Effects of dietary tannin on growth, feed utilization and digestibility, and carcass composition in juvenile European seabass (Dicentrarchus labrax L.)

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Effects of dietary tannin on growth, feed utilization and

digestibility, and carcass composition in juvenile

European seabass (Dicentrarchus labrax L.)

Marie-Hélène Omnes, Julien Le Goasduff, Hervé Le Delliou, Nicolas Le Bayon,

Patrick Quazuguel, Jean H. Robin

To cite this version:

Marie-Hélène Omnes, Julien Le Goasduff, Hervé Le Delliou, Nicolas Le Bayon, Patrick Quazuguel, et

al.. Effects of dietary tannin on growth, feed utilization and digestibility, and carcass composition in

juvenile European seabass (Dicentrarchus labrax L.). Aquaculture Reports, Elsevier, 2017, 6, pp.21-27.

�10.1016/j.aqrep.2017.01.004�. �hal-01510334�

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ContentslistsavailableatScienceDirect

Aquaculture

Reports

j ou rn a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / a q r e p

Effects

of

dietary

tannin

on

growth,

feed

utilization

and

digestibility,

and

carcass

composition

in

juvenile

European

seabass

(Dicentrarchus

labrax

L.)

Marie-Hélène

Omnes

∗

,

Julien

Le

Goasduff,

Hervé

Le

Delliou,

Nicolas

Le

Bayon,

Patrick

Quazuguel,

Jean

H. Robin

Ifremer,UMRLEMAR,Univ.Brest,CNRS,IRD,LEMAR,IUEM,Z.I.PointeduDiable,F-29280Plouzané,France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6September2016

Receivedinrevisedform26January2017 Accepted30January2017

Availableonline6March2017 Keywords: Dicentrarchuslabrax Polyphenolictannin Growth Digestibility Carcasscomposition

a

b

s

t

r

a

c

t

Plant-basedproductsinfishdietsarevaluableproteinalternativestofishmealfortheaquafeedindustry. Manyplantfeedingredientscontainpolyphenoliccompounds,includingtannins,whichcanhave bene-ficialoradverseeffects.Thetolerablethresholdofingestedtanninsisunknownformarinecarnivorous fishes.Westudiedtheeffectsoftannicacid(TA)supplementationtothedietofjuvenileEuropeanseabass (Dicentrarchuslabrax)bymeasuringgrowth,feedutilizationanddigestibility,andcarcasscomposition. Werandomlyallocatedgroupsoffish(initialmeanbodyweightof10.2±0.7g;n=40fishpertank)to 12replicatecylindrical-conicaltanks(threepertreatment).Thefishwereassignedtooneoffourdietary treatmentsforfiveweeks:controldiet(C)withtannin-freeproteinsources(mostlyfishmealasthebase diet,containing55.7%drymatter(DM)crudeprotein,grossenergy22.3kJg−1DM)andthree experi-mentaldietssupplementedwith10,20,or30gTAkg−1_(called_TA1,_TA2,_and_TA3,_{respectively).}_Tannin

ingestionresultedinsignificantlydecreasedcumulativefeedintake,growth,feedandproteinefficiencies, apparentdigestibilitycoefficients,hepatosomaticindex,andcarcasslipids.Theproteindigestibilityin fishfedthe10gkg−1_{tannin-containing}_diet_was_{significantly}_lower_than_that_in_fish_fed_the_control_diet.

Thisthresholdshouldbetakenintoaccountwhenusingnovelterrestrialandaquaticplantingredients fortemperatemarineﬁshes.

1. Introduction

Theuseof plantprotein sourcesin ﬁshfeeds hasexpanded considerablyinrecentyearstomeetthedemandforfeedsand sus-tainthedevelopmentofworldwideaquacultureproduction(Tacon

andMetian,2015).Manytrialshavetestedtheinclusionofnovel

unconventionalorunderutilizedplantspeciesafteradequate pro-cessing.Thesustainabledevelopmentofaquaculturesystemsin someregionsdependsontheirregionalintegrationandthelocal availability of agriculturalby-products (Dongmeza et al., 2009;

Chebbaki et al.,2010; Altanand Korkut, 2011).Thus, feed

for-mulationscontainingabroadrangeofpromisingingredientsare beingdevelopedforfarmedtropicalherbivorous/omnivorousand temperatecarnivorousﬁshes.Theseingredientsincludeplantoils,

∗ Corresponding author at: Ifremer, Institut Franc¸ais de Recherche pour l’ExploitationdelaMer,UnitéPhysiologieFonctionnelledesOrganismesMarins, Z.I.PointeduDiable,CS10070,F-29280Plouzané,France.

E-mailaddress:mhomnes@ifremer.fr(M.-H.Omnes).

legumeseeds,fruitsandtheirrespectiveby-products(Emreetal.,

2008;Azazaetal.,2009b),plantleaves(Dongmezaetal.,2009),

aquaticplants(MandalandGhosh,2010a,b),seaweeds(Güroyetal.,

2013;Peixotoetal.,2016)andalgalbiomass(Tullietal.,2012;

Tibaldietal.,2015).

Plant-basedproductsused topartially or completely substi-tutefishmeal,havebeenassociatedwithsmallamountsofvarious endogenoussecondarycompoundsandimbalancednutrient pro-files, resulting in poor nutritional value, whereas amino acid supplementsleadtobetterutilizationofplant-basedfeed.These secondary compounds include heat-labile (protease inhibitor, lectin)andheat-stable(phyticacid,phenoliccompounds,tannin, andfiber)antinutritionalfactors(Drewetal.,2007).Theproperties ofsuchantinutrientsarelikelytoimpairdigestivefunctionand themetabolicutilizationofnutrients,andthereforefish produc-tion.Onlyafewcompoundshavebeenstudiedinteleostspecies tobetterunderstandtheantinutritionaleffectsonfishphysiology

(Francisetal.,2001;Krogdahletal.,2010;Coutoetal.,2015).

Severalplantfeedingredientscontainappreciableamountsof polyphenolicsubstances,broadlyreferredtoastannins.

Polyphe-https://doi.org/10.1016/j.aqrep.2017.01.004

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22 M.-H.Omnesetal./AquacultureReports6(2017)21–27 nolictanninsshowbothpositiveandnegativeeffects;theypossess

antinutritive properties, but are also beneﬁcial for health due to theirrole as antioxidants and theirability to stimulate the immunesystemandvariouseffectors(Chungetal.,1998;Makkar

et al., 2007; Quideau et al., 2011). Tannic compound has also

beenreportedtohaveantimicrobialactivityagainstﬁshpathogens

(Schrader,2008).

Polyphenolictanninsrepresentmostofthesecondary metabo-litesfoundinterrestrialplants(Makkaretal.,2007)andseveral aquaticmacrophytes(ArnoldandTargett,2002).Threeclassesof tanninshave beenestablished(Hagerman,2002;Quideauetal., 2011): 1) hydrolysable tannins (HTs), gallo- and ellagi-tannins derivedfromgallicandellagicacids,esterifiedtoacorepolyoland galloylgroup,thatcanbeesterifiedoroxidizedtomorecomplex HT;2)condensedtannins(CTs),proanthocyanidinpolymeric fla-vanoids;and3)phlorotannins(PTs),monomersofphloroglucinol foundinbrownalgae.Tanninlevelsinfeeddependontheplant sourcesandvarieties(Créponetal.,2010),plantmaturity,andthe season,aswellasthetechnologicalprocessesusedforfeed manu-facturing(Drewetal.,2007).TheproportionofHTinfeedfrequently exceedsthatofCT(Gaber,2006;Dongmezaetal.,2009),andcan varywidely.Thetannincontentofingredientsusedforanimalfeed hasbeendeterminedtobebetween5and16gkg−1 drymatter (DM).Itishighestinoilseedmeals(Heuzéetal.,2015a,b;Sauvant etal.,2015),followedbyvariouslegumestestedfortemperatefish production,whichcontainbetween4.9and11.4gtanninkg−1DM

(Adamidouetal.,2009a;Collinsetal.,2013).Estimatedtannin

con-tentinnaturalfoodsandplantingredientsfortropicalcarpspecies rangesfrom5.4(coconutoilcake)to34.3(phytoplankton)gkg−1 dryweight(DW,MandalandGhosh,2010a).Thetotalphenolic con-tentofalgalbiomasswasshowntobebetween6.0and20.0gkg−1

DM(Tibbettsetal.,2015).Thenumberofvarietiesandthe

propor-tionofplantproteinsourcesinﬁshdietsaresteadilyincreasing.Itis thusimportanttostudytheeffectsoftanninsinorganismsthatare currentlyorlikelytoingesttannin-containingfood,orpolyphenolic tanninsupplements.

Ingredients containing tannin give feed an unpleasant taste andreduceconsumptionduetodecreasedpalatability(Beckerand

Makkar,1999).Inaddition,tanninsaffectanimalsthroughseveral

mechanisms,includingtheformationof strongcomplexeswith feedcomponents, suchas proteinsand minerals (Chung et al., 1998),thelossofendogenousproteins(MansooriandAcamovic, 2007),andinactivationofdigestiveenzymes,thusinterferingwith digestion(Chungetal.,1998;MandalandGhosh,2010b).

Thereis limited literatureontheeffectofpolyphenolic tan-ninsonintensiveﬁshproduction.Manypreviousstudiesonthe effectofdietarytanninontropicalﬁshspecies(BeckerandMakkar,

1999;Prustyetal.,2007;AiuraanddeCarvalho2007)andanimals

(MansooriandAcamovic,2007)usedtannicacid(TA)asamodel

compoundforHT.Theeffectsoftanninhavebeenlittlestudiedin carnivorousﬁsh,exceptforrecentstudiesrelatedtotheinﬂuenceof antinutrientsfromvariousdietsonthephysiologyofrainbowtrout (Oncorhynchusmykiss,Collinsetal.,2013)andpolyphenol-enriched feedonthatofseabass(Magroneetal.,2016).

European seabass (Dicentrarchus labrax) is one of the most importantmarinefinfishspecies rearedinEurope,especially in the Mediterranean basin, with a production similar to that of giltheadseabream(Sparus aurata),comprising145–150,000tons in2014suppliedbyTurkey,Greece,andSpainamongEuropean countries(FEAP,2015).Europeanseabassisthusoneofthemost highlystudiedtemperatemarine fishspecies(Sanchez-Vazquez

andMu ˜noz-Cueto,2014).Itsnutritionalrequirementsandfeeding

managementhavebeenrecentlyestablished,highlightedbythe nearreplacementofﬁshmealbyavarietyofplant-based

ingredi-ents(Kousoulakietal.,2015).

However,thetolerablethresholdofingestedtanninhasnotyet beendetermined.Theobjectiveofthisﬁve-weekshort-terminvivo studywastoevaluatetheeffectsofvariousamountsofTA,added tothebasediet,ongrowth,feedintakeanddigestibility,andbody compositioninjuvenileEuropeanseabass.

2. Materialsandmethods

Thisstudycompliedwithethicalguidelines(ASAB/ABS,2006) andregulationsconcerninganimalexperimentation,andwas car-riedoutwithpermissionfromtheFrenchnationalboardforanimal experimentation.

2.1. Fishandrearingconditions

Europeanseabassweresuppliedbyafishfarm(Aquastream, Ploemeur,France).Thetrialwasconductedintheexperimental indoorfacilitiesatIfremerCentreBretagne(Plouzané,France).The fish wereinitially fed Neo Start ½ commercial pellets for pre-growingmarinefish(LeGouessant,Lamballe,France)containing crudeprotein:52%DM,crudefat:17%DM,andanegligibleamount oftanninestimatedtobebelow0.2gkg−1DM.Afterafour-week acclimationperiod,thetrialwassetupin acomplete random-izeddesignwith480fish.Thefishweredividedintobatchesof tenandrandomlydistributedintoreplicatetanks(threetanksper treatment,fourtreatments)atadensityof40fishpertank.The12 cylindrical-conicalfiberglasstanks(75Lcapacity)wereexposedto an8hlight:16hdarkcycle.Theyweresuppliedwithacontinuous flowoffilteredseawater(5.4Lmin−1).Anindividualstonediffuser pertankensureddissolvedoxygenlevelsabove90%saturation.The salinityaveraged35.3gL−1andthetemperaturewasheldconstant at20.8±0.2◦C.Thefishwerefastedforoneday,anaesthetizedwith 2-phenoxyethanol(ataconcentrationof0.125mLL−1 seawater, withinafewminutesofexposure),andbulkweighedatthe begin-ning,aftertwoweeksofacclimationinthetanks,andattheendof thetrial.Forsampling,thefishwerehumanelykilledbyablowto thehead.

2.2. Experimentaldiets

Followingacclimation,thefishhadanaveragebodyweightof 10.2±0.7gandwerefedtheexperimentaldiets(Table1).Four iso-nitrogenous(crudeprotein: 55.7% DM)and iso-energetic(gross energy:22.3kJg−1DM)dietswereprepared.Theyincluded fish-mealastheprincipalproteinsource,fishproteinhydrolysate,small amountsofwheatglutenmeal,fishoil,andwheatstarchasthe baseorcontroldiet(C).Thebasedietwassupplementedwith10, 20,or30gkg−1 tannicacid(Chinesegallnutgrass,Sigma-Aldrich T0200,Saint-QuentinFallavier,France),toconstitutethe experi-mentaldiets,calledTA1,TA2,andTA3,respectively,andcellulose wasusedtoreplacethetannininthecontroldiet.Vitaminsand mineralswereaddedaccordingtothenutrientrequirementsof Europeanseabass.Forthedigestibilitystudy,10gkg−1 ofstarch wasreplacedbythesame amountof theinertmarkerchromic oxide(Cr2O3)ineachdiet.Allingredientsweregroundfor

homo-geneity(<800_␮m),mixedwithwater,andpelletized.Thepellets weredriedinaforced-airdryer,at80◦Cfor10min,toreducethe moisturecontenttounder10%DW.Thepelletswerepackedand storedat−20◦_C_until_use_as_feeds_or_samples_for_chemical

analy-sis.Theﬁshwerehand-fedadailyrationof3%bodyweight,which wasthenvisuallyadjustedtoapparentsatiety,threetimes(09:00, 13:00,and16:00)onworkingdays,andtwiceonweekends,forﬁve weeks.Thefeedingestedwasrecorded.

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Table1

Compositionofexperimentaldiets:control(C)dietanddietscontainingvariouslevelsoftannicacid.

Experimentaldiets

Ingredients(gkg−1_dry_weight) _C _TA1 _TA2 _TA3

Fishmeala ₆₀₀ ₆₀₀ ₆₀₀ ₆₀₀

CPSP90b ₅₀ ₅₀ ₅₀ ₅₀

Wheatglutenmealc ₅₀ ₅₀ ₅₀ ₅₀

Marineﬁshoila ₈₃ ₈₃ ₈₃ ₈₃ Pregelatinizedstarchd,j ₁₄₇ ₁₄₇ ₁₄₇ ₁₄₇ Bindere ₁₀ ₁₀ ₁₀ ₁₀ Vitaminpremixf ₂₀ ₂₀ ₂₀ ₂₀ Mineralpremixg ₁₀ ₁₀ ₁₀ ₁₀ Celluloseh ₃₀ ₂₀ ₁₀ ₀ Tannicacidi ₀ ₁₀ ₂₀ ₃₀

Proximatecomposition(%dryweight;%DM)

Drymatter(DM) 96.43±0.03 95.09±0.06 94.40±0.01 95.03±0.06 Crudeprotein 55.21±0.78 55.17±0.38 56.01±0.75 56.46±1.00 Crudefat 14.01±0.27 13.69±0.23 13.28±0.24 13.02±0.03 Starch 15.52±0.10 14.71±0.08 13.36±0.12 13.94±0.10 Ash 11.69±0.15 11.52±0.06 11.75±0.08 11.61±0.12 Grossenergy(kJg−1_DM) _22.27_±_0.15 _22.25_±_0.06 _22.44_±_0.04 _22.34_±_0.02 a_Norvik_LT_70,_blue_whiting_meal_(CP_70%_DM;_CF_12%_DM)_and_cod_liver_oil,_La_Lorientaise,_Lorient,_France.

b_Soluble_ﬁsh_protein_hydrolysate_(CP_82%_DM;_CF_10%_DM;_ash_6%_DM)_Sopropêche,_Boulogne,_France. c _Vitalor,_Chamtor,_Bazancourt,_France.

d _Pregeﬂo,_Roquette,_Lestrem,_France. e_Na_Alginate_Agrimer,_{Plouguerneau,}_France.

f _Vitamin_premix_composition_(g_kg−1_):_retinyl_acetate,_1;_thiamin-HCl,_0.1;_riboﬂavin,_0.5;_niacin,_1;_d-calcium_{panthothenate,}_2;_{pyridoxine-HCl,}_0.3;_{mesoinositol,}_30;

d-biotin,1;folicacid,0.1;cyanocobalamin,1;ascorbicacid(ascorbylpolyphosphate),14.2;cholecalciferol,0.5;dl-a-tocopherylacetate,10;menadione,2;choline,167; UPAE,Inra,Jouy-en-Josas,France.(Allingredientsweredilutedwith␣-cellulose).

g_Mineral_premix_composition_(g_kg−1_):_calcium_phosphate_(bicalcic),_500;_calcium_carbonate,_215;_sodium_chloride_(salt)_40;_potassium_chloride,_90;_magnesium_oxide,

124;ferroussulfate,20;zincsulfate,4;manganesesulfate,3;coppersulfate,3;cobaltsulfate,0.02;potassiumiodide,0.04;sodiumselenite,0.03;sodiumﬂuoride,1.00; UPAE,Inra,Jouy-en-Josas,France.

h _Arbocel,_J._Rettenmaier_&_Söhne_(JRS),_{Saint-Germain}_en_Laye,_France. i _Natural_extract_T0200,_{Sigma-Aldrich,}_Saint_Quentin_Fallavier,_France.

j _In_the_diets_used_for_the_{digestibility}_study,₁₀_g_kg−1_of_starch_was_replaced_by_Cr₂_O₃_as_an_inert_marker_(Merck_Millipore,_Guyancourt,_France). Table2

GrowthperformanceandfeedutilizationinjuvenileEuropeanseabassfedexperimentaldietsfor5weeks.

C TA1 TA2 TA3 R2 _P

Initialbodyweight(g) 10.2±0.7 10.5±0.7 10.4±0.8 10.1±0.9

Finalbodyweight(g) 19.8a_±_0.7 _18.9a_±_0.9 _16.7b_±_0.6 _14.8c_±_0.5 _y₌_20.15₋_0.22_X _0.97 _<0.014

SGR(%d−1) 1.8a_±_0.2 _1.6ab_±_0.3 _1.3bc_±_0.1 _1.0c_±_0.0 _y₌_1.83₋_0.04_X _0.99 _<0.005

FI(gkg−1d−1) 15.5a_±_0.4 _15.0ab_±_1.3 _14.7ab_±_1.3 _13.7b_±_1.6 _y₌_15.59₋_0.07_X _0.94 _<0.030

FE(gg−1₎ _1.1a_±_0.1 _1.0ab_±_0.2 _0.9ab_±_0.0 _0.7b_±_0.1 _y₌_1.12₋_0.02_X _0.96 _<0.018

PER(%) 2.0a_±_0.2 _1.8ab_±_0.4 _1.6ab_±_0.1 _1.3b_±_0.2 _y₌_2.02₋_0.03_X _0.99 _<0.006

Dataareexpressedasthemean±S.D.(n=3).Valuesinthesamerowwithdifferentsuperscriptlettersaresignificantlydifferent(P<0.05,andP<0.10,FI).Therelationship betweenthetanninconcentrationinthediets(X,explanatoryvariable)andtheresponse(Y,dependentvariable)isexpressedbylinearpolynomialregressionequations, coefficientofdetermination(R2₎_and_probability_(P)._Specific_growth_rate_(SGR);_feed_intake_(FI);_feed_efficiency_(FE);_and_protein_efficiency_ratio_(PER).

2.3. Samplecollection,chemicalanalysisanddigestibilitystudy Forinitialcarcasscomposition, 20fishfromtheinitialstock wererandomlysampled.Attheendofthestudy,sixfishpertank wererandomlycollectedandindividuallymeasured.Fishorgans wereremoved,weighedforsomaticindexcalculations,and car-cassesstoredforlatercompositionanalysis.Beforeanalysis,feed andfishsampleswerehomogeneously groundup,freeze-dried, and thecomponents determined (duplicated sub-samplesfrom diets,n=4, or fromfish mass, n=3×4) bystandard laboratory methods:DMat105◦Cfor 24hbyoven drying,ashcontentat 550◦Cfor 12hbycombustionin amuffle furnace(Thermolyne 30400Furnace,ThermofisherBioblock,Illkirch,France),crude pro-tein content (N×6.25) bythe Dumasmethod, usingautomatic flash combustion, followed by gas chromatographic separation anddetectionbythermalconductivity(NitrogenAnalyser2000, FisonInstruments,Arcueil,France),lipidcontentbyextractionwith dichloromethaneusingtheSoxhletmethod(Avanti2050 extrac-tionsystem,Bezons,France),andgrossenergycontentusingan adiabaticcalorimeter (IKA-CalorimeterSystem-C4000 Adiabatic, Fontenay-sous-Bois,France).

The digestibility trial,to determinenutrient utilization, was started after the first weekof adaptation to thediet and con-ductedfortwoweeksunderthesameconditionsasforthefish groups(n=40)bysubstitutingtheexperimentaldietsbythediets includingthedigestibility marker.Sodiumalginateas a dietary binderensuredcohesionofthefecalmatter.Feceswereharvested dailyusinganautomaticsystemwithcontinuouswaterfiltration

(GuillaumeandChoubert,2001).Thesieveswereplacedinthe

sys-temcollectorafterthelastmealoftheday,thefecescollectedfor 15hd−1 priortothenextmorningmealandpooledpertankin testtubestoobtainasufﬁcientmass(30g)foranalysis,andstored at−20◦_C._Samples_were_lyophilized_at₄₀◦_C_and_homogenized_for

analysis.Chromiumcontentinthedietandfeces(n=3×4)was determinedinduplicateusinganatomicabsorption spectropho-tometer(PerkinElmer,Villebon-sur-Yvette,France)withamixture ofperchloricacidfordigestionandsulfuricacidforconcentration. Thedichromateconcentrationwasmeasuredatawavelengthof 440nmusingastandardCr2O3solution.

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24 M.-H.Omnesetal./AquacultureReports6(2017)21–27 2.4. Calculations

Theapparentdigestibilitycoefﬁcients(ADCs)werecalculated, basedontheratioofthemarkerinthedietsandfeces,accordingto theformula(GuillaumeandChoubert,2001):

ADCDM= 100×[1−(Crdiet/Crfeces)]

ADCnutrientorenergy=100×[1−(Crdiet/Crfeces)×(nutrient or

energyfeces/nutrientorenergydiet)];Crdiet/feces:chromiumcontent

indietorfeces,respectively;nutrient_diet/feces:speciﬁcnutritional variable,DM,proteinorenergycontentindietorfeces.

Growth and feed utilization parameters were calcu-lated based on the following criteria: live weight gain (%)=100×[(Wf−Wi)/Wi], where Wi, Wf are initial and final averagefishbodyweight(g);specificgrowthrate(SGR, %body weightday−1)=100×[(lnWf−lnWi)/t],witht,durationindays of the feedingperiod; feedintake (FI, gkg−1day−1)=feed con-sumed/biomass/t,biomass:averagefishbodyweight(Wi+Wf)/2; feedefficiency (FE,gg−1)=weightgain/feedconsumed; protein efficiencyratio(PER,gg−1)=weightgain/crudeproteinconsumed ona DM basis; percent protein or energy retained (PR or ER, %)=100×(proteinor energy gain/crude protein or energy con-sumedonaDMbasis).Fulton’sconditionfactor(K)wascalculated as:K=Wf/Lfexp3×100,Wf,finalweightoffish(g),Lf,totallength (cm).Somatic indices were calculated as: organ-somatic index (%gg−1)=100×(weight of organ/total weight of fish); hepato-(HSI),spleno-(SSI),nephro- (NSI),viscera-(VSI)and intestinal-(ISI) somaticindex referring tothe relative proportionof liver, spleen,kidney,viscera,andemptiedintestine,respectively, (vis-cera,calculatedasthedifferencebetweenthecarcassweightand totalweightoffish).

2.5. Statisticalanalysis

Theexperimentaldataareexpressedasthemean±standard deviation(SD).Data transformationwasappliedifnecessaryfor thestatisticalanalysis.Theresultsofthetannineffectswere ana-lyzedbyone-wayanalysisofvariance(ANOVA)followedbythe Newman-Keulspost-hocmultiple-rangetest,whenevertherewere statisticallysigniﬁcantdifferencesbetweenthemeans(atP<0.05, orP<0.10).Simplelinearregressionwasappliedtodeterminethe relationshipbetweentheﬁshparameters(dependentvariable)and includeddietaryTAlevels(independentvariable).Statistical anal-ysiswasperformedusingStatistica,version6software(StatSoft France,Maisons-Alfort,France).

3. Results

Nodiet-relatedmortalityoccurredduringtheexperiment in anygroup.However,twofishescapedandthislosswastakeninto accountforthecalculations.Alldietswerereadilyeaten,butfish fedtheTA2andTA3dietsmanifestedearlysignsofreducedfeed consumptionthatpersisteduntiltheendoftheexperiment.The cumulativefeedintakewasaffectedbytheexposuretodietary TA:fishshowedasignificantlossofappetitefromthe17thday intheTA3groupandthe21stdayintheTA2group,respectively (Fig.1), whereas therewasnodifferencebetweentheTA1 and controlgroups.FeedintakewassignificantlydifferentatP<0.10 betweengroups.

Bytheendoftheexperiment,thegrowthoftheTA2andTA3 groupswassigniﬁcantlylessthanthatofthecontrolgroup;their bodymasswas15.6and25.6%lowerthanthatoftheﬁshfedthe Cdiet,respectively,whichdoubledtheirweightduringthestudy

(Table2).ThecorrespondingsigniﬁcantdecreasesofSGRwere27.8

and44.4%,respectively.TheTA3dietsigniﬁcantlyaffectedFEand

Fig.1.Cumulativefeedintake(gﬁsh−1₎_in_juvenile_European_seabass_fed

experi-mentaldietscontainingvariouslevelsoftannicacid.Differencesamonggroupswere recorded(verticallines)fromthe17thday(betweentreatmentTA3andtheothers) andthe21stday(betweentreatmentTA2,TA3,andtheothers)untiltheendof thetrial(ﬁnalpoint).Dataareexpressedasthemeanvalues±SEM(n=3groups pertreatment);thedifferentlettersabovetheverticallinesindicatesigniﬁcant differencesbetweenthemeans(P<0.05).

Table3

Apparentdigestibilitycoefﬁcient(ADC)ofdrymatter(DM),crudeprotein(CP),and energy(E)ofexperimentaldiets.

ADC(%) C TA1 TA2 TA3

ADCDM 80.2a_±_0.4 _78.2ab_±_0.6 _77.7ab_±_0.3 _75.2b_±_3.5

ADCCP 95.8a_±_0.2 _94.7b_±_0.3 _93.4c_±_0.2 _91.6d_±_0.2

ADCE 92.8a_±_0.3 _92.2a_±_0.2 _91.7ab_±_0.7 _90.2b_±_1.2

Dataareexpressedasthemean±S.D.(n=3).Valuesinthesamerowwithdifferent superscriptlettersaresigniﬁcantlydifferent(P<0.05).

PER,whichwere36.4and20%lowerthanthoseofthecontrolgroup, respectively.

The diets were highly digestible, even when supplemented withTA, but the digestibility of DMwassigniﬁcantly different betweenTA3andtheothertreatments,asweretheproteinADCs, whichwerebetween91.6and95.8%,dependingonthequantity

ofTA(Table3).TheproteinADC(Y)andTAconcentrationinthe

diets(X)wererelatedbyasimplelinearregressionbythemodel, Y=95.38₋0.153X(determinationcoefﬁcientR2₌_0.89_at_P_<_0.036).

Thesubsequentenergy ADCwassigniﬁcantlyaffected(P<0.05) onlybytheTA3dietrelativetotheCandothertwodiets.

TheK index of fishreared ontheTA3 diet (1.17) was non-significantlylowerthanthatoffishfromtheC(1.29)andTA1(1.31) groups,showingthattheytendedtonotbeinasgoodconditionas thefishfromtheothergroups.Somaticindicesandtheproximate compositionofthefishfedtheCandTA-containingdietsare pre-sentedinTable4.Thehepatosomaticindex(HSI)was38.5%lower infishoftheTA3groupthanincontrolfish,andthedifferencewas significant.Therewerenosignificantdifferencesbetweentheother somaticindicesofthedifferentgroups.

OnlyTAsupplementationatthehighestconcentration signif-icantlyaffectedcarcasscompositionintermsoflipidandenergy content,whichdecreasedby13.6and9.3%,respectively,whereas proteincontentwasnotalteredbyTA(Table4).However,PRvalues weresignificantlylowerinfishfromtheTA2andTA3groupsthan thoseintheCgroup(24and39.5%,respectively).Energyretention wasalsosignificantlylowerinfishfedtheTA2andTA3dietsthan thosefedtheCdiet(32.4and58.7%,respectively).

4. Discussion

ThesuccessfuluseofplantmealsasfeedsinEuropeanseabass dietshasbeendemonstratedbygoodgrowthperformance,butthe effectsof TAinclusionhave notbeenstudiedyet.Thetolerable thresholdofthis polyphenoliccompoundmerited investigation,

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Table4

Somaticindices(%)andproximatecarcasscomposition(%wetweight)ofjuvenileEuropeanseabassfedexperimentaldietsfor5weeks,andnutrient/energyretention efﬁciency(%ofintake).

C TA1 TA2 TA3

Somaticindices K 1.29±0.25 1.31±0.18 1.22±0.16 1.17±0.10 HSI 2.83a_±_0.43 _2.67ab_±_1.02 _2.45ab_±_0.56 _1.74b_±_0.33 SSI 0.04±0.01 0.06±0.02 0.06±0.03 0.05±0.05 NSI 0.28±0.13 0.25±0.12 0.34±0.13 0.27±0.19 VSI 19.10±7.12 19.13±5.37 18.40±6.95 18.20±2.86 ISI 2.00±3.60 2.51±0.27 2.30±0.90 2.50±1.03

Proximatecarcasscomposition Initial

Moisture 67.8 67.5±0.1 67.7±0.6 68.3±0.7 68.9±0.6 Protein 17.3 18.0±0.6 17.8±1.0 17.5±0.3 17.4±0.6 Fat 11.0 11.0a_±_0.2 _10.8a_±_0.6 _10.1ab_±_0.4 _9.5b_±_0.6 Ash 3.6 4.0±0.1 3.8±0.3 4.2±0.2 4.2±0.2 Energy(kJg−1_w._w.) _8.5 _8.6a_±_0.2 _8.6a_±_0.3 _8.3a_±_0.3 _7.8b_±_0.2 Retentionefﬁciency PR 36.1a_±_4.0 _34.3a_±_3.7 _27.4b_±_1.0 _21.8b_±_3.4 ER 13.8a_±_1.5 _12.9a_±_2.6 _9.3b_±_0.9 _5.7c_±_0.8

especiallygiventheincreasingsupplyofmiscellaneousplant-based proteinsourcesbythefeedindustry.TheupperTAconcentration testedinthepresentstudywashigherthanthelowtomoderate levelsnormallyfoundinmostformulations.Thiswastoincrease theaccuracyoftheresultsobtainedinvivoandtoenablesmaller antinutritionaleffectstobemoreclearlydetected(Griffith,1989). Allfishgrewduringtheexperimentalperiodandnodiet-related mortalityoccurred.TheresultsdemonstratethatTAadditiontothe dietlowerednutritionalquality,leadingtoasignificantreduction ingrowth.

Tanninsarewidelypresentinmostplantspeciesusedas pro-teinsubstitutesforfishmeal,includingsoybean,canola-rapeseed, wheat,sunflower,fieldpea,fababean,chickpea,sorghum,hazelnut seed,microalgal biomass,andseaweedmeals(Oliva-Telesetal.,

1998; Valenteet al., 2006; Emre et al.,2008; Adamidou et al.,

2009a,b;Chebbakietal.,2010;AltanandKorkut,2011;Messina

etal.,2013;Collinsetal.,2013).Severalstudieshavebeen

pub-lished,butmanywithoutanalyticalmeasurements,andonlyfew reportthetannincontentofthediets.

Ahighlevelofincorporationofsoybeanprotein(580gkg−1DM,

Oliva-Telesetal.,1998),andofdifferentlyprocessedsoybeanmeals

(480and530gkg−1,Tibaldietal.,2006)wasshowntoaffectthe

growthofseabass.Inclusionofchickpeasand fababeansinthe diets(340and350gkg−1,withatannincontentinrawingredients of between 5.9 and 11.4gkg−1 DM) lowered the feed conver-sionratioandgrowthrate(Adamidouetal.,2009a).However,the growthofseabasswasnotadverselyaffectedbylowinclusionof legumesinthediet(160gkg−1,Adamidouetal.,2009a).Dietsfor troutcontainingsoybean,canola,andfieldpeamealsortheir pro-teinconcentrates(300gkg−1,withtanninlevelsbetween4.9and 10.6gkg−1DM),didnotaffectgrowthrates(Collinsetal.,2013). Currentlyunderutilized fat-extractedhazelnutoilmealcontains upto7.5gtanninkg−1 tanninsamongthetotalphenolicsinthe rawmaterial(XuandHanna,2011).Seabassfedadietcontaining thismeal(from75to300gkg−1,whichincludesfrom0.6to2.3g estimatedtanninkg−1),exhibitedgrowthcomparabletothatoffish receivingthecontrolfishmealdiet(Emreetal.,2008).

Theincorporationof greenalgae(50and100gkg−1,Valente etal.,2006)orseaweed(25and75gkg−1,Peixotoetal.,2016)in theseabassdiets,andtheinclusionofseaweed(upto100gkg−1,

Güroyetal.,2013),inthetroutdietsalsodidnotadverselyaffectthe

growth.However,incorporationofUlvasp.meal(100–300gkg−1 thatbroughttanninlevelsto1.6–2.2gkg−1DM)toreplacesoybean mealin thediet ofNile tilapia(Oreochromisniloticus),impaired growth(Azazaetal.,2008),whereasadietcontaining2.9g tan-ninkg−1comingfromtheuppervolumeof300gwastedatemeal

didnot(Azazaetal.,2009b).Inclusionat15gkg−1and25gkg−1

ofeitherhydrolysableorcondensedtanninsintodietsimpaired tilapia growth, with a greater negative effect for HT than CT

(Buyukcaparetal.,2011).

Furthermore, diets including soybean meal, sunﬂower, and coconutoilcakes,whichcontain6.3gtanninkg−1 (Xavieretal., 2012),orsupplementationwithtannicacidupto20gkg−1 diet

(Prustyetal.,2007),didnotnegativelyaffectthegrowthofrohu

(Labeorohita,Hamilton)ﬁngerlings.

Impairedfishgrowthappearstobeduetoreducedpalatability, resultinginlowerfoodintake.Inthepresentstudy,thehighdietary tanninlevelsthatreducedvoluntaryconsumptionbyseabassarein agreementtothe20gtanninkg−1thatloweredfeedintakeofthe commoncarp(CyprinuscarpioL.)forasimilarfeedingperiod rela-tivetofishfedCTforalongerperiodoftime(BeckerandMakkar, 1999).Incontrast,theadditionoftannintotroutdietshadasmall positiveeffectonfeedintake(Collinsetal.,2013).Palatabilityisan importantcriterionforfeedinginecosystemsandinanimal nutri-tion,affectingwhetherthefoodisingestedorrejected.Forexample, extractsofpolyphenoliccompounds(>20gkg−1DW)from temper-atebrownalgaeareeffectivefeedingdeterrentsforsometropical herbivorousfishes(VanAlstyneandPaul,1990).Inanimals, sup-plementationoffeedswith30gkg−1oftannin-richextractreduced pigfeedintake(Candek-Potokaretal.,2015).Substantial quanti-tiesofpolyphenolictanninspresentiningredients,combinedwith otherantinutrients,contributetotheastringencyandbittertaste ofthemealsthatcontainthem(Mwachireyaetal.,1999).Aside fromtheunpleasanttaste,tanninsloweredtheappetiteof rain-bowtroutfedcanolameal(Mwachireyaetal.,1999),tilapiafedV.

fabameal(Gaber,2006),andseabassfedahightannin-containing

diet(presentstudy).

Theavailabilityofnutrientstoﬁshisessentiallyassociatedwith theirdigestibility.Inthepresentstudy,theproteinsourceinthe basediet wasmostlyblue whiting(crude protein:70% ofDM), whichishighlydigestibleandshowedthehighestADCforprotein (95.8%),inthesamerangeofADCvaluesfoundinprevious stud-iesforseabassdiets(Adamidouetal.,2009b).Incontrast,there wasacorrelationbetweenthegradientofdietarytanninandADC forprotein,thatshowedaninversedose-responserelationshipand ADCforDMandenergywerealsoaffected.AltanandKorkut(2011)

reportedahighADCforprotein(90.1%)inseabassjuvenilesfed 100gkg−1 plantproteinsourcesindietsusingvarious combina-tionsoflowcostfeeds.However,CTsinthesorghumcontroldiet, notnoticedby theauthors,probablyresulted inlowernutrient digestibilitythanfortheothermeals.Indeed,lowinvitro digestibil-ityofV.fabacontainingCT,hasbeenreportedfortrout(Grabner

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26 M.-H.Omnesetal./AquacultureReports6(2017)21–27

andHofer,1985).Incontrast,incorporationof150and300gkg−1

fababeanasa proteinsourcein extrudeddietsfor on-growing seabasssignificantlyimprovedtheADCforprotein,fat,andenergy relativetothecontroldiet(Adamidouetal.,2009b),whereas sim-ilarorlowerADCvalueswereobtainedwhenthedietsincluded chickpeasand fieldpeas.Inclusion of360gkg−1 fababeanmeal (tannin21.4gkg−1;CT 20.4gkg−1)impairedthegrowthof Nile tilapia(Azazaetal.,2009a).TanninsfromaFabaceaeplantextractat levelsequaltoorgreaterthan6.3gkg−1alsosignificantlyaffected DMandproteindigestibilityintilapia(Pintoetal.,2000).Thus, dif-ferencesinnutrientdigestibilityappeartobespeciesspecific,and dependonthetoleranceofthefishtotannin-containingplant pro-teinsourcesandtanninsupplements.Tannin-proteincomplexes are formedwithexogenous dietaryproteinscoming fromfeed

(BeckerandMakkar,1999),inparticularthosecontainingproline,

butotheraminoacidsandphospholipidscanalsointeractwith tan-nin.Inaddition,reduceddigestibilityhasbeenpartiallyascribedto endogenousnitrogenorproteinandaminoacidlossespreviously observedinsmallanimals(ratorchickens)feddietscontaining tannin-richfood (Skopec et al., 2004; Mansoori and Acamovic, 2007),aswellasinpigsfedacorn-richhydrolysabletannin(Cappai etal.,2013).Inthisstudy,theamountofTAthatboundprotein moleculesmayhavereducedthedigestibilitybyloweringgut pro-teinbioavailability.Furthermore,inhibitionofdigestiveenzymes bytanninmayhavealsolowereddigestibilityandimpairedgrowth

(MandalandGhosh,2010b).

DietaryTAdidnotaffectedproteinlevelsinthecarcasswhereas lipiddeposition,energycontent,andHSIwereproportionallylower withincreasingTAinclusionlevels.Here,thehighHSIvalueswere probablyrelatedtotheproportionofﬁshmealusedinthebasediet. Lowpalatabilityofthediets(LanariandD’Agaro,2005),reduced dietarystarch (Tibaldi et al.,2006; Adamidou etal., 2009b), or incorporationofdriedmicroalgaeintothediet(Tullietal.,2012) havediminishedfeedintakeandinducedsubsequentdepletionof lipidstoresandliverglycogen,andhavereducedHSIandADC val-ues,probablyduetothemodulatingeffectsofdietaryelementson seabasslipidmetabolism.Theseobservationsmaypartiallyexplain ourresultsontheeffectsofTAonHSI,whereasthedepletionof lipidsaftertanninexposuremaybeduetoinhibitionoflipid syn-thesisand/orlipidmobilization,resultingfromtheformationof complexesbetweentanninand enzymesinvolved inthese pro-cesses(AiuraanddeCarvalho,2007).Tanninsupplementationalso reducedproteinandenergyretentioninthisstudy.Different pat-ternsofcarcassfatstoragehavebeenobservedinotherﬁshspecies.

AiuraanddeCarvalho(2007)showedthatNiletilapiafeddiets

con-tainingfrom0.8to6gTAkg−1hadmorebodylipiddepositionthan whenfedlowtannin-containingsorghumandthanthosefedhigh tannin-containingsorghum.However,gradedlevelsofTAinthe dietofrohudidnotaffectthelipidstores,butsigniﬁcantlyincreased

theHSI(Prustyetal.,2007).

WeusedcommerciallyavailableTAinthisstudytoinvestigate theeffectsoftannin.However,therearemanynaturalmolecules classiﬁedastannins,ofwhichCTswouldprobablybelesstoxicto

ﬁshthanTA(BeckerandMakkar,1999;Buyukcaparetal.,2011).

Currently,dietsformulatedwithamixtureofvariousplant pro-teinsourcesmustcompensatefornutrientdeﬁcienciesandavoid undesirablecompoundeffectstoproducethebestperformance. Indeed,tanninsarealsoknowntointeractwithother antinutri-ents,removingtheinhibitoryactionoftanninsorthedeleterious effectsofothers(Francisetal.,2001).Geneticimprovementshave beenattemptedtoselecttannin-freeorlow-tanninplantvarieties

(Créponetal.,2010).Varioustreatments(Xavieretal.,2012;Bhat

etal.,2013)andbiotechnologicalapplicationssuchasfermentation usingﬁshguttannaseenzyme,havealsobeentestedwiththeaim

toeliminateorreducetheamountoftannininfeedstoensuretheir nutritionalvalue(Mandal,2012;GhoshandMandal,2015).

5. Conclusion

Tannin supplementation above 10gkg−1 in diets decreased proteindigestibility,andabove20gkg−1growthperformancein Europeanseabassjuveniles.Theseresultsfurthersupporttheuse ofavarietyoflowtannin-containingplantproteinsourcesas nutri-tionallydigestibleingredientsforcarnivorousfishes,andhighlight theneedtotakeprecautionswhenincludingtannin-richfeedsin diets.Furthermore,amongthepotentiallybeneficial phytochem-icalsfor fishfarming, polyphenolextractsshowpromisedueto theirbiologicalproperties.However,theeffectoftheirhydrolyzed derivativesonthehealthoffishremainstobedetermined.

Acknowledgements

ThisworkwasconductedwithintheAquacultureresearch pro-grams‘Qualityofprocessandproducts’(IfremerPlouzané,France) and ‘Functionalfoodand health ofﬁsh’(InraURNuMeA Saint-Pée-sur-Nivelle,France).TheauthorsthankF.J.Gatesoupe(Ifremer, Plouzané;InraURNuMeA),thetwoanonymousreviewersandW. Hempel,forcriticalreading andcomments,andamendmentsto thearticlethathavebeenappreciable,theBibliothèqueLaPérouse andS.Gros(Ifremer,Plouzané),forresourcecollectionand previ-ousassistanceonacontributiontoaconference.Thisresearchwas notsupportedbyaspeciﬁcgrant.

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