Individual differences in cortisol levels and behaviour of Senegalesesole (Solea senegalensis) juveniles: Evidence for coping styles

Patrıcia Isabel Mota Silva a,c,*, Catarina I.M. Martins a, Sofia Engrola a,Giovanna Marino b, Øyvind Øverli c, Luis E.C. Conceicao a

a Centro de Ciencias do Mar (CCMar), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugalb Institute for Environmental Protection and Research (ISPRA), Via di Casalotti 300, I-00166 Rome, Italyc Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Aas, Norway

Applied Animal Behaviour Science 124 (2010) 75–81

A R T I C L E I N F O

Article history:

Accepted 14 January 2010

Available online 12 February 2010

Keywords:

Individual variation

Personality

Feeding motivation

Activity

Stress response

Flatfish

A B S T R A C T

Individual variation in stress physiology and behaviour has been previously reported in

several fish species. As seen in other vertebrates, existence of stress coping styles seems to be

reflected by the presence of individual variation. Aggressive behaviour, amongst others, is

one of the most commonly used parameters to characterize coping styles. However, not all

fish species exhibit aggressive behaviour, such as the flatfish Senegalese sole, Solea

senegalensis (Kaup, 1858). Therefore, the goal of this study was to determine the magnitude

of individual variation in behavioural parameters other than aggression (feeding motivation

and activity during stress) as well as in growth and stress response in Senegalese sole. The

relationship between these variables was investigated to determine whether they could be

used as indicators of coping styles. Thirty-six juvenile fish (9.9� 2.2 g) were individually

housed for 73 days. Feeding motivation, measured as the time (in s) taken by each fish to react to

feed, was determined on days 10, 17, 24 and 31. Blood samples for plasma cortisol were collected

on days 51 and 71 for determination of undisturbed and stress levels, respectively. The stress test

consisted of holding each fish individually in a net, outside the water, for 3 min. Duration of

escape attempts, i.e. the time taken by each fish to stop struggling (in an attempt to escape) in the

net, was quantified. The results showed a pronounced individual variation in both control

(CV = 54%) and acute stress (CV = 71%) cortisol levels. Senegalese sole also exhibited high

coefficient of variation in the behavioural parameters: 75% in feeding latency and 96% in duration

of escape attempts. Growth (RGR = 1.17� 0.38) showed to be the parameter with lower

variation of only 32% and was not correlated with any of the measured parameters. A significant

correlation between undisturbed cortisol levels and duration of escape attempts was found.

Undisturbed cortisol levels (8.08� 4.36 ng/ml) were negatively correlated with duration of

escape attempts (P = 0.009, rs =�0.503). Correlations between plasma cortisol levels after stress

(398.45� 282.67 ng/ml) and the behavioural parameters were not found. The observed

individual variation in behaviour and stress physiology as well as their relationship suggests the

existence of coping styles in Senegalese sole where proactive individuals exhibit shorter feeding

latency, higher duration of escape attempts and lower undisturbed cortisol levels than passive

individuals.

� 2010 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

Applied Animal Behaviour Science

journa l homepage: www.e lsev ier .com/ locate /applan im

* Corresponding author at: Centro de Ciencias do Mar (CCMar),

Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal.

Tel.: +351 916489641.

E-mail address: tishamaria@gmail.com (P.I.M. Silva).

0168-1591/$ – see front matter � 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.applanim.2010.01.008

1. Introduction

Individual variation in growth, stress response andbehaviour has been described in several fish species(Jobling and Reinsnes, 1986; Jobling and Koskela, 1996;

P.I.M. Silva et al. / Applied Animal Behaviour Science 124 (2010) 75–8176

Carter et al., 1998; Carter and Bransden, 2001; Martinset al., 2005, 2006; Øverli et al., 2006). Until recently, thisvariation was considered to be a consequence of theestablishment of social hierarchies, with dominant fishexhibiting superior growth and lower response to stress.However, recent studies demonstrate that individualvariation is not simply a consequence of social hierarchiesbut also of inherent (i.e. genetic) factors (Øverli et al., 2002;Martins et al., 2005; Schjolden and Winberg, 2007).Several studies showed that, in the absence of socialhierarchies, fish still exhibit a large individual variation ingrowth, stress response and behaviour and that thisvariation is not only consistent over time (e.g. Martinset al., 2005; van de Nieuwegiessen et al., 2008) but alsoacross generations (Pottinger and Carrick, 1999). In fact,like in mammals, fish seem to express differential copingstyles or personalities, i.e. ‘‘coherent set of behavioural and

physiological stress responses which are consistent over time

and which are characteristic to a certain group of individuals’’(Koolhaas et al., 1999).

Coping styles include two major types, the proactive(active coping or fight-flight or bold personalities) andreactive (passive coping, or conservation-withdrawal orshy personalities) responses (Koolhaas et al., 1999). Onone hand, high levels of aggression, territorial control,active avoidance of a negative stimulus and otherbehavioural responses that suggest active efforts to offseta negative stimulus characterize a proactive response. Onthe other hand, low levels of aggression and avoidance of anegative stimulus characterize behaviourally a reactiveresponse (Koolhaas et al., 1999; Øverli et al., 2007).Individual differences in behaviour are associated withdifferences in the physiological stress response. Studies inrainbow trout demonstrate that stress responsiveness interms of confinement induce changes in plasma cortisolwhich are negatively correlated with aggressive behaviour(Øverli et al., 2004). This finding supports that proactivecoping responses are related with low secretion of cortisolin plasma under stress conditions while passive copingresponses are related with an enhanced secretion.Differences in behaviour have been shown not to be justassociated to physiological stress response but also withcortisol levels under undisturbed conditions. Øverli et al.(2007) showed that fish that obtained a high total feedingscore, i.e. fish that resume feeding quicker after beingisolated, showed high aggression (related to proactivecoping styles) and low levels of plasma cortisol underundisturbed conditions. These findings suggest thatindividuals with low levels of undisturbed plasma cortisoldisplay proactive coping styles.

In fish, coping styles have been described in rainbowtrout (Øverli et al., 2002, 2006), brown trout (Kristiansen,1999; Brelin et al., 2005), sticklebacks (Ward et al., 2004),fighting fish (Verbeek et al., 2008), halibut (Kristiansenand Ferno, 2007) and in African catfish (Martins et al.,2006; van de Nieuwegiessen et al., 2008). The under-standing of coping styles in fish is of major importance, notonly under an evolutionary perspective but also underpractical situations as in aquaculture. Coping styles canbe a helpful model in understanding individual adaptivecapacity and vulnerability to stress-related disease

(Koolhaas et al., 1999). Furthermore, when fish arecultured in intensive husbandry systems, characterizedby high stocking densities and fixed feeding locations, it isexpected that reactive fish gain little feed over time andgrow weakly which will result in loss of production andcompromised welfare (Huntingford and Adams, 2005).Understanding this individuality can be used to approachremedial measures, such as selection programs andalteration of husbandry systems to optimize fish farming.

Senegalese sole, Solea senegalensis is a flatfish with higheconomic and commercial potential for aquaculture and isbeing exploited in some southern European countries, suchas Portugal and Spain (Dinis et al., 1999; Imsland et al.,2003). Despite the commercial importance of Senegalesesole very little is known on its individual variation ingrowth, stress response and behaviour. Aragao et al.(2008) reported a coefficient variation (CV) in growthbetween 24 and 29% in juvenile Senegalese sole. However,it is not known whether this variation in growth is relatedto the existence of coping styles. In sole, which is a non-aggressive species (Salas-Leiton et al., 2008) it may bemore difficult to assess coping styles, as one of the methodsused is to determine the outcome of fights. Also measuringindividual feed intake (or residual feed intake) that hasbeen shown to predict coping styles in African catfish(Martins et al., 2005) seems to be more difficult than inother fish species as Senegalese sole present a sluggishfeeding behaviour with a swallow-and-spit behaviouroften being observed (Dinis et al., 2000). Therefore, the useof behavioural observations (others than aggression andfeed intake) to predict coping styles in Senegalese sole maybe especially interesting.

The aim of this study was to determine individualvariation in growth, feeding behaviour and stress responsein Senegalese sole and whether such individual differencesand their relationships could be used as predictor of copingstyles in a non-aggressive species.

2. Material and methods

2.1. Fish, housing and feeding

The experiment was carried out at the LEOA ResearchFacility (University of Algarve, Faro, Portugal) using aclosed seawater system (temperature: 19.46� 2.15 8C(mean� SD); salinity: 29.59� 1.26 ppt) with a photoperiodof 12 h light:12 h dark. This experiment lasted for 73 days.

Thirty-six Senegalese sole S. senegalensis juveniles withan initial weight of 9.9� 2.2 g were obtained from INRB-IPIMAR Pilot Station (Olhao, Portugal). All fish were kept in21 l flat-bottom beige fiberglass tanks, 0.21 m2 area, with awater flow rate of 73.5 l h�1. Each tank was divided in threeequal compartments (0.07 m2 of area) using plastic nets with1 cm2 holes covered with sponge to allow continuous waterflow rate inside the tank. Fish were housed individually ineach compartment, without having physical and visualcontact with each other. Tanks were daily cleaned and waterparameters daily measured. Fish were fed every day threetimes a day (11:00, 15:00, and 17:00). The commercial dietMigas 4 (2.0 mm) (A. Coelho e Castro, Povoa de Varzim,Portugal) used had 53% protein, 11% lipid, 15% carbohydrate,

P.I.M. Silva et al. / Applied Animal Behaviour Science 124 (2010) 75–81 77

according to the manufacturer’s data. Diets were hydrated toallow feed to sink quickly. Fish were initially fed with 2.6% ofbiomass and this quantity was daily adjusted based on visualinspection of the feed remaining, so that fish were fed tosatiation. Water quality parameters were maintained accord-ing to the species.

2.2. Experimental procedures

Before the start of the experiment fish were held inRamalhete facilities at the University of Algarve (Faro,Portugal) on a semi-closed system at a rearing density of0.5 kg/m2 and fed ad libitum.

Fish were individually weighted (to 0.001 g) at thestart and end of the experiment. Feeding motivation wasmeasured as the feeding latency (s) defined as the timetaken by each fish to start swimming towards the feed.This behaviour was measured directly using a stopwatchfor the 3 meals on days 10, 17, 24 and 31. Day 10 wasused as the first day to measure feeding motivationbecause it was thought it would represent the feedingmotivation of fish already adapted to isolation. Previousstudies with African catfish showed that a period of 15days was sufficient as an adaptation period to isolation(Martins et al., 2005). Afterwards measurements wererepeated once per week for an extra 3-week period toallow the determination of how consistent feedingmotivation was. Individual feed intake was not mea-sured since this specie presents a swallow-and-spit andsluggish feeding behaviour that could be translated intoerroneous results.

Blood samples for determination of control (i.e.undisturbed) plasma cortisol were collected on day 51.On day 71 all fish were subjected to an acute stress byholding each fish individually in a net outside the waterfor 3 min (Aragao et al., 2008). Afterwards fish werereturned to their holding tanks and after 30 min bloodsamples were taken (based on Ruane et al., 2001). Fishwere fasted for 24 h prior to blood sampling. Prior toblood sampling fish were anaesthetized with 2-phenox-yethanol (Sigma, Madrid, Spain; 200 ppm). Blood waswithdrawn from the caudal vein using heparinisedsyringes. This procedure was finalized within 3 minafter fish were anaesthetized. For 5 fish, this proceduretook longer than the 3 min and the respective bloodsamples were not considered for further analysis. Thecollected blood was centrifuged at 1500� g for 2 min atroom temperature and plasma was stored at �25 8C forfurther cortisol analyses. In four samples, the resultingplasma from centrifugation was reddish and was notconsidered for analysis. This resulted in a total of 27blood samples for cortisol determination. The timebetween blood samplings was long since 15 days areusually used to allow the recovery of the fish after bloodsampling (van Zutphen et al., 2001).

During the acute stress induction, the time taken byeach fish to stop moving was measured using astopwatch. This behaviour was defined as duration ofescape attempts (s).

At the end of the experiment fish were kept alive forfurther experimental work.

2.3. Cortisol analysis

Plasma cortisol was measured in 25 ml samples usingcommercially available solid phase 125Iodine RIA mea-suring the total amount of hormone in unextracted serum(Coat-A-Count Cortisol1, D.P.C. Los Angeles, CA). Thesensitivity of the assay was 2 ng ml�1 and the intra-assaycoefficients of variation were 4.7 and 6.4 (n = 3), respec-tively. Data was obtained using a gamma counter(Kontron Analytical MDA 312) and analyzed using RIAsoftware.

2.4. Data analysis

All results are expressed as means� standarddeviation (SD). Growth, expressed as relative growth rate(RGR, %/day), was calculated, between final and initialbody weight, using the formula: (eg� 1)� 100 withg = [(ln final weight� ln initial weight)/time] (Ricker,1958). Coefficient of variation (CV, %) was calculated asfollows: [(standard deviation/mean)� 100]. Pearson cor-relation was used to verify the consistency of individualfeeding latency on different days. The feeding latency of anindividual was calculated as the average of the 3 mealsmeasured on days 17, 24 and 31 (day 10 was excluded as itfailed on consistency test). Differences in feeding latencyover time were tested using repeated measured design.Mauchly’s test was used to check the sphericityassumption and the Bonferroni test for making pairwisecomparisons (Field, 2000). A paired t-test (dependentsamples) was used to verify whether the netting and airexposure induced a stress response in Senegalese sole. Therelationship between feeding latency (log transformed),undisturbed cortisol levels (log x + 1 transformed) andcortisol levels obtained after an acute stress was investi-gated using Pearson correlation. The duration of escapeattempts and growth rate failed to achieve normality evenafter transformation and their relationship with othervariables was investigated using Spearman correlation.Statistical analyses were performed using SPSS (version15.0) for windows. Statistical significance was taken atP< 0.05.

3. Results

3.1. Individual variation in behaviour, plasma cortisol and

growth

Duration of escape attempts (CV = 96%), feeding latency(CV = 75%) and the cortisol levels obtained after an acutestress (CV = 71%) showed the highest individual variation(Table 1). Feeding latency decreased significantly overtime (repeated measured ANOVA, F1,35 P< 0.001, Fig. 1).On day 10, feeding latency was significantly higher than allthe other sampling days (Bonferroni pairwise compar-isons, P< 0.001) and not correlated with other days, whichsuggested an adaptation period. Therefore, day 10 was notconsidered in further analyses.

The netting and exposure of Senegalese sole to airduring 3 min resulted in a significant increase of plasmacortisol levels (paired t-test, t(26) =�7.369, P< 0.001).

Table 1

Means, coefficient of variation (CV), minimum (min.) and maximum (max.) of feeding behaviour (feeding latency), duration of escape attempts, plasma

cortisol (control and after an acute stress) and growth of Senegalese sole housed individually.

Parameter Mean� SD CV (%) Min. Max.

Behaviour

Feeding latency (s) 38.30� 28.62 74.73 7.33 117.00

Duration of escape attempts (s) 7.20� 6.91 95.98 1.00 27.00

Plasma cortisol

Control (ng/ml) 8.08� 4.36 54.01 0.80 17.60

Acute stress (ng/ml) 398.45� 282.67 70.94 21.00 949.00

Growth (%/day) 1.17� 0.38 32.72 0.28 1.71

Fig. 1. Feeding latency over time for Senegalese sole juveniles housed

individually. Values are means (+SD). Different letters indicates

significant difference (n = 36).

Fig. 2. Relationship between individual differences in behaviour (feeding

latency and duration of escape attempts) and cortisol levels obtained in

isolated and undisturbed Senegalese sole juveniles. (A) Relationship

between feeding latency and undisturbed plasma cortisol and (B)

Relationship between duration of escape attempts and undisturbed

plasma cortisol (Pearson r2 and P values).

P.I.M. Silva et al. / Applied Animal Behaviour Science 124 (2010) 75–8178

3.2. Correlations

There was a trend towards a significant positivecorrelation between undisturbed plasma cortisol levelsand feeding latency (P = 0.0778, r2 = 0.119, Fig. 2A). Theundisturbed plasma cortisol levels were significantlycorrelated with duration of escape attempts (P = 0.009,rs =�0.503, Fig. 2B): individuals with lower undisturbedcortisol levels seemed more motivated to eat and weremore responsive towards an acute stressor. No correlationwas found between plasma cortisol levels obtained after anacute stress and the behaviour parameters (P> 0.05).Feeding latency and duration of escape attempts were alsonot correlated (P> 0.05).

Growth was not correlated with any of the measuredparameters (P> 0.05).

4. Discussion

This study reports for the first time individualdifferences in behaviour, stress response and growth ofS. senegalensis, a flatfish with increasing interest for theaquaculture sector.

Over the past years, there has been an increased intereston studying individual differences in behaviour and stressphysiology and their relationships, as these seem to reflectthe existence of coping styles in fish (Øverli et al., 2007).Individual variation in cortisol responsiveness (as one ofthe most common methods to assess the stress response infish, Wendelaar Bonga, 1997; Ruane et al., 2001; Martinset al., 2006) has also been addressed in this study.Senegalese sole exhibited a pronounced individual varia-

tion in plasma cortisol levels before (54%) and after anacute stress (71%) was applied. Undisturbed cortisol levelsobtained in this study (8.08� 4.36 ng/ml) differed fromthose reported by Aragao et al. (2008; approx. 2 ng/ml) inSenegalese sole groups. However, differences could be due todifferent cortisol analytical methods as well to individualhousing condition, which can act as a stressful situation infish (Øverli et al., 2002). Nevertheless, it must be noticed thatin Senegalese sole the response to an acute stress in bothindividually (present study) and grouped housed conditions(Aragao et al., 2008; Costas et al., 2008) has a considerable

P.I.M. Silva et al. / Applied Animal Behaviour Science 124 (2010) 75–81 79

variation, up to 73%. Other studies with African catfish andsea bass (Dicentrarchus labrax) have also reported compar-able individual variation in both undisturbed levels of plasmacortisol (Martins et al., 2006; Marino et al., 2001), and post-stressor levels (Martins et al., 2006).

In terms of behaviour the present study reportspronounced individual variation in feeding and durationof escape attempts (75 and 96%, respectively). Thevariation obtained in the duration of escape attempts iscomparable with the variation of another variable that alsoreflects locomotory activity during stress reported byØverli et al. (2006) in female rainbow trout that had anindividual variation in locomotor activity during confine-ment test of approximately 46%. Also in the same studypronounced individual variation was observed in feedingscore, another way of measuring feeding motivation.

The observed individual variation in behaviour andstress physiology suggests differences in coping styles inSenegalese sole. This is supported by the type of relation-ships found between plasma cortisol, feeding motivationand duration of escape attempts. The relationship foundbetween undisturbed plasma cortisol levels and durationof escape attempts was never reported in fish. In pigs, thenumber of escape attempts when individuals are exposedto a backtest has been shown to reflect the existence ofcoping styles (Bolhuis et al., 2004). The number of escapeattempts was shown to be related with baseline cortisollevels obtained in stall-housing gilts (Geverink et al.,2003). These authors found a relationship similar to therelationship found in this study: individuals with lowerescape attempts exhibit higher baseline cortisol levels.However, this relationship was not observed when giltswere grouped housed. If this lack of relationship wouldalso apply in grouped housed fish is still to be determined.Nevertheless, lower escape attempts suggest a passivecoping style, which has been associated with higher basalcorticosteroid concentrations (Korte et al., 1997).

The trend found between undisturbed plasma cortisoland feeding motivation in sole juveniles is similar to therelationship reported by Øverli et al. (2007) for rainbowtrout. Individuals with a higher feeding score had lowerundisturbed cortisol levels until a certain level after whichthe relationship was inverted however, in the presentstudy a linear trend line yielded a better fit than thecurvilinear approach. Øverli et al. (2006, 2007) reported arelationship between feeding motivation and activityduring acute confinement stress. In the present study arelationship between feeding motivation and duration ofescape attempts (movement during stress) was not found.Nevertheless it must be noticed that in the studies byØverli et al. feeding motivation was measured immedi-ately after transfer to isolation (reflecting a behaviour ofadaptation to isolation) and not after the end of anadaptation period, which was the case in the present study.In combination, the results suggest that individuals withlow undisturbed cortisol levels, a short feeding latency andactive avoidance after stress are proactive individuals. Onthe contrary, individuals that present high-undisturbedcortisol levels, higher feeding latency and short duration ofescape attempts seem to be passive individuals. However,no correlation was found between post-stress cortisol

levels and feeding motivation or duration of escapeattempts. Such relationship has been reported in otherstudies suggesting coping styles in fish. Animals thatshowed reduced locomotion during stress test and highmotivation towards feed exhibited lower post-stresscortisol levels (Øverli et al., 2006). These authors used adifferent stress test in which fish were kept in a small,immersed box. Such test may have allowed individuals toexpress their coping strategy. In the stress test used in ourstudy fish were kept in a net outside the water, which mayhave limited the individual’s ability to cope.

Feeding motivation, cortisol levels obtained before andafter stress and the duration of escape attempts obtainedduring the stress test were collected at different timepoints. Obtaining and correlating variables at differenttime points may seem questionable. However, bothfeeding motivation and cortisol levels have been shownto be consistent over time in several fish species (Pottingerand Carrick, 1999; Tort et al., 2001; Martins et al., 2005;Schjolden et al., 2005) making the difference in samplingtimes less relevant. Nevertheless, it is important tounderstand how consistent these parameters are forSenegal sole and subsequently evaluate the possibleimplications of correlating variable collected at differenttime points.

The consistency of the individual differences found inthe present study should be further studied. As defined byKoolhaas et al. (1999), copying styles are defined as‘‘coherent set of behavioural and physiological stress

responses which are consistent over time. . .’’. Thereforeone need to understand whether the individual variation instress physiology and behaviour as well as the relation-ships found in this study are consistent over time andgenetically linked. If these individual differences prove tobe genetically based then selection programs can beadopted. It has been already demonstrated in strains ofrainbow trout selected for consistently high (HR) or low(LR) responses to stress that there is a heritable associationbetween increased cortisol production, anxiety-like beha-viour, brain monoaminergic function and altered cognitivefunction (Pottinger and Carrick, 1999, 2001; Øverli et al.,2001; Moreira et al., 2004). A recent study even show thatyolk-sac larvae originating from the HR strain were moresensitive to environmental stressors than LR yolk-saclarvae, suggesting that coping styles are inherent sincesocial experience or variable access to food resources couldnot modify behavioural strategy in this case (Hoglund etal., 2008).

Most of the previous studies on individual differencesare related to individual differences in growth since theconsequences of individual variation in growth for the fishfarming industry are extensive. One of the main reasons forthese studies was because until recently, growth variationwas thought to be the result of social interactions resultingin that dominant fish consumed more feed and thereforegrew faster than subordinates. However, if the presence ofsocial interactions would be the main reason for growthvariation it would be expected that in the absence of socialinteractions (such as in the case of individual housing) thisvariation would be reduced. This seems not be the case inthe present study. Senegalese sole housed individually

P.I.M. Silva et al. / Applied Animal Behaviour Science 124 (2010) 75–8180

exhibited an individual variation in growth of 33%,suggesting that these individual differences may beinherent. Similar results were observed by Martins etal. (2005) that reported a variation in growth of 53% inAfrican catfish Clarias gariepinus individually housed.Studies performed with Senegalese sole housed in groupsreported an individual variation in growth ranging from21% till 29% (Aragao et al., 2008; Costas et al., 2008)showing values in fact somewhat smaller than the valuesobtained in this study using individual housing. Thissuggests that in Senegalese sole the presence of socialinteractions does not explain the presence of individualdifferences in growth. Growth variation was observed inother flatfish both individually housed such as Greenbackflounder (CV 19%, Carter and Bransden, 2001) and ingroups such as Atlantic halibut (CV approx. 21%, Kristian-sen et al., 2004).

Furthermore, to which extend coping styles differ inperformance traits such as feed efficiency as shown inMartins et al. (2005, 2006) and van de Nieuwegiessen etal. (2008) would be an interesting point to study withinthe context of aquaculture. In fact, the link betweenperformance traits and coping strategies is largelyunknown for aquaculture species. This is mainly due toexperimental protocols that are limited in time anddespite allowing a characterization of individual beha-viour and stress response do not allow a propercharacterization of performance traits that need longerexperimental periods.

5. Conclusion

In conclusion, this study quantifies for the first time theindividual variation in growth, undisturbed and post-stress cortisol levels, feeding behaviour and activity duringacute stress in Senegalese sole. The relationships betweenthese variables support the existence of coping styles.Behaviour traits like feeding latency and duration of escapeattempts are easier, faster and cheaper to measure andwere shown to predict undisturbed cortisol levels. Whenlinked with performance traits, these behaviour traits,together with physiological measurements, may be ofinterest as a discriminatory tool in genetic selectionprograms.

Acknowledgments

We wish to thank H. Teixeira for practical assistance.This study benefited from funding by Project PROMAR -SP5.P117/03 (programme INTERREG III A, co-funded byFEDER, European Commission), SFRH/BPD/49051/2008and SFRH/BD/44103/2008 from ‘‘Fundacao para a Cienciae Tecnologia’’ (Portugal).

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