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Applied Animal Behaviour Science 151 (2014) 110– 116

Contents lists available at ScienceDirect

Applied Animal Behaviour Science

journa l h omepa g e: www.elsev ier .com/ locate /applan im

he preference of 0+ and 2+ gilthead seabream Sparus aurataor coloured substrates or no-substrate

lkisti Batzinaa, Kyriaki Sotirakogloub, Nafsika Karakatsouli a,∗

Department of Applied Hydrobiology, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Iera Odos 75,1855 Athens, GreeceDepartment of Mathematics and Statistics, Agricultural University of Athens, Athens, Greece

a r t i c l e i n f o

rticle history:ccepted 27 November 2013vailable online 5 December 2013

eywords:ravel substratereference testparus aurata

a b s t r a c t

A major component of animal welfare is to provide for the behavioural and psychologicalneeds of captive animals. A means to estimate these needs is preference tests. However,what an animal chooses may not prove to be beneficial for its long-term health and welfare.The long-term effect (for approximately three months) of the presence of coloured sub-strates on gilthead seabream Sparus aurata growth performance and aggressive behaviourhave been previously examined. In particular, positive results for the blue substrate(enhanced growth performance, reduced aggressive behaviour), ambiguous results for thered-brown substrate (reduced aggressive behaviour, but failed to improve growth) and nobenefits for the green substrate (no differences from fish in tanks without substrate) wereobserved. The aim of the present study was to “ask” if and what substrate gilthead seabreamprefer among those that were used in previous experiments. Binary preference tests weredesigned among four treatments [Blue (BS), Red-Brown (RBS), Green (GS) Substrate or nosubstrate-Control (C)]. Trials were performed for two age classes (0+ and 2+) and fish weretested individually (10 fish per trial) or in groups of 7 fish (in triplicate). Fish choices weresimilar either tested individually or in groups. Older gilthead seabream preferred the BSin all dual combinations, chose the RBS only in cases that the BS was not present and didnot prefer the GS. Younger gilthead seabream chose the BS over the C, preferred the RBS in

certain combinations and did not prefer the GS. Moreover, in the combinations of two sub-strates an increased frequency of changing compartments was observed. Obtained resultsindicate that gilthead seabream choices were not always in line with long-term effectsof substrates. However, these outcomes could be strengthened if motivational tests wereperformed to confirm that these choices are of some value for gilthead seabream.

. Introduction

A major component of animal welfare is to provideor the behavioural and psychological needs of captive

nimals (Dawkins, 2004; Shepherdson et al., 1998). In gen-ral, several indicators have been used to estimate welfaree.g. body damage, life expectancy, disease level; Broom,

∗ Corresponding author. Tel.: +30 210 5294429.E-mail address: nafsika@aua.gr (N. Karakatsouli).

168-1591/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.applanim.2013.11.013

© 2013 Elsevier B.V. All rights reserved.

1991). Moreover, the “animal needs index” was developedto merge several components of the animal environment(Bartussek, 1999). According to Dawkins (2004), there isno single measure that can be used to assess animal wel-fare, while behaviour can integrate all indicators and givean overall picture of animal state. A means to estimateanimal needs is preference tests (Broom, 1991; Dawkins,

2004). Although they help to assess the choice of an animalbetween two or more options, they provide no informa-tion on how important this preference is for the animal(Dawkins, 2004). To evaluate how hard an animal may be

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A. Batzina et al. / Applied Animal

willing to work in order to gain access to or avoid a certainchoice, motivational tests are necessary to be performed(Dawkins, 2004; Gonyou, 1994).

Preference tests should be carefully designed toensure the un-biased choice of an animal. The finaloutcome of a test may depend on many factors likeage, previous experience, the actual number of choicesoffered, sex, reproductive state, water temperature etc.(Bateson, 2004; Dawkins, 2004; Luchiari and Pirhonen,2008; Widowski, 2009). For example, juvenile and adultgammarids Pontogammarus robustoides showed differenthabitat preference (Czarnecka et al., 2010), previous expe-rience of birds on certain housing conditions biased theirpreference (Petherick et al., 1990), while the gypsy mothLymantria dispar made a clear preference when two dif-ferent concentrations of isopimaric acid were offered butfailed to choose among five concentrations (Raffa et al.,2002). Taking these into account, it is indicated that theinterpretation of results is a multifaceted issue.

In the case of fish, preference tests have been used toobtain knowledge for behavioural patterns important tofish and for appropriate housing conditions. For examplepreference tests have been performed to identify habi-tat use, nesting, mate or shoalmate choice, anxiety (e.g.Bierbach et al., 2011; Blakeslee et al., 2009; Blaser andRosemberg, 2012; Inui et al., 2010; Pappal et al., 2009), foodpreference (e.g. Fortes-Silva et al., 2011) or preference forstructured environment (e.g. Benhaïm et al., 2009; Kistleret al., 2011; Luchiari et al., 2007).

An important issue that concerns the interpretation ofthe outcomes of preference tests is that what an animalchooses may not prove to be the most beneficial for itslong-term health and welfare (Dawkins, 2004; Widowski,2009). Similarly, fish may indeed choose what is beneficialfor them in long-term experiments (Luchiari and Pirhonen,2008) or have different choices (Ullmann et al., 2011).

In the case of gilthead seabream Sparus aurata the effectof coloured substrates in long-term experiments has beenpreviously examined. In particular, it has been shown thatthe long-term (84 days) presence of blue or red–brownsubstrate on the tank bottom can act as a means of envi-ronmental enrichment for gilthead seabream (age 1+) sinceit enhanced growth and suppressed aggressive behaviour(Batzina and Karakatsouli, 2012). On the other hand, greensubstrate cannot be considered as environmental enrich-ment for 1 + gilthead seabream since its effects weresimilar to that of tanks without substrate (Batzina andKarakatsouli, 2012). Moreover, in a subsequent experi-ment younger S. aurata (age 0+) was reared (for 98 days)under two different stocking densities in tanks with blue,red–brown or without substrate. Regardless the density,the previously mentioned positive results of the bluesubstrate were once more observed while red–brown sub-strate reduced aggressive behaviour but failed to improvegrowth (Batzina et al., 2013).

Taking into consideration the possible discrepanciesbetween fish choices and long-term benefits, the aim of the

present study was to “ask” if and what substrate giltheadseabream prefer among those that were used in previousexperiments. Preference tests were used and performed fortwo different age classes (0+, 2+) to account for previous

ur Science 151 (2014) 110– 116 111

long-term experiments (Batzina and Karakatsouli, 2012;Batzina et al., 2013). Fish were tested individually or ingroups in order to evaluate the possible involvement ofsociality on fish preferences.

2. Materials and methods

2.1. Fish

One hundred twenty three fish of mean body weight84.8 ± 1.9 g (age 2+) and mean total length 17.7 ± 0.1 cmcame from our stock populations. Fish were acclimated tolaboratory conditions (see Section 2.5) in tanks (glass, rect-angular, height × width × length: 41 × 49 × 67.5 cm, watervolume 135.6 L; all sides, apart from the front and top ones,externally covered with light blue styrofoam). One hundredtwenty three fish of mean body weight 21.9 ± 0.5 g (age 0+)and mean total length 11.5 ± 0.1 cm were obtained from acommercial Greek farm and acclimated in laboratory con-ditions (see Section 2.5) for at least six month in glass tanks(135.6 L). All tanks were part of an indoor recirculating sea-water system, provided with mechanical (polyester filterpad) and biological filters (submerged gravel biofilter), UVsterilization and compressed air supply. All fish used werenaïve without previous experience of the substrate whilethey had not participated in any other experiment.

2.2. Trials and experimental tanks

Binary preference tests were designed among the threesubstrates previously used, that is blue, red-brown andgreen single colour glass gravel (Hermes S.A. DecorativeMaterials, Koropi, Greece; gravel size: 6–12 mm) and thecontrol environment in which our fish were used to live in(tanks without substrate; glass bottom)-Control (BS, RBS,GS and C, respectively). Thus, the six sets of choices wereBS–RBS, BS–GS, RBS–GS, BS–C, RBS–C and GS–C.

Fish were tested individually (10 fish/set) and in groupsof seven fish (3 groups/set). In the individual trials all sixcombinations were examined, while in group trials, thecombinations including the GS were excluded based onthe preceding individual trials and the results of Batzinaand Karakatsouli (2012) concerning GS long-term effectsfor S. aurata.

Four observation tanks were used (glass, rectangular,height x width x length: 42 × 49 × 83 cm, water volume171 L; all sides, apart from the front and top ones, exter-nally covered with light blue styrofoam). In each tank,the bottom was divided in two equal compartments witha transparent glass partition of 5 cm height. Each com-partment was filled with one of the substrates used (5 cmheight) or remained plain as control. In the individual tri-als, the position of the substrate in the tank changed everyalternate fish to account for possible position bias on fishpreference. Side bias for each set was tested by t-test and inall cases fish preference was not affected (0+ fish, p > 0.090;2+ fish, p > 0.107). Thus, in group trials the position of the

substrate was not changed. In each compartment an airstone was placed along the side glass wall of the tank.Light intensity was adjusted to 200 lx at water surface,which was the intensity used for long-term experiments

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Batzina and Karakatsouli, 2012; Batzina et al., 2013).ight source (cool white fluorescent lamps, OSRAM DULUX/E 26W/840 G24Q-3) was placed above and at the cen-

re of each tank. Light manipulation was controlled withinDim 4.0e PC software and light intensity was measured

y means of digital light meter (RS 180-7133, RS Compo-ents Ltd, Corby, Northants, UK). During observation time,ater inlet was stopped to provide for similar water cur-

ents in both compartments. However, water was totallyenewed after each set completion. Water quality parame-ers were maintained within the welfare requirements forilthead seabream (Poli, 2009) as follows: temperature,8.9 ± 0.02 ◦C; dissolved oxygen, 7.1 ± 0.03 mg/L (96 ± 0.7%aturation); pH, 7.54 ± 0.015; salinity, 35 ± 0.0 g/L.

.3. Fish handling and video recording

Experimental fish were fed to satiation, a commercialelleted diet for gilthead seabream, on the morning (08:30)f the day before observation in their stock tanks and at7:00 of the same day they were transferred in acclima-ion tanks (height x width x length: 42 × 49 × 49 cm; waterolume 101 L) which were set up exactly as the observa-ion tanks with the difference that the water inlet was nottopped. The next day, fish were transferred in the obser-ation tanks where they remained undisturbed for 1 h toecover from the netting. Two video recordings took placeor each fish (20 min duration) or fish group (30 mi dura-ion), one and two hours after transfer to the observationank (from the front side of the tank). After a fish or ash group was tested, it was anaesthetized (2-phenoxy-thanol; 0.4 ml/L), subjected to individual body weighing0.1 g precision) and body measurements (vernier caliper,recision 0.1 mm) (see Section 2.5).

.4. Calculations and data analysis

In individual trials, the time (in seconds) fish spentn each compartment per visit was measured. Prefer-nce was estimated by calculating the total time spentn each compartment (% of total observation time). More-ver, the frequency of changing compartments (times/min)as estimated as an indicator of fish swimming behaviourhen confronted with the tested environment. In group

rials, preference was estimated by counting the numberf fish in each compartment at 15 s intervals (% of total fishumber). A fish was considered to be in a compartmenthen the 2/3 of fish body was within a compartment. Each

ideo recording was analyzed for 18 min (individual trials)r 28 min (group trials). The first two minutes of each videoere excluded to eliminate possible disquiet of fish by the

etting of the camera.Preference data were analyzed with one-tail paired t-

est or Wilcoxon signed-rank test according to normalistribution results. Time spent per visit (s) in each com-artment was analyzed with one-way ANOVA followed byuncan multiple range test when p < 0.05. When the nor-

ality assumption was not fulfilled Kruskal–Wallis testas used to identify significance. A two-way repeatedeasures ANOVA (General Linear Model) was performed

o analyze the effect of recording time on fish preference

ur Science 151 (2014) 110– 116

and on the frequency of changing compartments. All val-ues presented in the text and the table are untransformedmeans ± SE.

2.5. Ethical note

Gilthead seabream S. aurata stock populations of ourdepartment were offered from commercial hatcheries atan early life stage (1.5–2 g) and were acclimated in recir-culating water systems where water quality and rearingconditions (density 5–10 kg/m3) were always controlled tobe within the welfare limits of the species (EFSA, 2008).Photoperiod was 12 h light: 12 h dark and temperaturefluctuated in accordance with natural temperature. Fishwere fed three times per week in one early meal to sati-ation, a commercial pelleted diet for gilthead seabream. Toensure the minimum distress of fish, standard procedureswere followed for fish transportation between tanks andanaesthesia. These included gentle netting (without chas-ing) from a tank, slight anaesthetization (stage 4 within3 min) in a water bath with good aeration and filled withwater from the fish tank. Fish were anaesthetized individ-ually or in groups of less than 10 specimens. Once stage 4was reached fish were individually weighed. Fish immer-sion in the anaesthetic solution was managed to last lessthan 5 min. Fish recovered (within 1.5 min) in a water bathfree of anaesthetic solution with good aeration and, oncefully recovered, they were transferred to their tank by gen-tle netting. All of the fish used in the present study werekept alive for future research. At the time our researchwas conducted there was no institutional licensing of ani-mal research (i.e. ethics committee). However, to ensurethe welfare of the fish in our experiment, care and useof animals complied with national laws (PD 160/91) andDirective 2010/63/EU. Fish presented no signs of adversereactions to the experimental procedures.

3. Results

In individual and group trials no effect of observationtime (1 h or 2 h after transfer to observation tank) wasobserved on fish preference (in all cases p > 0.985). Dur-ing preference testing, neither territorial behaviour noraggressive acts were observed in grouped fish, while all fishinteracted with gravel (fish grabbed gravel in the mouthand ejected it or “chewed” it and then ejected it or rarelyswallowed it).

In individual trials, in the combinations of a substratewith the C compartment, 2+ fish significantly preferredthe BS (W = 164, n = 18, p = 0.0001), did not prefer the GS(W = 210, n = 20, p = 0.0001) while they made no choicebetween the RBS and the C compartment (t = 1.09, n = 17,p = 0.146; Fig. 1). In the combinations of two substrates, 2+fish preferred the BS whenever present (BS–GS, W = 148,n = 18, p = 0.003; BS–RBS, t = 1.79, n = 20, p = 0.045), while inthe absence of the BS they preferred the RBS over the GS(t = 2.62, n = 18, p = 0.009; Fig. 1). On the other hand, 0+ fish

significantly preferred the BS or the RBS over the C com-partment (BS–C, t = 2.55, n = 19, p = 0.010; RBS–C, W = 183,n = 19, p = 0.0001) and did not prefer the GS (W = 166, n = 19,p = 0.002; Fig. 2). In the combinations of two substrates fish

A. Batzina et al. / Applied Animal Behaviour Science 151 (2014) 110– 116 113

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Fig. 1. Preference (time spent as % of total observation time) of age 2+ gil-thead seabream in individual trial (n = 10 fish per combination) for Blue(BS), Red-Brown (RBS), Green (GS) Substrate or no substrate-Control (C).Results represent means ± SE. *p < 0.05; **p < 0.01; ***p < 0.001; ns, non-significant.

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Fig. 2. Preference (time spent as % of total observation time) of age 0+

Table 1Time spent per visit (s) in each compartment of age 2+ and 0+ giltheadseabream (individual trial, n = 10 fish per combination).

Pairedcombinations

p

2+ gilthead seabreamBS–GS BS: 42.7 ± 19.3 GS: 8.5 ± 1.1 **

BS–RBS BS: 41.8 ± 26.4 RBS: 11.2 ± 2.3 nsRBS–GS RBS: 12.6 ± 2.2 GS: 8.2 ± 1.5 **

GS–C GS: 2.2 ± 0.7 C: 570.4 ± 111.3 ***

RBS–C RBS: 9.9 ± 0.8 C: 11.1 ± 2.0 nsBS–C BS: 355.0 ± 101.4 C: 7.8 ± 1.6 **

END>[5pt] 0+ gilthead seabreamBS–GS BS: 9.3 ± 0.7 GS: 9.3 ± 1.3 nsBS–RBS BS: 14.2 ± 2.4 RBS: 10.8 ± 1.8 nsRBS–GS RBS: 25.8±4.9 GS: 4.0 ± 0.2 ***

GS–C GS: 5.0 ± 0.9 C: 44.9 ± 9.4 ***

RBS–C RBS: 336.7 ± 105.8 C: 6.6 ± 1.3 ***

BS–C BS: 57.8 ± 19.5 C: 11.9 ± 2.2 *

BS: Blue Substrate; GS: Green Substrate; RBS: Red-Brown Substrate; C:Control-no substrate. Results represent means ± SE.ns, non-significant.

gilthead seabream in individual trial (n = 10 fish per combination) for Blue(BS), Red-Brown (RBS), Green (GS) Substrate or no substrate-Control (C).Results represent means ± SE. **p < 0.01;***p < 0.001; ns, non-significant.

made no choice between the BS over the GS or the RBS(BS–GS, t = 1.09, n = 18, p = 0.146; BS–RBS, W = 120, n = 20,p = 0.294) while they preferred the RBS over the GS (t = 9.87,n = 20, p = 0.0001; Fig. 2).

In the combinations of two substrates 0+ and 2+ fishshowed equally higher frequency of changing compart-ments than in the combinations of a substrate with the Cenvironment (F5,230 = 10.22, p = 0.0001; Fig. 3). However, in

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Fig. 3. Frequency of changing compartments (times/min) of 0+ and 2+gilthead seabream (n = 10 fish per combination) in tanks with Blue (BS),Red-Brown (RBS), Green (GS) Substrate or no substrate-Control (C) in allcombinations. Results represent means ± SE. Bars with the same letters(a-d) are not significantly different (two-way ANOVA).

* P<0.05.** P<0.01.

*** P<0.001.

the combinations of a substrate with the C compartment,0+ fish made more frequent changes over the BS or the GSthan 2+ fish, while the opposite was observed in the RBS–Ccombination (F5,230 = 2.70, p = 0.022; Fig. 3).

Visits of 0+ fish in each compartment of the BS–GSand BS–RBS combinations were equally short (BS–GS,F1,34 = 0.09, p = 0.766; BS–RBS, F1,38 = 0.76, p = 0.389;Table 1). In the other combinations of two substrates[0 + (RBS–GS, Kruskal–Wallis test, p = 0.0001) and 2+(BS–GS, Kruskal–Wallis test, p = 0.005; BS–RBS, F1,38 = 1.33,p = 0.255; RBS–GS, Kruskal–Wallis test, p = 0.002)], meanvalue of time spent per visit in the compartment that thefish chose was less than one minute (Table 1). On the otherhand, in the combinations of the BS and the RBS with theC environment, visits of 0 + (BS–C, Kruskal–Wallis test,p = 0.037; RBS–C, Kruskal–Wallis test, p = 0.0001) and 2+(BS–C, F1,34 = 11.25, p = 0.002) fish in the compartment oftheir choice lasted from 1 to 6 min, with the exception of 2+fish in the combination RBS–C where differences were notsignificant (F1,32 = 1.31, p = 0.260; Table 1). In the case ofthe GS–C combination, fish of both age classes visited the Ccompartment for longer periods. In the case of 2+ fish, meanvisit time lasted approximately 9 1/2 min (0+, F1,38 = 25.74,p = 0.0001; 2+, Kruskal–Wallis test, p = 0.0001; Table 1).

In group trials, 2+ fish preferred the BS and the RBSover the C compartment (BS–C, W = 20, n = 6, p = 0.030;RBS–C, t = 2.36, n = 6, p = 0.032), as well as the BS overthe RBS in their combination (t = 7.79, n = 6, p = 0.0001;Fig. 4). On the other hand, 0+ fish preferred the BS orthe RBS over the C compartment (BS–C, t = 11.53, n = 6,p = 0.0001; RBS–C, t = 7.67, n = 6, p = 0.0001) while theymade no choice in the BS–RBS combination (t = −0.03, n = 6,p = 0.512; Fig. 5).

4. Discussion

Fish choices, either tested individually or in groups,were quite similar. Thus, social effect on fish preference

114 A. Batzina et al. / Applied Animal Behavio

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Fig. 4. Preference (number of fish as % of total fish number) of age 2+gBr

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ilthead seabream in group trial (n = 3 groups of fish per combination) forlue (BS), Red-Brown (RBS) Substrate or no substrate-Control (C). Resultsepresent means ± SE. *p < 0.05; ***p < 0.001.

s not indicated, at least under the present experimentalonditions where neither territorial behaviour nor aggres-ive acts were observed. In accordance with this result,ebrafish Danio rerio preference to shoal was similar eitherested individually or in groups (Polverino et al., 2012).hese results may indicate that individual tests couldeflect the choice of at least small fish groups.

When fish were tested in the combinations of a sub-trate with the C compartment, a clear preference for theS or RBS was observed, despite fish familiarity with the

environment. Previous experience, or else familiarityith a choice, is a strong ignition and determinant of the

utcome of a preference test (Gómez-Laplaza and Fuente,007; Petherick et al., 1990). Interestingly, in the presenttudy fish chose the alternative environment. This prefer-nce may indicate fish attraction towards an environmentloser to a natural one or fish exploratory behaviour for

novel environment. The presence of the compartmentithout substrate, with which fish were already familiar,ay have promoted fish boldness to visit the alternative

hoice and permitted for a definite preference (Galhardot al., 2012). Whatever the reasons that triggered the cer-ain choices, they do not seem to stand in the case ofhe GS, since fish did not prefer it. Instead, an implica-ion of substrate colour apart from its physical presence

s indicated. In other studies investigating fish preferenceor ambient colours (tank or light colour), tested species

ade clear choices for specific colours (Avdesh et al., 2012;

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ig. 5. Preference (number of fish as % of total fish number) of age 0+ilthead seabream in group trial (n = 3 groups of fish per combination) forlue (BS), Red-Brown (RBS) Substrate or no substrate-Control (C). Resultsepresent means ± SE. ***p < 0.001; ns, non-significant.

ur Science 151 (2014) 110– 116

Luchiari and Pirhonen, 2008; Ullmann et al., 2011). How-ever, gilthead seabream colour preference has not beeninvestigated.

In the case of the combinations of two substrates, fishof both age classes had a clear preference for the RBS overthe GS. However, older fish did make a choice between theBS over the RBS or GS, while younger fish did not. Theseresults are in contrast with what was observed for Atlanticcod Gadus morhua (age 0+ vs. 1+; Fraser et al., 1996) or win-ter flounder Pseudopleuronectes americanus (age 0+ vs. 1+;Pappal et al., 2012), where fish presented similar choicesdespite their age difference. In the present study, thegreater age difference of tested fish may have contributedto preference variation. Moreover, other factors such assex and/or visual abilities of gilthead seabream could havebeen involved. It is known that gilthead seabream is aprotandrous hermaphrodite species. The time scale of sex-ual inversion of males into females depends on fish bodylength/mass, age or environment. Gilthead seabream hasbeen reported to become female at a minimum body lengthof 26 cm and after the second year of age (Basurco et al.,2011; Bruslé-Sicard and Fourcault, 1997; Chaoui et al.,2006; Emre et al., 2009). However, in the present study fishwere not killed and sex could not be identified. Acknowl-edging that in other animals sex has been reported to affectpreferences for housing conditions (Blom et al., 1995), theimplication of sex in present results cannot be excluded. Onthe other hand, age-related differences in visual abilitiesof a species may also have interfered with present prefer-ences. Fish continue to grow through lifetime and the samehappens to their eyes with modifications in retinal struc-ture and visual acuity (Fernald, 1991). Despite the lack ofknowledge concerning gilthead seabream visual abilities,younger and older fish may have perceived each substratedifferently. It is known that gilthead seabream, as otherSparidae species, is found over both rocky and soft bottomswith varying grounds (sand, gravel, small blocks or seagrassbeds) in shallower waters when young while adults grad-ually move to deeper waters, where the blue wavelengthprevails (Basurco et al., 2011). This may account for theabsence of a clear preference of age 0+ fish for those com-binations where the BS was contrasted with other colouredsubstrates.

In the combinations of two substrates, an increasedfrequency of changing compartments for fish of bothage classes was observed. Increased swimming activitywhen fish first encounter a novel environment has beenpreviously suggested to indicate increased exploratorybehaviour which tends to subside with time (Mikheev andAndreev, 1993; Ward, 2012). However, the overnight accli-mation in the experimental conditions does not justifya similar approach. Present results may possibly indicatethat fish were more anxious over two novel substrates(Benhaïm et al., 2013) than in the combinations of a sub-strate with the C environment. On the other hand, it hasbeen reported that low shuttling and/or freezing of fish mayequally indicate higher anxiety (Ahmad and Richardson,

2013; Blaser and Rosemberg, 2012). However, althoughfish changed compartments less frequently in the combi-nations of a substrate with C, they swam loosely within thecompartment of their choice.

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The evaluation of present fish choices under the scopeof long-term effects of substrates reveals certain notablepoints. First, older fish of the present study chose the BSwhenever present in a combination and long-term rearingof similarly sized gilthead seabream with the BS proved tobe the most beneficial for fish and its intensive aquaculture(Batzina and Karakatsouli, 2012). In addition, long-termrearing of younger gilthead seabream with the BS provedto be equally favourable to that observed for the olderfish (Batzina et al., 2013), despite the fact that young fishdid not chose the BS over the other coloured substrates.Second, in almost all present tests, fish did not prefer theGS while long-term rearing of gilthead seabream with theGS was neither detrimental nor beneficial (Batzina andKarakatsouli, 2012). Third, in this study, older fish chosethe RBS only when the BS was not a choice, while fishperformance and behaviour under long-term rearing wassimilar with both substrates (Batzina and Karakatsouli,2012). Younger fish of the present study preferred the RBSonly in certain combinations, while they made no choicebetween the RBS and BS. On the other hand, long-term rear-ing of younger fish with the RBS proved to be beneficial forfish (reduced aggression) but not for its intensive aquacul-ture (no growth enhancement) (Batzina et al., 2013).

Similar juxtapositions of fish preferences versus long-term effects of their choices have also been reported forother fish species. For example, growth enhancement wasobserved for rainbow trout Oncorhynchus mykiss whenreared (for 8 weeks) under the ambient light colour thatfish chose during a preference test (Luchiari and Pirhonen,2008). In contrast, barramundi Lates calcarifer growth per-formance (after 9 weeks) was better in tanks with a colourother than fish choice (Ullmann et al., 2011).

5. Conclusion

In conclusion, under present experimental conditions,gilthead seabream made choices that were not always inline with long-term effects of substrates. Thus, the cautioususe of preference tests to ensure welfare is further sup-ported. The preference of fish for the BS and the aversionto the GS may imply that these choices are strong enoughto be taken into consideration for laboratory or land-basedaquaculture facilities. However, these outcomes could bestrengthened if motivational tests were performed to con-firm that these choices are of some value for giltheadseabream.

Acknowledgements

We are most grateful to G. Konstantinou for his valuabletechnical assistance and to Abotis S.E. for providing the fish.

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