ContentslistsavailableatScienceDirect
Applied
Animal
Behaviour
Science
jo u r n al ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / a p p l a n i m
Effects
of
multiple
daily
litter
applications
on
the
dust
bathing
behaviour
of
laying
hens
kept
in
an
enriched
cage
system
Hye-Won
Lee
a,1,
Helen
Louton
a,
Angela
Schwarzer
a,
Elke
Rauch
a,
Amrei
Probst
a,
Shuai
Shao
b,
Paul
Schmidt
c,
Michael
H.
Erhard
a,
Shana
Bergmann
a,∗aChairofAnimalWelfare,Ethology,AnimalHygieneandAnimalHusbandry,DepartmentofVeterinarySciences,FacultyofVeterinaryMedicine,LMU
Munich,Veterinärstr.13/R,80539Munich,Germany
bStatisticalConsultingUnit,DepartmentofStatistics,LMUMunich,Akademiestr.1,80799Munich,Germany cStatisticalConsultingforScienceandResearch,Jessnerstr.6,10247Berlin,Germany
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received28May2015
Receivedinrevisedform2February2016 Accepted7February2016
Availableonline27February2016
Keywords:
Chicken Animalwelfare Dustbath
a
b
s
t
r
a
c
t
Conventional‘battery’cagesforlayinghenswithoutperches,nestsandlitterareashavebeenbanned bylawthroughouttheEuropeanUnionsince1January2012.Asanalternativesolution,enrichedcage systemswereintroduced.Ouraimwastoinvestigatehowmanyapplicationsoflittersubstrateperday arenecessarytomotivatelayinghenstoperformdustbathingbehaviour,andtowhatextentthehensuse theseofferedlitterareasinaspecies-appropriatemanner.Eachofthetwoconsecutiveexperimentswas conductedfor12months,duringwhich20(experiment1)and40(experiment2)layinghensofthestrains LohmannSelectedLeghorn(LSL)andLohmannBrownClassic(LB)werehousedin10unitsoftheenriched cagesystemHÜK125/80(2hens/unit[(experiment1],4hens/unit[experiment2],samestrainperunit). Indefineddailyapplicationfrequenciesfromonetofourtimes(11:00a.m.,1:00p.m.,3:00p.m.and5:00 p.m.)overthelayingperiod,50gconventionalfeedperapplicationwereappliedaslittersubstrateonto each925cm2sizedmat.Thehenswererecordedweeklybydigitalvideosystems.Resultsshowedthatthe meandurationofdustbathingbehaviourlasted05:58mininexperiment1and04:59mininexperiment 2.Litterapplicationfrequencyhadasignificanteffectonthedustbathingdurationaswellinexperiment 1(P<0.05)asexperiment2(P<0.01).Forbothexperimentsthenumberofdustbathsincreasedwith themaximumnumberoflitterapplicationsperdayandthislineartrendissignificantforexperiment 2(P<0.001).Ahighpercentageofinterruptedandearlyterminateddustbathingboutswereobserved, mostlyinducedbyaconspecific.Themeaninterruptiondurationwas00:18minforexperiment1and 00:45minforexperiment2.Thedailylitterapplicationfrequencyhadnosignificanteffectontheduration ofinterruptionsforneitherexperimentbutonthenumberofinterruptions.Accordingtotheresultsof thisstudy,litterapplicationsatleasttwiceadaycanberecommended,andsufficientlysizeddustbathing matsshouldbeofferedtolayinghenshousedincagesystems.Evenwithgenerouslyprovidedresources, thehensshoweddeficitsinspecies–specificbehaviour.
©2016TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Following the Council Directive 1999/74/EC (European Commission,1999),thehousingoflayinghensinunenrichedcage
∗ Corresponding author at: Chair of Animal Welfare, Ethology, Animal Hygieneand Animal Husbandry,Veterinärstr.13/R, 80539 Munich, Germany. Fax:+4989218078333.
E-mailaddresses:[email protected](H.-W.Lee),[email protected] (S.Bergmann).
1 Presentaddress:ChoonghyunAnimalHospital,GangnamguNonhyunro621, Seoul,Korea.
housingsystems(batterycages)wasprohibited asof1 January 2012throughouttheEuropeanUnion.Forenrichedcagesystems thatwereapprovedorinstalleduntil13March2002,atransition perioduntiltheendof2020wasprovidedbytheGerman‘Orderon theProtectionofAnimalsandtheKeepingofProductionAnimals’ (German designation: Tierschutz-Nutztierhaltungsverordnung, 2006). Enriched colony housing systems were accredited as alternative housing systems, and the requirements nowadays include,amongstothers,alitterareaforscratchingandpecking. AccordingtotheCouncilDirective1999/74/EC,litterisdefinedas ‘anyfriablematerialenablingthehenstosatisfytheirethological needs’. This sentence leaves widespread possibilities for the useof differentkindsofmaterialsbutgivesnosuggestionfora
http://dx.doi.org/10.1016/j.applanim.2016.02.001
0168-1591/©2016TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4. 0/).
thehens’ purpose tomaintain their plumagecondition and to eradicateparasites(Engelmann,1983;MartinandMullens,2012). Henskeptinfreerangeordeeplittershowdustbathingbehaviour everyotherdaythewholeyearroundwithameandustbathing durationof20min(Fölsch,1981;Vestergaard,1982;vanNiekerk andReuvekamp,2000)to27min(Engelmann,1983;Petermann, 2006).Thisbehaviourusuallyisperformedinthemiddleoftheday withapeakbetween12:00p.m.and1:00p.m.(Vestergaard,1982) and therefore shows some kindof diurnal rhythm. Hens often dustbathetogetheratthesametime(Abrahamssonetal.,1996; Sewerin, 2002) and are highly motivated (Lindberg and Nicol, 1997).Dustbathingcanthereforebeseenasa socialbehaviour (van Rooijen, 2005). Hens show explicit frustration and stress reactionsduetoforceddeprivationofdustbathingpossibilityand littermaterial(GuesdonandFaure,2008).Aclassificationofdust bathingbehaviourintosequencesandstageshasbeenrecognized and described by numerous authors suchas van Liere (1991),
Fölschetal.(1992)andvanRooijen(2005).
Infurnishedcagesystems,littermaterialusually isgivenon matsmadeofsyntheticmaterialsthatarepositionedonthewire meshfloor.Thedistributionofthelittermaterialvariesaswell infrequencyasinamountandconsistence, andlitterisapplied eitherbyatime-controlledautomatictransportsystemor,insmall businessoperations,byhanddirectlyontothemats.Because sub-strateslikesand,woodshavingsorsawdustandstrawturnedout tobeuneconomic,incompatiblewiththeexistingmanuredisposal ortechnicalsystemor tobeharmfultothelayinghens,it isat presentcommonpracticeinGermanytoprovidefoodparticlesas littermaterialincagesystems,although‘foodparticlesmaynotbe asuitablelittersubstrateduetoitscontentoflipids’(Scholzetal., 2011).Theadvantageoffoodparticlesoverothersubstratesliesin thecontinuousavailabilityonthefarm.However,feednowadays alsorepresentsthemostexpensivefactorineggproduction,and farmmanagershesitatetoprovidefoodparticlesasloosesubstrate onadailybasis.Ouraimwastoinvestigatetowhatextentlaying hensusetheofferedlitterareasinaspecies-appropriatemanner whenhousedatmoderatestockingrates,andhowmanysubstrate applicationsperdayarenecessarytomotivatethehenstoperform dustbathingbehaviourbecausethefrequencyoflittersubstrate applicationmayleadtofrustration(GuesdonandFaure,2008).
2. Animals,materialsandmethods
Thisstudywasconductedwithethicalapprovalfromthe Bavar-iangovernmentandreceivedanexceptionalpermission(reference number:55.2-1-54-2531.8-189-09)accordingtoArticle9(1) Sen-tence1oftheGermanAnimalWelfareAct(TierSchG)from2009.To examinetheeffectsofvariedlitterapplicationfrequenciesonthe dustbathingbehaviouroflayinghens,twoseparateconsecutive experiments(experiment1andexperiment2)werecarriedout. Adifferentstockingdensityperexperimentwaschosento eval-uateapossibleeffectofprovidedspaceandusagepossibilitiesof theofferedenrichmentelementssuchasdustbathingmats. Ani-mals,housingandexperimentalconditionarethesameforboth experimentsunlessstated.
ticspiralringoneachleg(SiepmannGmbH,Herdecke,Germany; innerdiameterLSL:16mmandLB:18mm)markedeachhen indi-viduallyforhealthandperformanceparameters.Henscouldnotbe identifiedindividuallyduringvideoobservation.Afterthisstudy, thehenswereslaughteredattheBavarianStateResearchCentre forAgriculture,SpecializationinPoultryManagement,Kitzingen, Germany.
2.2. Animalhousingandexperimentalconditions
The hens were housed in a separate section at the Chair of AnimalWelfare, Ethology, Animal Hygiene and Animal Hus-bandryoftheLudwig-Maximilians-UniversityMünchen,Munich, Germany.Thefacilitymeasured24.7m2.Theclimateandair
sup-plywerecontrolledbyanairconditioningsystemofRosenberger, Künzelsau,Germany.Theflowofincomingandoutgoingairwas approximately1.500m3/h.Duringthewholelayingperiodof12
months,temperatureandhumidityweremeasuredhourlywitha datalogger(LogBoxRHT,TemperatureandHumidityDataLogger, B+BThermo-TechnikGmbH,Donaueschingen,Germany),which recordedanaverageenvironmentaltemperatureof19.3◦Canda humidityof55.1%.Aslightingsystem,aSunlight-Simulator SLS-1(iLOXGmbH&Co.,KG,Vechta,Germany)wasinstalledbyBig DutchmanInternationalGmbH,Vechta,Germany,withaltogether threehigh-performancetubes(Osram,Lumilux®Warmwhitewith
reflector80P/80P,58W,1.15cm,dimmable).Thesewereinstalled inahanging positionandwithadistanceof80cm towardsthe compartments and were equipped with a perforated plate in ordertoavoiddazzling.Thedailylightperiodlasted14hinboth experiments,eachfrom4a.m.to6p.m.(wintertime)and5a.m.to 7p.m.(summertime)withlightintensitiesrangingfrom0.13lux (nest)to39.40lux(dustbathingmats).Atthebeginningandthe endofeachdailylightperiod,twilightphasesof30mineachwere added.TheenrichedcagesystemsoftheformercompanyEBECO (since1September2011BioscapeEbecoGmbH,Castrop-Rauxel, Germany)weretheso-calledHÜK125/80withawidthof128cm, adepthof65cmandaheightof80cm(doublecompartment).A partitiongridalloweddividingthedoublecompartmentintosingle compartmentsifrequired.Thetotalfloorspaceofeachcagesystem measured8.320cm2.Altogether,10ofthesedouble-compartment
systemswereinstalled.Thedistancebetweentheopposing com-partmentswas160cm.Thesystemandallaccessories,exceptthe perches,nestmaterialanddustbathingmats,weremadeof stain-lesssteel.Thefloorconsistedofwiremeshandwasmountedabove amanurepan.Atthebacksideofeachdoublecompartment,two nestboxeswereattachedthatmeasured27×39cm (1.053cm2)
and were equippedwith a matmade of coconutfibre (Kokos-matteEuropa,SiepmannGmbH,Herdecke,Germany).Eachdouble compartment contained two feeding (31×2.5×13cm, 2.6L capacity) and two drinking troughs (1L capacity) and two woodenperchesof55cmlength.Feed,Premium-Alleinfuttermittel Korngold®LAM38&LAM40(RKWSüd),BayWaAG,Bockhorn,
Germany(ingredientsperkg:11.4MJ,17.0–17.5%crudeprotein, 3.80–4.10%Ca,0.50%P,0.15%Na,0.38–0.40%Met)andwaterwere availableadlibitum.Asyntheticmat(AstroturfPoultryPad,Grass Tech,Louvain-la-Neuve,Belgium;size:37×25cm,925cm2)was
mountedattheeachouterfaceofthedoublecompartments(two matspercompartment)toallowdustbathingbehaviour,pecking andscratching.Afteranadjustmentperiodofatleast1week,feed
Fig.1. Operatingschedule(summertime)concerningbehaviouralobservationsandassessmentpointsofvideorecordingswithamaximumdailysubstrateapplication frequencyoffourtimes(11:00a.m.,1:00p.m.,3:00p.m.and5:00p.m.)
(50gperservingandmat)wasprovidedmanuallyoneach mat (equivalentto100goffeedperunit).Thefrequencywasincreased inascendingorderfromonetimedailyat11:00a.m.toamaximum offourtimesdailyat11:00a.m.,1:00p.m.,3:00p.m.and5:00p.m. Thenthefrequencywasreduced inreverseordertoa one-time dailylitterapplication(11:00a.m.)attheendofthelayingperiod. Everylitterintervallastedupto7weeksuntilitchangedtothe nextone.Altogethersevenobservationintervalswereassessedper experiment.
2.3. Videorecordingsandbehaviouralobservations
Thehens’behaviourwasrecordedbydigitalvideoequipment. Onetriangle-shapedcamera(typeVTC-E220IRPwithIR-LED, San-tecSecuritySolutions/SanyoVideoAG,Ahrensburg,Germany)per doublecompartmentwaspositionedinthesameuppercornerof each compartment and allowedan overlookof thewholecage areaof this unit. Altogether 10 cameraswere connected toan inputtransmitter(H.264EncoderboxEPoE,ISO-Norm14496-10 H.264/MPEGAdvanced VideoCoding for Virtual Matrix Utiliza-tion,IndigoVisionLtd.,Edinburgh,UK,throughIppiGmbH,Munich, Germany) and then to a standard computer. To evaluate the receivedvideomaterial,thesoftwareprogramIndigoVisionControl CenterClient,Version‘3.16build9’(IndigoVisionLtd.,Edinburgh, UK,throughIppiGmbH,Munich,Germany)wasused.Video obser-vationtookplaceduring48consecutivehourspercalendarweek. Videorecordingswereconductedduringthelightphaseandthe darkphasefollowingacertainschedule(Fig.1)includingscan sam-pling(20mininterval)andcontinuousrecording(60min)methods (MartinandBateson,2007).Dustbathingwasdefinedascombined preeningandscratchingbehaviourduringwhichthehenpecksand scratchesatthedustbatharea,thensquatsdownandfollowsan organizedsequence ofbehaviourpatternssuchasheadrubbing andverticalwingshaking(vanLiere,1991;Fölschetal.,1992;van Rooijen,2005).Becauseoftheblack-and-whitequalityofinfrared video materialduringnight observation, severalhens oftheLB strain(darkbodycolour)couldnotbeobservedduringsome inter-vals.Theanalysiswascarriedoutaccordingtothesamplingand recordingrulesofMartinandBateson(2007).
2.4. Differencesbetweenexperiments1and2 2.4.1. Experiment1
Forthefirstexperiment,altogether20layinghens(10LSLand 10LB)werehousedinrandomizedorderalwaystogetherwithhens ofthesamestrain.Thehenshadhatchedsimultaneouslyon5May 2009,hadbeenraisedinadeep-litter systemand werehoused atthe19thweekofageon9 September2009.4.160cm2 were
providedperhen.Videoobservationtookplacefrom3February
2010until28August2010(30weeksofevaluatedvideomaterial, 2hens/cagesystem).
2.4.2. Experiment2
Forthesecondexperiment,40layinghens(20LSLand20LB) thathadhatchedon24June2010andbeenrearedinabattery cagesystemwerehousedatthe18thweekofageon27Oct2010 and2.080cm2perhenwereprovided.Videoobservationtookplace
forexperiment2from3November2010until6October2011(14 weeksofevaluatedvideomaterial,4hens/cagesystem).
2.5. Statisticalanalyses
Effectsofexperimentalconditions(cagesystem,strain, maxi-mumnumberoflitterapplicationperday)ontheprobabilityof observing acertain behaviour(dustbathingonwireframeand grooming)wereanalysedbybinomiallogisticregressionmodels. Here,cagesystemswereusedasexperimentalunits.Analyseswere performedseparatelyforexperiment1andexperiment2aswell as jointlyby taking intoaccount interactioneffects in order to measuredifferencesbetweenbothexperiments.Fordustbathing behaviour(durationandnumberofdustbathsandinterruptions aswellas number ofaxial bodyshaking) analysesofvariances wereusedtomeasuretheeffectsofexperimentalconditions.As experimentalunitsaggregated dataof onedaywereused. This datawascorrectedbythenumberofhensineachcageinorderto permitacomparisonbetweenbothexperiments.Cagesystemhas beenconsideredasanadditionalcovariableinallanalysesinorder toaccountforheterogeneityalongcagesand,thereforetoobtain morepreciseestimandsfortheeffectsofinterest.Validityofmodel assumptionswascheckedbyresidualdiagnosticsandplots.Again, analyseswerefirstperformedseparatelyforbothexperimentsand then jointlywithinteractioneffects inorder tomeasure differ-encesbetweenbothexperiments.ForallanalysestheRlanguage forstatisticalcomputing(RCoreTeam, 2015)wasused.Results wereconsideredsignificantiftheP-valuewassmallerthan0.05. Rawdata(mean±standarddeviation,standarderrorunlessstated) wereusedinthetextandtables.
Becausethehensof experiment1 wereraisedindeeplitter andthehensinexperiment2wereraisedinbatterycages, pos-sibleeffectsonthedustbathingbehaviourneededtobeanalysed. Therefore,beforeanalysingthedifferencesbetweenexperiment1 andexperiment2withrespecttotheeffectofcageunits,strains andlitterapplicationsondurationofdustbaths,numberofdust bathsandinterruptionsitwascheckedifbothexperimentshadthe samestartingconfigurationwithrespecttothesevariables.Thatis, comparingthesevariablesalongbothexperimentsforthefirstdays afterhousingthehensintothecagesystems,withonlyone-time dailylitterapplication.Forthisanalysis,Welch’stwo-samplet-test wasused.
Fig.2.Meannumberofperformeddustbathingboutswithinthefirsthourofalitterapplicationontheprovidedmats(2hensinexperiment1;4hensinexperiment2)with respecttothedailylitterapplicationfrequencyandexperiment.*:P<0.05andP>0.01;**:P<0.01andP>0.001;***:P<0.001.
3. Results 3.1. Experiment1 3.1.1. Dustbathingbouts
Withinthefirsthour aftera litterapplication, altogetherup to722dustbathingboutswerecountedduringtheobservation time in experiment 1. The averagenumber of performed dust bathingboutsperhenanddaythroughoutthelayingperiodwas 2.41±1.70. Themeannumber ofperformeddustbathingbouts increasedwiththemaximumdailylitterapplicationfrequencyand showedsignificantdifferencesconcerningthelitterapplication fre-quency(P<0.001;Fig.2).Thetwo-time,three-timeandfour-time applicationsdifferedsignificantlyfromtheone-timelitter applica-tion(allP<0.001;Fig.2).Thereforethenumberofperformeddust bathingboutswascountedlowestduringtheone-timedailylitter applicationwithanaverageof1.49±1.38boutsandhighestduring thefour-timelitterapplicationwith3.19±1.79boutswithinthe firsthouroflitterapplication.Forthemeancountofdustbathing
boutspercagesystem/layerstrainduringthewholeobservation timeoverthelayingperiodwithinthefirsthourafterlitter applica-tionseeTable1.Whilethenumberofperformeddustbathsdiffered significantlybetweenindividualcagesystems(P<0.001),no signif-icantdifferenceoccurredbetweenthetwolayerstrainsLSLandLB (P=0.505).
3.1.2. Dustbathingduration
Thenumberofdailylitterapplicationhadasignificanteffect onthedustbathingduration(P<0.05).Thethree-timeversusthe two-time(P<0.05)andthefour-timeversusthetwo-time(P<0.05) differedsignificantly(Fig.3).Theaveragedustbathingduration measured05:58±05:53min.Comparingthetwostrainswitheach otherthemeandustbathingdurationwas05:34±05:50minfor LSLand06:27±05:53minforLBinexperiment1(Table1).Forthe averagedustbathingdurationpercagesystem/layerstrainwithin the60minafterlitterapplicationinexperiment1seeTable1.The longestobserveddustbathingdurationwas49:44min(LSL)and 45:50min(LB).A significantdifferencewasfoundbetweenthe
Fig.3.Meanaveragedustbathingduration(s)withinthefirsthourofalitterapplicationontheprovidedmats(2hensinexperiment1;4hensinexperiment2)withrespect tothedailylitterapplicationfrequencyandexperiment.*:P<0.05andP>0.01;**:P<0.01andP>0.001;***:P<0.001.
Table1
Meanvalues±SD(standarddeviation)oftheresponsevariables“numberofdustbathingbouts”,“dustbathingduration”,“numberofinterrupteddustbaths”and“duration ofdustbathinterruptions”performedwithinthefirsthourafterlitterapplicationontheprovidedmats(LSL:LohmannSelectedLeghorn;LB:LohmannBrownClassic;with respecttoexperiment1andexperiment2,cagesystem(1–10)andlayerstrain.
Experiment1 Experiment2 Cage system/layer strain Numberofdust bathingbouts Dustbathing duration(min) Numberof interrupted dustbaths Durationof dustbath interruptions (min) Numberofdust bathingbouts Dustbathing duration(min) Numberof interrupted dustbaths Durationof dustbath interruptions (min) 1-LSL 3.63±1.81 05:18±06:55 0.52±0.63 00:10±00:07 1.80±1.04 05:08±o3:55 0.79±0.80 00:35±00:33 2-LB 1.00±1.06 07:39±05:51 0.18±0.48 00:24±00:17 3.30±1.84 04:08±03:43 0.18±0.33 00:37±00:19 3-LB 2.04±1.24 05:38±06:12 0.41±0.68 00:22±00:17 2.15±1.22 06:39±06:46 0.38±0.46 00:33±00:52 4-LSL 2.20±1.33 04:29±04:13 0.16±0.31 00:17±00:09 0.93±0.71 06:05±5:59 0.80±1.22 01:16±01:23 5-LB 3.36±1.97 06:36±05:02 0.91±0.83 00:22±00:17 1.48±0.86 06:36±05:05 0.25±0.34 00:38±00:26 6-LSL 1.86±1.25 05:20±04:27 0.09±0.24 00:20±00:03 1.12±1.46 04:11±05:28 0.76±1.01 01:01±01:09 7-LSL 2.61±1.32 06:59±07:12 0.34±0.47 00:19±00:12 3.64±2.34 02:43±04:13 1.23±1.37 00:35±00:34 8-LB 1.48±1.02 09:46±08:00 0.09±0.24 00:22±00:17 2.05±1.50 05:23±05:15 0.56±0.75 00:53±01:17 9-LB 3.30±2.07 04:56±04:35 0.12±0.26 00:18±00:18 1.11±0.83 07:52±07:14 0.18±0.37 00:36±00:19 10-LSL 2.63±1.67 05:37±04.21 0.25±0.40 00:15±00:14 1.96±1.16 05:24±04:27 0.12±0.19 00:32±00:29 MeanLSL 2.58±1.59 05:34±05:50 0.27±0.45 00:15±00:10 1.89±1.74 04:12±04:45 0.75±1.06 00:47±00:55 MeanLB 2.24±1.79 06:27±05:53 0.34±0.62 00:22±00:17 2.00±1.47 05:42±05:35 0.31±0.49 00:42±00:54
individualcagesystems(P<0.001)andalongermeandustbathing durationwasseenintheLBstraincomparedwiththeLSLstrain throughout the layingperiod (P<0.01). The longestmean dust bathingdurationwasobservedat11:00a.m.andreached05:24min (LSL)and06:10min(LB).
3.1.3. Interruptionsandearlyterminationofdustbathing behaviour
Concerningthelitterapplicationfrequency,nosignificant dif-ferencesinthedurationofaborteddustbathingboutswerefound (P=0.409;Fig.4).Theaveragedurationofsuchinterruptions mea-sured 00:18±00:14min. Comparing the two strains with each otherthemeandurationofinterruptionswas00:15±00:10min forLSLand00:22±00:17minforLBinexperiment1witha sig-nificantdifference(P<0.01;Table1).Thedurationofinterruptions alsodifferedsignificantbetweenindividualcagesystems(P<0.05). The litter application frequency had a significant effect onthe numberof interruptions(P<0.05). Thenumber ofinterruptions seemedtoincreasewiththenumberoflitterapplication frequen-cies,althoughonlythecombinationone-time(0.18±0.36)versus thefour-time(0.45±0.66)applicationfrequencyshoweda seri-ousdifference(P<0.05;Fig.5).Themeannumberofinterruptions
was0.31±0.54(Fig.5).Interruptionswereinvolvedin9.3%(LSL) and12.6%(LB)ofallobserveddustbathingboutsduring experi-ment1.Nosignificantdifferencesoccurredbetweenthetwolayer strains(P=0.267).Theinterruptionsoriginatedfromstandingup withoutleavingthedustbathingmatandfromfeedingoutofthe trough.Duetodisturbances,thedustbathingbehaviourwas ter-minatedearlyintheLSLlayers(14.3%)andtheLBlayers(36.2%)in experiment1.Litterapplicationfrequencyhadasignificanteffect onthenumberofaborteddustbaths(P<0.05)duringexperiment 1.Therefore thenumbersofaborted dustbathsdecreased with increasinglitterfrequency from0.26to0.16. Thepercentageof earlyterminateddustbathingboutsdifferedsignificantlybetween thestrains(P<0.01).Themainreasonforearlyterminationwasan interventionfromconspecifics,whichwasobservedfor65.1%(LSL) and74.8%(LB)oftheboutsinexperiment1.Slidingfromthe pro-videdmats during a dustbathing bout onto thewire meshby accidentwasthereasonforearlyterminationin32.0%(LSL)and 20.0%(LB)ofbouts.Litterapplicationfrequencyhadnosignificant effectondustbathingonwiremesh(2=3.20,df=3,P=0.362)but
onthegroomingfrequency(2=636.34,df=3,P<0.001).Grooming
frequencydecreasedwithincreasinglitterapplicationfrequency. The mean number of hens showing axial body shaking after a
Fig.4. Meanaveragedurationofdustbathinginterruptionswithinthefirsthourofalitterapplicationontheprovidedmats(2hensinexperiment1;4hensinexperiment 2)withrespecttothedailylitterapplicationfrequencyandexperiment.*:P<0.05andP>0.01;**:P<0.01andP>0.001;***:P<0.001.
Fig.5. Meannumberofdustbathinginterruptionswithinthefirsthourofalitterapplicationontheprovidedmats(2hensinexperiment1;4hensinexperiment2)with respecttothedailylitterapplicationfrequencyandexperiment.*:P<0.05andP>0.01;**:P<0.01andP>0.001;***:P<0.001.
dustbathingboutwas0.08±0.21duringexperiment1.Because thenumberofaxialbodyshakingwasquitelowonlyexplorative analysiswasperformed.
3.2. Experiment2 3.2.1. Dustbathingbouts
Withinthefirsthourafteralitterapplicationinexperiment2, altogetherupto2330dustbathingboutswerecountedduringthe observationtime.Theaveragenumberofperformeddustbathing boutsthroughoutthelayingperiodwas1.95±1.61.During exper-iment2,themeannumberofdustbathingboutsincreasedwith thenumberofdailylitterapplications(P<0.001)from0.81±0.74 boutsduringtheone-timeapplication to3.56±2.05 bouts dur-ingthefour-timeapplication(Fig.1).Allapplicationfrequencies differedfromeachotherwiththerespecttothenumberofdust bathingbouts(Fig.2).Forthemeancountofdustbathingbouts percagesystem/layerstrainduringthewholeobservationtime overthelayingperiodwithinthefirsthourafterlitterapplication seeTable1.Thenumberofperformeddustbathingboutsdiffered significantlybetweencagesystems(P<0.001).Nosignificant dif-ferenceoccurredbetweenthestrainsLSLandLB(P=0.505).
3.2.2. Dustbathingduration
The number of daily litter application had a significant effect on the dust bathing duration (P<0.01). The four-time (04:13±05:09min) versus the one-time (05:56±06:07min) (P<0.05)andthefour-timeversusthethree-time(05:00±04:56) (P<0.05) differed significantly (Fig. 3). Dust bathing duration decreasedwithincreasingdailylitterfrequency.Theaveragedust bathingdurationmeasured04:59±04:15min.Fortheaveragedust bathingdurationpercagesystem/strainwithinthe60minafter lit-terapplicationinexperiment2seeTable1.Thelongestobserved dustbathingdurationwas33:28min(LSL)and46:48min(LB).A significantdifferencewasfoundbetweentheindividualcage sys-tems(P<0.001)andalongermeandustbathingdurationwasseen intheLBstraincomparedwiththeLSLstrainthroughoutthe lay-ingperiod(P<0.01).Thelongestmeandustbathingdurationwas observedat11:00a.m.andreached04:17min(LSL)and05:35min (LB).
3.2.3. Interruptionsandearlyterminationofdustbathing behaviour
Concerningthelitterapplicationfrequency,nosignificant dif-ferences in the duration of aborted dust bathing bouts were found (P=0.502). The average duration of interruptions mea-sured 00:45±00:55min. Comparing the two strainswith each other the average duration of such interruptions measured 00:47±00:55minforLSLand00:41±00:54minforLBin exper-iment2andalsoshowednosignificantdifferencesbetweenthe layerstrains(P=0.448;Table1),whilethedurationofinterruptions differedsignificantlybetweenindividualcagesystems(P<0.01). Litterapplicationfrequencyhadasignificanteffectonthenumber ofinterruptions(P<0.001).Thereforethenumberofinterruptions seemedtoincreasewithraisingnumberofdailylitterapplication frequencies.Allcombinationsoflitterfrequenciesshowed signif-icantdifferences exceptforthethree-timeversusthetwo-time combinationandthefour-timeversusthethree-timecombination oflitterfrequencies(Fig.5).Themeannumberofinterruptionswas 0.53±0.85.Significantdifferencescouldbedetectedbetweenthe layerstrains(P<0.001).Interruptionswereinvolvedin19.7%(LSL) and10.8%(LB)ofallobserveddustbathingboutsduringexperiment 2.Theinterruptionsoriginatedfromstandingupwithoutleaving thedustbathingmatandfromfeedingoutofthetrough.Crowding outandpeckingatdustbathinghensbyconspecificsasmain rea-sonsledtointerruptionsof60.2%(LSL)and60.7%(LB)ofthebouts. Slidingfromtheprovidedmatsduringadustbathingboutonto thewiremeshbyaccidentwasthereasonforearlytermination in1.4%(LSL)and3.8%(LB)ofbouts.Unfortunatelytheinformation abouttheeffectoflitterapplicationsonthenumberofaborteddust bathingboutsdoesnotexistforexperiment2.14.8%oftheLSLand 18.4%oftheLBlayershadatleasttemporarilymorethanhalfof theirbodyonthewirefloorinsteadofthematduringdustbathing behaviour.Litterapplicationfrequency had nosignificanteffect ondustbathingonwiremesh(2=5.35,df=3,P=0.148)buton
thegroomingfrequency(2==27.08,df=3,P<0.001).Grooming
frequencydecreasedwithincreasinglitterapplicationfrequency. The mean number of hens showingaxial body shaking after a dustbathingboutwas0.23±0.27duringexperiment2.Because thenumberofaxialbodyshakingwasquitelowonlyexplorative analysiswasperformed.
Table2
Differencesbetweenexperiment1andexperiment2concerningtheinteractioneffects“cagesystem”,“layerstrain”and“litterapplicationfrequency”onthenumberof performeddustbathingbouts,dustbathingduration,durationofdustbathinterruptionsandnumberofdustbathinterruptions(df=degreesoffreedom).Resultswere consideredsignificantiftheP-valuewassmallerthan0.05.
System:experiment(df=9) Layerstrain:experiment(df=1) Experiment:litterapplication(df=3)forsystem(layerstrain)
Numberofdustbathingbouts P<0.001 P=0.093 P<0.001(P<0.01)
Dustbathingduration P<0.001 P<0.001 P<0.05(P<0.001)
Durationofinterruptions P=0.323 P<0.05 P=0.865(P=0.847)
Numberofdustbathinginterruptions P<0.001 P<0.001 P<0.01
3.3. Differencesbetweenexperiment1andexperiment2
Concerning weather experiment 1 and experiment 2 had the same starting configuration at the trial station due tothe different rearing conditions during the first 18 weeks of life (deep litter versus cage rearing) before housing, the analy-sis performed with the Welch Two sample t-test resulted in no significant differences for the variables dust bathing dura-tion(t=0.66,df=301.77,P=0.510)andnumber ofinterruptions (t=0.54,df=77.598,P=0.591).Forthevariablesnumber of per-formed dust baths (t=3.28, df=57.68, P<0.01) and length of interruptions(t=−3.06,df=39.57,P<0.01)significantdifferences couldbeobserved.Forexperiment1moredustbaths(1.91±1.66) wereobservedthanforexperiment2(0.95±0.84)andfor experi-ment2(00:09±0.38)thelengthofinterruptionswaslongerthan forexperiment1(00:02±00:06).
3.3.1. Dustbathingbouts
Analysisofinteractioneffectsshowedthattheeffectsofcage system(P<0.001)andlitterapplicationfrequency(P<0.001) dif-feredsignificantly betweenthetwoexperimentsandshowthat thenumberofperformeddustbathingboutsdifferedsignificantly betweenthetwoexperiments.Whenreplacingcagesystemwith layerstrainthenonlytheinteractioneffectlitterapplication fre-quencyremainssignificant(P<0.01;Table2).
3.3.2. Dustbathingduration
Theinteractioneffectsofcagesystem(P<0.001)andlitter appli-cationfrequency(P<0.05)weresignificantandshowthatthedust bathingduration differedsignificantlybetweenthetwo experi-mentsconcerningtheseeffects. Whentheeffectcagesystemis replacedbylayerstrainthentheeffectofthelayerstrainalsohad asignificanteffectonthetwoconductedexperiments(P<0.001). Theeffectoflitterapplicationfrequencystillholdsafterthe replace-ment(P<0.001;Table2).
3.3.3. Interruptionsandearlyterminationofdustbathing behaviour
Analysisofinteractionsshowednodifferenceintheeffectsof cagesystem(P=0.323)anddailylitterapplication(P=0.865)on thedurationofinterruptions.Furthermore,effectsofcagesystems (P<0.001)andlitterapplicationfrequency(P<0.01)onthe num-berofdustbathinginterruptionsdifferedsignificantlybetweenthe twoexperiments,thisfindingalsoholdswhenthecagesystemis replacedwithlayerstrain(Table2).Theinteractioneffectscage systems(P<0.001)andlitterapplicationfrequency(P<0.01)were significantforthenumberofdustbathinginterruptionsandshow thatthenumberofdustbathinginterruptionsdifferedsignificantly betweenthetwoexperiments,thisfindingalsoholdswhenthecage systemisreplacedwithlayerstrain(Table2).
Dustbathingboutsthatendedwithaxialbodyshakinglasted longer(LSL:07:57min;LB:11:04min)thanboutsthatended with-out(LSL:02:56min;LB:04:29min).Layersofbothstrainsshowed moreaxialbodyshakingwhentheboutendedwithoutany distur-bance.
4. Discussion
Thepresent studydealswiththeresultsof two consecutive experimentsconcerningthepossibleinfluenceofvarieddaily lit-terapplicationfrequenciesonthedustbathingbehaviouroflaying henskeptinacagesystem.Becausethelayinghensofthepresent studyhadbeenrearedunderdifferentcircumstances(deeplitterin experiment1vs.wirefloorinexperiment2),apossibleinfluenceof rearingconditionswasconsidered.Thereforehensinexperiment2 performedfewerdustbathingboutscomparedtothehensin exper-iment1duringtheinitialphase(firstfewdaysafterhousing)ofthe experiments.FewerdustbathingboutswerealsoseenbyJohnson etal.(1998)inchicksrearedwithoutlittercomparedwithchicks rearedwithsandorstrawanddifferentlyrearedjunglefowlchicks inthestudyofVestergaardetal.(1990)showedthesamedust bathingbehaviourconcerningnumberofdustbathingbouts inde-pendentoftherearingsituation.Becausebothexperimentsduring thepresentstudyfollowedthesametendencieswecontributethis findingtootherinternalorexternalfactorsthanmererearing dif-ferences.AccordingtoFölsch(1981),vanNiekerkandReuvekamp (2000)andOlssonandKeeling(2005)dustbathingbehaviour usu-allyoccurseveryotherdayallyearround.Theresultsofthemean counteddustbathingboutsduringbothexperimentsrangedabove theaveragenumberof0.8and0.5performeddustbathingbouts perhenperdaystatedbyVestergaard(1982)andSewerin(2002), respectively.Thenumberofdustbathingboutsincreasedduring bothexperimentswithincreasingdailylitterapplicationfrequency. Thereforeamorefrequentpresenceoflittermaterialcanmotivate thehenstoperformahigherdustbathingactivityoralsobeasignof frustration(Odenetal.,2002).Sincethenumberofthedustbathing boutsincreasedmorenoticeableduringexperiment2withahigher litterfrequencythelackofspacemayhaveledtofrustration(Oden etal.,2002)becausethehenswerenotcompletelysatiated. There-forethismayhave resultedinacompensatoryresponsewitha highernumberofdustbathingbouts.AuthorssuchasApplebyetal. (1993),Abrahamssonetal.(1996)andDöring(2012)alsodescribe adirectimpactbetweentheperformeddustbathingactivityand sizeofprovidedlitterareas.Infurnishedcagesystems,the num-berofperformeddustbathingboutsisquadrupledtooctuplicated comparedwithspecies-specificbehaviourinnature(vanRoojien, 1996;LindbergandNicol,1997;Brieseetal.,2004).Thiscouldalso beobservedinthepresenttwoexperiments.Externalstimulisuch astemperature,lightintensityandthesubstratehavegreat influ-enceonthedustbathingbehaviour.AccordingtoDuncanetal. (1998),temperaturehasanimpactonthenumberofdustbathing bouts.Thereforehigherfrequenciesofdustbathingactivitywere observedattemperaturesaround22◦Ccompared with10◦C.In thepresentstudy,theaveragetemperatureof19.3◦Cpossibly con-tributedtothenumberof observedboutsin bothexperiments.
Hoganand vanBoxel(1993)showedthat bathingdurationand frequencycouldbeexperimentallyincreasedwiththeprovision oflightsource.Averagelight intensityduringbothexperiments was39.4luxmeasuredonthedustbathingmatsandpossiblyalso contributedtothenumberofobservedbouts.Sincenosignificant differencesoccurredbetweenthetwodifferentlayerstrainsin nei-therofthebothexperiments,geneticdifferencescanbeneglected.
hensrearedonwireflooranddeeplitter.AccordingtoVestergaard etal.(1990)2-to3-month-oldjunglefowlchicksrearedwithand withoutaccesstolitteronwirefloorshowedthesamedustbathing duration.Inthepresentstudy,thelitterapplicationfrequencyhad significantimpactonthedust bathingdurationof both experi-ments.Thedecreasingdurationandincreasingdustbathingonwire meshbytendencywithinexperiment2mightbearesultofsocial facilitation(Duncanetal.,1998).Otherhensbecomemore moti-vatedbyobservingalreadydustbathinghenstosynchronizedust bathingbehaviour.Thisvisualstimulusalonecaninfluencethedust bathingduration(Duncanetal.,1998).Sincethegroupsizewas largerinexperiment2thepossibilitytodustbatheatthesame timeontheprovidedmatswaslimitedandcouldhaveleadtoa decreaseddustbathingduration.Telle(2011)observedthe short-estdustbathingsequencesinanenrichedcolonyhousingsystem withthesmallestprovidedAstroturflittermats.Inliterature,the durationofacompletedustbathaveragesbetween20and30min (Vestergaard,1982;vanLiereetal.,1990).Inthepresentstudy, dustbathsonlyoccasionallyreachedthisdescribedvalue.Appleby etal.(1993)foundshortdustbathingdurationsof5min,Sewerin (2002)ofabout8minandOrságetal.(2011)of6.6mininenriched cagesystems.Fragmenteddustbathingsequencescanalso indi-cateaninadequatesubstrate(WidowskiandDuncan,2000)and feed,asusedinbothexperiments,maynotbeasuitablelitter sub-stratebecauseitcontains ahighamountoflipids(Scholzetal., 2010).Substratedepthcouldalsoplayanimportantroleondust bathingbehaviour.Theamountofprovidedfeedontothematswas thesameforbothexperimentsandwaspossiblynotsufficientfor 4hensandthereforeledtoanabbreviateddustbathingduration. AlthoughMoestaetal.(2008)foundunderexperimental condi-tionsthatthedepthofwoodshavingsaslitter(2cmversus20cm) playedasmallerroleondustbathingbehaviour.Theamountof substratemightneedstobeincreasedwithincreasingnumberof layinghenspercagesystemwhenusingfeedassubstrate.Since significantdifferencesoccurredbetweenthetwo differentlayer strains(LB>LSL)inbothexperiments,geneticdifferencesneedto betakenintoaccountforthedustbathingduration.Thelitter appli-cationfrequencyshowedasignificantimpactonthenumber of interruptionswithahigherfrequencyperday.Mostoftheearly terminateddustbathswereinterruptedbyaconspecific,as pre-viouslydescribedbyDeJongetal.(2007).Againsocialfacilitation andalackofsufficientsizeddustbathingmatscanexplainthese findings.Krujit(1964)statedthatalower-rankedhengiveswayto ahigher-rankedhenbythemerepresenceofthelatter.Because therankingofhenswasnotsubjectofthepresentstudyand there-forenotexamined,wecanneitherincludenorexcluderankingas onepossiblereasonfortheinterruptedandaborteddustbathing bouts.AccordingtovanLiereetal.(1990),hensthatshow abbre-viated dustbathingsequences reachonly theinitialphase of a sequenceandaborttheirdustbathingbehaviourbecauseoflacking effectiveness.Duringthepresentstudy,thelayersofbothstrains andexperimentsshowedmoreaxialbodyshakingwhenthebout ended without any disturbance. However, axial body shaking, knownasasignforacompletedustbathingbehaviour,wasseen relativelyseldom,suggestingearlyaborts.Severalauthors(Appleby etal.,1993;Telle,2011;Döring,2012)describedadirect relation-shipbetweenthedustbathingactivityandthesizeofavailable dustbathingfacilities.AccordingtoOdenetal.(2002),enriched cagesystemsrarelyprovidesufficientspaceinthelitterareafor
theresultsofMerrill(2005),where74outof80henshousedin fur-nishedcagespreferredtodustbatheontheAstroturfmatsinstead ofthewirewhen partofthewirecagefloorwasreplacedwith perforatedAstroturf.Also,inlinewiththeresultsofApplebyand Hughes(1995),alldustbathingbehaviourtookplaceinthe desig-natedlitterareasoffurnishedcages.Thetimeofdayalsoneedstobe takenintoaccountforanimpactontheperformanceofdustbathing behaviour.Dustbathingbehaviourcouldbeobservedespecially aroundmiddayat11a.m.and1p.m.Thisfindingagreeswiththose ofTelle(2011),whoreportedadustbathingmaximumat10:30 a.m.,and thoseofVestergaard(1982)andHoganandvanBoxel (1993),who reporteda peak indust bathingbehaviouraround 6hafterthelighthadbeenturnedon.AccordingtoWichmanand Keeling(2009)henspreferthemiddleofthedayandOrságetal. (2011)foundthemaximumdustbathingvaluebetween10a.m. and4p.m.
5. Conclusions
Basedontheresultsofthisstudythenumberoflitterapplication frequencyhad animpactondustbathingbehaviourconcerning numberofperformeddustbathingbouts,dustbathingdurationand thenumberofinterruptions.Numberofaborteddustbathingbouts decreasedwithnumberoflitterapplications.Furtherinvestigation concerningthis findingisdesirable.Duetotheresultsanupto four-timedailylitterapplicationfrequencyatleastduringthemain dustbathingtimecanberecommendedtomotivatelayinghensto dustbatheonAstroturfmats.Insufficientsizedlitterareascanlimit species-specificdustbathingbehaviour.
Conflictofinterest
Theauthorsconfirmthatnoconflictsofinterestareassociated withthispublicationandnofinancialsupportwasgiventhatcould haveinfluencedtheoutcomeofthepresentstudy.
Acknowledgements
WethankChristianStroblfortechnicaladviceandsupportand ClaudiaSchweizerforthevaluableassistance.
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