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Applied Animal Behaviour Science 138 (2012) 118– 124

Contents lists available at SciVerse ScienceDirect

Applied Animal Behaviour Science

journa l h o me pag e: www.elsev ier .com/ locate /applan im

rom fright to anticipation: Reward conditioning versus habituation to moving dip net in farmed Atlantic cod (Gadus morhua)

onatan Nilsson ∗, Lars H. Stien, Jan Erik Fosseidengen, Rolf Erik Olsen, Tore S. Kristiansennstitute of Marine Research, P.O. Box 1870 Nordnes, NO-5817 Bergen, Norway

r t i c l e i n f o

rticle history:ccepted 12 February 2012vailable online 8 March 2012

eywords:tressavlovian conditioningearningognitive interpretation of stressors

a b s t r a c t

Using reward conditioning to change the cognitive interpretation of stressors has beensuggested as a method to reduce stress in farmed fish. We studied the modification ofbehavioural and physiological responses of Atlantic cod to a stressor. Eight groups of codwere exposed to a moving dip net six times daily over 7 days. Half were rewarded withfood 10 s after dip net exposure (trace reward conditioning), while the remaining groupswere fed 30 min after dip net exposure (unrewarded). The swimming speed and oxygenconsumption responses after dip net exposure in the unrewarded groups declined at a rateof 13% and 12% trial−1, respectively. The swimming speed response was lost completely,while 37% of the initial oxygen hyper consumption response remained after habituation.The rewarded groups learnt to associate dip net exposure with food delivery. When thedip net stopped moving, they approached it before gathering in the feeding area. Due to

this anticipatory behaviour, swimming speed and oxygen consumption remained elevatedafter dip net exposure in the rewarded groups. Plasma cortisol and glucose levels remainedlow after dip net exposure on the last day in both groups. The experiment demonstratesthat a stimulus that initially evokes a fright response can become a positive signal of aforthcoming reward.

© 2012 Elsevier B.V. All rights reserved.

. Introduction

Many sudden stimuli, for instance loud noises or mov-ng objects, release inherent fright responses in fish (Fernöt al., 2011). Wild fish can flee or hide when they encounterrightening stimuli, while for farmed fish this possibil-ty is usually limited. Although these stimuli are rarelyarmful in aquaculture, they will nevertheless evoke aeneralized stress response. When exposed repeatedly ton initially stressful stimulus that is never followed byarm, the strength of the response may decline, a pro-

ess known as habituation (Thompson and Spencer, 1966).owever, a variety of different stimuli and low predictabil-

ty in the aquaculture environment may make habituation

∗ Corresponding author. Tel.: +47 55238500.E-mail address: jonatan.nilsson@imr.no (J. Nilsson).

168-1591/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.applanim.2012.02.014

slow or absent. Reward conditioning has been suggested asa method to change the appraisal of stimuli from negativeto positive and thereby reduce the level of stress inducedby husbandry routines (Schreck et al., 1995; Bratland et al.,2010; Fernö et al., 2011).

In reward Pavlovian conditioning, a certain event (con-ditioned stimulus, CS) repeatedly announces the arrival ofa reward, e.g. food (unconditioned stimulus, US). By feed-ing the fish when they are exposed to a frightful stimulus,i.e. using that stimulus as a CS and food as a US, the fish maylearn to associate the stimulus with a positive event. Pre-vious studies on reward conditioning as a stress reducingprocedure (Schreck et al., 1995; Bratland et al., 2010) haveused delay conditioning, the standard Pavlovian condition-

ing paradigm. In delay conditioning the CS is temporallyoverlapping with the US, and this is a simple form of learn-ing that may occur unconsciously (Clark and Squire, 1998).Trace conditioning, in which there is a stimulus-free time

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J. Nilsson et al. / Applied Animal

interval between the CS and the US, is regarded as a moredifficult learning task than delay conditioning (Lieberman,2000). In contrast to delay conditioning, trace conditioninginvolves the hippocampal brain structures (Solomon et al.,1986; Broglio et al., 2005), and has been shown to be depen-dent on an awareness of the CS–US relationship (Clark andSquire, 1998).

We have previously found that Atlantic cod (Gadusmorhua) can rapidly learn to associate a neutral CS(light flashes) with a food US when the CS and the USwere separated with as much as a 60-s interval (Nilssonet al., 2008a). In the present study we investigate howthe physiological and behavioural responses of Atlanticcod are modified over repeated exposures to an initiallystrong stressor, a moving dip net, when the stressor isrewarded with food (reward conditioning) versus unre-warded (habituation). To ensure that reward conditioninginvolved an awareness of the CS–US relationship (Clarkand Squire, 1998), i.e. that food followed dip net expo-sure, a trace conditioning paradigm was used with the foodreward separated from dip net exposure with a 10-s traceinterval.

2. Materials and methods

2.1. Subject fish

The subject fish were cod of the 6th generation in cul-ture, with origin from the southwest of Norway. Theywere hatched in late March 2008, released into a sea-water pond shortly after hatching and transferred fromthe pond to sea cages in June. In September, 3 monthsbefore the start of the experiment, they were moved to

indoor tanks at the Research station at Austevoll south-west of Bergen, Norway, where the experiment was carriedout. Two days prior to the experiment 680 cod were ran-domly distributed to eight experimental tanks (85 fish

Fig. 1. Fish distribution 5 s before (left), 10 s after (middle) and 25 s after (right) don the first trial on Day 7 (trial 37). The fish were not fed after dip net exposure insquare marks out the three stripes defined as feeding area in the image analysis.

ur Science 138 (2012) 118– 124 119

in each tank). The average weight of a subsample of 24fish at the termination of the experiment was 129 ± 30 g(±SD).

2.2. Experimental setup

The tanks were 150 cm × 150 cm wide with roundedcorners, 65 cm high and filled with 35 cm of seawater(0.75 m3). The water flow was 15 l min−1, temperature8.2 ± 0.1 ◦C (mean ± SD) and salinity 35‰. Each tank wascontinuously illuminated (24:0 L:D regime) by a 35 W spot-light positioned 180 cm above the water surface. The eighttanks were placed in two rows. An aluminium pipe wasattached parallel with the water a few cm above each tankrow and a 25 cm × 20 cm dip net was attached perpen-dicularly to the pipe above each tank, 25 cm horizontallyfrom the tank wall. The net was cut open in order to avoidinadvertent netting of fish. An electric motor controlledby programmable relays rotated the pipes around, dippingthe dip nets in and out of the water. The horizontal rangeof the dip net in the water was 57 cm, and at its deepestpoint the dip net was 10 cm below the surface. Automaticfeeders (ArvoTec TD2000, ArvoTec, Huutokoski, Finland)positioned 100 cm above each tank delivered food 15 cmfrom the tank wall in the downstream corner of the dipnet side of the tank. A video camera (VNSVUC-Z10, ScanSecure AS, Horten, Norway) was mounted 210 cm abovethe surface of each tank, with the field of view coveringthe tank floor. The cameras were controlled via the GeoVi-sion GV-800 Video Capture Card and the GV-800 MulticamSurveillance System (GeoVision Inc., Taipei, Taiwan). Seven

strips of grey duct tape were used to give a striped patternto the floor of the tank. The stripes were used during imageanalysis of the behaviour of the fish (see below). The setupof the tanks can be seen in Fig. 1.

ip net exposure in (A) a rewarded group and (B) in an unrewarded group this trial. The arrow marks out where food dropped down and the dotted

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.3. Experimental procedure

The fish were exposed to the moving dip net stressorCS) six times per day at 1-h intervals (10:00 to 15:00)or 7 days. At each exposure the apparatus was switchedn for 6 s, resulting in the dip net rotating twice throughhe tank, and then stopping above the water. Four of thesh groups (rewarded fish) were fed (US) after each dipet exposure, with feeding starting 10 s after the dip netpparatus had stopped and lasting for 30 s (around 300ellets Amber Neptun 30, 3 mm, Skretting, per feeding,orresponding to 0.5% of the biomass day−1). The remain-ng four groups (unrewarded fish) were fed in the same

anner 30 min after dip net exposure to ensure that theyid not associate dip net exposure with feeding. All tri-ls were recorded on video for later analysis. To eliminatehe effect of feeding upon behaviour and oxygen consump-ion in response to the dip net, food was omitted on threeest trials. As too many trials with food omission couldotentially influence learning, the number of test trialsas limited to trials 1, 19 and 37, i.e. the first dip net

xposure on Days 1, 4 and 7. The omitted feeding in theest trials was not compensated for, so that on these dayshe fish were fed a total of 0.4% of the biomass instead of.5% as on the remaining days. Oxygen levels in the efflu-nt water of each tank were recorded at 1-min intervalsOxyguard Commander, Oxyguard International, Denmark,ww.oxyguard.dk).

.4. Fish distribution

If cod expected food after dip net exposure they shouldave gathered in the feeding area. In order to estimatehe change in density of fish in the feeding area after dipet exposure the video recordings of the behaviour of theod were analysed with image analysis using a modifiedersion of the procedure described by Stien et al. (2007).uring feeding when fish gathered around the feeder toat, fish density was highest over the three first tape stripesownstream from the feeder (see Fig. 1). Therefore, thisart of the tank was defined as feeding area in the analysis.he tape stripes were necessary in order to make the darkod more visible against the dark tank bottom. In short,or each image frame an automatic image analysis proce-ure identified those parts of the tape stripes that wereot covered by fish from the field of view of the camera.his was then compared with the known extent of thesetripes, and the percentage coverage in the feeding areaas then calculated for each image. The baseline density ofsh was estimated from a 20-s period immediately beforeip net exposure and the post-exposure density during theeriod from 10 to 40 s after exposure, i.e. the period dur-

ng which the rewarded fish were fed except in the testrials.

.5. Oxygen consumption

Oxygen consumption rate per replicate group (VO2,g O2 kg−1 min−1) was measured by using the experimen-

al tanks as open respirometers. The oxygen consumptionate per tank was calculated by measuring the difference in

ur Science 138 (2012) 118– 124

oxygen content between influent and effluent water. Thefollowing equation was used:

VO2t = Vol · Sol · Satt − Satt−1

100ıt+ Flow · Sol · Satin − Satt

100,

(1)

where Vol is the tank volume, Sol is the solubility of oxygenat prevailing temperature and conductivity conditions, Satt

is the oxygen saturation at time t, and Satin is the oxygensaturation in the influent water, in this case 100%. Exper-iments in similar tanks and similar oxygen consumptionrates suggest that the diffusion of air oxygen into the sys-tem is negligible compared to consumption of the fish (T.Torgersen, Institute of Marine Research, unpublished data).In order to study if oxygen consumption changed after dipnet exposure, we calculated the average consumption dur-ing a 10-min period immediately before exposure (baselineconsumption) and during the 10 min immediately afterexposure. Estimating oxygen consumption during a shorterinterval, e.g. the period during which the rewarded fishwere usually fed, was impossible due to the time lag(approximately 5 min) between oxygen being consumedby the fish and water reaching the drain with the oxygenprobe.

2.6. Swimming speed

To study if dip net exposure affected the swimmingspeed of the fish, swimming speed (body length s−1) wasmeasured 20 s before exposure (baseline level) and, toexclude the immediate startle response to the dip net, 30 safter exposure. Swimming speed was estimated for six ran-dom individuals before and six individuals after dip netexposure in each tank. The average speed of the six indi-viduals was treated as one data point. Due to the timeconsuming nature of this analysis, not all trials were ana-lysed. All trials were analysed on Days 1–3 (trials 1–18),where habituation was expected to be most rapid, and later(Days 4–7, trials 19–42) every second trial was analysed.

2.7. Blood analysis

In order to assess the hypothalamic–pituitary–adrenal(HPA) response to dip net exposure in conditioned andhabituated cod, the fish were exposed to the dip net oncewith food being omitted on Day 8 and blood sampleswere taken from 12 fish of each procedure starting 30 minafter the dip net exposure. Fish from the rewarded andunrewarded groups were sampled simultaneously by twoexperimenters. Three fish from each tank were percussivestunned, and 500 �l blood was drawn from the caudal veininto heparinised syringes. To reduce stress during sam-pling, tanks were sampled one at the time so that fish in thenext tank were netted after all samples in the previous tankwere taken. The sampling of all 24 fish, from dip net expo-sure of the first fish until the last blood sample was taken,took 13 min. Plasma was prepared by immediate centrifu-

gation at 13,400 rotations min−1 for 5 min and stored at−80 ◦C until analysed. Plasma glucose was assessed usingthe COBAS C111 autoanalyzer (Roche Diagnostics, Indi-anapolis, USA). Plasma cortisol was analyzed by ELISA using

Behaviour Science 138 (2012) 118– 124 121

Fig. 2. Fish density in the feeding area estimated as mean ± SE percentstripe coverage in (A) rewarded fish, and (B) unrewarded fish. Open cir-

J. Nilsson et al. / Applied Animal

producer’s recommendations (IBL International, Hamburg,Germany, kit RE52061).

2.8. Statistics

Modification of the oxygen hyper consumption andswimming speed responses in the unrewarded fish wasevaluated by fitting the data to a non-linear least squaremodel:

Response = a + b · e(trial−1)c (2)

using the “nls” method in R software system Version 2.9.0(Copyright 2009, The R Foundation for Statistical Com-puting, Vienna, Austria). Here, a + b corresponds to themodelled initial response (trial 1), a is the modelled resid-ual response after the modification is completed (trial ∞),b is the portion of the initial response that eventually islost, and c is the exponential decay rate (trial−1), i.e. howsteeply the line drops towards a. Welch Two Sample t-testwas used to test for differences between rewarded andunrewarded fish in the change of oxygen consumption,swimming speed and fish density after dip net exposure.Data on blood chemistry did not meet the assumptions ofnormal distribution for parametric tests. Hence, the non-parametric Mann–Whitney U-test was used to test fordifferences in concentration of plasma cortisol and glu-cose between rewarded and unrewarded fish, using dataon individual fish with replicate groups for each proce-dure pooled. For plasma cortisol, concentrations below thedetection level (2 ng ml−1) were set to be 2 ng ml−1 in thestatistical analysis. All tests were two-tailed, and the levelof significance set to 0.05.

3. Results

3.1. Fish distribution

In all tanks from both the rewarded and unrewardedprocedure, fish did not gather in any part of the tank afterthe first exposure, and this pattern remained throughoutthe experiment in the unrewarded groups. In contrast, thebehaviour of the rewarded fish changed with experiencewith the dip net–food relationship. After the dip net hadstopped moving the rewarded fish approached the dip netand gathered below it for a few seconds, before they movedto the feeding area. It was not possible to accurately useimage analysis to quantify the fish density in the nettingarea because it was partly obscured by the dip net. How-ever, a clear increase in fish density in the vicinity of the dipnet immediately after the dip net stopped moving (mid-dle panel in Fig. 1A) was observed in all rewarded groupson the test trials 19 and 37, while this pattern was neverobserved for the unrewarded fish. After the approach to thedip net, the fish in the rewarded groups moved to the feed-ing area. The fish density in the feeding area during feeding,estimated as coverage of the tape stripes (Fig. 1), was lowin the first trials, and increased from trial to trial during

the first days (Fig. 2A). Also on the test trials when foodwas omitted most fish gathered downstream the feederafter dip net exposure. Fish spread out over a larger areabehind the feeder on test trials (Fig. 1A), and the change

cles: before dip net exposure (baseline), grey circles: the period 10–40 safter exposure on rewarded trials, i.e. during feeding, black circles: theperiod 10–40 s after exposure on unrewarded trials.

in fish density in the defined feeding area was less thanduring rewarded trials (Fig. 2A). No increase in fish den-sity in the feeding area after exposure was observed in theunrewarded groups (Fig. 2B). The change in fish densitywas higher in the rewarded (n = 4) than in the unrewarded(n = 4) groups on trial 37 (Welch Two Sample t-test, t = 3.40,p < 0.05, Fig. 2), while the difference on trial 19 was notsignificant (t = 1.56, p > 0.1).

3.2. Oxygen hyper consumption

The hyper consumption on the rewarded trialsin the rewarded groups was 1.06 ± 0.21 mg kg−1 min−1

(mean ± SD), and did not change during the course ofthe experiment (Fig. 3A). The hyper consumption of therewarded (n = 4) and unrewarded (n = 4) fish did not dif-fer on the first test trial (trial 1: Welch Two Sample t-test,t = −0.6371, p > 0.1, Fig. 3A and B), i.e. before they hadthe possibility to associate dip net exposure with reward.On the two later test trials, the hyper consumption washigher in the rewarded than in the unrewarded groups,though not significantly so on trial 37 (trial 19: t = 2.92,p < 0.05; trial 37: t = 2.16, p = 0.075, Fig. 3A and B). Modi-fication of the oxygen hyper consumption response after

dip net exposure (i.e. increase from baseline level) wasmodelled for the unrewarded fish (Fig. 3C). On trial 24 thehyper consumption was for some unknown reason unusu-ally high in all four unrewarded tanks (see Fig. 3C), and

122 J. Nilsson et al. / Applied Animal Behaviour Science 138 (2012) 118– 124

Fig. 3. Mean ± SE oxygen consumption of (A) rewarded fish, and (B)unrewarded fish. Open circles: the 10 min prior to dip net expo-sure (baseline consumption), grey circles: the 10 min followingexposure on rewarded trials, black circles: the 10 min followingexposure on unrewarded trials. (C) shows the hyper consumption,i.e. the increase from baseline, during the 10 min following expo-sure, of the unrewarded fish. The line indicates the modelled hyperc( .

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Fig. 4. Mean ± SE swimming speed of (A) rewarded and (B) unre-warded fish. Open circles: 20 s prior to dip net exposure (baselinespeed), grey circles: 30 s after exposure on rewarded trials, black cir-cles: 30 s after exposure on unrewarded trials. (C) shows the changefrom baseline speed 30 s after exposure of the unrewarded fish. The line

onsumption for the unrewarded fish: hyper consumption = 0.26610±0.02991) + 0.46133(±0.06619) × exp((trial − 1) × −0.13054(±0.04085))

his trial was excluded from the model. Under the modelhe initial (trial 1) oxygen hyper consumption responseas 0.727 mg kg−1 min−1 (26.8% increase from the aver-

ge baseline level), which declined at a rate c (p < 0.01, Eq.2)) corresponding to 12.2% trial−1. The estimated resid-al oxygen hyper consumption response a at trial ∞as 0.266 mg kg−1 min−1, i.e. 36.6% of the initial response

p < 0.0001, Eq. (2)). Thus, 63.4% of the initial response wasventually lost (b, p < 0.0001, Eq. (2)), of which 90.4% and9.5% of the response loss was achieved on trials 19 and 37test trials), respectively.

indicates the modelled change in swimming speed for the unrewardedfish: change = 0.04147(±0.03067) + 0.26560(±0.05834) × exp((trial − 1) ×−0.14022(±0.06855)).

3.3. Swimming speed

On rewarded trials, the swimming speed of therewarded groups after dip net exposure (i.e. during feed-ing) was always higher than baseline level, most markedlyfrom Day 2 onwards (Fig. 4A). On the test trials, the changein swimming speed after dip net exposure of the rewarded(n = 4) and unrewarded (n = 4) fish did not differ on trial

1 (Welch Two Sample t-test, t = 0.18, p > 0.1), while it washigher for the rewarded fish on trial 19 (t = 3.56, p < 0.05)and trial 37 (t = 3.82, p < 0.01, Fig. 4A and B). The modifi-cation of the swimming speed response was modelled for

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J. Nilsson et al. / Applied Animal

the unrewarded fish (Fig. 4C). Under the model the initial(trial 1) response was an increase in swimming speed of0.307 body lengths (bl) s−1 (50.0% increase from the aver-age baseline level), and the response declined at a rate c(p < 0.05, Eq. (2)) corresponding to 13.1% trial−1 (Fig. 4C).The estimated residual response a at trial ∞ of 0.0415 bl s−1

did not differ significantly from 0 (p > 0.1, Eq. (2)). Thus,the swimming speed response was eventually fully lost(model estimate: b = 0.266 bl s−1, p < 0.0001, Eq. (2)). On tri-als 19 and 37 (test trials), 92.0% and 99.4% of the modelledresponse loss was achieved, respectively.

3.4. Blood chemistry

There was no significant difference in plasma cor-tisol level between the rewarded and unrewardedfish (Mann–Whitney U-test, U = 76, nrewarded = 11,nunrewarded = 12, p > 0.1). The mean plasma cortisol concen-tration of the rewarded fish was 14.4 ng ml−1, of whichtwo individuals had concentrations much higher (39.3and 83.8 ng ml−1) than the mean for the unrewardedfish (5.0 ng ml−1). Six out of 11 individuals (data forone individual missing) in the rewarded groups and8 out of 12 individuals in the unrewarded groups hadplasma cortisol concentration below the detection level(<2 ng ml−1). There was no significant difference in plasmaglucose level between rewarded and unrewarded fish(rewarded fish: 4.24 ± 1.84 mmol l−1, unrewarded fish:4.87 ± 2.53 mmol l−1, mean ± SD, U = 70, nrewarded = 12,nunrewarded = 12, p > 0.1).

4. Discussion

The first dip net exposure resulted in increased swim-ming speed and oxygen consumption, a typical responseto acute stress (Martins et al., 2012). With experience withthe dip net exposure the unrewarded fish largely becamehabituated to the stressor, with most of the initial oxygenhyper consumption response and all of the swimmingspeed response gone at the end of the experiment (42trials). The oxygen consumption and swimming speedwere generally more elevated in the rewarded groups, alsoon unrewarded test trials. These observations could beinterpreted as if the fright response to the dip net exposureremained for the rewarded fish. However, a closer lookat the behaviour of the fish revealed that the increase inoxygen consumption and swimming speed on the twolater test trials reflect food anticipation rather than fright.The fish density in the feeding area during feeding wasrelatively low on the first trials, but increased from trial totrial during the first 3 days. This suggests that during thefirst trials the fright response to the dip net reduced feed-ing motivation, while feeding motivation increased as thefright response decreased. Eventually, the rewarded fishapproached and gathered below the dip net immediatelyafter it stopped moving, and then moved to the feedingarea, even on trials where food was omitted. Approaching

the CS, i.e. the predictive signal, rather than the location ofthe forthcoming US (reward) is referred to as sign-trackingin the psychological literature (Hearst and Jenkins, 1974),and assumed to be due to the CS becoming a substitute for

ur Science 138 (2012) 118– 124 123

the US. Goal-tracking (Boakes, 1977) is when the responseis due to the CS bringing an explicit expectation of, andbehaviour directed towards, the forthcoming US. Thus,unlike sign-tracking, goal-tracking relies on an aware-ness of the CS being a predictor of the US (Lieberman,2000). In previous studies with a neutral light signal asa CS announcing food delivery, conditioned cod alwaysapproached the signal at announcement (sign-tracked)and remained gathered near the CS light if the CS was givenin the feeding area (Nilsson et al., 2008a) or goal-trackedto the feeding area after sign-tracking if the CS was givenon the opposite side of the tank (Nilsson et al., 2008b). Thebehaviour of the rewarded cod in the present study is thusvery similar to that of cod trained in an appetitive condi-tioning procedure with a neutral signal, with sign-trackingfollowed by goal-tracking, which is a strong indication thatthe behaviour observed here reflects reward anticipation.Our finding that the initially stressful dip net exposurecould be turned to a positive signal for reward is in line withthe study by Bratland et al. (2010), where the response ofAtlantic salmon to light flashes in the feeding area changedfrom fright (escape) to anticipation (approach) duringthe course of the experiment. That reward conditioningcan reduce stress level is also supported by the study bySchreck et al. (1995), who found the physiological stressresponse and mortality of chinook salmon conditioned tolowering of the water to be lower than that of controlswhen exposed to subsequent stressors.

In the present study the fright response of the unre-warded fish habituated rapidly, with the swimming speedresponse eventually fully habituated, and the levels of cor-tisol and glucose in the blood plasma 30 min after dip netexposure the last day were low, with cortisol concentrationbelow detection levels (<2 ng ml−1) in most individuals.Plasma cortisol will increase rapidly in cod following acutestress. Using a severe acute stress model (lowering of waterlayer and chasing for 15 min) Olsen et al. (2008) foundthat cortisol increased from 15 ng ml−1 before stress to48 and 81 ng ml−1 after 15 min and 1 h, respectively. Inthe present study, the generalized stress response of bothrewarded and unrewarded fish on the last day can beassumed to be negligible. The residual oxygen hyper con-sumption response may be due to a startle response whenthe dip net splashed into the water, which was not lostdespite the habituation to the stressor. Similarly, the oxy-gen hyper consumption response of salmon parr to thetransition from darkness to light was not completely lostafter habituation, with the residual hyper consumptionbeing 31% of the initial response (Folkedal et al., 2010).Nevertheless, it appears that some fish in the rewardedpopulation responded to the treatment with a significantstress response as seen in highly elevated plasma corti-sol levels. In particular, one fish having a level of morethan 80 ng ml−1 is regarded very high in cod (Olsen et al.,2008). This may indicate variation in habituation to stress-ors among individuals (Koolhaas et al., 1999) with somenot habituating at all. An alternative explanation for high

cortisol responses after dip net exposure in some cod couldbe that these individuals had been subjected to aggressionfrom others. However, aggressive interactions were rarelyobserved and never of high intensity.

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In the present study the dip net presentation was iden-ical on all trials, exposure occurred frequently at regularntervals and had never any harmful impact on the fish,nd thus the stimulus was more predictable than whatould usually be the case in aquaculture. Therefore, habit-ation and conditioning may have been more rapid herehan in a more realistic situation, which would be moreomplex. Still, this study clearly shows that the appraisal of

stimulus can be changed from negative (inducing a frightesponse) to positive (inducing an anticipatory response)n cod.

Under farming conditions fish are exposed to severaltimuli that are inherently stressful, such as large objectsoving in the water. The actual consequences of such stim-

li may vary from near nothing (e.g. cleaning of tanks,emoval of dead fish) to highly stressful events (e.g. con-nement, handling). If the fish are unable to discriminateetween stimuli with neutral consequences and stimuliith negative consequences, the “average consequence”ill be negative and habituation may be slower or absent

Fernö et al., 2011). This may be of particular impor-ance in farm conditions where fish may not be subjectedo single repeated intense stressors, but rather severaltressors of mild intensity with unpredictable sequences.

hile repeated stress might allow for habituation, aseen in a decline in the hypothalamic–pituitary–adrenalHPA) response, a variable stress paradigm that com-ines various low-intensity stressors in unpredictableequences, may lead to the maintenance of a high HPAtimulation and consequently high levels of glucocor-icoids (Magarinos and McEwen, 1995). Such chronictimulation will have many secondary and tertiary con-equences including the well-known effects on growth,mmunity, etc. (Kristiansen et al., 2004; Weyts et al.,999).

The present study demonstrates that the appraisal of atimulus may change from negative to positive when it isssociated with a reward. Introduction of routines whereotentially stressful but harmless stimuli are followed by

food reward should be a simple but effective way to turnncertainty and fright to positive anticipation, which in

tself will be rewarding and improve welfare (Spruijt et al.,001).

cknowledgements

This study was funded by the Research Council oforway (no. 173538, Physiological and behavioural indi-ators for fish welfare assessment in aquaculture andsh capture), and complies with Norwegian regulationsn animal experimentation. We would like to thankwo anonymous referees for valuable comments on the

anuscript, Thomas Torgersen for calculations of oxygenonsumption and advice on statistical modelling, and Stigve Utskot and Svanhild Lohne Gokstad for assistance dur-

ng blood sampling.

ur Science 138 (2012) 118– 124

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