Aquaculture 318 (2011) 101–108

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Increasing dietary plant proteins affects growth performance and ammonia excretionbut not digestibility and gut histology in turbot (Psetta maxima) juveniles

Alessio Bonaldo a,⁎, Luca Parma a, Luciana Mandrioli a, Rubina Sirri a, Ramon Fontanillas b,Anna Badiani a, Pier Paolo Gatta a

a Dipartimento di Scienze Mediche Veterinarie, Università di Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia (BO), Italyb Skretting Aquaculture Research Centre, Box 48, N-4001 Stavanger, Norway

⁎ Corresponding author. Tel.: +39 0547 338931; fax:E-mail address: alessio.bonaldo@unibo.it (A. Bonald

0044-8486/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.aquaculture.2011.05.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 January 2011Received in revised form 2 May 2011Accepted 3 May 2011Available online 12 May 2011

Keywords:TurbotPsetta maximaPlant proteinsGrowthAmino acidGut histology

A trial on turbot (Psetta maxima) juveniles was undertaken to evaluate the effect of replacing dietary fishmeal(FM) with a complementary mixture of plant proteins (PP) consisting of soybean meal (SBM), wheat glutenmeal (WGM) and corn gluten meal (CGM). Four practical diets were formulated to progressively replace 25%(PP25), 39% (PP39), 52% (PP52) and 66% (PP66) of FM protein. Forty animals per tank (initial weight 24.2±4.4 g) were randomly distributed into twelve 450-liter square tanks connected to a closed recirculationsystem (temperature 18±1 °C). The diets were tested in triplicate for 77 days. Final weight ranged from126.6 g (PP25) to 99.5 g (PP66). Voluntary feed intake of group PP66 (55.5 gfish−1) was significantly lowerthan in PP25. Specific growth rate in fish fed diet PP25 (2.14% day−1) was significantly higher than thosefound both in PP52 (2.01% day−1) and PP66 (1.82% day−1) groups. Feed conversion ratio (FCR) in groupsPP25 and PP39 (0.66–0.67) was lower in comparison with those of the other groups (FCR 0.70–0.74). None ofthe diets affected whole-body composition and hepatosomatic index. Condition factor significantly decreasedwith increasing dietary PP (2.06, 2.02, 1.97 and 1.91) whereas a significant increase in viscerosomatic indexwas observed in fish fed PP52 and PP66 (6.70–6.95) in comparison with the other two groups (6.31–6.21).Reduced protein retention was found in groups fed diet PP52 and PP66, with protein efficiency ratio (PER)ranging from 2.71 to 2.63 and gross protein efficiency (GPE) from 40.3 to 40.0, in comparison with the othertwo groups (PER 2.83 and GPE 45.5–45.8), even though apparent digestibility coefficients were not reduced.Ammonia excretion, (g total ammonia nitrogen 100 gprotein intake−1), was significantly higher for groupPP25 (3.41) than group PP52 (2.61). Gut histology examined in four different sites of intestine (pyloric caeca,proximal, intermediate and distal intestine) revealed no noticeable differences among fish of the various dietgroups. In conclusion, substitution of a mixture of SBM, WGM and CGM for up to 52% of FM protein did notreduce feed intake, and at 39% substitution, turbot maintained optimal growth rate and nutrient utilization.Worsened FCR of fish fed diets containing higher plant protein levels was not associated with a reduceddigestibility of ingredients or alterations of gut histology.

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1. Introduction

The turbot Psetta maxima is one of the most important marinefish species in European aquaculture. However, as with the culture ofother carnivorous species, the long-term sustainability of turbotfarming may be threatened by its present over-dependence onfishmeal (FM) as the main source of dietary protein. Numerousstudies have dealt with the use of plant proteins (PP) in diets forcarnivorous species showing several problems, which includereduced growth (Carter and Hauler, 2000; Gomes et al., 1995;

Gómez-Requeni et al., 2004; Kaushik et al., 1995; Kikuchi, 1999; Limet al., 2004; Storebakken et al., 1998), increased feed conversionratio (FCR) (Krogdahl et al., 2003; Refstie et al., 1998), reducedprotein utilization (Opstvedt et al., 2003; Refstie et al., 2000;Robaina et al., 1995) and morphological changes in the intestine(Storebakken et al., 2000; van den Ingh et al., 1991). Some of theseproblems are due to the presence of anti-nutritional factors (ANFs)(Francis et al., 2001) or associated with unbalanced amino acid (AA)composition (Krogdahl et al., 2003) of vegetable feedstuffs. Anumber of previous turbot studies investigated reducing the FMcontent in diets using single vegetable ingredients at increasinginclusion levels or mixtures of different plant feedstuffs. Soybeanconcentrate can substitute up to 25% FM protein (Day and González,2000), whereas turbot fed a diet containing 20% of CGM hadcomparable growth performance as those fed a FM based diet

Table 1Formulation and composition of the experimental diets (%).

Diet PP25 PP39 PP52 PP66

Wheata 9.75 8.27 6.58 5.05Wheat glutenb 8.70 13.06 17.66 22.35Fishmeal LTc 55.00 45.00 35.00 25.00Soybean meald 8.70 13.06 17.66 22.35Fish oile 14.44 15.19 15.94 16.70Corn glutenf 3.00 5.00 6.50 7.00Phosphateg . . 0.27 0.84L-Lysineh . . . 0.33Vitamin and mineral premixi 0.30 0.30 0.30 0.30Yttrium premix 0.10 0.10 0.10 0.10Proximate composition (analyzed)

Moisture 7.0 6.9 7.2 7.1Crude protein (N×6.25) 53.6 52.6 52.4 51.4Crude fat 19.6 17.9 18.8 18.1Ash 9.1 7.9 6.8 6.2Gross energy (MJ/kg) 22.3 22.3 22.1 22.5

a Statkorn, Norway.b Cerestar Scandinavian AS, Denmark (crude protein CP: 81.9% as it is; crude fat CF:

4.6% as it is).c Consortio, Peru (CP: 71.2% as it is; CF: 8.1% as it is).d Denofa, Norway (CP: 49.4% as it is; CF: 2.4% as it is).e Nordsildmel, Norway.f Cargill, USA (CP: 61.3% as it is; CF: 1.5% as it is).g Tessenderlo Chemie, The Netherlands.h SvedaKjeni AS, Norway.i Proprietary formula of Skretting Aquaculture Research Centre, Norway.

102 A. Bonaldo et al. / Aquaculture 318 (2011) 101–108

(Regost et al., 1999). Rapeseed meal can be incorporated at levels upto 30% following a preliminary heat treatment (Burel et al., 2000).Diets of up to 50% extruded lupin, produced no adverse effects ongrowth performance (Burel et al., 2000) and allowed more than 30%reduction of the FM content in the diet. However, the same authorsobserved a decrease of feed intake when diets contained 30% and50% of lupin for the first 3 weeks of experiment. According toFournier et al. (2004), lupin was responsible for the decline of feedintake and oscillation in daily feed consumption when combinedwith wheat gluten meal (WGM) and corn gluten meal (CGM). In thiscontext, it was of interest to further explore the feasibility of FMreplacement by PP ingredients in turbot diet. In the absence of asingle FM analog, it is important to evaluate the nutritional value ofalternative ingredients and formulate diets based on a mixture ofsuch ingredients which can collectively replace FM in the diet of fish(Gomes et al., 1995). In particular, the use of PPmixtures reduces thepotential inhibition of feed consumption and macronutrient utili-zation resulting from the presence of ANFs (Fournier et al., 2004)and provides a more adequate AA profile. Soybeanmeal (SBM), CGMand WGM are good candidates to substitute FM in turbot diets, asthese are readily available in high quantities at low prices and arerich in protein. SBM is one of the most attractive alternatives to FMdue to its favorable availability, price and AA composition. On theother hand, the use of soybean products may pose a series ofproblems that may affect palatability (Rackis, 1974), proteindigestibility (Glencross et al., 2004) and may reduce feed utilization.Furthermore, other fish species have shown variable sensitivity tosoy ANFs. In Atlantic salmon and rainbow trout, SBM causesimpaired growth and protein utilization in addition to distinctmorphological alterations in the intestine that become morepronounced with increasing dietary level (Krogdahl et al., 2003).These changes are characterized by a shortening of the primary andsecondary mucosal folds, a widening of the lamina propria which isinfiltrated by a mixed population of inflammatory cells identified aslymphocytes, macrophages, eosinophilic and neutrophilic granularcells, and diffuse immunoglobulin M (Baeverfjord and Krogdahl,1996; Bakke-McKellep et al., 2000), and concurrent changes inenterocyte structure, including a decrease, or even a loss, of thesupranuclear vacuoles present in normal enterocytes (Baeverfjordand Krogdahl, 1996; Bakke-McKellep et al., 2000), a shortening ofthe microvilli and increased formation of microvillar vesicles (vanden Ingh et al., 1991). To our knowledge, no studies have beenreported on the use of SBM inclusion in diets for turbot. Wheatgluten meal and CGM have a high protein level, are low in fiber, richin vitamins B and E and are known to contain no ANFs. They havebeen already included in diet for turbot at increasing levels, showinga good potential in substituting FM (Fournier et al., 2004; Regost etal., 1999). On a protein basis, WGM and CGM are especially low inlysine while SBM is low in methionine when compared with FM andthe recognized requirements of fish (National Research Council(NRC), 1993). Unbalanced AA composition can result in reducedretention and a higher portion of the protein directed towardscatabolic processes (Von Der Decken and Lied, 1993; Walton et al.,1984) and furthermore a decrease in the indispensable/dispensableamino acid (IAA/DAA) ratio may result in increased proteincatabolism (Gómez-Requeni et al., 2004).

In this general context, a trial was undertaken to evaluate theeffects of replacing FM in practical diets of turbot with a complemen-tary mixture of PP sources consisting of SBM, WGM and CGM. Thespecific objectives of the present study were (1) to evaluate growthperformance and protein utilization of turbot fed diets containinggraded levels of a blend of these three ingredients, (2) to evaluate ifany decrease in growth performance resulted from a reduceddigestibility of the experimental diets and (3) to evaluate potentialnegative effects of the plant protein mixture on gut histology as acause of possible retarded growth.

2. Materials and methods

2.1. Diets

Experimental feed was manufactured by Skretting AquacultureResearch Centre (Stavanger, Norway) using extrusion technology andcommon feed ingredients. A control diet (PP25) was formulated withpractical ingredients to contain 52% crude protein, 18% crude fat and aPP inclusion level of 25%. This inclusion level was chosen on the basisof previous studies on FM substitution in turbot juvenile (Burel et al.,2000; Day and González, 2000; Regost et al., 1999). On the grounds ofthese studies, significant differences in growth performances wererecorded starting from a 30% level. Furthermore, this inclusion levelrepresents the standard level of PP currently used in commercialturbot culture. The other three diets were formulated in order to beisoproteic and isolipidic to the control diet and contain 39% (PP39),52% (PP52) and 66% (PP66) of PP by increasing the level of SBM,WGM, and CGM. The ratio of the three ingredients was chosen inorder to balance the AA content according to IAA levels suggested byKaushik (1998) and their increase was in the same proportion at eachstep in order to maintain constant the AA ratio of the PP mixture. DietPP66 was supplemented with lysine. In the absence of specific data onvitamin, mineral and trace mineral requirements of turbot, require-ment data for other species were applied (National Research Council(NRC), 1993). All diets contained yttrium oxide as an inert marker fordetermining apparent digestibility coefficient (ADC). The ingredients,the proximate and the AA composition are given in Tables 1 and 2.

2.2. Fish, experimental set-up and sampling

The experiment was carried out at the Laboratory of Aquaculture,Faculty of Veterinary Medicine, Cesenatico, Italy. Turbot (P. maxima)juveniles with an initial average weight 24.2±4.4 g were obtainedfrom the hatchery France Turbot, Noirmoutier, France. Before theexperiment, the fish were acclimatized for 4 weeks to the experi-mental facilities and fed commercial FM-based diets (Europa 22,Skretting, Cojóbar Burgos, Spain; crude protein 55%, crude fat 22%). At

Table 2Amino acid profile of the diets and requirements of turbot (g 16 g N−1).

Amino acids PP25 PP39 PP52 PP66 Essential amino acidrequirementsa

Methionine 1.77 1.47 1.73 1.56 {2.7Cysteine 0.93 1.15 1.23 1.30Lysine 6.50 5.87 5.30 5.21 5.0Threonine 4.17 3.95 3.73 3.67 2.9Arginine 6.77 6.57 6.16 5.91 4.8Isoleucine 4.50 4.45 4.38 4.30 2.6Leucine 8.15 8.10 8.03 7.77 4.6Valine 5.17 5.02 4.91 4.78 2.9Histidine 2.28 2.26 2.28 2.25 1.5Phenylalanine 4.72 4.95 5.07 5.07 {5.3Tyrosine 4.67 4.10 4.20 3.74Glycine 5.93 5.49 5.01 4.64Serine 4.97 4.99 5.01 4.93Proline 6.70 7.44 7.87 8.21Alanine 6.33 6.12 5.72 5.68Aspartic acid 7.83 7.58 7.27 7.19Glutamic acid 17.14 19.16 20.91 22.68Hydroxyproline 0.78 0.63 0.47 0.38Tryptophan 0.70 0.71 0.70 0.72 0.6IAA/DAA 0.95 0.88 0.85 0.79

a From Kaushik (1998).

103A. Bonaldo et al. / Aquaculture 318 (2011) 101–108

the start of the trial, 40 animals per tank were randomly distributedinto twelve 450-liter square tanks (bottom surface: 0.56 m2) and fedthe experimental diets for 77 days. Tanks were provided with naturalseawater and connected to a unique closed recirculation systemconsisting of a mechanical sand filter (Astralpool, Spain), anultraviolet light (Philips, the Netherlands) and a biofilter (Astralpool,Spain). The water exchange rate per tank was 100% every 2 h. Theoverall water renewal of the system was 5% daily. Temperature wasmaintained constant at 18±1 °C throughout the experiment; photo-period was held constant at a 12-hour day length through artificiallight (200 lx at the water surface — Delta Ohm luxmeter HD-9221;Delta-Ohm, Padua, Italy). Water temperature and dissolved oxygen(≥7 ppm) were monitored daily in each tank. Ammonia (totalammonia nitrogen, TAN ≤0.1 ppm), nitrite (NO2

- ≤0.2 ppm) andnitrate (NO3

- ≤50 ppm) were determined spectrophotometricallyonce aweek (Spectroquant Nova 60, Merk, Lab business) at 12.00 p.m.At the same time, pH (7.8–8.2) and salinity (28–33 gl−1) weredetermined. Animals were hand-fed to apparent satiation twice a day(at 9.00 a.m. and 5.00 p.m.), 6 days per week and once on Sundays.Feed losses were minimal throughout the trial but, when necessary,remaining feed was estimated and deducted from feed intake foroverall calculations. At the beginning and at the end of theexperiment, all the fish of each tank were individually weighed andtotal length was recorded. Carcass proximate composition wasdetermined at the beginning and at the end of the trial. In the formercase, one pooled sample of five fish was sampled to determine initialproximate composition while in the latter case one pooled sample offive fish from each tank was collected to determine final proximatecomposition. Furthermore, at the end of the trial, wet weight, visceraand liver weight were individually recorded from five fish per tank fordetermination of visceral and hepatosomatic indices. At this time,three fish per each tank were also randomly sampled for histology. Allexperimental procedures were evaluated and approved by theEthical-scientific Committee for Animal Experimentation of theUniversity of Bologna, in accordance with the European CommunityCouncil directive (86/609/ECC).

2.3. Ammonia excretion

In order to evaluate nitrogen metabolism, after the end of thegrowth trial, ammonia excretion was measured in all tanks during a

24-hour cycle and repeated collected values were integrated accord-ing to the following formula given by Kaushik (1980):

Et = V0 × ΔC + Ct × ΔW:

Where

V0 volume of water in the tankΔC variation in TAN (Ci–Ci–t)Ci mean of TAN concentration between two consecutive

intervals (Ci+Ci− t/2)ΔW flow rate/unit of time “t”t unit of increment in time in which concentration variation

is considered minimalEt TAN excreted by fish per unit of time retained

Fish were fed once at time zero to satiation and the amount ofeaten feed was registered for each tank to calculate the protein intake.The water inflow was kept constant at 250 l/h in each tank the daybefore and during ammonia sampling. Water of each tank wassampled from the outlet at 0, 2, 4, 6, 8, 10, 12, 18, and 24 h after themeal. A tank without fish was used to collect control samples. Watersamples were immediately stored at −32 °C until analysis. TANconcentration in samples was measured by the indophenol method(Koroleff, 1983) and overall data were expressed as g TAN100 gprotein intake−1.

2.4. Digestibility experiment

At the end of the growth trial and following all associatedsamplings, the remaining groups of turbot were used to determinethe ADC of dry matter, protein and energy, by the indirect methodwith diets containing yttrium oxide. The experiment was conducted,using the identical facilities and environmental conditions used in thegrowth trial. Fish were fed once to satiation the experimental dietswhich were assigned to three tanks each. Feces were collected 12 hafter feeding by stripping the animals. This procedure was performedtwice with an interval of 2 days between sampling. Immediately afterthe collection, feces were pooled for each tank and kept at −20 °Cuntil analysis for yttrium, dry matter, protein, and energy content.ADC was calculated as follows:

ADCð%Þ = 100 × 1−dietaryY2O2 levelfaecalY2O2 level

×faecalnutrient or energyleveldietarynutrient or energylevel

� �:

2.5. Analytical methods

Analyses of experimental diets, carcasses and freeze-dried fecalsamples were made using the following procedures: dry matter wasdetermined after drying to constant weight in a stove at 105 °C; crudeprotein was determined by the Kjeldahl method; fat was determinedaccording to Folch et al. (1957); ash content wasmade by incinerationto a constant weight in a muffle oven at 450 °C; gross energy wasdetermined by calorimetric bomb (Adiabatic Calorimetric Bomb Parr1261, PARR Instrument, IL). Amino acid analyses of diets were madeusing the method of Cunico et al. (1986); tryptophan was analyzed bythe method of Garcia and Baxter (1992). Yttrium oxide levels in dietsand feces were determined using Inductively Coupled Plasma-AtomicEmission Spectrometry (Perkin Elmer, MA, USA). Prior to analyses, thesamples were first mixed with 6 ml ultrapure nitric acid 67%, 1 mlultrapure hydrogen peroxide 31% (Merk, Darmstadt Germany), and50 ml distilled water, then heated in a microwave oven (Microwave3000, Perkin Elmer, Massachusetts, USA).

Table 3Growth performance and feed utilization of turbot fed experimental diets for 77 days.

PP25 PP39 PP52 PP66 Linear regression line R2 P

Initial weight g 24.4±0.8 23.9±0.8 23.7±1.4 24.6±0.5 N.A. . .Final weight g 126.6±4.0c 119.4±5.1bc 111.2±8.3b 99.5±3.1a Y=144.1−0.656 x 0.81 ≤0.0001Weight gain g 102.2±4.2c 95.5±4.5bc 87.5±7.0b 75.0±2.9a Y=120.0−0.659 x 0.83 ≤0.0001SGR, % day−1 2.14±0.06c 2.09±0.03bc 2.01±0.04b 1.82±0.04a Y=2.36−0.008 x 0.81 ≤0.0001VFI g fish−1 67.3±2.3b 64.1±3.2ab 61.8±6.0ab 55.5±2.7a Y=74.7−0.276 x 0.59 ≤0.01FCR 0.66±0.01a 0.67±0.01a 0.70±0.01b 0.74±0.02c Y=0.60+0.002 x 0.80 ≤0.0001

Different subscript letters ab in the same row denote significant (P≤0.05) differences among treatments. Linear regression lines (where Y is the response and x is the % level of PP indiet, ranging from 25 to 66), R2 and P are also given. Each value is the mean (±S.D.) of three replicates. N.A. = not applicable.SGR=Specific growth rate (% day−1)=(ln final weight− ln initial weight)/days×100; VFI=Voluntary feed intake (g)=feed intake (g)/fish; FCR=Feed conversion ratio=g feedgiven/g live weight gain.

104 A. Bonaldo et al. / Aquaculture 318 (2011) 101–108

2.6. Histology

At the end of the trial, three fish per each tank (nine per diet) wererandomly sampled for routine histology. Samples of intestinal tract(pyloric caeca, proximal, intermediate and distal intestine) werecollected, fixed in 10% buffered formalin, dehydrated in a gradedethanol series and embedded in paraffin. Sections of 4 μm werestained with hematoxylin and eosin (H&E). Sections of each portionwere evaluated under light microscopy for the presence of degener-ative changes of epithelial cells or inflammation according to thecriteria suggested by Krogdahl et al. (2003).

2.7. Statistical analyses

All data were analyzed by one-way ANOVA with Newman–Keulspost-hoc test, using GraphPad Prism version 4.00 for Windows(GraphPad Software, San Diego, CA, USA). A linear regression analysiswas also conducted to analyze weight gain, SGR, VFI and FCR againstPP level, using the statistical package R version 2.11.1 for Windows(Revolution analytics, Palo Alto, CA, USA). A significant level ofP≤0.05 was adopted for all parameters.

3. Results

During the feeding trial no mortality was recorded. While all fishaccepted the experimental diets, voluntary feed intake (VFI) of fishfed diet P66was significantly lower in comparisonwith that of fish feddiet PP25 (Table 3). Fish fed diet PP25 scored significantly higher finalweight, weight gain and SGR than those found in fish fed diet PP52and PP66, while fish fed PP66 showed significantly lower values for allthese parameters in comparison to the other fish groups. Feedconversion ratio of fish fed diet PP25 and PP39 was significantly lowerin comparison to that of fish fed diets PP52 and P66. Final weight,weight gain, SGR, VFI and FCR were linearly related to the PP level, thelast parameter being the only one to be directly proportional.

Table 4Carcass proximate composition (% as it is) and biometric parameters of turbot fed theexperimental diets for 77 days.

PP25 PP39 PP52 PP66

Proximate compositionMoisture 73.9±0.1 73.7±0.6 73.6±0.4 73.9±1.1Protein 16.2±0.2 16.2±0.2 15.2±0.5 15.5±0.4Lipid 7.8±0.6 8.7±0.6 8.5±0.2 8.6±0.6Ash 3.9±0.3 3.7±0.4 3.4±0.3 3.2±0.2

Biometric parametersCF, g (cm3)−1 2.06±0.14d 2.02±0.16c 1.97±0.14b 1.91±0.13a

VSI (%) 6.31±0.34a 6.21±0.56a 6.70±0.46b 6.95±0.45b

HSI (%) 2.21±0.36 2.12±0.25 2.37±0.41 2.39±0.45

CF=Condition factor=(body weight/total lenght3)×100.VSI=Viscerosomatic index=(viscera weight/body weight)×100.HSI=Hepatosomatic index=(liver weight/body weight)×100.

Whole-body composition and biometric parameters are shown inTable 4. No significant effect of inclusion level of PP was apparent onwhole-body composition (moisture, protein, lipid and ash). Conditionfactor was significantly lower when PP levels increased whereas asignificant increase in VSI was observed in fish fed PP52 and PP66 incomparison to the other two groups. Dietary treatment did not affectHSI.

Data on nutrient retention efficiency and ammonia excretion arepresented in Table 5. Protein efficiency ratio and GPE significantlydecreased in fish fed diets PP52 and PP66. Gross lipid efficiencyranged from 78.5 to 64.8%, but was not significantly affected bydietary treatments. Ammonia excretionwas statistically different onlybetween groups fed diets PP25 and P66.

Apparent digestibility coefficients of dry matter, protein andenergy (Table 6) did not present any statistical differences amonggroups. Histopathological gut examinations revealed no noticeabledifferences in the appearances of the intestines among any of the dietgroups. The histological evaluation of pyloric caeca and the threeintestinal segments from all fish (Fig. 1) showed scattered cells mainlycomprising lymphocytes and macrophages together with a lessernumber of eosinophilic granular cells in the capillary rich laminapropria, and distal loose connective tissue. Inflammatory anddegenerative changes were not detected in any fish from all dietgroups. The mucosa of all the three segments showed two types offolds: complex folds, characterized by multiple branches, and simplefolds which regularly alternated. In pyloric caeca, the folding wascloser than in proximal and intermediate tracts, contributing to areduction of the lumen. On the other hand, in the distal portion thefolds were lower and less numerous and the lumen was wider.Epithelial cells of the mucosa in all the segments showed variousdegrees of cytoplasmic supranuclear vacuolation but the presence ofvacuolated and non-vacuolated cells was seen within groups and wasnot related to the treatment. The nuclei were evenly polarized andbasally located. Among epithelial cells there were numerous goblet

Table 5Nutrient retention efficiency and ammonia excretion of turbot fed experimental diets.

PP25 PP39 PP52 PP66

PER 2.83±0.03b 2.83±0.05b 2.71±0.05a 2.63±0.06a

GPE 45.5±1.2b 45.8±0.8b 40.3±1.0a 40.0±2.5a

GLE 64.8±5.7 78.5±4.9 70.0±1.1 71.8±7.0Ammonia excretion

g total ammonia nitrogen100 g protein intake−1

2.61±0.19a 2.88±0.28ab 3.09±0.23ab 3.41±0.34b

Different subscript letters ab in the same row denote significant (P≤0.05) differencesamong treatments.PER=Protein efficiency ratio=body weight gain g/protein intake g.GPE=Gross protein efficiency=((% final body protein×final body weight)−(% initialbody protein×initial body weight)/protein intake×100).GLE=Gross lipid efficiency=((% final body lipid×final body weight)−(% initial bodylipid×initial body weight)/lipid intake×100).Ammonia excretion=100×(total ammonia nitrogen (g)/protein intake (100 g)).

Table 6Apparent digestibility coefficient (%) of dry matter, protein and energy of theexperimental diets.

PP25 PP39 PP52 PP66

Dry matter 75.8±5.5 69.2±9.0 65.9±16.0 68.6±8.4Protein 90.0±1.9 84.7±4.9 83.8±4.2 86.3±3.8Energy 94.3±4.2 93.2±1.8 91.4±3.7 92.9±3.7

Dry matter and protein values are means of six replicates, energy values are means oftwo replicates.

105A. Bonaldo et al. / Aquaculture 318 (2011) 101–108

cells and scattered intraepithelial lymphocytes. The apical surface ofepithelial cells was covered by a microvillous border.

4. Discussion

One reason for unfavorable outcomes with PP based diets has beendeficient feeding activity. The reduction of feed intake with PP baseddiets has been reported in turbot (Fournier et al., 2004) as well as inEuropean sea bass (Dias et al., 1997) or rainbow trout (Gomes et al.,1995). Low palatability, poor IAA profiles and complex synergisticinteractions among ANFs are perhaps the most plausible responsiblefactors for an altered feeding activity in diet characterized by an highinclusion of PP. Data of the present study indicate that feed ingestionrates were not significantly affected up to 52% of FM proteinsubstitution and that only the maximum incorporation of PP in dietPP66 led to a reduction in VFI in comparison with fish fed PP25.Fournier et al. (2004), registered a decreased feed intake in fish fed adiet containing approximately 55% of PP in comparison with the

Fig. 1. Intestinal segments coming from fish fed diet PP66 (A, E, I, O), PP52 (B, F, L, P), PP39segment; I, L, M, N intestinal intermediate segment; O, P, Q, R intestinal distal segment. All thevenly polarized and basally located. The lamina propria is normal in architecture in all segtissue. (H&E, bar=100 μm).

group fed the reference diet containing approximately 43% of PP.However, the PP mixture in our study differed from that used by theseauthors, since we did not include extruded lupin, which was indicatedas the possible ingredient reducing feed intake. Several researchershave suggested that the poor palatability of diets containing SBM canbe responsible for the limited consumption and thus reduced growthobserved in many species (Arndt et al., 1999), and this could be thecause of the reduced feed intake in fish fed PP66, where SBM waspresent at the maximum level of 22.35%. In the study by Day andGonzález (2000) in turbot, the soy protein concentrate affected feedingestion rate at 75% and 100% protein replacement levels corre-sponding to an inclusion of 55% and 73.5% of this ingredient in thediet.

In general, growth rates recorded in our trial were higher orsimilar than those observed by other authors (Fournier et al., 2004;Regost et al., 1999). Fish fed diets PP52 and PP66 grew more slowlyand showed a lower SGR than those fed on PP25. This was not only aconsequence of reduced feed intake, as fish fed diets PP25, PP39 andPP52 consumed similar amounts of feed. Hence, the difference waslikely to be a result of poorer utilization of consumed N, whichnegatively influenced FCR in fish fed PP52 and PP66 in comparisonwith those fed PP25 and PP39. The increase of FCR in fish fed dietsPP52 and PP66 coincided with reduced protein utilization (PER andGPE) and agrees with some earlier studies evaluating PP inclusion inturbot diets (Day and González, 2000; Regost et al., 1999). The findingthat PER and GPE decreased significantly in fish fed diets PP52 andPP66 while the protein ADC remained almost constant, suggests thatthe proportion of dietary protein used for catabolic processes (energy

(C, G, M, Q) and PP25 (D, H, N, R). A, B, C, D pyloric caeca; E, F, G, H intestinal proximale segments of the four tested diets show a regular columnar epithelium with the nucleiments and in the distal intestine only is rich in capillary network and loose connective

106 A. Bonaldo et al. / Aquaculture 318 (2011) 101–108

production), instead of anabolic ones (protein synthesis), increaseswith the level of FM replacement, as already stated by Day andGonzález (2000). Such results were not observed in the study byFournier et al. (2004) where N retention decreased, whereas ADC ofdry matter, nitrogen and energy increased at increasing FMsubstitution. In most cases, a decrease in protein efficiency is coupledwith an increase in ammonia excretion (Fournier et al., 2004) as foundin our study in fish fed PP66.

Ammonia is an end product from amino acid (AA) catabolismcontributing 60 to 90% of the nitrogen excreted (Cowey and Walton,1988) and excretion of ammonia is found to be high when proteinsynthesis is low (Lied and Braaten, 1984). Therefore, increased TANexcretion can be an indicator of reduced protein synthesis, expressedas lower growth and protein retention. An increase in TAN excretionin response to elevated level of plant origin meal was previouslyreported in European sea bass (Ballestrazzi et al., 1994), rainbow trout(Médale et al., 1998) and gilthead sea bream (Robaina et al., 1995).

High TAN excretion and plasma TAN levels observed in turbot andrainbow trout fed plant meal-based diets are also indications ofindispensable AA (IAA) imbalance of plant meal-based diets, resultingin the low protein efficiency calculated in fish fed these diets(Fournier et al., 2003). This could indicate that differences in AAprofiles of the experimental dietsmay explain the reduction in growthand the increase in FCR in PP66 and PP52 groups compared with thePP25 group. Currently, there are few data available on AA re-quirements in turbot and the inclusion of high levels of some PPsources may result in deficiencies of IAA with respect to theseundetermined levels. An estimation of IAA requirements of turbot hasbeen made by Kaushik (1998) and, more recently, by Peres and Oliva-Teles (2008). Diets used the current trial were formulated taking intoaccount the levels of IAA suggested in these two studies. Nevertheless,the AA compositions were different among groups. In particular, thediet PP66 and PP52 contained 5.21 and 5.30 g16 gN−1 of lysine,respectively, which were little over the turbot requirement of5.00 g16 gN−1 suggested by Kaushik (1998) and Peres and Oliva-Teles (2008). Lysine is generally considered the first limiting AA inmost fish species and is highly required for body protein deposition.Another point to be considered is the dietary IAA/dispensable AA(DAA) ratio, which decreased with increased inclusion of PP from 0.95in diet PP25 to 0.88, 0.85 and 0.79 in diets PP39, PP52 and PP66,respectively. The decrease in IAA/DAA ratio with increasing dietary PPfound in our study, could determine a corresponding increase inprotein catabolism and a reduced protein synthesis, expressed aslower growth and protein retention as already showed in gilthead seabream (Gómez-Requeni et al., 2004), European sea bass (Peres andOliva-Teles, 2006) or Atlantic cod (Hansen et al., 2007). This indicatesthat differences in AA profiles may explain the reduction in growth inPP66 and PP52 groups compared with the PP25 group.

Whole-body composition of animals was not affected by PPinclusion level. These results are in accordance with those of Burelet al. (2000) where final body composition remained unchangedirrespective of dietary treatment. However, these results do not agreewith those of Regost et al. (1999) and Fournier et al. (2004), whoreported an influence of dietary treatment on carcass composition. CFdecreased with increasing FM substitution and this was also found inDay and González (2000) where turbot was fed diets containingincreasing amounts of soybean protein concentrate. This data could bedue to a reduced development of muscle in these animals, leading to alower thickness of fillets. Conversely, increased VSI in fish fed dietsPP52 and PP66 was probably due to a reduction of growth that led to ahigher weight percent of viscera in comparison with the whole body.

Histology of aquaculture species is important in the understandingof pathological alteration related to nutritional sources (Gargiulo etal., 1998). Descriptions about turbot histology found in the literaturemainly focused on histopathology of developing larvae (Cousin andBaudin-Laurencin, 1985; McFadzen et al., 1994; Padrós et al., 1993).

Moreover, histological investigations in juvenile turbots can be foundrelated to experimental parasitic infestations (Bermúdez et al., 2010;Redondo et al., 2002) or in support to nutritional trials. The groupsimilarity of gut segment histology does not support the growthreduction observed in groups fed diets PP52 and PP66. The presence ofnumerous lymphocytes and macrophages in the lamina propria andscattered intraepithelial lymphocytes in all animals are normalfeatures of the gut-associated lymphoid tissue. Leucocytes occur inall parts of the teleost digestive system, most extensively in theintestine, where lymphocytes, plasma cells, granulocytes and macro-phages are present in and under the epithelium. Although largelymphoid centers are lacking, many lymphoid cells, either scattered orin small groups, were reported to be present in the epithelium andlamina propria (Abelli et al., 1997). Considering diet formulations,SBM was included at increasing levels up to 22.35% in diet PP66.Previously, researchers have evaluated the intestinal histology of fishfed SBM and have observed that various fish species can have differenttolerance limits to the presence of ANFs, which may be the cause ofenteritis (Heikkinen et al., 2006; van den Ingh et al., 1991). Forexample, in salmon, a dose-dependent effect was observed onhistopathologic changes in intestine of salmon (Krogdahl et al.,2003) beginning from a SBM inclusion of 10%. In contrast, otherspecies, such as Atlantic halibut (Grisdale-Helland et al., 2002),channel catfish (Evans et al., 2005), Egyptian sole (Solea aegyptiaca)(Bonaldo et al., 2006) or European sea bass (Bonaldo et al., 2008), didnot exhibit any inflammatory response of intestinal mucosa to theinclusion of SBM in the diet. Based on the results of this study, a SBMinclusion of up to 22.35% fed for 77 days did not exert anymorphological changes in the intestine of turbot and this mightindicate that the species is able to accept high levels of this ingredientwithout negative effects on intestinal mucosa. Concerning theutilization of CGM and WGM as single ingredients, there are fewreports regarding intestinal histology of Atlantic salmon. Storebakkenet al. (2000) showed minor inflammatory changes only in onespecimen of those fed a diet with 35% of PP from WGM, whileAslaksen et al. (2007) didn't find any intestinal changes in the samespecies fed diets containing 20.4% of CGM. Studies on other fishspecies, in which the three plant ingredients used in our study werepresent in different mixtures of PP, didn't find significant histopath-ologic changes in intestine of rainbow trout (Barrows et al., 2007),gilthead sea bream (Benedito-Palos et al., 2008; Sitjà-Bobadilla et al.,2005), Atlantic cod (Hansen et al., 2006) and grey mullet (Luzzana etal., 2005). Only in one case, a mild enteritis was described in Atlanticcod fed diet with 100% PP consisting of a mixture of WGM, SBM andsoy protein concentrate (Olsen et al., 2007) while Santigosa et al.(2008) showed some epithelial changes without enteritis in seabream fed a diet with 100% PP consisting of a mixture of CGM andWGM, extruded peas, rapeseed meal, sweet white lupin and extrudedwhole wheat.

5. Conclusions

While a FM protein substitution of up to 52% with a mixture of SBM,WGM and CGM did not reduce feed intake, a 39% substitution alsomaintained optimal growth rate and nutrient utilization. A worsenedFCR in fish fed diets containing higher PP levels was not supported by areduced digestibility of ingredients or alterations of gut histology andthe reduced growth, PER and GPE values can partially be explained bydiets not balancing AA thus resulting in increased protein turnover.

Acknowledgements

This research was supported by grants from the Italian RegionEmilia-Romagna. We thank Marina Silvi and Sara Giuliani for technicalassistance and Clive Naylor for English editing. Diets were kindlyprovided by Skretting Aquaculture Research Centre, Stavanger, Norway.

107A. Bonaldo et al. / Aquaculture 318 (2011) 101–108

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