Submitted 24 May 2016Accepted 14 November 2016Published 03 January 2017

Corresponding authorDidone Frigeriodidonefrigeriounivieacat

Academic editorJohn Ringo

Additional Information andDeclarations can be found onpage 14

DOI 107717peerj2792

Copyright2017 Frigerio et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

Social and environmental factorsmodulate leucocyte profiles in free-livingGreylag geese (Anser anser)Didone Frigerio12 Sonja C Ludwig13 Josef Hemetsberger12 Kurt Kotrschal12

and Claudia AF Wascher14

1Core Facility Konrad Lorenz Forschungsstelle for Behaviour and Cognition University of Vienna Gruumlnau imAlmtal Austria

2Department of Behavioural Biology University of Vienna Vienna Austria3Game ampWildlife Conservation Trust The Coach House Eggleston Hall Barnard Castle United Kingdom4Department of Life Sciences Anglia Ruskin University Cambridge United Kingdom

ABSTRACTBackground Blood parameters such as haematocrit or leucocyte counts are indicatorsof immune status and health which can be affected in a complex way by exogenous aswell as endogenous factors Additionally social context is known to be among the mostpotent stressors in group living individuals therefore potentially influencing haema-tological parameters However with few exceptions this potential causal relationshipreceived only moderate scientific attentionMethods In a free-living and individually marked population of the highly social andlong-lived Greylag goose Anser anser we relate variation in haematocrit (HCT) het-erophils to lymphocytes ratio (HL) and blood leucocyte counts to the following factorsintrinsic (sex age raising condition ie goose- or hand-raised) social (pair-bondstatus pair-bond duration and parental experience) and environmental (biologicallyrelevant periods ambient temperature) factors Blood sampleswere collected repeatedlyfrom a total of 105 focal birds during three biologically relevant seasons (winter flockmating season summer)Results We found significant relationships between haematological parameters andsocial as well as environmental factors During the mating season unpaired individualshad higher HCT compared to paired and family individuals and this pattern reversedin fall Similarly HL ratio was positively related to pair-bond status in a seasonallydependent way with highest values during mating and successful pairs had higherHL ratio than unsuccessful ones Also absolute number of leucocytes tended to varydepending on raising condition in a seasonally dependent wayDiscussion Haematology bears a great potential in ecological and behavioural studieson wild vertebrates In sum we found that HTC HL ratio and absolute number ofleucocytes are modulated by social factors and conclude that they may be consideredvalid indicators of individual stress load

Subjects Animal Behavior ZoologyKeywords Greylag geese Anser anser Haematology Age Social status Haematocrit Seasonalpatterns Differential leucocyte count Sex

How to cite this article Frigerio et al (2017) Social and environmental factors modulate leucocyte profiles in free-living Greylag geese(Anser anser) PeerJ 5e2792 DOI 107717peerj2792

INTRODUCTIONHaematocrit (HCT) and differential leucocyte count are important diagnostic tools ingaining information about an animalrsquos condition and health and may be regarded asindicators of individual responses to environmental and social conditions (Cooper 1975Gavett amp Wakeley 1986 Hellgren Vaughan amp Kirkpatrick 1989 Averbeck 1992 Saino etal 1997 Ots Murumaumlgi amp Hotilderak 1998 Bortolotti et al 2009 Vinkler et al 2010)

In wild birds HCT ie the relative volume of red blood cells compared to the totalblood volume (Harrison amp Harrison 1986) has been used to indicate the animalsrsquo physicalcondition (eg Saino et al 1997 Ots Murumaumlgi amp Hotilderak 1998) and is known to decreasein response to stressful conditions (Dickens Earle amp Romero 2009) However HCT alsovaries with sex age reproductive status as well as geographic distribution (Rehder etal 1982 Rehder Bird amp Lagueuml 1982 Dawson amp Bortolotti 1997a Dawson amp Bortolotti1997b Bearhop et al 1999 Potti et al 1999 Fair Whitaker amp Pearson 2007)

The differential leucocyte count may be regarded as a proper proxy of individualimmune function (Dufva amp Allander 1995 Zuk Johnsen amp MacLarty 1995 Johnsen ampZuk 1998) providing information on the relative occurrence of different leucocyte typesAbsolute leucocyte numbers generally scale negatively with body condition (as an indicatorof health see Verhulst Oosterbeek amp Bruinzeel 2002) In birds heterophils are ratherindicative of changes in the environment (Gross amp Siegel 1983 Maxwell amp Robertson1998) As lymphocyte numbers decrease while heterophil numbers increase in responseto stressful conditions the ratio of heterophilslymphocytes (HL ratio) is used as anindicator of physiological stress (eg Gross amp Siegel 1983 Gross amp Siegel 1986 McFarlaneamp Curtis 1989Maxwell 1993Vleck et al 2000 Lebigre et al 2012) Finallymonocytes arelong-lived phagocytic cells associated together with other leukocyte types (eg eosinophilsbasophils) with defence against infections and bacteria (Davis Maney amp Maerz 2008)

Social interactions are known to be among the most potent stressors in group livingindividuals (Von Holst 1998 De Vries Glasper amp Detillion 2003) Therefore it may alsoinfluence haematological parameters However with the exception of a few veterinarystudies (eg Arfuso et al 2016 Zakari et al 2016) this potential causal relationship(ie social context and haematology) received only moderate scientific attention InBlack-capped Chickadees (Poecile atricapillus) for example dominant males had higherhaematocrits compared to subordinates (Van Oort et al 2007) and in yellow baboons(Papio cynocephalus) frequent involvement in aggressive encounters was related withdecreased lymphocyte count (Alberts Sapolsky amp Altmann 1992) On the other sideseveral studies suggest a relationship between steroid hormones and haematocrit (eg apositive one with testosterone in White-plumed Honeyeaters Lichenostomus penicillatusand American Kestrels Falco sparverius Rehder Bird amp Lagueuml 1982 a negative one withcorticosterone in both sexes of two species of longspurs Calcarius ornatus and CmccowniiLynn Hunt amp Wingfield 2003 for a review see Fair Whitaker amp Pearson 2007) eventhough the exact mechanisms remain unclear

We investigated variation in haematocrit and blood leucocytes in relation toendogenous (age sex) and exogenous factors (social status season) in a free living and

Frigerio et al (2017) PeerJ DOI 107717peerj2792 221

individually marked population of the socially complex greylag geese Anser anser (Lorenz1988 Kotrschal Hemetsberger amp Weiszlig 2006 Kotrschal Scheiber amp Hirschenhauser 2010Hemetsberger Scheiber amp Weiszlig 2013) Social context is known to be among the strongestmodulators of the physiological stress responses in greylag geese (eg Wascher Scheiberamp Kotrschal 2008 Wascher Arnold amp Kotrschal 2008 Wascher et al 2009 Kralj-Fiser etal 2010) which in turn are alleviated via emotional social support by partners (Frigerioet al 2003 Scheiber et al 2005 Scheiber Kotrschal amp Weiszlig 2009 Wascher et al 2012)Across seasons males and females are faced with different demands (Kotrschal Scheiber ampHirschenhauser 2010) consequently physiological changes such as levels of corticosteroneco-vary with seasonal variation in behaviour (Hirschenhauser Moestl amp Kotrschal 1999aHirschenhauser Moestl amp Kotrschal 1999b Frigerio et al 2004a) The reproductive seasonof greylag geese starts in January Testosterone and corticosterone levels increase overthe season (Kotrschal Hirschenhauser amp Moestl 1998 Hirschenhauser Moestl amp Kotrschal1999a) as do agonistic encounters between individuals (Lorenz 1988) With the beginningof the breeding season (March to July) the flock disintegrates into pairs As for most birdspecies reproduction in geese is energetically costly especially for the females who layand incubate the eggs (Raveling 1979 Thompson amp Raveling 1988) Parental geese showelevated corticosterone levels (Kotrschal Hirschenhauser amp Moestl 1998) and synchronisetheir moult with the rearing period so that they start flying again together with theiroffspring when the goslings are approximately 10 weeks old (Lorenz 1988) In alignmentwith the parental phase (ie from hatching to fledging) androgen levels reach their annualminimum in both sexes The flock re-unites in August after moulting when androgensreach a second peak in unpaired males while in paired individuals the second peak isreached later in fall (Hirschenhauser Moestl amp Kotrschal 1999a Hirschenhauser Moestl ampKotrschal 1999b) This hints at the close interplay between physiology and social status ingreylag geese as suggested by further studies (eg stress response and social allies SachserDuerschlag amp Hirzel 1998 heart rate in the context of sociality Aureli Preston amp De Waal1999 steroid hormones and social status Wingfield Hegner amp Lewis 1991 GoymannVillavicencio amp Apfelbeck 2015)

We studied seasonal and individual variation in HCT and leucocyte counts We testedwhether (a) individual factors such as sex age or raising condition (b) social factors suchas pair-bond status (c) biologically relevant periods (mating season after moult autumn)or a combination of the above accounted for variation in haematological parametersWe hypothesized that individuals of different sexes and social status (females and malespaired versus unpaired) are faced with different social and energetic demands within theflock (eg Metcalfe Taylor amp Thorpe 1995 Senar et al 2000) which in turn will affectthe immune system and will be manifest in HCT and leucocyte count Since the matingseason is socially and energetically more challenging than moult and the stable winterflock (Drent amp Daan 1980) we predict that both haematological measurements will peakduring this period particularly in the high-ranking parental individuals Furthermore weexpect to find seasonal differences between males and females contingent with behaviouraland physiological differences between the sexes depending on an individualrsquos socialenvironment (Wingfield Hegner amp Lewis 1991 Kotrschal Hirschenhauser amp Moestl 1998

Frigerio et al (2017) PeerJ DOI 107717peerj2792 321

Hirschenhauser Moestl amp Kotrschal 1999a Hirschenhauser Moestl amp Kotrschal 1999bOwens amp Hartley 1998 Wascher et al 2012 Lees et al 2012) Additionally as hand-raised individuals generally differ from parent-raised conspecifics in glucocorticoid stressreactivity (Hemetsberger et al 2010) we finally predict that hand-raised geese will showhigher values of both haematological parameters during socially challenging periods thanparent-raised ones

MATERIAL AND METHODSEthical statementThis study complies with all current Austrian laws and regulations concerning the workwith wildlife Catching and blood sampling of focal individuals was permitted underAnimal Experiment License Nr 660060010-II10b2010 by the Austrian Federal Ministryfor Science and Research

Study area and focal animalsThe Konrad Lorenz Research Station (KLF) is situated at 550 m above sea level in thevalley of the river Alm in the Northern part of the Austrian Alps (4748primeE 1356primeN) Thenon-migratory flock of greylag geese we studied was introduced by Konrad Lorenz andco-workers in 1973 (Lorenz 1988) The geese are unrestrained and generally spend the dayclose to the research station where they are provided with supplemental food (pellets andgrain) twice a day year-round At night the birds roost on a lake approximately 10 km tothe South (Almsee) As in wild populations natural predators (mainly red foxes Vulpesvulpes and golden eagles Aquila chrysaetos) may account for losses up to 10 of the flockper year (Hemetsberger 2001 Hemetsberger 2002) All geese are marked with coloured legrings and are habituated to the close presence of humans They neither show increasedimmunoreactive corticosterone metabolites in their droppings nor heart rate increaseswhen approached by familiar humans (Scheiber et al 2005 Wascher et al 2012) Socialbehaviour and individual life-history data have beenmonitored since 1973 Such long-termobservations provide reliable information about an individualrsquos social relationships withinthe flock (ie paired or not) Approximately 20 of the flock members are carefully hand-raised in the frame of specific research projects Details about the hand-raising traditionof the KLF are published elsewhere (Hemetsberger Scheiber amp Weiszlig 2013) During theperiod of data collection (autumn 2010 to spring 2013) the number of geese in flock variedbetween 153 and 157 individuals

Focal animals of the present study were 105 greylag geese of different age (at the time ofsampling ranging from 016 to 2281 years mean age plusmn SE = 506 plusmn 036) sex (67 males49 females) social status (68 paired individuals 30 family individuals 18 unpaired ones)and different raising condition (71 goose-raised and 45 hand-raised individuals)

Data collectionData were collected during three phases representing biologically relevant periods of theyear (Hemetsberger Scheiber amp Weiszlig 2013) (1) in summer after moulting (2) in autumn(winter flock) and (3) during the mating season before the beginning of the laying period

Frigerio et al (2017) PeerJ DOI 107717peerj2792 421

(approx mid-March) Focal geese were caught by hand up to three times once in eachperiod either by a familiar human observer who approached the geese from the backand picked them up when being close enough or in a lsquotrapping enclosurersquo which thegeese entered voluntarily during feeding In this way chases or strong wing flapping wereavoided To control for diurnal variation of the focal parameters all geese were sampled inthe morning between 0730 and 0930

Weather data were provided by a weather station in Gruumlnau (4751primeE 1357primeN) operatedby Max Rauscher (httpwwwgruenautv last accessed 25 December 2014) Temperaturedata were recorded every 5 min for which we calculated daily means during our analysis

Blood samplesA total number of 169 blood samples (N = 105 individuals Table 1) were collected bypuncturing the tarsal vein with a sterile needle (24 microm diameter) and collecting bloodin two heparinized micro-haematocrit capillaries (75 mm) Furthermore we measuredtarsus beak and wing length and body weight The whole procedure lasted less than 7 minper individual

In order to determine an individualrsquos blood cell count (Prinzinger Misovic amp Nagel2012) one drop of blood was smeared onto a microscope slide air-dried and stored untillater identification of leucocytes at the Clinical Pathology Platform of the Universityof Veterinary Medicine in Vienna (Austria) Differential blood cells count providedinformation on absolute leucocyte numbermicrol (LEUCO) and the relative occurrenceof different leucocyte types (heterophils lymphocytes monocytes basophils andeosinophils Prinzinger Misovic amp Nagel 2012) Blood smears were Romanowsky-stainedwith Haemaquick (E Lehmann GmbH Salzburg Austria) and microscopically evaluatedThereafter 100 white blood cells were differentiated into heterophilic eosinophilic orbasophilic granulocytes monocytes and lymphocytes at 1000times magnification using oilimmersion Results were provided in percentages

The haematocrit capillarieswere then sealedwith plasticine at the bottomand centrifugedat 8000 rpm for 5 min in order to determine the HCT Volumes of red blood cell andplasma respectively were measured on the capillaries to the nearest 05 mm with calipersHCT was then calculated as ratio as follow (Prinzinger Misovic amp Nagel 2012) red bloodcell volume(red blood cell volume + plasma volume) The individual arithmetic mean ofthe two HCT values was then taken into further analyses

Statistical analysisWe ran linear mixed effect models (LMMs) fitted with maximum likelihood using thenlme package Pinheiro et al 2015) model formulation and computational methods aredescribed in Lindstrom amp Bates 1990) in R (R Core Team 2015) Haematocrit heterophilsto lymphocytes (HL) ratio leucocyte numbers and percentage of monocytes wereconsidered each in turn as response variable and four LMMs were calculated for each ofthe three data subsets Subset (1) included all 169 samples collected from 105 individualsWe tested 7 competing hypotheses (a) individual factors accounting for variation inhaematological parameters with sex age (ie days of life) and raising condition (hand-raised goose-raised) included as fixed factors (b) social factors (pair-bond status unpaired

Frigerio et al (2017) PeerJ DOI 107717peerj2792 521

Table 1 Details about the number of sampled individuals and the number of collected samples per season and category (ie sex social status rearing condition)

Season Nr ofsamples

Nr ofsampledindividuals

Nr of sampledindividuals by sex

Nr ofsamplesby sex

Nr of sampledindividualsby rearingcondition

Nr ofsamples byrear cond

Nr ofsampled

individualsby social status

Nr ofsamples bysoc stat

Males 10 10 Goose-raised 23 23 Single 0 0Females 13 13 Hand-raised 0 0 Paired 0 0After Molt 23 23

Family 23 23Males 37 40 Goose-raised 37 37 Single 8 8Females 36 42 Hand-raised 36 45 Paired 39 48Autumn 82 73

Family 26 26Males 40 42 Goose-raised 39 39 Single 16 18Females 21 22 Hand-raised 22 25 Paired 30 31Mating 64 61

Family 15 15

Frigerioetal(2017)PeerJD

OI107717peerj2792

621

paired family) explaining variation in haematological parameters (c) biologically relevantperiod (mating season after moult autumn) accounting for variation in haematologicalparameters (d) Social factors (pair-bond status) explaining variation in a seasonallydependent way (the interaction between season and pair-bond status was additionallyincluded in the model) (e) individual factors (sex age raising-condition) season and theinteractions between season and individuals were included in the model (f) individualfactors pair-bond status and the interaction between individual factors and pair-bondstatus (g) null model Subset (2) included only data from malendashfemale pair bondedindividuals (68 samples from 50 individuals) We tested three hypotheses (a) pair-bondduration defined as lsquoestablishedrsquo if the pair spent already one breeding season togetheror lsquonewlyrsquo if this was not the case or (b) parental experience ie if the pair previouslysuccessfully raised fledged offspring (yesno) accounting for variation in haematologicalparameters (c) null model

The third subset of data only included individuals from which morphologicalmeasurements were collected besides blood samples (116 samples from 70 individuals) Amodel including the body size index (BSI) calculated as ratio weighttarsus (as indicator forbodyrsquos structural size sensu Green 2001) as fixed factor was tested against the null modelIn order to account for repeated measures for each observed individual the individualidentity was included as random factor We based our model selection on correctedAkaikersquos Information Criterion values (AICc) We calculated the difference between thebest model and each other possible model (1AICc) and ranked the model combinationsaccording to their 1AICc which provides an evaluation of the overall strength of eachmodel in the candidate set Different competing models tested are presented in Table 2If multiple models qualified as the similarly good models ie 1AICcle 2 (Burnham ampAnderson 2002 Burnham 2004) we applied a model averaging approach which calculatesmodel averaged parameters using the MuMIn package (version 1156) In both data subset(2) pair-bonded individuals and (3) morphological measures for some of the parametersthe candidate models did not qualify as better as compared to the null model (Table 2) andare therefore not presented in the results section

RESULTS(1) Full dataset

The best candidate model explaining variation in haematocrit included individualfactors (sex age raising condition) season as well as the interactions between season andindividual factors HCT significantly increased with age in autumn but not during themating season or after moult (Table 3) The opposite pattern was found for percentage ofmonocytes which tended to decrease with age in autumn The HL ratio varied dependingon pair-bond status in a seasonally dependent way (Fig 1)

Leucocyte counts differed depending on pair-bond status in a seasonally dependentway Generally leucocyte counts were highest after moult (family mean plusmn SD 11086956plusmn 4494067) and in autumn (unpaired 13125 plusmn 630051 paired 13175 plusmn 4948741family 951923 plusmn 3548184) and decreased during the mating season (unpaired 7933333

Frigerio et al (2017) PeerJ DOI 107717peerj2792 721

Table 2 Model selection of analyses examining factors affecting (a) haematocrit (HCT) (b) haeterophily to lymphocytes ratio (HL ratio) (c)leucocyte count and (d) percentage of monocytes Individual identity was fitted as a random term Three subsets of data have been tested (1) thefull-dataset included all 169 samples collected from 105 individuals subset (2) included data from malendashfemale pair bonded individuals (68 samplesfrom 50 individuals) subset (3) included only data from individuals from which morphological measurements were collected (116 samples from 70individuals) LogLik log-likelihood AICc second order Akaikersquos Information Criterion 1AICc difference between the best model and each otherpossible model

Models df LogLik AIC 1AICc

(1a) Full-dataset HCTSex age raising history season seasonsex seasonageseasonraising-status

10 242574 minus463757 0

Pair-bond status season seasonpair-bond status 6 233649 minus45478 8976Season 4 230983 minus453722 10034Sex age raising history 6 219471 minus426423 37333Pair-bond status 4 215376 minus422508 41248Sex age raising history pair-bond status pair-bondstatussex pair-bond statusage pair-bond statusraisingstatus

8 218177 minus419454 44302

Null model 3 207589 minus409032 54724(1b) Full-dataset HL ratioPair-bond status season seasonpair-bond status 6 minus196356 40523 0Sex age raising history season seasonsex seasonageseasonraising-status

10 minus198064 417521 1229

Season 4 minus205476 419196 13965Sex age raising history 6 minus207444 427406 22176Sex age raising history pair-bond status pair-bondstatussex pair-bond statusage pair-bond statusraisingstatus

8 minus205462 427824 22593

Pair-bond status 4 minus209898 42804 2281Null model 3 minus213424 432994 27763(1c) Full-dataset leucocyte countPair-bond status season seasonpair-bond status 6 minus1650015 3312549 0Sex age raising history season seasonsex seasonageseasonraising-status

10 minus1646151 3313715 1166

Season 4 minus1656422 3321087 8538Null model 3 minus1660982 332811 1556Sex age raising history 6 minus1658534 3329586 17036Pair-bond status 4 minus1660977 3330199 17649Sex age raising history pair-bond status pair-bondstatussex pair-bond statusage pair-bond statusraisingstatus

8 minus1657008 3330917 18367

(1d) Full-dataset percentage of monocytesSex age raising history season seasonsex seasonageseasonraising-status

10 minus480402 982197 0

Pair-bond status season seasonpair-bond status 6 minus485006 982531 0334Season 4 minus491375 990995 8797Sex age raising history 6 minus491792 996104 13906

(continued on next page)

Frigerio et al (2017) PeerJ DOI 107717peerj2792 821

Table 2 (continued)

Models df LogLik AIC 1AICc

Pair-bond status 4 minus494597 997439 15241Null model 3 minus496611 999369 17171Sex age raising history pair-bond status pair-bondstatussex pair-bond statusage pair-bond statusraisingstatus

8 minus493017 1002935 20737

(2a) Pair-bonded individuals HCTNull model 3 9011 minus173846 0Pair-bond duration 4 9084 minus173046 08Parental experience 4 90276 minus171918 1928(2b) Pair-bonded individuals HL ratioParental experience 4 minus55067 118769 0Pair-bond duration 4 minus55911 120458 1688Null model 3 minus58245 122865 4095(2c) Pair-bonded individuals LeucocytesNull model 3 minus665648 1337672 0Parental experience 4 minus665424 1339484 1812Pair-bond duration 4 minus66549 1339616 1944(2d) Pair-bonded individuals MonocytesNull model 3 minus191664 389704 0Pair-bond duration 4 minus19102 390676 0972Parental experience 4 minus191293 391222 1518(3a) Morphological measurements HCTBSI 4 175545 minus342731 0Null model 3 14671 minus278206 55525(3b) Morphological measurements HL ratioBSI 4 minus146399 301158 0Null model 3 minus148494 303203 2045(3c) Morphological measurements LeucocytesNull model 3 minus114287 2291954 0BSI 4 minus1142073 2292506 0552(3d) Morphological measurements MonocytesBSI 4 minus345286 698932 0Null model 3 minus34929 704794 5862

plusmn 2651303 paired 10500 plusmn 5154748 family 9200 plusmn 2111194) This decrease was mostpronounced in unpaired individuals as compared to paired and family individuals Furtherleucocyte counts tended to vary seasonally depending on individual raising conditionIn the winter leucocyte counts were higher in hand-raised as compared to goose raisedindividuals (hand-raised 13722222 plusmn 524824 goose-raised 10050 plusmn 3645861) and thispattern reversed during the mating season (hand-raised 946875plusmn 4870810 goose-raised9715909plusmn 3901993) In summer (after moult) only hand-raised individuals were sampled(11086956 plusmn 4494067)(2) Pair-bonded individuals dataset

Frigerio et al (2017) PeerJ DOI 107717peerj2792 921

Table 3 Results of the full statistical models Models for each response variable of all three data subsets(1= full dataset 2= paired individuals 3= body measures) are presented Factors in bold are significant(ple 005)

Response variable Estimateplusmn SE t value p

(1) HaematocritRaising history minus0078plusmn 0049 minus1574 0118Sex 0021plusmn 0032 0669 0504Age 0plusmn 0 3815 lt0001Season 0656plusmn 0011 5715 lt0001Seasonraising history 003plusmn 0019 1564 0123Seasonsex minus002plusmn 0014 minus1425 0159Seasonage 0plusmn 0 minus3572 lt0001

(1) HL ratioPair-bond status 1245plusmn 0346 3588 lt0001Season 1192plusmn 023 5177 lt0001Seasonpair-bond status minus0563plusmn 0135 minus4528 lt0001

(1) LeucocytesPair-bond status minus1052plusmn 0293 3545 lt0001Season minus0555plusmn 0418 132 0186Raising history 0801plusmn 0384 2057 0039Sex 0087plusmn 0253 0339 0734Age 0687plusmn 0409 1644 01Seasonpair-bond status 0868plusmn 0265 3212 0001Seasonraising history minus0689plusmn 0372 1812 0069Seasonsex minus0161plusmn 0252 0623 0532Seasonage minus0691plusmn 0416 1624 0104

(1) MonocytesRaising history minus0645plusmn 0375 1698 0089Sex 0053plusmn 0249 0212 0831Age 0516plusmn 0405 1247 0212Season 0374plusmn 0154 2369 0017Pair-bond status 0319plusmn 029 1085 0277Seasonraising history 0471plusmn 0362 1276 0201Seasonsex minus0014plusmn 0246 0056 0955Seasonage minus0807plusmn 041 1924 0054Seasonpair-bond status minus0004plusmn 0261 0016 0986

(2) HL ratioPair-bond duration 0268plusmn 0123 2111 0034Parental experience 0315plusmn 0123 2493 0012

(3) HCTBSI 0plusmn 0 10281 lt0001

(3) HL ratioBSI 0003plusmn 0001 2048 0046

(3) MonocytesBSI 0026plusmn 0008 3248 0002

Frigerio et al (2017) PeerJ DOI 107717peerj2792 1021

Figure 1 Haematocrit in relation to season and pair-bond statusGray circles represent individuals withoffspring (ie family) black circles represent paired individuals without offspring plain circles representunpaired individuals Circles indicate median error bars represent interquartile ranges (lower 25th andupper 75th percentile)

HCT leucocyte number and percentage of monocytes were not significantly affectedby pair-bond duration or parental experience HL ratios were higher in pairs with anlsquoestablishedrsquo pair-bond duration compared to those with a lsquonewlyrsquo formed one (established1228 plusmn 0593 newly 0819 plusmn 0418) and were higher in reproductively successful ascompared to unsuccessful pairs (Fig 2)(3) Morphological measures

The body size index (BSI) was positively related to HCT percentage of monocytes andHL ratio

DISCUSSIONOur results show that social and environmental factors interact with individual physiologyin a complex way We found that in the free-living greylag geese investigated haematocrit(HCT) and differential leucocyte counts are seemingly contingent with a suite of endoge-nous (ie sex age raising condition) social (ie pair-bond status pair-bond durationand parental experience) and environmental factors (ie biologically relevant seasons)

Interestingly pair-bond status showed a seasonal dependent relationship with severalhaematological parameters unpaired individuals had the highest HCT during the matingseason and HL ratio was significantly higher in individuals who successfully raised youngas compared to individuals which failed to fledge offspring that year Our results hint ata complex relationship between an individualrsquos social status within the flock the seasonalpatterns of corticosterone and individual behavioural investment in order to optimizethe own fitness as discussed by Kotrschal Hirschenhauser amp Moestl (1998) In fact during

Frigerio et al (2017) PeerJ DOI 107717peerj2792 1121

Figure 2 Heterophil to lymphocyte (HL) ratio in relation to individualsrsquo reproductive success Circlesindicate median error bars represent interquartile ranges (lower 25th and upper 75th percentile)

summer and winter the paired males with offspring had significantly higher corticosteronethan both pairedmales without offspring and singletons whereas during themating seasonsingletons had marginally higher corticosterone than paired males Furthermore stresslevels of all three male categories were significantly higher during the mating season thanduring the rest of the year Even though we did not measure levels of corticosterone in thepresent study a number of studies confirm the close relationship between stress physiologyand haematology (eg Gross amp Siegel 1983 Dickens Earle amp Romero 2009)

Therefore we suggest that high HCT levels in unpaired individuals during the matingseasonmirror social stress causedmainly by competition for amate as well as by constrainedaccess to mates whereas the haematological differences related to reproductive successmight reflect the costs of reproduction in successful geese Such costs are one of themost significant components of life-history trade-offs and the immune system has beenproposed as an important link between reproductive investment and survival (Sheldon ampVerhulst 1996 Deerenberg et al 1997 French Denardo amp Moore 2007 French Moore ampDemas 2009 Harshman amp Zera 2007 Cox et al 2010)

Although earlier avian studies found higher HCT levels in males than in females (egcormorant Phalacrocorax carbo Balasch et al 1974 kestrel Falcus tinnunculus RehderBird amp Lagueuml 1982 sparrowhawk Falcus sparverius Rehder amp Bird 1983) our results arein line with a recent meta-analysis based on 36 published studies did not provide evidencefor sex differences in HCT (Fair Whitaker amp Pearson 2007)

Hence the seasonal differences in relation to pair-bond status we found in our studymayreflect differences among greylag geese with different bonding status and energetics (DeGraw Kern amp King 1979 Jenni et al 2006) High HL ratio may reflect physiological stress

Frigerio et al (2017) PeerJ DOI 107717peerj2792 1221

produced by competition for partners during the mating season and frequent agonisticinteractions among the unpaired individuals which may not enjoy the stress bufferingeffect of emotional social support by social allies notably by a pair partner (Scheiber et al2005) On the other hand high HL ratio may represent the costs of parental commitmentfor successfully breeding individuals

Heterophils are the predominant immunological cell type within the Anseriformes(Lucas amp Jamroz 1961) They form the first line of cellular defence against invadingmicrobial pathogens (Maxwell amp Robertson 1998) Findings in poultry suggest the HLratio to be a reliable indicator of social stress (eg Gross amp Siegel 1983 Vleck et al 2000)This is supported by other studies in birds which have shown that HL increases in responseto a wide variety of stressors including long-distance migration (Owen amp Moore 2006) andparasitic infection (Davis Cook amp Altizer 2004 Lobato et al 2005 for a review see DavisManey amp Maerz 2008)

We suggest that the decrease of the percentage of lymphocytes and the increase ofheterophils (ie high HL ratio) may also reflect an increase in susceptibility to infectionswith aging (Uciechowski amp Rink 2014) which affects several haematological parameters(Maxwell et al 1990 Maxwell amp Robertson 1998 Prinzinger Misovic amp Nagel 2012) Infact glucocorticoids and oestrogens may reduce T-cell production (ie low lymphocyteslevels) whereas androgens may increase susceptibility to infections via elevated heterophilslevels (Nelson et al 2002) However as we did not directly measure glucocorticoidhormones we can only speculate about the relation between haematology and healthNotwithstanding our results confirm haematologyrsquos great potential in studies of ecologyand in wild vertebrates as recently suggested byMaceda-Veiga et al (2015)

Finally our results showed that raising condition did affect haematological parameterssuggesting that goose-raised individuals could cope better with the stressful mating seasonthan hand-raised individuals as the latter showed significantly lower levels of leucocytesas compared to goose-raised geese Although raising history does not seem to affect otherreproductive parameters such as number and weight of eggs laid hatching success ornumber of young fledged (Hemetsberger et al 2010) this intrinsic aspect may deservefurther attention in the future when considering focal individuals

In conclusion our results indicate that the way social and intrinsic factors modulatehaematological parameters varied between seasons Seasonal activities such as reproductionor migration need to fine-tune physiology with weather conditions (eg Dorn et al 2014Frigerio et al 2004b Romero Reed amp Wingfield 2000) By and large the investigatedhaematological parameters varied with individual behavioural investment and stress loadTherefore they may be considered as valid indicators of social burden

ACKNOWLEDGEMENTSWe gratefully acknowledge Lara Cibulski Oliver Elsaesser and Alexander Karl for helpingwith catching geese in autumn 2012 Blood samples were analysed at the ClinicalPathology Platform of the University of Veterinary Medicine in Vienna (Head ProfDr I Schwendenwein)

Frigerio et al (2017) PeerJ DOI 107717peerj2792 1321

ADDITIONAL INFORMATION AND DECLARATIONS

FundingFinancial support was provided by the FWF-Project P21489-B17 (DF SL) the Universityof Vienna the lsquolsquoVerein der Foumlrderer der Konrad Lorenz Forschungsstellersquorsquo and the lsquolsquoHerzogvon Cumberland Stiftungrsquorsquo The funders had no role in study design data collection andanalysis decision to publish or preparation of the manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsFWF-Project P21489-B17University of ViennaHerzog von Cumberland Stiftung

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull Didone Frigerio conceived and designed the experiments performed the experimentsanalyzed the data wrote the paper reviewed drafts of the paper

bull Sonja C Ludwig conceived and designed the experiments performed the experimentsreviewed drafts of the paper

bull JosefHemetsberger conceived anddesigned the experiments performed the experimentscontributed reagentsmaterialsanalysis tools reviewed drafts of the paper

bull Kurt Kotrschal contributed reagentsmaterialsanalysis tools wrote the paper revieweddrafts of the paper

bull Claudia AF Wascher analyzed the data wrote the paper prepared figures andor tablesreviewed drafts of the paper

Animal EthicsThe following information was supplied relating to ethical approvals (ie approving bodyand any reference numbers)

This study complies with all current Austrian laws and regulations concerning the workwith wildlife Catching and blood sampling were performed under Animal ExperimentLicense Nr 660060010-II10b2010 by the Austrian Federal Ministry for Science andResearch

Data AvailabilityThe following information was supplied regarding data availability

The raw data has been supplied as Data S1

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj2792supplemental-information

Frigerio et al (2017) PeerJ DOI 107717peerj2792 1421

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Frigerio D Dittami J Moestl E Kotrschal K 2004b Excreted corticosterone metabolitesco-vary with ambient temperature and air pressure in male Greylag geese (Anseranser) General and Comparative Endocrinology 13729ndash36DOI 101016jygcen200402013

Frigerio D Hirschenhauser K Moestl E Dittami J Kotrschal K 2004a Experimentallyelevated testosterone increases status signalling in male Greylag geese (Anser anser)Acta Ethologica 79ndash18

Frigerio DWeiszlig B Dittami J Kotrschal K 2003 Social allies modulate corticosteroneexcretion and increase success in agonistic interactions in juvenile hand-raisedGreylag geese (Anser anser) Canadian Journal of Zoology 811746ndash1754DOI 101139z03-149

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GoymannW Villavicencio CP Apfelbeck B 2015 Does a short-term increase intestosterone affect the intensity or persistence of territorial aggressionmdashAnapproach using an individualrsquos hormonal reactive scope to study hormonal effects onbehaviour Physiology amp Behavior 149310ndash316 DOI 101016jphysbeh201506029

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