcorrelate(P>0.05)toanyoftheGPAdimensionsaftercastrationare:stand,scrotalarea

grooming,hindlegstompingandtailswish.

ThedescriptivetermsthatshowedthestrongestcorrelationwitheachGPAdimension,for

eachsessionareshowninTable4.2.

4.7 4.7Discussion

Thisstudyusedbothquantificationandqualificationmethodstodeterminetreatmentaffects

forcattleundergoingsurgicalcastrationandwhetheranalgesiareducespainassociatedwith

castration. Results showed that in GPA dimension 1, observers could distinguish NC and

CLMpost from the restof thegroupduring castration.Cattle in those two (2) groupswere

58

describedasmore‘calm’and‘relaxed’inGPA1.Thisindicatedthatreceivingbothtypesof

analgesia (Lignocaine at the site and Meloxicam post-castration) may have significantly

reducedpain forCLMpostgroupthusmakingtheirbehaviour like theNCgroup.OnGPA1,

observersdescribedCandCMpostcattletobemore‘calm’and‘relaxed’astheyexitedthe

crush,post-castration.Thiswassurprisingasweexpectedthesecattletobeshowingvisible

signsofpain,likeaggression.Thesecattleweredescribedasmorerelaxedandcalmerperhaps

duetotheirslowermovementbecauseofthepain,whichalsocorrelatedwiththewalking-

forward behaviour in the quantication behaviour list. NC, CLA, CLMpost, CMpre and CMpost

groupsweredescribedasmore‘agitated’and‘anxious’thantheCgroup,whichcorrelated

withwalking backwards and running behaviour. Thus, in both scenarios, observers could

differentiatecattlethatwereexperiencingthemostpainfromthosethatmayhavebeenin

reduced, or nopain. This proved that analgesia (CLMpost) can reducepain anddiscomfort

associatedwithcastration,butthetermsusedtodescribecattlebehaviourmaynotalways

haveadirectmeaningandneedtobecarefullyanalysed.

QBAresultsobtainedfromthisresearchindicatethattheremaybeanalleviationofsome

painanddiscomfortbyprovidingacombinedpainreliefmethod.Beingabletoreducesome

unpleasantstimulitothesepreyanimalsiscrucialasnotonlydoesitimprovetheirwelfare

outcome,butalsomakes thestockperson’s jobeasier.Past studieshaveshownthat local

anaesthesiaremindseffectivefortwo(2)tothree(3)hourspost-administered(McMeekanet

al.,1998a;McMeekanetal.,1998b)andNSAIDsformuchlonger(uptoforty-eight(48)hours

post-administration (Dumka and Srivastava, 2004)) following a procedure (Weary et al.,

2006).Bycombiningthesetwo(2)typesofpainrelief,itwouldprovideamoreeffectivepain

control method. Administering Meloxicam prior to castration, may benefit more than

administeringpost-procedure.Staffordetal. (2002)conductedvariouscastrationmethods

59

usingsimilarpainreliefprotocolsandfoundthatwhenlocalanaestheticandketoprofenwere

givenbeforecastration,thecortisolresponsetoallmethodswasvirtuallyeliminated,being

likethatofcontrolcalves.Peakplasmacortisolconcentrationwasthirty(30)minutespost-

surgicalcastrationaccordingtoStaffordetal.(2002) whichwassupportedbyDumkaand

Srivastava(2004), inthattheminimumtherapeuticplasmaconcentrationforMeloxicamis

maintained from thirty (30) minutes to forty-eight (48) hours of being administered.

Therefore,bygivingananti-inflammatorypriortocastrationmaybemorehelpfulwithpain

control. However, in this study, CMpre group (GPA dimension 1) post-castration were

describedas‘agitated’,’anxious’,‘restless’.Whichweresimilardescriptiontothatofother

treatmentgroupsexceptforCgroup. Cwastheonlygroupdescribedas ‘calm’, ‘relaxed’,

‘comfortable’thatmostlikelyindicatedtheyweresorehencemovingslowly.,

Duringcastration,calvesthatreceivedthecombinedpainreliefshowedsimilarbehaviourto

thatofNCgroup,beingdescribedas‘calm’and‘relaxed’.However,aftersurgery,calvesthat

receivedapainreliefprotocolweredisplayingamore‘active’typeofbehaviour,thantheC

group.Painanddiscomfort,especiallyinthegroinregion,candetercalvesfrommovingtoo

quickly therefore, those in the C group were less active. Being described as ‘calm’ and

‘relaxed’maynotnecessarilymeanthattheywerecomfortable,butrathermovingslower

due to pain. Therefore, how we interpret these terms is important as one needs to

understand the bigger picture rather than drawing direct conclusions. By performing

quantitative analysis, it validated howobservers described the cattle and validated those

terms.Forexample,whencalvesweredescribedas‘agitated’and‘anxious’,therewerealso

morewalking(backwards)andrunningtypeofbehaviour,whichcorrelatedwiththeterms

used.PerformingtimebudgetanalysismayhelpwithclarifyingQBAtermuse.Forexample,

during castration,QBA terms suchas ‘agitated’, ‘restless’ and ‘frightened’ correlatedwith

60

behavioursuchas,stand(struggling),headshaking/strugglingandheadloweringtoground.

QBA terms described the emotional state of the animal whereas time-budget analysis

describedthephysicalstateoftheanimalattheverysamemoment.Therefore,thecombined

analysismadethescenariomuchclearer.

However,someresultswereunexpectedanddidnotappeartosupporttheuseofpainrelief.

Forexample,thebehaviouraldisplayofCcalveswereinterpretedsimilarlytothatofCLMpost

andNCcalvesinGPAdimension2and3,duringandpost-castration.Duetotheirnatureof

beingapreyspecie,cattlebehaviourcanbehardtodistinguishsoitiscrucialnottooverlook

anyambiguousbehavioursbuttocarefullyinterpretthemtodeterminewhysuchbehaviour

wasondisplay.Standing, lyingandwalking forwardcanbedifficult to interpret regarding

pain,butareluctancetomoveisknowntobeindicativeofpain(Molonyetal.,1995;Stafford

andMellor,2005a).Hence,mostofthecattledisplayedanaturalfearresponse,andranout

ofthecrushuponbeingreleased,includingthosethathadnosurgery,butthosethatwerein

themostpain(C),maskedthatpainsomewhat(weredescribedas‘calm’)andonlywalked

outslowly.

Molonyetal.(1995)reportedthatinsurgicallycastratedcalves,standingimmobileisapain-

relatedresponse.Capturinganimalbehaviourbothimmediatelyafterapainfulprocedureas

wellaslaterpointwillenableresearcherstocomparedifferencesintheirfindingsandbea

betterwaytoaccomplishaccurateresults.Post-operativepain,occurringhoursanddaysafter

the procedure, is less often treated (Walker et al., 2011) as the focus sometimes is

immediatelyafter theprocedure.Due to thenumberofanimalsona farm, it canalsobe

difficultforstockmentoidentifythoseinpainastheymaybewellhiddenamongsttheirherd

mates.Ontheotherhand,abnormalbehaviourcanalsobehardtoidentifyasanimalsare

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oftenrestrainedduringsurgery,makingitdifficulttoseeorinterpretanybehaviourresponses

(Johnson et al., 2005). There are various studies thatmonitored cattle for days following

castration, especially with bloodless castration such as using Burdizzo or rubber ring

techniques(Thüeretal.,2007),asthesemethodstakemuchlongertimeforthespermatic

cords to retractoratrophy.However, insurgicalcastration,eventhoughtheprocedure is

quick,theintensityofpainanddiscomfortandthetimeforwoundhealingareimportantand

theseanimals still requiremonitoringpost-procedure.Hence the importanceof capturing

their behaviour within the optimal time frame (example; seventy-two (72) hours of

continuous monitoring) to be able to perform a detailed analysis. This will also enable

researcherstodifferentiatebetweenpainfulorfearfultypeofbehaviour.

Cattle that are unhabituated to handling, restraint andphysiologicalmeasurement for an

extensive period in the crush might also exhibit fearful behaviour that make it hard to

determinetreatmentdifferences.Toavoidtheseissues,itwouldbebeneficialtobothanimals

andresearcherstospendextratimehabituating(moredaysspentonthepropertypriorto

castrationwithregularhumancontact)andrestrictonephysiologicalmeasurementatatime.

4.8 Conclusion

UsingbehaviourasatoolforassessingpaininBosindicuscattlehasbeenuseful,butthere

weresomelimitations.QBAshowedpainassociatedwithsurgicalcastrationwaspresentin

thecalves.However,itcanbequitechallengingforobserverstodifferentiatebetweenpainful

and normal cattle behaviour. As Rutherford (2002) mentioned, pain is a complex

multidimensional phenomenon and the responses of animals to it are also complex. To

properlyassessbehaviouralchangesinducedbypain,anexperiencedobservermaybethe

onlymethodtrulycapableofcapturingthiscomplexity(Rutherford,2002).However,inthis

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study,wefoundthatinexperiencedobservers,thosenotexpertinthefieldofanimalsciences,

coulddetectcertainbehavioursthatarenotspecifictocattleassumedtobeinpain,using

terms such as ‘sore’, ‘uneasy’ or ‘in_pain’ to describe cattle. This was validated against

quantitative measures. Using a combination of qualitative and quantitative methods to

evaluatepaincouldproduceabetteroutcomeatunderstandingtheunderlyingmeaningof

behaviour. Behaviour is likely to be the most practical tool for assessing pain in clinical

situations (Hansenetal.,1997). It is themost convenient tool,because it isnon-invasive,

inexpensive and does not require special equipment. It can be used in conjunction with

physiological measurements to obtain the most accurate results or on its own where

physiologicalmeasurementsarenotavailable.

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4.9 Appendix

Table4.1Alistofbehaviourdevelopedforquantification(a)duringcastrationand(b)postcastration

(a).

(b).

Category Behaviour Description

States Stand(still) Stationary-torsouprightposition

Stand(hunched) Stationary–shoulderslightlylowered

Stand(struggling) Notstationary–torsomovingvigorously

Events Earsflick Movementofoneorbothears

Swallowingsaliva Visibleswallowingreflex

Producingsaliva Producingclearoralsecretion

Head(shaking/struggling) Vigorouslymovinghead

Eyeswidening Wideningofeyelids/scleravisible

Head(loweringtoground) Headbelowbrisket,almosttouchinggroundwhilestanding

Category Behaviour Description

States Walk(forward) Forwardlocomotion

Walk(backwards) Backwardslocomotion

Stand Nomovementofbody,stationary

Run Continuousmotionofforwardmovement

Events Scrotalareagrooming Lickingscrotalarea

Hindlegstomping Stompingofeitherhindfootonground

Weightshifting Liftingeitherfootwhilemaintainingbalance

Tailswishing Movementoftailfromsidetoside

Urinate/Defecate Excretionofbodilyfluid/solid

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Table 4.2 Termsusedbyobservers todescribequalitativebehaviour expressionof calves

duringandimmediatelyaftersurgicalcastration

TermsCorrelatedwitheachendoftheGPAdimensionaxisGPADimensions

Lowvalues Highvalues TreatmentEffect(MANOVA)§

(a)duringcastration1(66.2%)

Calm(8),Relaxed(5),

Contented(3),Comfortable,

Laid-back,Happy,Carefree,

Docile

Agitated(7),Restless(5),

Frightened(3),Frustrated(2),

Unsettled(2),

Anxious(2),Irritated(2),

Stressed(2),Uncomfortable

(2),In_pain,Hurt,Sore,

Disquieted,Excited,Aware,

Alert,Defensive,Bothered,

Cautious,Nervous,Afraid,

Scared

*Stand(struggling)

*Headshaking/struggling

*Headloweringtoground

F5,95=46.35,P<0.0001

2(10.5%)

Playful,Uneasy,Timid

Trapped,Sad,Inpain

Tired

*Stand(hunched)

Unsure

*Swallowingsaliva

F5,95=5.98,P<0.0001

3(5.1%) Sore,Frustrated,Anxious,

Listening,Impatient

Sleepy(2),Depressed,Tired

*Headloweringtoground

F5,95=14.90,P<0.0001

(b)post-castration1(50.1%) Agitated(4),Anxious(4),

Restless(3),Nervous(2),

Frightened(2),Excited(2),

Startled,Energetic,Stressed

Alert,Irritated,Anxious,

Scared,Annoyed,Disturbed

Tensed,Edgy

*Walk(backwards)

*Running

Calm(5),Relaxed(3),

Comfortable(2),Contented,

Chilled,Settled,Quiet,Happy,

Patient,Aimless

*Walk(forward)

F5,145=21.91,P<0.0001

2(7.9%) Relaxed(2),Confused,Tired,

Calm,Lonely

Watchful,Frightened

Amused,Sociable

Unsure,Angry

F5,145=4.89,P<0.0001

3(5.4%) Angry(3),Annoyed(2),

Inquisitive,Uncomfortable,

Anxious,Unpleasant

Aggressive,Confused

Discontented,Restless,

Stressed

Affectionate,Comfortable

Playful,Motivated

Excited

*Running

F5,145=4.87,P<0.0001

65

GPADimensions

Lowvalues Highvalues TreatmentEffect(MANOVA)§

*Weightshifting

*Urinate/Defecate

GPA=GeneralisedProcrustesAnalysis

†ThepercentageofvariationexplainedbyeachGPAdimensionisshowninbrackets.

‡Termsthathad75%ofthemaximumabsolutecorrelationvalue(Mardiaetal.,1979)are

shownforeachendoftheGPAdimensionaxis.Termsorderisdeterminedfirstbythenumber

of observers to use each term (in brackets if >1), and second byweighing of each term.

*indicates time budget categories behaviour that significantly correlated with the GPA

dimensionscores(*P<0.05);shownoneitherleftorrightcolumn.

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Session(a)duringcastration Session(b)post-castration

(a).

(d).

(b).

(e).

(c).

(f).

Figures4.1(a-f).ComparisonofQualitativeBehaviouralExpressionduringcastration(a,b,c)

andpostcastration(d,e,f).

NOTE:Letterslinktreatmentgroupsthatwerenotsignificantlydifferenttoeachother.

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5 Qualitative Behavioural Assessment to distinguish surgically

castratedcattlefromnon-castratedandanalgesiacontrols

5.1 Abstract

Therearevariouswaystoperformcattlecastration,butsurgicalcastrationisadoptedbymost

practitioners.Althoughitiswelldocumentedthatsurgicalproceduresinflictpain,analgesic

use is routinely omitted in surgical castration in food animals. This study compares the

behavioural responses of castrated cattle (C) and non-castrated (NC) controls with those

castratedandgivenlignocaineandmeloxicam(CLMpost)inthecontextofthehomepaddock

andfeederyard.Eight(8)Brahmanbullcalvesineachtreatmentgroupwerefilmedinthe

morningpre (day -1) andpost (day +1) castration in thepaddock and feeder yard.Video

footagewasshowntovolunteerobserversforanalysisoverfoursessionsusingQualitative

BehaviourAnalysis(QBA).UsingFreeChoiceProfilingmethodology,theconsensusprofileof

the variation among observers for the analysis explained 37.4% and 40.6% for paddock

sessionsand34.7%and38.7%forfeederyardsessions.ThereweresignificantTreatmentx

DayinteractioneffectsforGPAdimension2(P=0.007)and3(P<0.001)forthepaddock;and

GPAdimension1(P=0.004)and2(P=0.025)forfeederyardsessions.Inthepaddock,calves

inCgrouponDay-1,GPA2weredescribedasmore‘happy’,‘contented’and‘relaxed’than

NC,andonDay+1calvesinCgroupweredescribedasmore‘lethargic’,‘disinterested’and

‘bored’thanNC.InGPA3,onDay-1calvesinCgroupweremore‘curious’,‘itchy’and‘lonely’

thanNC,butweremore‘alone’onDay+1thanNCgroup.Intheyard,calvesinCgroupon

Day-1,GPA1weredescribedasmore‘agitated’,‘anxious’and‘nervous’thanNC,andonDay

+1calvesinCgroupweredescribedasmore‘calm’,‘relaxed’and‘comfortable’thanNC.In

GPA2,andonDay-1calvesinCgroupweredescribedasmore‘relaxed’,‘confused’and‘tired’

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thanNCbutwereless‘watchful’,‘frightened’and‘amused’onDay+1thanNC.Quantitative

behaviourmeasureswererecordedinthepaddockusingCompositebehaviourscoring(CBS).

Therewas somecorrelationbetweenqualitativeandquantitativemeasuresofbehaviour;

ruminatinghadsignificantcorrelationwithGPAdimensions3(paddock)andGPA2(feeder).

QBAshowedthereweresignificantdifferencesbetweentreatments;Cwerelessactivethan

NCandCLMpost.ThisstudydemonstratesthatBosindicusbullsarelikelytobenefitfromthe

administrationofanalgesiaatthetimeofcastration.Thestudyhighlightsthecomplexities

andchallengesofidentifyingpainresponsesinBrahmancattle.

Keywords: Cattle, Castration, Analgesia, Pain, Qualitative Behavioural Analysis (QBA),GeneralisedProcrustesAnalysis(GPA)

69

5.2 Introduction

Castration ofmale calves is a routine husbandry procedure that is commonly performed

withoutanalgesiaaroundtheworld(LomaxandWindsor,2013).Itisconsiderednecessary

foreconomic,safetyandmanagementreasons(LomaxandWindsor,2013).Therearethree

(3)commonmethodsofcastration(Phillips,2010):1)surgicalremovaloftestes,2)applying

arubberringaroundthescrotumtocutoffthebloodsupplytothetestes,and3)crushing

thespermaticcordwithaBurdizzoinstrument(Phillips,2010).Surgicalcastrationisthemost

common method used in Australia and the USA, making up 60% and 57% of castration

procedures,respectively(Coetzeeetal.,2010).

Ithasbeenwell-documentedthatcastrationispainful(Molonyetal.,1995;Fisheretal.,1996;

Petherick,2006;Coetzee,2013a).Nevertheless,thereisnolegalrequirementforproviding

analgesia for castrationof cattle under 6monthsof age in or under 12months if at first

muster,accordingtothecurrentAustralianCodeofPractice(PISC,2004).IntheUSA,there

norequirementforpainmanagementforcattlecastration,andtherearenoanalgesicdrugs

approvedforpainrelief incattle(FDA,2015).Withtheincreasingpublic interestinanimal

welfareandproduction,itissuggestedthatfurtherrefinementofsuchacommonprocedure

iswarranted.

Paincansubstantiallyreduceanimalwell-beingandprolongthetimeneededforrecovery

fromtheunderlyingcondition(Hellyer,1998;MuirandWoolf,2001).Protractedpainresults

whenanalgesiaisnotusedforsurgicalprocedures,suchascastrationanddehorning(Molony

et al., 1995; Stafford andMellor, 2005a). Some veterinary practitioners who do not use

analgesiabelievethatyounganimalsgenerallydonotrequireanalgesiaforroutineelective

surgeriessuchascastrationordehorning(Hewsonetal.,2007a;b).However,studieshave

70

shownthatnomatterwhichmethodwasusedtheseproceduresstillcausesprotractedpain

lastingseveraldaystoweeks(Hayetal.,2003;StaffordandMellor,2005a).

Perioperativeanalgesicsareregularlyadministeredtocompanionanimalssuchascatsand

dogs,butnotforlivestock.Hewsonetal.(2007a)surveyedlivestockveterinarypractitioners

whomostlyagreedthatthereisnolong-acting,costeffectiveanalgesicavailableforusein

livestockandthelongorunknownwithdrawalperiodsofsomedrugsoutweighedthebenefits

of using them. Analgesic agents available for livestock use aremainly non-steroidal anti-

inflammatoryanalgesicdrugs(NSAIDs)suchasFlunixin,Meloxicam,andKetoprofen.Procaine

andLignocainearethemostfrequentlyusedlocalanaesthetics(HuxleyandWhay,2006).It

wouldbebeneficial touseanalgesiaon livestockduringpainfulhusbandryprocedures for

theirwelfare,anditservestokeeptheanimalstill,thusprovidingsafetyforthepractitioners

involved(Hewsonetal.,2007b).

Painwarnsananimalthattissuedamagemightoccur,isoccurringorhasoccurred,thereby

elicitingorallowingimmediateescape,withdrawalorotherbehaviour(Melloretal.,2000).

Painisthereforeanimportantaspectofhowanimalsprotecttheirbodyandmaintaintheir

health.However,thereisalsoselectionagainsttheobviousexpressionofpaininpreyspecies,

whichmightsinglethemoutasvulnerable.Consequently,thepresenceofpainincattlemay

bedifficulttodetermineorquantify.Fitzpatricketal.(2002)indicatedthatcattlepractitioners

wouldfinditusefultohaveaformalmethodofassessingpaininpractice,andseveralprevious

studieshaveusedbehaviouralresponsesasanindicatorofpainassociatedwithcastration

(Thüeretal.,2007;Pethericketal.,2014).

Onemethodofassessinganimalbehaviour is throughQualitativeBehaviouralAssessment

(QBA),whichusesawhole-animalmethodologytoassessestheexpressivequalitiesofanimal

71

demeanour,usingdescriptorssuchas“content”,“relaxed”or“anxious”(Wemelsfelderetal.,

2000;Wemelsfelderetal.,2001).QBAusesaFree-ChoiceProfiling(FCP)methodologythat

reliesonhumanobserversgeneratingdescriptorsorafixedlistoftermsprovidedtoobservers

toscoretheanimals(Wemelsfelderetal.,2001).

TheaimofthisstudywastodetermineifBosindicuscattleexhibitpaininbehaviourwhen

undergoingsurgicalcastration,withandwithoutanalgesia.Wecomparedthebehavioural

expression of cattle exposed to different analgesic regimes, using video footage of bulls

collected in paddock and in the feeder-yard pre (Day -1) and post-castration (Day +1).

Specifically,wecompared:

(i) Cattlecastratedwithoutanalgesia(C)withcattlethatwerenotcastrated(NC)

(ii) Cattlecastratedwithoutanalgesia(C)withcattlethatreceivedlocalanaesthesia(LA-

Lidocaine)andanon-steroidalanti-inflammatorydrug(NSAID–Meloxicam)(CLMpost)

Eachtreatmentgroupcontainedeight(8)Bosindicusbullcalves(n=8).Videofootagewere

showntovolunteerobservers forscoringusingQBA.Wepredicted thataftersurgery, the

cattle castratedwithout any analgesia (C) would showmore pain related behaviour than

cattlenotcastrated(NC)andthencattlegivenanalgesia(CLMpost).Inaddition,weexpected

castratedcattlewouldshowanincreaseinthefrequencyofpainrelatedbehaviourstheday

aftercastrationwhencomparedtothedaybefore.

5.3 Methods

ThisstudywasapprovedbytheAnimalEthics(PermitNumberR2551/13)andHumanEthics

(PermitNumber2008/021)committees,MurdochUniversity,WesternAustralia.

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5.3.1 Animalsandvideorecording

Forty-eight(48),six(6)toeight(8)month-oldBosindicus(Brahman)bullcalveswithamean

weightof165.5(±17.5)kgweresourcedfromanextensivecattlestationinthenorth-west

regionofAustralia(Pilbararegion).Theywerenotusedtocontactwithhumansandwere

transportedtoMurdochUniversityfarm,SouthStreet,WesternAustraliainwinter2013for

thedurationoftheproject.Thecalveswereheldinthesamepaddockforeight(8)daysfor

acclimationpost-arrival,duringwhichtimetheyweremovedthrougharace,andthenheld

individually in a crush to be for habituation and sampling of other physiological baseline

measures.Thesemeasuresincludedbodyweight,pedometry,bloodcortisolandnociceptive

threshold testing andhavebeendescribedelsewhere (Laurence et al., 2016;Musk et al.,

2016).Eight(8)individualswererandomlyallocatedtoeachofthetreatmentgroups:

• Non-castrated (NC) calves were held in the crush for the same duration (five (5)

minutes)asthattakenforsurgicalcastration.

• Castrated(C)calvesweresurgicallycastratedwhilestandinginthecrush.

• Castrated with analgesia (CLMpost) calves were administered Lignocaine (2mg/kg,

Lignocaine 20, Ilium, Troy Laboratories, Glendenning, NSW, Australia) into each

testicle five minutes pre-castration, surgically castrated and then administered

Meloxicam subcutaneously (0.5mg/kg, Meloxicam 20, Ilium, Troy Laboratories,

Glendenning,NSW,Australia)immediatelyaftercastrationasdescribedinLaurence

etal.(2016).

CalveswereidentifiedbynumericalIDtagsintheirrightearsandnumeralsspraypaintedon

their rumps. Animals were filmed daily using a handheld digital Panasonic SDR-H250

camcorders intwocontexts.Eachwasrecordedfortwo(2)minutesfirstly, inthepaddock

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(07:00to08:00)whentheywereundisturbed,andagainatthetimeofmorningfeedingina

smallyard,nearthefeeder(11:00to13:00)aftertheyhadbeenmovedthroughtheracefor

handling.Binocularswereusedtoalloweaseofidentificationofanimalsinthepaddock.One

(1) observer recorded quantitativemeasures of behaviour pre-and post-castration in the

paddockasdescribedinchapter3.

Table5.1Treatmentgroupsandlocationsusedinthestudy.

Sessionseries: Treatmentgroups: Location: Numberofobservers:

(A). • CvsNC

• CvsCLMpost

Paddock(Day-1&+1) 20

(B). • CvsNC

• CvsCLMpost

Feeder(Day-1&+1) 30

5.4 QualitativeBehaviouralAssessment(QBA)

5.4.1 Observers

Detailsregardingobserverscanbeobtainedfromsection2.4.2above.

ObserverswererecruitedbyadvertisementsonnoticeboardsaroundtheMurdochUniversity

campus,andbyusinguniversitysocialmedia;andincludeduniversitystudents,staff,primary

producersandthegeneralpublic.

Videofootagewasrecordedandedited(AdobePremierProCS3andAdobeAfterEffectsCS3,

Chatswood,NSW,Australia)intoforty(40)tofifty(50)secondclips.Two(2)seriesofviewing

sessionswereheldinseparatemonths;eachseriescomparedtwo(2)pairsoftreatmentsin

either the paddock or feeder yard location (Table 5.1). All observers were naïve to the

experimentaltreatmentsorthecontextinwhichtheanimalshadbeenfilmed.Eachobserver

attendedthree(3)viewingsessions,oncampus;atermgenerationsession(wherethescoring

process was explained, and terms generated) and two (2) video scoring sessions (where

74

observersscoredcattleintheclips).Inthefirstscoringsession,theobserverswereshown

clipsofthesixteen(16)calvesfilmedonday-1and+1fromtheCandNCgroups.Onthe

secondscoringsession,observerswereshownclipsfilmedonday-1and+1fromtheCand

CLMpostgroups.FreeChoiceProfiling(FCP)methodologywasusedforscoringthevideoclips

(Flemingetal.,2016),whichreliesonobserversgeneratingtheirownuniquesetofdescriptive

terms(Wemelsfelderetal.,2001).Observersattendedtermgeneratingsessionwherethey

wereshownaseriesofclipsthatshowedcattleexhibitingarangeofbehaviouralexpressions

andtheywereaskedtolistdescriptivetermsthattheybelieveddescribedthecattle.During

subsequentscoringsessions,eachobserverusedtheirownsetofuniquedescriptivetermsto

scorethebehaviourofthecattleusingavisualanaloguescalesetuponanExcelspreadsheet,

withobserversaskedtomarkonthescaletheintensityofthebehaviourexpressionforeach

oftheirdescriptivetermsrangingfrom0=minimumto100=maximum.Excelfiles(Microsoft

Excel2013,NorthRyde,NSW,Australia)foreachindividualobserverwerethenanalysedby

Generalised Procrustes Analysis (GPA) using a specialised software edition written for

FrancoiseWemelsfelder (GenStat,2008).AdetaileddescriptionofGPAprocedurescanbe

foundin(Wemelsfelderetal.,2000;Wemelsfelderetal.,2001).

GPAdevelopsaconsensusprofiledescribingallobservers’scorebytransformingallthedata

and identifying complex patterns. A Procrustes Statistic is calculated, quantifying the

percentage of variation between observers that is explained by the consensus. A

randomisationtest (DijksterhuisandHeiser,1995) is thenusedtodeterminewhetherthis

consensusisasignificantfeatureofthedataset,andnotsimplyanartefactoftheProcrustean

calculation procedures. The Procrustes Statistic is compared with the result of this

randomisationtestbyone-wayt-testtodeterminewhethertheactualobserverconsensus

profilefallssignificantlyoutsidethedistributionoftherandomisedprofiles(Flemingetal.,

75

2013).Subsequently,thenumbersofdimensionsoftheconsensusprofileifreducedthrough

PrincipleComponentAnalysis(PCA).

ThetermsusedbyeachobservertoscorecattlebehaviourwerecorrelatedwitheachGPA

dimension to identify terms that show the strongest correlations (>75% of the highest

absolutecorrelationcoefficientvalues)forthatdimension(Mardiaetal.,1979).Mixed-model

ANOVAwasused to test for significant treatmentdifferences in theGPAscores (StatSoft,

2007)withdayandtreatmentasfixedfactorsandobserverasarandomfactor.

5.4.2 QuantitativeAnalysis

Eachmorning (07:00-08:00h), behavioural scoringwas carried out in the paddock by one

observer for two (2)minutesperanimalonpre-andpost-castrationdaysusing the listof

behaviourdescribedinTable3.1(Chapter3).

5.5 Results

5.5.1 QualitativeBehaviouralAssessment(QBA)

Twenty(20)observersfromSeriesA(paddock)generatedninety-seven(97)uniqueterms;14

±5termsperobserver,(7-23).ThirtyobserversfromSeriesB(feederyard)generatedone

hundredthirty-seven(137)uniqueterms;15±5termsperobserver,(7-28).TheProcrustes

statisticindicatedthattheGPAconsensusprofileexplained34.65%(CvsNC)and38.72%(C

vsCLMpost)ofvariationamongobserversforpaddockanalysis,whichvariedsignificantlyfrom

themeanrandomisedprofile(t99=32.07;P<0.001)forCvsNCand(t99=45.12;P<0.001)for

CvsCLMpost.

Forthefeederyardanalysis,theProcrustesstatisticindicatedtheconsensusprofileexplained

40.56% (C vsNC) and 37.37% (C vs CLMpost) of the variation among observers for feeder

76

analysis,whichvariedsignificantlyfromthemeanrandomisedprofile(t99=32.07;P<0.001)

forCvsNCand(t99=45.12;P<0.001)forCvsCLMpost.

5.5.2 Terms

ThelistoftermsassociatedwitheachoftheGPAaxesisshowninTable5.2(paddock)and

Table5.3(feederyard).ForthepaddockcontextinCvsNC,themostfrequentlyusedterms

forGPAdimension1wererelaxed/calmonthelowendandcurious/alertonthehighend

oftheaxis.ForGPA2,happy/contentedwerethemostcommontermsonthelowendand

bored/lethargiconthehighend.ForGPA3,curious/itchywereonthelowendandalone

onthehighendoftheaxis.InCvsCLMpost,comparison,themostfrequentlyusedtermsfor

GPA1wereuncomfortable/sleepyonthelowendandhappy/relaxedonthehighendof

theaxis.ForGPA2,curious/alertwereonthelowendanddocileonthehighend.InGPA3,

friendly/tenderwereassociatedwiththelowendandcurious/awareonthehighendofthe

axis.

ForthefeederyardcontextinCvsNC,themostcommonusedtermsforGPAdimension1

were,agitated/anxiousonthelowendandcalm/relaxedonthehighendoftheaxis.For

GPA2,relaxed/confusedonthelowendandwatchful/frightenedonthehighend.ForGPA

3,angry/annoyedonthelowendandaffectionate/comfortableonthehighend.InCvs

CLMpost comparison, forGPA 1, alert / tensedwas associated on the low end and calm /

relaxedonhighend.ForGPA2,tired/sadonthelowendandhungry/alertonthehighend.

Finally,forGPA3,startled/unsurewasassociatedonthelowendandfreedom/comfortable

onthehighend.

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5.5.3 Treatmenteffects

ThepaddockanalysisshowedsignificanttreatmentbydayeffectforCvsNCinGPA2(F1,19=

9.01,P=0.007)and3(F1,19=33.45,P<0.0001),andforCvsCLMpostinGPA2(F1,19=4.69,P

= 0.043) andGPA3 (F1,19 = 26.91,P < 0.0001). For the feeder analysis, treatment by day

interactioneffectswereshownforGPA1(F1,29=10.01,P=0.004)and2(F1,29=5.57,P=

0.025)forCvsNCandonlyinGPA2(F1,29=12.95,P=0.001)forCvsCLMpostcomparison.

Castratedcattle(C)inthepaddockscoredsignificantlyhigherforGPAdimension2onday-1

thanday+1;cattlewerescoredasmore‘happy’and‘contented’onday-1andscoredmore

‘boredand‘lethargic’onday+1comparedwithNCcattle(Figure5.1A).ForGPAdimension3,

Ccattlewerescoredmore‘curious’onday-1andmore‘alone’onday+1comparedtoNC

cattle,whichscoredmore‘curious’onday+1.

Inthepaddock,CcattleinGPAdimension2weredescribedasmore‘bored’and‘unsure’on

day-1andmore‘docile’onday+1.ThiscomparedtoCLMpostcattlethatdescribedasmore

‘docile’onday-1andmore‘bored’and‘unsure’onday+1(Figure5.2B).InGPAdimension3,

Cwerescoredmore‘friendly’and‘tender’onday-1andmore‘curious’and‘aware’onday

+1comparedtoCLMpostdescribedasmore‘curious’and‘aware’onday-1,butmore‘friendly’

and‘tender’onday+1(Figure5.2C).

Inthefeederyardanalysis, forGPAdimension1,Ccattlewerescoredasmore‘calm’and

‘relaxed’onday-1andmore‘agitated’and‘anxious’onday+1comparedtoNC,thatwere

scoredasmore‘anxious’and‘agitated’onday+1comparedtoNCthatwerescoredasmore

‘startled’and‘agitated’onday-1butmore‘comfortable’and‘contented’onday+1.InGPA

dimension 2, both C and NC were described as ‘confused’ and ‘relaxed’ on day -1 and

‘watchful’and‘frightened’onday+1(Figure5.3B).

78

Inthefeederyard,forGPAdimension2,CandCLMpostcattlewerescoredasmore‘tired’and

‘sad’onday-1butonday+1,CLMpostcattlewerescoredasmore‘hungry’and‘alert’thanC

(Figure5.4B).

5.5.4 Correlationsbetweenquantitativeandqualitativebehaviour

RuminatingwastheonlybehaviourtosignificantlycorrelatewithanyGPAdimensionscore.

RuminatingcorrelatedwithGPAdimension3(rs32=-0.50,P<0.05)inpaddock(CvsCLMpost)

andGPAdimension2(rs32=-0.38,P<0.05)infeederyard(CvsNC)(Table5.3).

5.6 Discussion

Studieshaveshownthatcastrationofcattle ispainful,yet fewstudieshave lookedat the

behaviouralresponsesofunhandledBosindicuscattlepostcastration.Inthisstudy,CandNC

cattleweredescribedas‘lethargic’and‘disinterested’onthedayafterhandlingandsurgery

in the paddock, indicating therewas no difference between their behaviour, despite one

groupundergoingcastration.BothCandNCwereinlowmoodpost-surgeryascomparedto

beingdescribedas‘happy’and‘relaxed’thedaybefore.Beingapreyspecieswithlittletono

interactionwithstockpersons,mostlikelymeanttheoverallhandlingandsurgerymayhave

increasedtheirpre-existingfear.Ccattleinthefeederweredescribedasmore‘comfortable’

onday+1andhencelessactivethanNCcattle.Lessactivemovementscanindicatecattle

wereexperiencingsomepainfromthesurgicalprocedure,oritmightrepresentafearfulor

‘frozen’state.WhencomparedwithCcattle,painreliefappearedtoprovidesomecomfort

as CLMpost cattle displayed amore sociable behaviour, described as ‘friendly’ on day +1.

However,therewerealsotermssuchas‘tender’and‘uncertain’beingusedinthesameGPA

dimension3whichisquitedifferentfrom‘friendly’.Thisvariationintermusecouldbedue

toanindividualobserverhavingadifferentviewontheobservedcattlebehaviourhencethe

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variation.Inthefeederyard,onGPA2,bothCandCLMpostgroupswerescoredsimilarlyon

day-1,howeveronday+1,Cweredescribedasless‘hungry’andmore‘tired’and‘sad’than

CLMpost.

Thisstudyshowedthattypicalpainrelatedbehaviourweredisplayedfromcattleacrossall

three(3)treatmentgroups.Thiscanbeexplainedbythefactthatdomesticatedcattlewere

descendedfromwildcattle,whichwerepronetoattackbypredators,andthereforehave

strongevolutionarypressurethatallowsthemtomaskanysignsofpainasthismayimply

weakness(Phillips,2002).Hence,cautionisneededwheninterpretingbehaviouralresponses

incattle,anditsuggeststhatcattledescribedas‘quiet’or‘calm’maynotnecessarilybefree

frompain.Thisdisplayofbehaviourwasnotconsistentthroughoutallthree(3)GPAanalyses

aseventheNCcattleexpectedtobe‘calm’and‘happy’didnotconsistentlyshowquietand

calmbehaviour.Thiscouldbeduetocattlenothabituatedandstillveryreactivetohandling.

Paddock-born calvesweredifficult to handle, spending greater time running, attempts to

escapeandbehavingaggressivelytowardshandlerscomparedtocalvesbornindoors,which

weremucheasiertohandleastheyhadfrequentexposuretohumans(Pethericketal.,2009).

Thecalves’interactionwiththeirenvironmentandhumanswillreflectintheirbehavioural

expressionandisoneofthekeyaspectsofstudyinganimalbehaviour.

Fleming et al. (2016) stated that QBA can provide an assessment of the animal’s whole

responsetoitsenvironmentandwhatishappeningtoit.QBAthereforemeasures‘outcomes’,

andcancontributetowelfareassessmentbecauseitcancapturevariationinhowanimals

respondtoanddealwiththeirenvironmentatthatinstant(Flemingetal.,2016).Thisstudy

showedthatQBA,isaflexibleassessmenttoolasitcanbeappliedindifferentcontexts,such

asthepaddock,feederyardandincrush.

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Interestingly,notallbehaviourdisplayedbythecalvesthathadsurgerywasnegative.There

werevariousdescriptivetermsusedbyobserversdescribingcattleas‘excited’and‘chilled’

oneitherpre-orpost-castrationday.Thisdisplayofpositiveaspectsofanimalbehaviourcould

beexplainedbythefactthatthesecalveswerekeptwiththepresenceoftheirherdmates,

whichreducestheiremotionalresponse(Phillips,2010).Beingplacedinthefeederyardwith

otherswithfreshfoodpelletsdailymayhaveprovidedapositiveenvironmentaldistraction

forthem.Especiallyasmostwouldbehighlyengagedineating,finditpleasurableandsome

showsignsofcuriositywiththefeederitself.QBAisoneofthefewcurrentmeasuresthat

capturepositiveaspectsofanimalwelfare, suchasanimalsbeingpositivelyengagedwith

theirenvironment,includingbeingactive,oralert(Keelingetal.,2013).

Cattle subjected to painful procedures will always be a challenge for both clinicians and

handlersasrecognisingthesignsofpaininthesestoicalspeciesisdifficult(Fitzpatricketal.,

1999;Barrett,2004).Rutherford(2002)statedtheimportanceofdevelopingmethods,such

asobservationsofbehaviourandmeasurementofphysiologicalresponses,whichcanbeused

toassesspainobjectively.QBAcanbeappliedunderarangeofconditionsandcanidentify

subtledifferencesinqualitativebehaviouralexpression(Flemingetal.,2016).Bodylanguage

isdynamic,soitallowsQBAallowstocapturesubtlechangesinananimal’sbodylanguage

that can be important for welfare assessment and may otherwise be overlooked when

individualbehavioursareisolatedandquantified(Wemelsfelder,1997;Wemelsfelder,2007;

Meagher,2009;WhithamandWielebnowski,2009).

Termsthattheobserversusedappearedtomakesense.Atthefeederyard,observersviewed

calvesinCgroupasmore‘tired’and‘sad’butcalvesinCLMpostasmore‘hungry’and‘excited’.

Atfirstglanceand inrelationtothedescriptiveterms, itmayseemthatthetermsdonot

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clearly describe cattle behaviour appropriately.However, these termswere a generalised

viewofbehaviouraldisplayoftheanimalincontextwiththeenvironmenttheywereplaced

in.Hence, the term ‘hungry’was used to describe the body languagedisplayed as calves

movedaroundthe feedtrough.Ccalves thatweexpectedtobe inpain,couldhavebeen

describedastiredandsadastheyshowedlessenergyordesiretoseekfoodpellets.

Uponanalysisof theseterms,theyappeardescribewhattheanimalmaybefeelingwhen

comparingthetwotreatmentgroups.Thoseinthecastratedwithanalgesiagroup(CLMpost)

were viewed on a more positive aspect of behaviour, for example various studies have

demonstratedthatindicatorsofpain,suchas,behaviouralandphysiologicalresponses,are

significantlyreducedifNSAIDareadministeredaspartofthetreatmentprotocolforsurgical

castration(EarleyandCrowe,2002;Staffordetal.,2002;Tingetal.,2003a;Tingetal.,2003b).

HuxleyandWhay(2006)revealedthatadministeringMeloxicamtocattleduringdehorning

showedareductioninpainanddistress.Therefore,itisquiteclearthatbyprovidingsome

form of analgesia, it does benefit the animal’swell-being, and in return, a saferworking

conditionforthepractitionersandhandlersisprovided.Therearesomeconstraintsastowhy

analgesiaisnotroutinelygiventolivestock,suchasthecostandtypeofanalgesiaapproved

forcattleuse.

It is important thatwe do not disregard any behavioural display thatmay be deemed as

‘unusual’or‘inappropriate’forcattleafterperformingpainfulhusbandryprocedures.Cattle

maybemaskingfearorpainbehaviourfromobserversorotherspeciesandmayhavefeltthe

predatorythreatduetotheirinstinctivenatureofbeingapreyspecies.Anysubtlebehaviour

displayneedstobetakenintoconsideration,andstudiedatthewholeanimalleveltofully

understandwhattheanimalisdisplaying.

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Thelackofcleartreatmenteffectmaybeduetosmallsamplesizeofthisstudy.However,

therewereevidencethatanalgesiapossiblyalleviatedsomeofthediscomfortofcastration.

ThiswasonlypossiblethoughtheapplicationofQBAandneedstofurtherthisstudywith

largernumberofanimalstoseetheextentofhowprovisionofanalgesiamayprovidemore

positiveexperiencesforcattle.Welfareisanimportantfocusoflivestockproduction,which

means that mitigating pain from any routine procedure performed on these animals

throughouttheirlifespanshouldbedesiredgoalforall.

5.7 Conclusion

Thebehaviouraldisplayofcattlecanbedependentontheconditiontheyareexperiencing,

aswellasinfluenceofthestrongancestralrootsofwildcattle.Ittakescarefulobservation

andbehaviouralanalysistounderstandthesignificanceoftheirbodylanguage.Accordingto

our observers, some cattle behaviour such as, being calm, happy and excitable may be

described as somewhat similar even the day after they have gone through unpleasant

procedures. Some stockmen underrate the pain caused by castration, because of the

traditionaldiscountingofpaininlivestock(Byrneetal.,2001)andtheattitudethattheyare

‘justanimals’andpossiblyunabletoexperiencesuchnegativeemotions,thereforenotmuch

attentiontopainreliefhastraditionallybeenwasgiven.

QBAmayholdthekeyinbreakingdownbarriersbetweentheassumptionthatanimalsdonot

feelpainandimprovingwaystobetterunderstandanimalbehaviour.QBAcanbeusedonits

ownorinconjunctionwithotherphysiologicalmeasures,suchasbloodcortisollevels,and

pedometerandhasthepotentialtobecomeausefulassessmenttool.Thefutureofanimal

welfareandeducatingbothprofessionalsandthegeneralpublicontheassessmentofpain

willbeenhancedusingQBAmethodology.Inconclusion,theapplicationofQBAinthisstudy

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havebeenvaluableandhavegivenustheopportunitytolookintopainrelatedbehaviourin

cattle.

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5.8 Appendix

Table5.2Termsusedbyobserverstodescribequalitativebehaviourexpressionofcalvesinpaddockpre-andpost-castration(SessionA).

TermsCorrelatedwitheachendoftheGPAdimensionaxis‡

TreatmentEffect(MANOVA)§

TreatmentGroups

GPADimensions Lowvalues Highvalues Significant Non-significant

CastratedvsNon-castrated(CvsNC)

1(36.95%)†

Relaxed(5),Calm(5),Laid-backChilled,Contented,QuietApathetic,Satisfied

Curious(3),Alert(3),Agitated(3),Stressed(2),Restless(2),Anxious,Disquieted,Distressed,Timid,Frightened,Cautious,Inquisitive,Nervous,Weary,Scared,Unsettled,Defensive,Lost,Uncomfortable

DayF1,19=20.87,P=0.0002

TreatmentF1,19=2.87,P=0.1066TreatmentXDayF1,19=0.055,P=0.818

2(18.8%)† Happy(2),Content,Relaxed,Active

Bored(3),Lethargic(2),Exhausted(2),Uncomfortable(2),Disinterested,SleepyTired,Sore

DayF1,19=22.12,P=0.0001TreatmentXDayinteractionF1,19=9.01,P=0.007

TreatmentF1,19=0.17,P=0.68

3(9.9%)† Curious(2),Itchy(2),Lonely(2),Anxious,Cautious,Bored,Impatient,Weary,Hesitant,Trapped,Afraid,AgitatedConfused

Alone DayF1,19=10.60,P=0.004TreatmentXDayinteractionF1,19=33.45,P<0.0001

TreatmentF1,19=0.63,P=0.438

CastratedvsCastratedwithanalgesia

1(26.8%)† Uncomfortable(4),Sleepy(2),Agitated,

Happy(4),Relaxed(4),Contented(3),Calm,Excited,Aware

DayF1,19=44.02,P<0.0001

TreatmentF1,19=1.36,P=0.259TreatmentXDay

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TreatmentGroups

GPADimensions Lowvalues Highvalues Significant Non-significant

(CvsCLMpost)

Unsettled,Depressed,Timid,Tired,Unsure,DisinterestedIn_pain,Stressed,SadNervous

F1,19=1.50,P=0.235

2(17.9%)† Curious(6),Alert(2),Inquisitive(2),Bored,Unsure,Excited,Distressed,Aware,Frustrated,Weary

Docile TreatmentF1,19=11.82,P=0.003TreatmentXDayinteractionF1,19=4.69,P=0.043

DayF1,19=0.36,P=0.556

3(13.3%)† Friendly,Tender,Uncertain,Tired,UncomfortableSatisfied,NotafraidComfortable,Calm*Ruminating

Curious,Aware,Dominant TreatmentF1,19=6.23,P=0.022TreatmentXDayinteractionF1,19=26.91,P<0.0001

DayF1,19=0.82,P=0.375

GPA=GeneralisedProcrustesAnalysis†ThepercentageofvariationexplainedbyeachGPAdimensionisshowninbrackets.‡Termsthathad75%ofthemaximumabsolutecorrelationvalue(Mardiaetal.,1979)areshownforeachendoftheGPAdimensionaxis.Termsorderisdeterminedfirstbythenumberofobserverstouseeachterm(inbracketsif>1),andsecondbyweighingofeachterm.ItalicsindicatestimebudgetcategoriesbehaviourthatsignificantlycorrelatedwiththeGPAdimensionscores(*P<0.05);shownontheleft-handcolumnastheywerenegativecorrelatedwiththeaxis.§Summaryof themixedmodelanalysis (MANOVA) foreachGPAdimensionshowingsignificant/non-significantresults forDay,TreatmentgroupsorDayandTreatmentgroupsinteractions.

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Table5.3Termsusedbyobserverstodescribequalitativebehaviourexpressionofcalvesinfeederpre-andpost-castration(SessionB).

TermsCorrelatedwitheachendoftheGPAdimensionaxis‡

TreatmentEffect(MANOVA)§

Treatment

GroupsGPADimensions Lowvalues Highvalues Significant

Non-significant

CastratedvsNon-castrated

(CvsNC)

1(30.7%)† Agitated(4),Anxious(4),Nervous(2),Restless(2),Frightened(2),Excited(2),Startled,Energetic,Stressed,Alert,Irritated,Scared,Annoyed,DisturbedTensed,Edgy

Calm(5),Relaxed(3)Comfortable(2),Contented,Chilled,Settled,Quiet,Happy,Patient,Aimless

TreatmentF1,29=9.13,P=0.005TreatmentXDayinteractionF1,29=10.01,P=0.004

DayF1,19=1.655,P=0.208

2(16.3%)† Relaxed(2),Confused,Tired,Calm,Lonely*Ruminating

Watchful,FrightenedAmused,SociableUnsure,Angry

DayF1,29=20.07,P=0.0001TreatmentF1,29=26.23,P<0.0001TreatmentXDayinteractionF1,29=5.57,P=0.025

3(8.8%)† Angry(3),Annoyed(2),Inquisitive,Uncomfortable,Anxious,UnpleasantAggressive,ConfusedDiscontented,Restless,Stressed

Affectionate,ComfortablePlayful,Motivated,Excited

DayF1,29=15.99,P=0.0004TreatmentF1,29=21.10,P<0.0001

TreatmentXDayF1,29=0.07,P=0.792

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TreatmentGroups

GPADimensions Lowvalues Highvalues Significant

Non-significant

CastratedvsCastratedwith

analgesia(CvsCLMpost)

1(39%)† Alert(3),Tense(2),Agitated(2),Excited,FrightenedStressed,NervousRestless,AnxiousUncomfortableDominant,IrritatedOn_edge,TensedScared,Edgy

Calm(4),Relaxed(4),Contented(2),Settled,Chilled,Comfortable,Quiet,Enjoying

DayF1,29=7.78,P=0.009

TreatmentF1,29=0.04,P=0.838TreatmentXDayF1,29=3.41,P=0.075

2(15.1%)† Tired(7),Sad(6),Bored,LonelyRestless,LethargicDepressed,DrowsyDeflated,DisorientedSick,Lifeless

Hungry(2),Alert,Excited

DayF1,29=22.55,P<0.0001TreatmentF1,29=8.08,P=0.008TreatmentXDayinteractionF1,29=12.95,P=0.001

3(9%)† Startled,Unsure Freedom,ComfortablePlacid,Happy,RelaxedAgitated

DayF1,29=14.77,P=0.0006TreatmentF1,29=18.94,P=0.0001

TreatmentXDayF1,29=0.01,P=0.935

GPA=GeneralisedProcrustesAnalysis†ThepercentageofvariationexplainedbyeachGPAdimensionisshowninbrackets.‡Termsthathad75%ofthemaximumabsolutecorrelationvalue(Mardiaetal.,1979)areshownforeachendoftheGPAdimensionaxis.Termsorderisdeterminedfirstbythenumberofobserverstouseeachterm(inbracketsif>1),andsecondbyweighingofeachterm.ItalicsindicatestimebudgetcategoriesbehaviourthatsignificantlycorrelatedwiththeGPAdimensionscores(*P<0.05);shownontheleft-handcolumnastheywerenegativecorrelatedwiththeaxis.§Summaryof themixedmodelanalysis (MANOVA) foreachGPAdimensionshowingsignificant/non-significantresults forDay,TreatmentgroupsorDayandTreatmentgroupsinteraction.

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Figure5.1QBASessionA(paddock)–CvsNCcattleforeachGPAdimensions

Figure5.2QBASessionA(paddock)–CvsCLMpostcattleforallGPAdimensionsNOTE:Letterslinktreatmentgroupsthatwerenotsignificantlydifferenttoeachother

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Figure5.3QBASessionA(feederyard)–CvsNCcattleforallGPAdimensions

Figure5.4QBASessionB(feeder)–CvsCLMpostcattleforallGPAdimensionsNOTE:Letterslinktreatmentgroupsthatwerenotsignificantlydifferenttoeachother

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6 GeneralDiscussion

TheAustralianlivestockindustryisoneoftheworld’slargestandmostefficientcommercial

livestock industries and Australia is a major exporter of redmeat and livestock; with an

estimatedtotalof26.3millionheadofbeefcattle(ABS,2013-14).Outbackcattlestationsare

prevalent, where cattle graze free range over extensive areas with minimal handling by

stockmen. Usually, cattle only encounter stockmen once yearly in themustering season.

Hence,mostcattleareneithermonitoredcloselynorhabituatedtohumans.Therefore,even

forsimpleroutinehusbandryproceduressuchasvaccinationandear-tagging,cattlewillbein

a stressed and fearful state.Bos indicus cattle aremore excitable thanBos taurus cattle

(HearnshawandMorris,1984;Fordyceetal.,1988)andcattlerearedinextensivesystems

areoftenmoreaggressivecomparedwithcattlerearedinintensiveconditionsbecauseof

lessfrequentinteractionwithhumans(Fordyceetal.,1985).

Thecalvesinthisstudyappearedtoexpressfearwhendirectlyhandledbyhumans,during

castrationandthetakingofphysiologicalmeasurements.Cattlewithexcitabletemperaments

mayhavereducedfeedintakecomparedwithcohortswithlessexcitabletemperaments(Fox

et al., 2004; Nkrumah et al., 2007) and heightened stress-related physiological responses

comparedwithcalmercohorts(Burdicketal.,2011).Inaddition,thesecattlehaveheightened

inflammatoryandacute-phaseresponsesfollowingastressstimulus(Hulbertetal.,2009),

whichisnotagoodwelfareoutcome.Havingexperiencedthepainofsurgicalcastrationand

the stress of being restrained in the crush, their behaviour was likely to have been a

demonstrationofa fear response.Hence, certaindescriptive termsdidnotalignwith the

behaviourexpectedfromtheiranalgesicgroups.Forexample,abulldescribedascalmcould

bekeepingverystillthusmaskinganypainbehaviourjusttocopewiththefearfulsituation

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orenvironment.Thetermsusedtodescribecattleinthisstudyhadpolaroppositemeanings;

from displaying positive type behaviours such as ‘calm’, ‘relaxed’ to negative type of

behaviourssuchas ‘annoyed’,agitated’couldverywellbe theircopingmechanism.Some

situationsmadeunderstandingthecomplexityoftheirbehaviouraldisplayhardtodecipher.

QuantifyingcattlebehaviourusingCBSinthefirstpartofthestudy(Chapter3)didnotreveal

differencesinwhatthecalveswereexperiencingwhileinthehomepaddockorduringsurgery

in thecrush.Surprisingly, therewasalsonovariance in treatmentwhenQBAwasapplied

during surgery (Chapter 4). Thus, we believe that the stress and fear of handling these

previously unhandled cattlemight havebeen anoverwhelming factor for their emotional

state. Observers were not able to distinguish between analgesic treatment groups while

cattlewereinthecrush,eventhoughwedobelievethatcastrationwouldhavebeenpainful.

Ina studydonebyBarrettetal. (2016)onpain-relatedbehaviour incattle following liver

biopsy,therewerenosignificantdifferencesinbehaviourbetweenanytreatmentgroupsand

itwaspostulatedthatonlymildpainexisted,whichwasnotdetected.However,cattlemight

bemaskingpainduetotheirbehaviouralevolutionaspreyspecies,toavoidanydisplaysof

abnormalbehaviourthatwouldmakethemvulnerabletopredators(Livingston,2010).

Ascattleexited thecrush, immediatelyaftercastration, thereweredetectable treatment

differences.Thismightbeinterpretedthattherewasasenseofimmediaterelief(noforeign

objectblockingtheirpath)whenthecrushgatewasopened,andwiththereducedstressand

fear,cattlecouldexhibittheirexpressionofpainbehaviour.Inaddition,observersmightalso

be context sensitive as they might have viewed all cattle in the crush as in a ‘negative’

situation and being ‘restrained’’ unlike cattle outside the crush that had possibility of

‘freedom’.Byconductingacomparisonoffree-choiceprofilingvs.fixedlistforQBAClarkeet

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al.(2016b),coulddetermineifobserversratingwasinfluencedbycontext.Performinginter-

andintra-observerreliabilitytestingandcross-validationwithothermeasuresarecrucial,as

theyareforallanimal-basedhealthandwelfareindicators(Tuyttensetal.,2014).

Animal behaviour is determined by both the physical andmental state of an animal. An

animal’s emotional state can be highlighted by its behavioural display such as decreased

rumination,which is regarded as a general indicator of pain in cattle (Stafford, 2013), or

abnormalstanding,whichhasbeendocumentedasapain-relatedresponse(McMeekanet

al.,1999;Sylvesteretal.,2004;Almeidaetal.,2008;Kolkmanetal.,2010;Pethericketal.,

2014).However, fearmightbesodominatingthatcattleremainmotionless,whichmeans

somesubtlestressbehavioursaremissed.Someobserversmightinterpretthis‘frozenwith

fear’ stateas cattle ina calmstateandnotexperiencinganypain.Reluctance tomove is

indicativeofpain(Molonyetal.,1995;StaffordandMellor,2005b),andstandingimmobileis

also a pain-related response that was previously reported in surgically castrated calves

(Molonyetal.,1995).

Severalfactorsmaylimitourabilitytointerpretpainbehaviour;removingfearresponsesand

increasingtimepointsformonitoringmayassistwithbetterinterpretingtheirbehaviour.Our

resultsaftersurgery,showedthattheanalgesiacouldmitigatepainassociatedwithsurgical

castration.ThiswassupportedbyresultsfromacompanionstudyconductedbyLaurenceet

al.(2016)whichshowedadecreaseinbloodcortisolconcentrationandweightgainincattle

thatreceivedaformofanalgesia.Cattlethatreceivedmeloxicameitherpreorpostcastration

weremoreactivewithfewerrestboutsaccordingtothepedometerresultsinLaurenceetal.

(2016),whichwere similar to our findings of cattle that received the combined form of

analgesiaweredescribedasmore ‘calm’ and ‘relaxed’ like thatof thenon-castrated (NC)

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group. Nociceptive threshold testing (NTT) was performed on these cattle for another

companion study (Musk et al., 2016), but the results were inconclusive. However, NTT

decreased during the course of study in all groups (Musk et al., 2016). This showed that

habituationforthesecattleiscrucialespeciallytothehandlingprocess.Ifcattlewereexposed

toroutinehandlinglongerbeforecastration,forexamplehavingarrivedattheuniversityfarm

amonthpriortosurgerywithregularexposuretopeopleaswellasbeingputthroughthe

raceandcrush, thenthere isapossibility that theirbehavioural responsetosurgery (pain

relatedbehaviour)couldbemuchclearertointerpretratherthanbeingoverwhelmbyfear

orstress.Musketal.(2016)

Itwaspostulatedthatpainandassociatedstresswiththeinjectionoflignocaine,followedby

theanticipationofthatpainfuleventateachsamplingoccasion, wasenoughtomaskthe

beneficialeffectsofmeloxicam(Laurenceetal.,2016).Thiswassuggestedbytheambiguous

resultsseenwhencomparingthebehaviouralobservationsofcattleexpectedtoshownopain

(NC),mildpain(CLMpost)ormaximumpain(C).

Perhaps, a comparison between two (2) herds of cattle exposed to different stimuli in a

longitudinalstudyintheirbehaviouraldisplaymightenableonetousethelistofbehaviour

displayedasaguidelineforfuturestudies.Forexample,observingcattlethathadnumerous

painfulorunpleasantprocedureswhileanothergroupofcattlewerehandled,withoutany

pain involved. Therefore, when pain relief is provided there might be a possibility to

distinguish differences in the behaviour of cattle experiencing painful stimuli. Another

important factorwhich couldbe corrected in future studies is toobserve asmuchof the

animalbodyratherthanjusttheheadorpartsofthebody,asthislimitviewinganglesfor

observerswhichcouldresultinincorrectjudgements.QBAisthestudyofthewholebodyand

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by applying QBA only on the head region will not make use of its full potential as an

assessmenttool.

Usingbehaviourassessmenttostudypainispracticalasitiscosteffectiveandnon-invasive,

contrary to physiological measurements. Pain is subjective and can only be measured

indirectly;yetusingbehaviour isamoresensitiveindicatorofpainthancortisolandother

physiologicalmeasures (Anil et al., 2002;Heinrich et al., 2009).QBAwasused to test for

effectsofanalgesiaandtovalidateitsaccuracybycomparingtwocontrolgroups(Cversus

NC)whileinpaddockandfeederyardforChapter5.IthighlightedthatBosindicusbullswere

likely to benefit from the use of analgesia and proved that QBA methodology for pain

assessment is useful.However, anybehaviour studies onprey species such as unhandled

cattlerequireseverallargersamplesizestoconfirmaccuracyoftreatmentspriortoon-farm

application.

The lackofaconsistentsignificanteffectbetweentheMeloxicam,Lignocaineandcontrol

groupsmaybecausedbylackofpower.ItisfeasiblethateffectiveplasmalevelsofMeloxicam

werenot present at the start of surgery, though further pharmacokinetic information for

MeloxicamuseinBosindicuscattlemustbegatheredtofullyunderstandthis.Arelatively

smallsamplesizecanmakedetectingsignificantdifferencesdifficult,particularlyinstudies

using largeanimalsaswehave,asoftenresourcesarerestrictedby logistics, facilitiesand

financialconstraints.

6.1 GeneralConclusion

QBAisarelativemeasurethatiscapableofdetectingsubtledifferencesinthebehavioural

expressionsofanimals(Flemingetal.,2016)andhasproventobeapplicableinseveralother

behavioural studies such as sow housing (Clarke et al., 2016a), sheep and cattle road

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transportation(Stockmanetal.,2011;Wickhametal.,2012),pigletweaning(Lauetal.,2014),

andcattletemperament(Stockmanetal.,2012).QBAcanalsobeusedtomeasurehandlers’

behaviour(Ellingsenetal.,2014),whichlookedatdairycalvesandtheirhandlers.Therefore,

it has the potential to be useful in assessing painful behaviour inBos indicus cattle since

resultsfromourstudyaswellasofLaurenceetal.(2016)foundpossiblebenefittowardsthe

welfareofthesecattlewhenanalgesiawasprovided.

Suggestions for improvement include having a larger sample size, longer periods of

observations with more time-points when monitoring cattle behaviour, less number of

treatmentgroupsandlongertimespenthabituatingthecattleonthepremisespriortoany

procedure.BeinghabituatediscrucialasMusketal.(2016)foundadecreaseinNTTovertime

andLaurenceetal.(2016)reportedtherewereevidenceofreducedcortisolconcentrations

post-operatively, increased activity and favourable weight gain over time regardless of

treatmentgroup.Givenplentyoftimeandresources,continuingtorecordandobservecattle

behaviourwellaftertheirhabituationperiodmighthavebenefitedthestudy.Wewouldbe

abletodrawaclearerconclusiononwhytheybehaveacertainwayaswellasachanceto

comparebeforeandafterhabituationbehaviours.Bymakingtheseadjustments,itishighly

likely to obtain desirable results that canbe translated into a useful on-farmbehavioural

assessmenttool.

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7 Appendices

7.1Behaviourscoringinpaddock(CompositeBehaviouralScoringchart)

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7.1 Post-graduatePosterDayNovember2013

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7.2 BehaviourConference-CairnsAugust2015

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7.3 QBAFlyer

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7.4 ParticipantInformationLetter

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7.5 InstructionSheet–ForQBAtermgenerationsession

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7.6 InstructionSheet–ForQBAviewingsessions

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7.7 QBAobserverssurveypart1

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108

109

110

111

112

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7.8 QBAobserversurveypart2

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116

7.9 QBAobserversurveypart3Aand3B

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