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Welfare status of cage farmed European sea bass (Dicentrarchus labrax):A comparison between submerged and surface cages

Giulia Maricchiolo a, Simone Mirto a,⁎, Gabriella Caruso a, Tiziana Caruso a, Rosa Bonaventura b, Monica Celi c,Valeria Matranga b, Lucrezia Genovese a

a Institute for Coastal Marine Environment (IAMC), CNR—Spianata S. Raineri, 86-98122 Messina, Italyb Institute of Biomedicine and Molecular Immunology “A. Monroy” (IBIM), CNR—via Ugo La Malfa 153, 90123 Palermo, Italyc Department of Animal Biology, University of Palermo, via Archirafi 18, 90123 Palermo, Italy

a b s t r a c ta r t i c l e i n f o

Article history:Received 1 June 2010Received in revised form 21 January 2011Accepted 1 February 2011Available online 4 March 2011

Keywords:Fish welfareGrowthSubmergenceSea-cageDicentrarchus labrax

Submersible sea-cages represent an alternative method to standard surface-based cages, and could help solveseveral production problems that exist in surface-based farming, such as heavy storms, algal and jellyfishblooms and attacks by predators.In this study, we investigated growth, haematological, biochemical and immunological parameters ofEuropean sea bass (Dicentrarchus labrax) farmed in surface and submersible cages, to compare thephysiological status under different rearing conditions. In addition, we evaluated the use of erythrocyte HSP70levels as a biomarker of fish welfare.The studywas conducted in two submerged and two surface cages filledwithD. labrax. No significant differenceswere observed in the growth rate and the relative condition factor between the two groups. Significantly higherlevels of serum cortisol, haematocrit plasma glucose and total plasma proteins were measured in surface-basedcages. Conversely,fish in submerged cages showedhigher haemolytic activity and lysozyme levels in bothmucusand kidney than surface reared sea bass.Results obtained in this study indicate that submergence could be a favorable condition for sea bass farming,suggesting mariculture in submerged net cages as a promising system that allows producers to minimize stress(included that from predation by fish-eating birds) and therefore favor animal welfare.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Offshore submersible fish farms may play an important role in theexpanding fish farming industry as inshore sites have reached fullcapacity, and offshore farming will likely greatly expand theproduction capacity of aquaculture (Ryan, 2004).

Submergence may solve several of the substantial operationalchallenges that exist in surface-basedfish farming, including those relatedto heavy storms, algal and jellyfish blooms, unsuitable temperatures, highpollutant levels and biofouling of net cages (Dempster et al., 2009;Fioravanati et al., 2004).Moreover, thehigh concentration offish confinedin surface net cage systems attracts predators (Sepùlveda and Oliva,2005), primarily sea birds (e.g. herons, gulls and cormorants) and aquatic

mammals (e.g. dolphins). Predation by fish-eating bird species is asignificant problem leading to death and injury in fish, resulting ineconomic damage to farmers (Beveridge, 2001; Carss, 1993). Further-more, the stress on cultured fish subjected to repeated attacks bypredators results in thepoor feed conversionefficiency and, hence,weightat harvest is not maximized (Nash et al., 2000).

The negative effects of stress in farmed fish regard, not only growth,but alsomanyphysiological functions (Barton, 2000, 2002; Iwama et al.,1997;Wendelaar Bonga, 1997). Fish react to stressful conditionsmainlythrough primary and secondary responses (Barton, 2002). Primaryresponses are characterized by the release of catecholamines from thechromaffin tissue (Pickering, 1981; Randall and Perry, 1992; Reid et al.,1998) and by the activation of the hypothalamus–pituitary–interrenal(HPI) axis, culminating in the release of corticosteroid hormones,mainly cortisol, into the circulation (Donaldson, 1981; Mommsen et al.,1999; Schreck, 1981; Wendelaar Bonga, 1997). Secondary responsesinclude the biochemical and physiological effects associatedwith stress,and mediated, to a large extent, by the above mentioned stresshormones. These include changes in plasma and tissue ions andmetabolite levels, haematological features and heat shock or stressproteins (HSPs), all of which are related to the physiologicaladjustments in metabolism, respiration, acid–base and hydromineral

Aquaculture 314 (2011) 173–181

⁎ Corresponding author at: Institute for Coastal Marine Environment (IAMC),CNR, Spianata S. Raineri, 86-98122 Messina, Italy. Tel.: +39 090669003; fax: +39090669007.

E-mail addresses: giulia.maricchiolo@iamc.cnr.it (G. Maricchiolo),simone.mirto@iamc.cnr.it (S. Mirto), gabriella.caruso@iamc.cnr.it (G. Caruso),bonaventura@ibim.cnr.it (R. Bonaventura), monica.celi@libero.it (M. Celi),matranga@ibim.cnr.it (V. Matranga), lucrezia.genovese@iamc.cnr.it (L. Genovese).

0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.aquaculture.2011.02.001

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balance, immunocompetence and cellular responses (Iwama et al.,1997, 1998; Mommsen et al., 1999; Pickering, 1981).

With regard to cellular response, HSPs in cells are constitutivelyexpressed and critical for protein metabolism but are also induced infish exposed to a wide variety of stressors, thus indicating theirprotective role during times of stress (Iwama et al., 1998, 1999;Segner, 1998). Amongst all, HSP70 is themost likely candidate proteinfor response to stressors, as it shows low constitutive expression,whilst it is strongly induced after stress (Liberek et al., 2008).

In recent years, interest in the protective role of HSPs in fish afterexposure to stressors has been increasing; this is also due to thepossibility of a functional relationship between the stress proteininduction and the neuro-endocrine system (Blake et al., 1991).Moreover, it has been shown that HSPs are significantly over-expressed in fish erythrocytes, known to be nucleated cells capableof synthesizing proteins (Fulladosa et al., 2006; Lund et al., 2003).

Finally, tertiary response occurs, which represents the whole animalchanges associated with the experience of stress. If fish are unable toacclimate or adapt to stress, and maintain the energy-repartitioningrequired for anabolic activities intact, animal performances, such asgrowth and gamete development, can be compromised. Thus, chronicexposure to stressors can lead todecreases in growth, disease resistance,reproductive success, smelting, swimming performances and othercharacteristics of the whole animal (Wedemeyer and Mc Leay, 1981;Wedemeyer et al., 1990).

On thebasis of the above scientific evidence, aquaticproducers knowthat controlling animalwelfare is absolutely essential for their economicsuccess and that the development of specific stress managementprotocols is essential for fish health and survival. Reducing stress andmaintaining fish welfare is, therefore, an important issue for theaquaculture industry for theproduction efficiency, quantity andproductquality (Poli et al., 2005) and, recently, it has also become important forboth the consumer perception andmarketing (Broom, 1998; FSBI, 2002;Southgate and Wall, 2001).

Subsurface technologies have been tested in several productionexperiments, e.g., the farming of yellowtail (Seriola spp.) in Japan(Brown, 1983) and Italy (Mazzola et al., 2000), Atlantic cod (Gadusmorhua) and haddock (Melanogrammus aeglefinus) in the U.S.A.(Chambers and Howell, 2006), cobia (Rachycentron canadum) in theCaribbean (Benetti et al., 2008, 2010) and Atlantic salmon (Salmosalar) in Norway (Dempster and Sanchez-Jerez, 2008, 2009; Korsøenet al., 2009). But, the growth and behavior of fish in submerged cages,relative to the standard surface systems, is largely unknown.

Objective comparisons of fish performance in commercial-scalesubmerged cages vs. surface cages have only been undertaken forshort-term, shallow submergences (Dempster and Sanchez-Jerez,2008, 2009), or long-term in Atlantic salmon (Korsøen et al., 2009). Inaddition, no previous experiment has investigated the effects ofsubmerged cage culture on the welfare of warm-water species.

The aim of this study is to investigate physical, haematological,biochemical and immunological parameters of European sea bass(Dicentrarchus labrax) farmed in submersible cages, in order to comparethe physiological status under different rearing conditions and to validatethe efficacy of submersible technology. In addition, because of theincreasing interest in the role of HSPs infish stress response,we evaluatedtheuse ofHSP70determination in blood cells of sea bass, in order to proveits amenability as a fish welfare biomarker, under farming conditions.

2. Materials and methods

2.1. Location and experimental design

The studywas conducted from January toDecember 2008 in theGulfof Castellammare (NW Sicily, Mediterranean Sea). Four fish cages(diameter 16 m,10 mdepth, approximately 1800–2000 m3)wereused:two for submergence and two for control treatments (surface). The two

control cages were a standard type used for commercial sea bream orsea bass production. The two submerged cages weremodified standardcages, with a roof of black netting, which used the same mesh as thecage, and were equipped with a submerging system. The tops of thesubmerged cages were approximately 5 m below the surface.

75,000 specimens of European sea bass (Dicentrarchus labrax) witha mean initial weight of 28.2±4.3 g were randomly distributed ineach cage in January 2008.

During the trial, fish were manually fed a commercial diet tosatiation twice a day according to the estimated live weight and watertemperature. In the submerged cages, feed was delivered through apipe (Ø=200 mm) with running water, 50 cm below the roof in thecenter of the cage. In order to obtain a comparable feed availability inthe two treatments (surface based and submerged cages), pelletswere distributed with the same frequency (in terms of Kg min−1).

The two submerged cages were sunk at the beginning of theexperiment and were raised monthly for a few hours to allow fishsampling collections. The submerging or re-surfacing of cages tookapproximately 40 min (speed=12.0 cm min−1), to avoid any possi-ble interference and effects on fish behavior. Moreover, the lifting ofcages from 5 m to the surface does not create a decrease in pressuregreater than 40%, considered the upper limits to avoid stress problemsin fish, as demonstrated by Korsøen et al., 2010.

2.1.1. Sampling protocolsFish feeding was stopped 24 h prior to sampling. To investigate

growth performances, fish (n=100) from each cage were randomlycollected on a monthly basis. Fish sampling was performed using acasting net pulled up from the bottom to crowd the fish at the surface;total and standard length (TL and SL) and wet weight (W) wereimmediately recorded for each specimen.

To measure welfare, additional fish (n=20) from each cage, wererandomly sampled on three occasions: late February (40 days afterseeding) June and September 2008. To avoid additional stress, fish forwelfare measurements were captured before the fish used for growthperformance determination.

After capture, fish were immediately anaesthetized using asolution of 0.1 g/l MS222 (Ethyl 3-aminobenzoate methanesulfonatesalt, Sigma-Aldrich, Milano, Italy). Careful netting and handling wereimplemented to minimize stress.

In addition, to avoid blood sampling-induced stress response,withdrawal was carried out simultaneously by different operators.Blood was withdrawn from the caudal vein in each fish, and,immediately, subdivided into different test tubes depending on theanalyzed parameter: sodium fluoride tubes for glucose and lactatedetermination, tubes with separating granules to extract serum andlithium heparin tubes to obtain plasma. Each test tube was stored oncrushed ice until all samples were collected.

Small volumes of heparinised blood were used for both theimmediate determination of haematocrit value and the HSP70 assay.The remaining fraction of the heparinised blood was centrifuged, at5000 g for 10 min, to obtain plasma for glucose, lactate and lysozymedeterminations. Unheparinised tubes were allowed to clot at 4 °C andcentrifuged for 20 min at 1500 g to obtain the serum for cortisol,proteins and spontaneous haemolytic activity (SH50) determinations.

Plasma and serum samples were stored at −20 °C and thentransferred to −80 °C on arrival at the laboratory. Mucus and kidneysamples for lysozyme measurements were collected after fisheuthanasia with a lethal dose of MS222 (0.8 g/l) to avoid any possibleperception of pain (Huntingford et al., 2006), as required by ethicalprinciples for the use of fish in scientific research.

2.2. Growth parameters

Specific growth rates (SGR) were calculated as [(ln W2− ln W1)/t]×100,whereW1 is theweight of thefish at the start,W2 is theweight at

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the end, and t is the duration of the growth period (Jobling and Koskela,1996).

Fulton's condition factor (Bolger and Connolly, 1989) was alsocalculated by using the following relationship: K=Wmeans/L

3×100where W represented our measured wet weights (g) and L the totallength (cm) of fish.

2.3. Haematological and biochemical parameters

All biochemical assayswere carried out using commercial diagnostickits.

Serum cortisol concentration was determined by the use of anenzyme-linked (ELISA) immunoassay kit (Alpha Diagnostic International,USA).

Plasma glucose and total protein levels were determined by kitsrespectively based on colorimetric Glucose Oxidase–Peroxidase (GOD–POD) and chemical biuret–tartrate methods (Sclavo Diagnostics, Italy).

Haematocrit value (% red blood cell) was determined in heparin-ized capillary tubes after centrifugation in a standard microhaema-tocrit centrifuge at 12,000 g for 10 min and the comparison of thecapillary tubes with a reference scale.

Plasma lactate was assayed by a kit that utilizes the enzymaticcolorimetric method of Lactate Oxidase–Peroxidase (LOD–POD)(Roche Diagnostics, Italy).

2.4. HSP70 assay

All procedures were performed at 4 °C. Samples were washed twiceusing 3 volumes of Hank's Balanced Salts Solution (HBSS) (SIGMA,H8264) by centrifugation at 1600 rpm for 5 min in a refrigeratedEppendorf centrifuge (5415R). After a third centrifugation at 2000 rpmfor 5 min, supernatants were discharged and pellets, containing bloodcells, were frozen in liquid nitrogen and maintained at −80 °C. Pelletswere subsequently lysed for 30 min/30 strokes in a glass homogenizerusing a small clearancepestle inRIPAbuffer containing: 50 mMTris–HClpH8.0, 150 mMNaCl, 1%NP-40, 0.5% sodiumdeoxycholate, 0.1% SDS. AnEDTA free-protease inhibitor cocktail (Roche) supplemented with0.1 mM PMSF was added to RIPA buffer. Lysates were centrifuged at12,000 rpm for 10 min; supernatants were collected and the totalprotein concentrations were determined by the Bradford method(1976).

Amounts of proteins corresponding to 40 mg were re-suspendedin the Laemmli SDS–PAGE buffer (Laemmli, 1970), denatured at100 °C for 5 min and separated by 10% SDS–PAGE minigels. High andlow molecular weight range markers (Bio-Rad) were used. Afterelectrophoresis, proteins were transferred to nitrocellulose mem-branes (Amersham) (Towbin et al., 1979) and blocked in 5% non-fatdried milk dissolved in 20 mM Tris, pH 7.6, 137 mM NaCl, and 0.1%Tween 20 (TBS-T). The primary antibodies used were diluted in 3%BSA-TBS-T as follows: monoclonal anti-heat shock protein 70 (SIGMA,H-5147) 1:1000 andmonoclonal anti-β-actin (SIGMA, A-5441) 1:500.The secondary antibody used was an anti-mouse peroxidase-conjugated (Amersham, NA931) diluted 1:000 in TBS-T. Proteinswere detected by chemi-luminescence (Supersignal West Pico,PIERCE) according to the manufacturer's instructions. Films werescanned and the band intensities quantified by the ChemiDoc system(Bio-Rad) equipped with the software Quantity One (4.2.1). The pixeldensities obtained from HSP70 bands scanned for each sample werenormalized using the corresponding β-actin values. It should bepointed out that the antibody used in this study recognizes both theconstitutive (HSC70) and inducible (HSP70) forms of the protein andtherefore the values represent the total combination of both forms; inthe following, this will be referred to as HSC70–HSP70 expressed inarbitrary units.

2.5. Immunological parameters

Non-specific haemolytic activity (SH50) against sheep erythro-cytes was determined in serum samples, according to Caruso et al.(2005). Haemolytic activity of the serum was assayed using a 2.5%suspension of sheep red blood cells (SRBC) in phosphate bufferedsaline (PBS) containing 10 mM Mg2+ and 2 mM Ca2+ which wasadded to the serum serially diluted in the same buffer. Afterincubation at 22 °C for 1 h, the unlysed erythrocytes were removedby centrifugation and the optical density of the supernatants wasmeasured at 540 nm (Sunyer et al., 1995). SH50 units were obtainedfrom the concentration of serum giving 50% haemolysis of SRBC,whilst 100% haemolysis was calculated by optical reading of thesupernatant from the same SRBC added to distilled water.

Aliquots of mucus, plasma and kidney were assayed for lysozymecontent. Lysozyme activity was measured by the radial diffusionmethod in 1% agarose plates containing 0.05% lyophilisedMicrococcuslysodeikticus (Sigma-Aldrich) as the substrate, dissolved in a pH 5.75phosphate buffer. Lysozyme content was evaluated by measuring thediameter of lysis produced after incubation at 30 °C for a period of22 h, which was converted into Units of lysozyme per ml of sample(U ml−1) using a standard of hen egg-white lysozyme (Sigma-Aldrich) (Möck and Peters, 1990; Ossermann and Lawlor, 1966).

2.6. Statistical analyses

Distance-based permutational multivariate analysis of variance(PERANOVA Anderson, 2001; McArdle and Anderson, 2001) wasemployed to test the null hypothesis of no significant differencesamong submerged and surface culture conditions. Analysis wasperformed on a Bray Curtis resemblance matrix (Bray and Curtis,1957) based on untransformed or square root transformed data andeach termof the analysiswas testedusing9999 randompermutationsofappropriate units (Anderson and ter Braak, 2003). The analysis wascarried out in two steps. First, the four cageswere consideredas separateunits, investigating the difference, with pair wise comparison, amongthe two surface cages and among the two submerged cages,respectively, with the PERMANOVA statistic and 9999 permutations,demonstrating the lack of difference between the two surface cages andbetween the two submerged cages. A further two way PERANOVA(maximum permutations=9999), was performed (using a uniquesurface and submerged conditions) on each parameter to test thedifference among treatments related to the following factors: site withtwo levels (submerged and surface-based fish cage) and time (witheleven levels for growthparameters and three levels for haematological,biochemical, immunological parameters and HSP70 assay).

All statistical analyses were performed using the PRIMER v6program (Clarke and Gorley, 2006), including the add-on packagePERANOVA+ (Anderson et al., 2008).

3. Results

3.1. Growth parameters

Surfacewater temperature ranged from12.95 °C in January to24.29 °Cin July and the salinity was between 37.5 psu (autumn–winter) and38.1 psu (summer). The thermocline was observed to be at a depth ofapproximately 15–17 m for the whole summer period until September.

Fig. 1a shows the monthly trend of the total wet weight of the twogroups of D. labrax during the experimental period. Although therewere no significant differences in the wet weight between the twotreatments (Table 1), fish, in December, reached 175.40±17.40 g and167.30±31.40 g, in submerged and surface cages, respectively.

No significant differences were observed in the specific growthrate (SGR, Table 1) between the two groups, and, during the trial, the

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groups showed positive values, except for the submerged fish for theperiod from April to May (−0.18%day−1, Fig. 1b).

Overlapping of the growth curves for the fish cultured in surfacecages and those cultured in submerged cages was also confirmed by a

non-statistically significant difference between the two treatments ofthe relative Condition Factor K (Table 1).

3.2. Haematological and biochemical parameters and HSP70 assay

Results of haematological and biochemical parameters suggestthat sea bass welfare is promoted by submergence.

Surfacefish showed significantly higher cortisol levels (Table 1) thanthe submerged fish throughout the entire sampling period. Meancortisol levels ranged from228.0±74.4 ng/ml to403.1±153.1 ng/ml insubmerged cages, and from594.1±115.1 ng/ml to 857.7±300.7 ng/mlin control cages (Fig. 2a).

As shown in Table 1, fish within the surface cages had significantlyhigher plasma glucose values than fish in submerged cages, only inJune. Mean plasma glucose values ranged from 91.7±32.4 mg/dl to101.9±22.3 mg/dl and from 95.9±20.3 to 156.1±48.8 in submergedand surface cages, respectively (Fig. 2b).

Haematocritwas also consistentwith the other analyzed parametersshowing significantly higher values in surface cages (Table 1). Meanhaematocrit values ranged from 29.8±6.3% to 30.6±12.5% and from35.2±3.7% and from 39.8±8.2% in submerged and surface conditions,respectively (Fig. 2c).

Total proteins infish cultured in submerged cages ranged frommeanvalues of 2.5±0.8 g/dl to 3.4±0.8 g/dl in February and September,respectively. Fish cultured in surface cages showed significantly highermean values of total proteins (Table 1), ranging from 3.5±0.5 g/dl to4.4±0.7 g/dl (Fig. 2d).

No significant differences in plasma lactate levels between treat-ments were observed in February and June (Fig. 2e), but a significantdifference was found in September when fish in submerged cagesshowed highermean values of plasma lactate than in surface cages. Thisparameter ranged from 10±1.1 mg/dl to 11±1.1 mg/dl in submergedcages and from 6±0.6 mg/dl to 12±0.10 mg/dl in surface conditions.

No significant differences in blood cell HSC70–HSP70 relative levelswere detectedwhen comparing surface and submerged cages (Table 1).The mean value of ratios between HSC70–HSP70 and actin in D. labraxcultured in surface cages were 1.95±0.87 and 1.62±0.47 (in February

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Fig. 1. (a) Monthly trends of wet weight (± standard deviation), relative to watertemperature changes during cultivation and (b) specific daily growth rate in wetweight of D. labrax cultured in submerged (sub) and surface (surf) cages (n=200 foreach sampling time).

Table 1Permutational analysis of variance testing the differences in fish growth and welfare between the two culture conditions (surf=surface cage; sub=submerged cage). Factors: site(two levels, submerged and surface-based fish cage) and time (with 11 levels for growth parameters and 3 levels for haematological, biochemical, immunological parameters andHSP70 assay). Results of the pair wise comparison are reported for significant differences. *=pb0.05; **=pb0.01.

Transformation df MS Pseudo-F Interaction Time Pair wise comparisons

Wet weight none 1 22.886 0.13548 none surf=subSGR none 1 2266.5 0.000001 none surf=subCondition factor none 1 12.567 0.43116 none surf=subCortisol square root 1 7057.2 78.896** none surfNsubHaematocrit none 1 1272.6 12.912** none surfNsubGlucose square root 2 194.59 3.071* site×time Feb

JunSept

surfNsubsurf=subsurf=sub

Total proteins none 1591.3 9.4285 none surfNsubPlasma lactate none 2 522.17 18.509** site×time Feb

JunSept

surf=subsurf=subsubNsurf

Haemolytic activity none 2 1486.7 10.715** site×time FebJunSept

surf=subsubNsurfsubNsurf

Mucus lysozyme none 2 254.4 6.1556** site×time FebJunSept

surf=subsurf=subsubNsurf

Kidney lysozyme none 1 321.08 5.9354* none subNsurfPlasma lysozyme none 2 182.21 28.018** site×time Feb

JunSept

surfNsubsurf=subsurf=sub

HSC70–HSP70 none 1 220.48 0.31796 none surf=sub

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and September, respectively), whereas, in fish cultured in submergedcages, they were 1.78±0.83 in February and 2.40±1.34 in September.

3.3. Immunological parameters

Submerged condition has a positive effect on some components ofinnate immune system in cultured sea bass.

Fish in submerged cages showed significantly higher values in non-specific haemolytic activity (SH50), in June and September (Table 1).The mean value of SH50 ranged from 7.6±1.8 U/ml to 26.7±3.8 U/mlin submerged cages, and from 8.7±2.3 U/ml to 11.7±4.1 U/ml insurface conditions (Fig. 2f).

Lysozyme content was different according to the different tissues ororgans (mucus, plasma and kidney). Significant differences were foundin the mucus lysozyme of sea bass cultured in submerged cagescompared to the surface ones, in September only (Table 1).Meanmucuslevels ranged from0.8±0.1 U/ml to 1.2±0.1 U/ml and from0.9±0.1 to1.0±0.3 U/ml in submerged and surface cages, respectively (Fig. 3a).

Kidney lysozyme ranged from mean values of 1.8±0.2 U/ml to2.0±0.2 U/ml in submerged cages and from 1.6±0.2 to 1.7±0.3 U/mlin surface cages (Fig. 3b); values measured in fish reared in submergedcondition were always significantly higher than those in surface cages(Table 1). Mean plasma lysozyme content was from 1.4±0.1 U/ml to1.8±0.1 U/ml and from 1.6±0.1 to 1.8±0.1 U/ml in submerged andsurface cages, respectively (Fig. 3c). Lysozyme content in plasma did not

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Fig. 2. Serum cortisol concentration (a), glucose concentration in plasma (b), haematocrit value (c), total plasma proteins (d), plasma lactate (e) and haemolytic activity (SH50) (f) inD. labrax cultured in submerged (sub) and surface (surf) cages. Valuesare expressedasmeans±standarddeviation. Reported are the results of thepair-wise tests: *=pb0.05, **=pb0.01,no indications is reported when the difference is not significant (n=40 for each sampling time).

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vary significantly between the two rearing conditions, except for theFebruary sampling, when values detected in sea bass reared in surfacecages showed significant differences from thosemeasured in fish rearedin submerged cages (Table 1).

4. Discussion

In this study, a range of welfare indicators, consisting of physical andphysiological parameters, were used to assess the effects of farmingconditions (surface vs. submergence) on the welfare of European seabass, Dicentrarchus labrax.

The research provides, for the first time, data on stress assessment insubmerged cages, a new farming technology tested in several salmonand cobia (Benetti et al., 2010; Korsøen et al., 2009) productionexperiments. In off-shore cages, where handling or other stressors,currently present in intensive practices, do not affect the quality of thefinal products, other factors can contribute to creating a stressfulenvironment, negatively influencing fish growth and welfare (Cottee

and Petersan, 2009). A negative factor that may influence fish reared inoff-shore cages, is a chronic stress condition induced by a progressiveincrease in stocking density (in terms of Kg m−3), a direct consequenceof the physiological fish growth during the rearing cycle. Anothersignificant problem is predation by sea birds and aquatic mammals thatare attracted by the high concentration of fish (Ashley, 2007;Huntingford et al., 2006).

In our study, submergence represents a favorable condition forsea bass, as evidenced by growth haematological, biochemical,immunological and cellular parameters that underline a clearrelationship between the observed changes in welfare indicatorsand farming conditions.

Submergence of full-scale sea-cages to shallow depths did notaffect the growth performances of European sea bass, as SGRs did notdiffer between treatments during the 12-month on-growing period.Our results are in agreement with previous studies on the effects ofsubmergence on cage farmed salmon, which have documented thatsubmergence does not negatively affect growth and welfare para-meters (Dempster et al., 2009). Also the feed conversion rates (FCR),based on sample weight, were very similar among the treatments,(FCR=1.93 and 1.94 in surface and submerged cages, respectively).As the environmental conditions and feeding regimes experienced bysea bass in both the surface and submerged cages were similar, weconclude that shallow submergence does not alter parameters of seabass production.

As in other vertebrates, fish experiencing stress show severalphysiological changes that are expressed through a number ofparticular indicators (Wendelaar Bonga, 1997) based on knowledgeof the natural responses of fish to adverse conditions.

In our study, all biochemical and haematological parameters usedas welfare indicators were significantly better in sea bass cultured insubmerged cages.

In particular, cortisol levels in submerged fish appeared signifi-cantly lower than in surface fish throughout the observation period,indicating a better welfare state.

Blood cortisol level is widely used as an indicator of the stresscondition in fish, due to the extreme sensitivity of the hypothalamic–pituitary–interrenal (HPI) axis, that is activated in response to mostforms of stress (Barton, 2002).

In this study, serum cortisol levels in D. labrax, were higher thanthose reported in literature. To our knowledge, this is the firstresearch that studies the serum cortisol levels of sea bass farmed innet cages. The only exception is the paper of Santulli et al. (1999) inwhich the biochemical response to acute stress, induced by anexperimental air gun blast exposition, in sea bass reared in cages, isexamined. The authors reported the cortisol values of 306.9±2.53 ng/ml for unstressed sea bass and clearly stated, in the paper, that theyused a particular technique to avoid stress during fish sampling; infact, fish sampling in cages might represent a procedure, which, initself, is stressful for fish.

Literature reports lower cortisol values for unstressed sea bass,ranging from15 to 200 ng/ml, for sea bass kept in experimental tanks orin laboratory, where the sampling procedures are easier and lessstressful for fish (Cerdà-Reverter et al., 1998; Di Marco et al., 2008;Lupatsch et al., 2010; Marino et al., 2001; Planas et al., 1990; Roche andBogè, 1996; Roche et al., 1989; Rotllant et al., 2003). Moreover, thecomparison of data reported in literature for this species, is not alwayseasy, due to the variability in the experimental design and in cortisolassay methods (RIA, ELISA, electrochemiluminescence immunoassay,etc.). Furthermore, European sea bass is considered more sensitive tostress than other species, as the cortisol levels of unstressed fish are inthe upper range across fish species (Acerete et al., 2009). On the otherhand, previous studies on the mechanisms of the cortisol release in seabass, demonstrate a continuous cortisol production under controlledconditions, but lower reactivity to adrenocorticotropin hormone(ACTH) pulses (Rotllant et al., 2003).

Mucus lysozyme

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Fig. 3. Lysozyme content in mucus (a), kidney (b) and plasma (c) of D. labrax culturedin submerged (sub) and surface (surf) cages. Values are expressed as means±standarddeviation. Reported are the results of pair-wise: *=pb0.05, **=pb0.01, no indicationsis reported when the difference is not significant (n=40 for each sampling time).

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In our study, due to the high stress sensitivity of this species, samplingprocedures, might have induced a rapid change in the hormoneconcentration in the blood, leading to such high serum cortisol values.However, having found a significant difference in the cortisol levelsbetween treatments,while the samplingprocedure appliedwas the same,leads us to suppose that there was a better welfare status of fish in thesubmerged environment. Certainly, a further study will be needed toconfirm the data reported and, also highlights the need to develop andstandardize a technique for fish sampling in a net cage.

Secondary response to stress includes changes in plasma andtissue ion and metabolite levels, haematological features and heat-shock proteins or stress protein (HSPs).

Lower values of plasma glucose, used as the secondary indicator ofstress in fish (Acerete et al., 2004; Arends et al., 1999; Erikson et al.,1999; Lowe et al., 1993), were observed in submerged fish, furtherconfirming a better welfare state. Blood glucose levels are known tobe affected by chronic and acute stress (Ellis et al., 2002). Indeed,stress is typically associated with increasedmetabolic rate and energysource mobilization (Barton et al., 1988). Therefore, increased glucoseconcentrations are commonly explained by the higher energyrequirement occurring during stressful conditions.

The positive effects of submergence are also evident in terms ofhigher haematocrit and total protein levels in sea bass reared insurface cages.

It is generally stated that in teleosts, an increase in haematocrit canoccur as a result of (a) the induction of splenic contraction and thesubsequent release of stored erythrocytes into the bloodstream (WellsandWeber, 1990; Yamamoto et al., 1980); (b) erythrocyte swelling dueto the activation of the Na/H+ exchange mechanism (Nikinmaa, 1983;Yamamoto et al., 1980); (c) increased muscle activity and theconcomitant movement of water from plasma to muscle (Jones andRandall, 1978). Moreover, haematocrit values rise in fish exposed tostress as a direct consequence of the increase in heart-beat and higheroxygen uptake (Poli et al., 2005). Similarly, high levels of total protein inplasma are indicators of hemoconcentration or impaired water balance(Wedemeyer and Mc Leay, 1981).

In our study, plasma lactate, in both treatments, indicates a nostressful condition, showing values in the range reported for unstressedD. labrax (Acerete et al., 2009). The low plasma lactate levels in bothtreatments suggest that, either fish groups have not experiencedsituations (or conditions) with a strong increase in anaerobic muscleactivity (burst swimming) or they do not release myotomal lactate intothe circulation (Dalla Via et al., 1997). In fact, during burst swimming,the bulk ofmuscle becomes functionally hypoxic and accumulates lacticacid as the end product of glycolysis (Goolish, 1991; Kauffman, 1990;Puckett and Dill, 1984, 1985; Weiser et al., 1985).

Previous studies have shown that farming at inappropriate (e.g. high)stocking densities can induce chronic stress, affecting the physiology andfish behavior (Ellis et al., 2002; Schreck et al., 1997;Wedemeyer, 1997). Itis known that high fish densities can affect the fish welfare throughcrowding stress and/or through changes in water quality (Santos et al.,2010). Ruane et al. (2002) report an increase (together with other stressparameters) in plasma lactate levels in common carp, Cyprinus carpio,subjected to high density stress. Our fish were reared at low densities, infact, at the end of the experiment, values of 12 Kg m−3 were reached,lower than the common stocking density for sea bass in commercial fishcages (over 30 Kgm−3, Paspatis et al., 2003).

Literature on the use of HSPs, as stress indicators in rearingconditions, is very limited and the results are contradictory. Someauthors show that stressors are responsible for the forced muscleactivity resulting in an increase of HSPs levels (Basu et al., 2002; Diniet al., 2006; Iwama et al., 2004). There is also evidence that some HSPsare inhibited in fish fed diets with the replacement of fish meal withplant protein sources (Martin et al., 2003).On thecontrary, other studiesreport a variety of stressors that do not involve a response of HSPs(Vijayan et al., 1997; Zarate and Bradley, 2003).

In our study, we did not find any significant differences in theblood cell HSP70 levels between submerged and surface cages. Thismight suggest a similar health status in both fish populations, but as itis the first report describing the use of erythrocytes as the tissue oforigin for HSPs assay in fish cultured in off-shore cages, these must beregarded as preliminary results. Nevertheless, we cannot exclude thepossibility of other stress protein variations, possibly detectable witha proteomic approach.

The non-specific or innate immune system is the most importantdefense mechanism in fish (Anderson, 1990; Ingram, 1980) and someof its components (e.g., lysozyme, haemolytic and haemagglutinatingactivity) are used as indicators of immunocompetence in fish exposedto stress (Sunyer et al., 1995; Tort et al., 2004). Recent studies havefocused particular attention on the physiological responses of fish toacute or chronic stressors in terms of their immunological parameters(Caruso et al., 2005, 2010; Maricchiolo et al., 2008; Tort et al., 1996).

Haemolytic activity values recorded in this study fell within a rangesimilar to those found for the samefish species farmed in off-shore cages(Caruso et al., 2005). Sea bass reared in submerged cages showedsignificantly higher haemolytic activity than those kept in controlsurface cages. To our knowledge, this is the first study where possiblevariations in immunological parameters, induced by submergence, havebeen examined. The homogeneity of environmental conditions underwhich the experiment was performed, lead us to suppose that thepatterns of haemolytic activity were not controlled by environmentalfactors, which are commonly reported as important modulators(Bowden, 2008), but rather by physiological factors. Since it is knownthat the activation of both pituitary–interrenal and sympathetico–chromaffin axes may result in a decrease in immune responses (Mazurand Iwama, 1993; Pickering and Stewart, 1984) and that cortisol inducesimmunosuppression as a typical stress response (Schreck et al., 2001;Weyts et al., 1999),wehypothesized that the cortisol levels, significantlyhigher infish reared in surface cages than in submergedones, could havean immunosuppressive effect on haemolytic activity, similarly to theresults reported inAtlantic salmonbyCarlson et al. (1993). Furthermore,since a reduction in the haemolytic activity was observed in response tostress conditions such as those related to crowding (Sunyer et al., 1995;Tort et al., 1996) and nutritional deficiency (Montero et al., 1998), highhaemolytic activity measured in fish kept in submerged cages couldsuggest a positive effect played by submergence on fish health,increasing protection against possible infectious diseases caused byfungi, bacteria andviruses, towhichfishmaybe exposedduring farming.

Similarly to haemolytic activity, also lysozyme levels were signifi-cantly higher in the mucus and in kidneys of sea bass reared insubmerged cages compared to those measured in fish reared in surfacecages. Lysozyme is a fundamental enzyme involved in the non-specificimmune response of many fish species (Tort et al., 2004); it acts as animportant factor in protectingfish against bacterial pathogens, due to itsantibacterial properties against both Gram positive and negativebacteria (Ingram, 1980; Möck and Peters, 1990), and because it islocated in areas that are in frequent contactwithpathogens (i.e., kidneysand skin mucus). Assuming that the lysozyme activity in the mucus isconnected to the defense function of this enzyme (Fast et al., 2002;Lindsay, 1986), decreased levels in themucus of surfacefish, as recordedin our study, suggest an impoverishment of this protective barrier,compared to fish farmed in submerged cages.

Surfacefish showedhigher levels of stress compared to submerged seabass (on the other hand, the cortisol levels found are indicative of poorwelfare conditions). A possible reason for this could be linked to the stressresponse from predation by fish-eating birds. In the Gulf of Castellam-mare, sea birds, and principally cormorants, are considered problematicfor aquaculture activities because of their high presence, particularlyduring the autumn and winter (fish farm manager Dr. Cascio, pers.comm.). Cormorants are voracious fish feeders and an adult specimenneeds a biomass intake per day of 300–500 g of fish (Leukona, 2002).Dieperink (1995) refers that a flock of cormorants are able, in about

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30 min, to consume 110 fishweighing a total of approximately 50 kg. Theincrease in the cormorant population registered in the last twenty years inthe area, as well as along the Mediterranean coasts (Consolo et al., 2009;Dìaz Lòpezet al., 2008), has led toan increaseof cormorant attacksdirectlyin the farm surface based cages (when not covered by anti-predator net),or onwildfish feedingvery close around the cages (Dempster et al., 2004).Inboth cases, cormorant attacks candetermine, other thanmortalities andinjury, also stress, in farmed fish, induced by the presence of predators(Sepùlveda and Oliva, 2005). This hypothesis is also supported by studieson neuroendocrine perception of stress in fish where it has beendemonstrated that a prey/predator encounter is a stressful situation andinduces a typical stress response with energy costs for escape (Bell et al.,2007).

5. Conclusions

Results obtained in this study indicate that fish held in submergedcages at industry-scale densities and numbers grow similarly and havehad similar FCRs to fish kept in surface cages, thus, none of the majorproduction parameters relevant to fish farming had been compromised.

Shallow depth submerged cages represent an optimal farmingenvironment, with regard to light, constant water temperature andagitation, favoring the fish welfare, as evidenced by good haematolo-gical, biochemical, immunological and cellular parameters.

Acknowledgements

The authors are particularly indebted with fish farm manager(Dr. F. Cascio) and the staff of the Ittica Trappeto s.r.l. (Trappeto, Italy) fortheir precious collaboration during sampling. Thanks are due to Dr. M.Sinopoli for the fundamental help in statistical analysis.

This work was supported by a grant of the Ministero dell'Uni-versità e della Ricerca Scientifica e Tecnologica and was conductedaccording to the ethical principles and guidelines currently in use foranimal experiments.

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