Rice protein-concentrate meal as a potential dietary ingredient in practical diets for blackspot...

Rice protein-concentrate meal as a potential dietaryingredient in practical diets for blackspot seabreamPagellus bogaraveo: a histological and enzymatic

investigation

F. DAPRA*†, F. GAI*, M. T. COSTANZO‡, G. MARICCHIOLO‡,V. MICALE‡, B. SICURO§, G. CARUSO‡, L. GENOVESE‡ AND

G. B. PALMEGIANO*

*Institute of Science of Food Production, Torino Division, National ResearchCouncil, Via L. da Vinci 44, Grugliasco (TO), Italy, ‡Institute of Marine CoastalEnvironment, Messina Division, National Research Council, Spianata Raineri 86,Messina, Italy and §Department of Animal Production, Epidemiology and Ecology,

University of Torino, Via L. da Vinci 44, Grugliasco (TO), Italy

(Received 12 February 2008, Accepted 10 November 2008)

Field and laboratory studies were conducted to evaluate the intestinal responses to partial

replacement of fish meal with rice protein concentrate (RPC) in practical diets for blackspot

seabream Pagellus bogaraveo. Two experimental diets were formulated to be isoproteic and

isoenergetic with an increasing level of RPC (20 and 35%, respectively) and were tested

against a fish meal-based control diet (RPC0). The diets showed similar features for growth

performances and both intestinal histology and digestive enzymes. This study confirmed

that RPC does not induce intestinal mucosa alterations in this fish. The dietary RPC

supplement caused a significant increase in trypsin activity, whereas lipase activity was

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Journal compilation # 2009 The Fisheries Society of the British Isles

Key words: digestibility; intestinal pattern; plant protein source; sparid.

INTRODUCTION

This experiment focused on blackspot seabream Pagellus bogaraveo (Brunnich),a novel species chosen to diversify Mediterranean aquaculture production, andwas a second step of a feeding trial for which the growth performance and thesomatic indices were already reported in a previous work by Palmegiano et al.(2007). This sparid is considered one of the most promising new species foraquaculture diversification (Genovese et al., 2004).

†Author to whom correspondence should be addressed. Tel.: þ39 116709231; fax: þ39 116709240;

email: [email protected]

Journal of Fish Biology (2009) 74, 773–789

doi:10.1111/j.1095-8649.2008.02157.x, available online at http://www.blackwell-synergy.com

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Journal compilation # 2009 The Fisheries Society of the British Isles

Previous studies have been focused on cereal concentrates that are generallyless expensive and readily available alternatives to substitute fish meal inmarine and freshwater fishes (Robaina et al., 1999; Opstevd et al., 2003).Rice protein concentrate (RPC) is a good raw material for fish nutrition due

to its high protein (75% crude protein) and lipid (11% ether extract content),and these nutrient values are comparable with fish meal (Palmegiano et al.,2006). In addition, the essential amino acid profile of RPC meets the nutri-tional requirements of sparids according to NRC (1993).The presence of anti-nutritional factors (ANF), however, has a limiting

effect; moreover, ANFs could cause patho-morphological changes in the intes-tinal epithelium of fishes (Krogdahl et al., 2003).The physiological and the biochemical features of the digestive system of

sparids have been investigated (Fernandez et al., 2001), but seems to be noinformation concerning the effect of plant protein sources on the digestive tractof P. bogaraveo.In this study, histological and biochemical (digestive enzymes) observations

were integrated to assess the possible changes induced by RPC introduced asa new protein source for the dietary formulation of P. bogaraveo.A panel of histochemical enzyme markers and biochemical assays were

selected and tested on P. bogaraveo. The histochemical enzymes selected inthe present study are components of the epithelial brush-border membrane[alkaline phosphatase (ALP) and 59Nucleotidase (59N)] and intracellular struc-tures [(acid phosphatase (ACP) and non-specific esterase (NSE)], which areimplicated in the digestion and the absorption of nutrients. The digestive en-zymes assayed in the present study included the detection of trypsin, chymo-trypsin, carboxypeptidases A and B, amylase and lipase.Preliminary studies examined the nature and distribution of digestive enzymes

along the gastrointestinal tract of P. bogaraveo, determining the occurrence ofproteolytic enzymes in the intestinal tract at concentrations similar to those mea-sured in other Pagellus species (Caruso et al., 1999; Caruso & Genovese, 2000).To date, however, the introduction of a new protein sources in dietary formula-tions for this fish has received little attention, and the metabolic effects are stillunknown, except for a few recent studies (Caruso et al., 2003, 2005). Nothing isknown of the effect of RPC on the intestine, either in histomorphology or diges-tive enzyme activity terms, particularly in P. bogaraveo. To fill this gap, the aimof this study was to investigate the intestinal histomorphology and activity pro-files of both the digestive and the marker enzymes of this species.

MATERIALS AND METHODS

EXPERIMENTAL PLAN

The experimental design of this study was monofactorial balanced, the experimentalfactor was the diet (three diets � four replications, 10 fish per tank) and the fish weredistributed in 12 0�5 m3 tanks. This experiment was carried out at the Marine CoastalEnvironment Institute experimental plant (Messina, Italy). Throughout the trial period,the average temperature of the sea water was 21�3° C, range � 2�3° C, the dissolvedoxygen was 4�9 ppm, range � 0�2 ppm the salinity c. 38 and the pH 8�2.

774 F. DAPRA ET AL .

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Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 773–789

Wild specimens of P. bogaraveo used in this experiment were harvested from the Mes-sina Straits in several catches. The fish were transferred to the experimental plant; theirhealth was checked and no pathologies were recorded during the acclimatization periodof 30 days in 1�9 m3 raceways. The fish were fed a commercial feedstuff formulated forgilthead sea bream Sparus aurata L. without any antibiotic treatments. After the accli-matization, 120 fish were individually weighed (mean � S.D. mass, M, 75�0 � 17�5 g)and then randomly distributed in 12 tanks.

A commercial rice-protein concentrate (RPC) (CBH Company Ltd; www.cbhcn.com)was nutritionally characterized by proximate analyses and amino acid composition ac-cording to official analytical methods (AOAC, 1995) and as described by Cavallarinet al. (2005), and the results are reported in Tables I and II.

Two experimental diets were formulated to be isoproteic (crude protein 47%) andisoenergetic (gross energy, 22 MJ kg�1 dry mass, MD) with an increasing level ofRPC (RPC 20 and 35, respectively) corresponding to a decreasing level of fish meal(36 and 20�5%, respectively). These diets were tested against a fish meal-based controldiet (RPC0). The feeds were manufactured in the laboratory at the Experimental Sta-tion of the Department of Animal Husbandry of the University of Torino and have

TABLE I. Ingredients and proximate composition of the raw rice protein concentrate(RPC) and experimental diets having increasing levels of RPC (n ¼ 3)

Ingredients (%) RPC RPC0 RPC20 RPC35

Fish meal 57�0 36�0 20�5Rice protein concentrate 0�0 20�0 35�0Dehulled barley meal 23�5 24�5 25�0Corn meal 9�0 9�0 9�0Fish oil 6�0 6�0 6�0Brewer’s yeast 2�0 2�0 2�0Bentonite 1�5 1�5 1�5Mineral mixa 0�5 0�5 0�5Vitamin mixb 0�5 0�5 0�5Proximate composition (%MD)MD (% fresh matter) 91�3 95�8 95�1 95�1Crude protein 75�5 48�1 47�1 46�9Ether extract 11�2 14�3 13�7 13�5Ash 4�3 10�1 8�1 6�4Crude fibre 3�0 2�0 2�3 2�9Nitrogen-free extractsc 6�0 25�5 28�8 30�3Gross energy (MJ kg�1 MD)

d 23�8 21�5 21�9 22�0

MD, dry mass; RPC0, control diet without rice protein; RPC20, diet with 20% of RPC on MW;

RPC35, diet with 35% of RPC on MW.aMineral mixture (g or mg kg�1): dicalcium phosphate 500 g, calcium carbonate 215 g, sodium salt

40 g, potassium chloride 90 g, magnesium chloride 124 g, magnesium carbonate 124 g, iron sulphate

20 g, zinc sulphate 4 g, copper sulphate 3 g, potassium iodide 4 mg, cobalt sulphate 20 mg,

manganese sulphate 3 g, sodium fluoride 1 g, (Granda Zootecnica, Cuneo, Italy).bVitamin mixture (IU or mg kg�1): DL-a tocopherol acetate, 60 IU; sodium menadione disulphate,

5 mg; retinyl acetate, 15000 IU; DL-cholecalciferol, 3000 IU; thiamin, 15 mg; riboflavin, 30 mg;

pyridoxine, 15 mg; B12, 0.05 mg; nicotinic acid, 175 mg; folic acid, 500 mg; inositol, 1000 mg;

biotin, 2.5 mg; calcium panthotenate, 50 mg; choline chloride, 2000 mg (Granda Zootecnica).cCalculated as 100-(% crude protein þ % ether extract þ % crude fibre þ % ash).dDetermined by bomb calorimetry.

PAGELLUS BOGARAVEO GUT ENZYMES AND HISTOLOGY 775

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been reported by Palmegiano et al. (2007). All the dry ingredients and the oil were thor-oughly mixed; water was then blended into the mixture to obtain a appropriate consis-tency for 3�5 mm diameter pellets. After the pelleting process, the diets were dried ina stove and stored at 4° C until used. The fish were fed twice a day, 6 days per weekfor a period of 15 weeks, according to the common rearing procedures utilized by theSicilian aquaculture farmers. This feeding scheme was utilized also by Kikuchi (1999)and Vergara et al. (1999) in similar feeding trials. The daily feeding ratio was 1�5%of the tank biomass; the daily ratio was updated every 15 days by bulk weighing. Mor-tality and feed consumption were carefully verified and recorded every meal, and nomortality occurred during the feeding period.

HISTOLOGICAL ANALYSIS

At the end of the growth trial, histological analyses were carried out to evaluate thestate of the intestine and three fish for each tank (mean � S.D. M 104�5 � 24�3 g),12 fish for each diet, were killed 4 h after their last meal using an anaesthetic over-dose (3-aminobenzoic acid ethyl ester, MS-222, 100 mgl�1; Sigma-Aldrich, www.sigmaaldrich.com). This time was chosen after previous investigations in order toobtain an adequate gut-fullness status. Rings of c. 5 mm in length were sampled fromthe proximal, mid and distal intestine and rinsed with a saline solution. Samples forhistomorphology were fixed in 4% phosphate-buffered formalin (pH 7�3) at 4° C, de-hydrated in graded alcohols and paraffin embedded. Transverse sections were cut ata 4 mm thickness and stained with haematoxylin and eosin–orange G for examinationunder a light microscope. Samples for histochemical enzyme assays were embedded in

TABLE II. Amino acid composition (% dry matter) of the rice protein concentrate (RPC)and experimental diets (see Table I) (n ¼ 12)

RPC RPC0 RPC20 RPC35

Cysteine* 1�4 n.d. 0�6 0�8Phenylalanine* 3�4 2�0 2�3 2�6Isoleucine* 6�0 2�3 2�3 2�4Leucine* 3�1 3�7 3�9 4�1Lysine* 2�7 3�6 2�8 2�4Methionine* 2�1 0�9 0�6 0�8Tyrosine* 3�2 1�1 1�5 1�8Threonine* 2�8 1�7 1�6 1�6Valine* 4�8 3�2 2�7 2�9Aspartic acid† 6�8 4�6 4�6 4�9Glutamic acid† 12�9 6�7 7�4 8�2Alanine† 4�1 3�1 3�1 3�1Arginine* 6�1 3�0 3�4 3�8Glycine† 3�3 2�8 2�6 2�5Histidine* 1�5 0�9 1�0 1�1Proline† 5�4 3�7 4�1 4�1Serine† 2�9 1�5 1�7 1�9AA 72�5 44�1 44�7 47�2IAA* 37�1 17�6 17�0 17�7DAA† 35�4 26�5 27�7 29�5

AA, total amino acids; n.d., not detected.

*IAA, indispensable amino acids.

†DAA, dispensable amino acids.


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an OCT solution (Bio-optica; www.bio-optica.it) in plastic vials. The sample vialswere frozen in isopentane, cooled by liquid nitrogen vapour and stored at �80° C.The samples were cut into sections (7 mm thickness) and stained to highlight the alka-line phosphatase [ALP, EC 3.1.3.1], acid phosphatase [ACP, EC 3.1.3.2],59nucleotidase [5N, EC 3.1.3.5] and non-specific esterase (NSE) reactivity. All sampleswere processed together in the same batch for each histochemical stain, in order toavoid differences in reaction.

ALP reaction was performed using a Sigma-Aldrich kit (alkaline phosphatase n 85reagents). Sections were fixed using citrate buffer acetone (1 ml of citrate solution;Sigma-Aldrich) for 30 s at room temperature and rinsed in distilled water (DW) for45 s. An incubation solution was obtained from 48 ml of DW, one capsule of fast blueRR salt (Sigma-Aldrich) and 2 ml of naphthol AS-MX phosphate alkaline solution(Sigma-Aldrich). The sections were placed in incubation for 30 min at room tempera-ture in a dark moist chamber and then rinsed in DW. They were then counter stainedin Mayer’s haematoxylin–eosin solution for 3 min, rinsed in DW, air-dried, dipped inxylene and mounted with DPX (Fluka BioChemika; Sigma-Aldrich). Heat inactivation(80° C water-bath for 5 min) was used for the controls.

ACP was performed using a Sigma-Aldrich kit. The incubation solution was pre-pared immediately before use with: 1 ml of NaNO2 solution 0�1 M, 1 ml of fast garnetGBC base solution (Sigma-Aldrich) in 38 ml of DW at 37° C, 5 ml of acetate buffer 2�5 MpH 5�2 (Sigma-Aldrich) and 5 ml of naphthol AS-BI phosphoric acid solution 4 g l�1

(Sigma-Aldrich). The slides were fixed in a citrate-buffer acetone–formaldehyde solution(25 ml citrate solution, 65 ml of acetone and 8ml of 37% formaldehyde) for 30 s andimmediately rinsed in DW for 1 min. The fixed sections were placed in incubation for90 min at room temperature and rinsed under tap water for 2 min, air-dried for 15min, counterstained with methylene blue 1:100 for 2 min rinsed in DW for 2 min andmounted as cited above.

The analysis of 5N was carried out using an incubation solution obtained with 40 ml oftris-maleate buffer 0�2 M (pH 7�2), 10 ml of MgSO4 0�1 M, 6 ml of Pb(NO3)2 2%, 50 mgof adenosine 59-monophosphate, 6 g of sucrose, 40 mg of L-tetramisole and 44 ml of DW.The sections were incubated for 60 min at 37° C. For the controls, the sections wereheated to 80° C for 5 min before addition of the incubation solution. Incubation wasstopped using a 4% formaldehyde solution for 1 min, and the sections were rinsed inDW, counterstained with toluidine blue 1:1000 for 10–20 s, rinsed in DW, air-dried, dip-ped in xylene and mounted with DPX.

The NSE incubation solution was prepared with 40 ml of warm DW, 1 ml of 0�1 MNaNO2 (Sigma-Aldrich), 1 ml of 15 mg ml�1 fast blue BB base solution (Sigma-Aldrich), 5 ml of 1 M Tryzmal� (Sigma-Aldrich) and 2 ml of 12�5 mg ml�1 a-naphthylacetate solution (Sigma-Aldrich). The sections were fixed in citrate buffer acetone form-aldehyde (as cited above) for 30 s at room temperature and rinsed in DW for 1 min.The fixed sections were incubated for 30 min at 37° C, light protected, rinsed 2 minin DW, air-dried and mounted as described above.

BIOCHEMICAL ASSAYS

Groups of five fish per tank were sacrificed 4 h after feeding to determine the enzymeactivities. This time was chosen after previous investigations in order to obtain anadequate gut fullness. From each individual, the whole intestine (tissues and content)was removed and homogenized with Potter-Ultraturrax (Kinematica GmBH; www.kinematica.ch) in 50 mM tris buffer pH 7�0 in a dilution ratio of 1:5 (w/v), then centri-fuged at 2000 g for 10 min, keeping the temperature at a value < þ5° C. The super-natant obtained was used as crude enzymatic extract; it was immediately stored at�80° C until assay according to conventional laboratory procedures. For the quantita-tive determination of trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), carboxypepti-dases A (EC 3.4.17.1) and B (EC 3.4.17.2), amylase (EC 3.2.1.1) and lipase (EC 3.1.1.3)activities, biochemical assays using specific substrates were performed in duplicate ac-cording to the methods reported below.


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Trypsin activity was measured according to Hummel’s (1959) method, which relies onthe ability of trypsin to hydrolyse the substrate N-toluen-sulphonyl-L-arginine methylester (TAME; Sigma-Aldrich) into toluenesulfonyl-L-arginine. The reaction mixturewas 1�3 ml of tris buffer (46 mM trizma base; 11�5 mM CaCl2; pH 8�10) and 150 mlof TAME (10 mM) incubated with 50 ml of enzymatic extract. Absorbance measure-ments were performed at 247 nm using a Cary Varian spectrophotometer (Unicam,Cambridge, U.K.) after incubation at 25° C for 1, 2 and 3 min.

Chymotrypsin activity was determined according to Hummel’s (1959) method, whichmeasures the hydrolysis of the substrate N-benzoyl-L-tyrosine-ethyl ester (BTEE,Sigma-Aldrich) into N-benzoyl-L-tyrosine. The reaction mixture was prepared 750 mlof tris buffer (80 mM trizma base; 0�1 M CaCl2; pH 7�80) mixed with 700 ml of BTEE(1�07 mM) and 50 ml of enzymatic extract. After incubation at 25° C for 1, 2 and 3 min,absorbance was measured at 256 nm.

Carboxypeptidase activities (A and B) were determined according to Appel’s (1974)method, which relies on the use of L-hippuryl-L-phenylalanine (Sigma-Aldrich) or L-hippuryl-L-arginine (Sigma-Aldrich) as specific substrates, respectively. The biochemical assaywas performed mixing 150 ml of enzymatic extract with 1�35 ml of a 1�1 mM solutionof substrate in tris buffer (27�5 mM trizma base; 0�11 M NaCl; pH 7�60); followingincubation at 25° C for 1, 2 and 3 min, absorbance was measured at 254 nm.

Amylase activity was measured by modified Bernfeld’s (1955) method, 1% solutionof soluble starch was used as substrate, hydrolysed in maltose. A mixture of 67 ml ofenzymatic extract with 333 ml of the substrate then incubated at 25° C for 10 min. Asolution containing 400 ml of dinitrosalicilic acid was added and boiled for 5 min. Aftercooling, distilled water was added to bring 5 ml final volume. The blanks without sub-strate and a control containing no enzyme extract were run simultaneously with thereaction mixture. Absorbance was measured at 540 nm to calculate net absorbance.

Lipase activity was measured by the lipase diagnostic kit (Sigma-Aldrich) based onthe titrimetric Tietz & Fiereck’s (1966) method, which determines the ability of thisenzyme to hydrolyse an emulsion of olive oil (as the substrate) into glycerol and fattyacids. After incubation of the enzymatic extract with the substrate at 35° C for 3 h ac-cording to the manufacturer’s instructions, the enzymatic reaction was stopped by addi-tion of 3 ml of a 1:1 ethanol–acetone solution. The blanks were run simultaneouslywith the reaction mixture without the incubation. A few drops of 1% phenolphthaleinin ethanol were added to the reaction mixture. The fatty acids released were measuredby titration with 0�01 M NaOH.

The enzyme activity was expressed as the total enzyme activity of the total intestinehomogenate, as reported by Krogdahl & Bakke-McKellep (2005), and was calculated asfollow: total activity ¼ [Enzyme activity (U ml�1) volume of homogenate (ml)] bodymass�1 (kg). The results are reported as enzyme units released kg�1 of body mass(M) (units kg�1 M), where one enzyme unit corresponded to the amount of enzyme thatcatalysed the release of 1 nmol of product min�1.

DIGESTIBILITY TRIAL

An in vivo digestibility experiment was performed in order to determine the appar-ent digestibility coefficients (ADC) of the diets. The tanks utilized for the growth trialwere equipped with a settling column for faeces collection as described by Cho (1992).The apparent digestibility coefficients were measured using the indirect acid-insolubleash (AIA) method; 1% Celite� (Sigma-Aldrich) was added to the diets as the inertmarker. After each meal, the tanks and the settling columns were cleaned to preventthe faeces from being contaminated by uneaten pellets. The faeces were collected dailyand frozen (�20° C) for three consecutive weeks. At the end of the collection period,the amounts of each unit (diet) were pooled and freeze-dried before analysis. The appar-ent digestibility coefficients of the dry matter (ADCDM), crude protein (ADCCP) andgross energy (ADCGE) were calculated using the Maynard & Loosly (1969) equations.

In order to evaluate fish growth the protein efficiency ratio (EPR), specific growth rate(G) and condition factor (K) were calculated as follows: EPR ¼ MGP

�1T ; where MG is the


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fish biomass gain, and PT is the total protein fed (MD) in the feedstuff for each tank,G ¼ 100ðln MF � ln M1Þt �1; where MF and M1 are the final and the initial biomassesrecorded in each tank, and t is the number of feeding days and K ¼ 100ML �3

S ; whereLS is standard length.

The proximate composition of fillets and faeces samples was determined for a grosschemical characterization according to standard methods for crude protein, ether extractand ash (Association of the Official Analytical Chemists, 1995). The total nitrogen con-tent was determined using a nitrogen analyzer (Rapid N III; Elementar AnalysensystemeGmbH; www.elementar.de) according to the Dumas method and the crude protein wascalculated as total N � 6�25. The ether was determined gravimetrically using for theextraction ‘Soxtec system-HT 1043 extraction units’ (Foss; www.foss.dk). The gross energycontent was determined using an adiabatic bomb (IKA C7000; www.ika.de).

STATISTICAL ANALYSIS

The enzyme activities were analysed with a t-test using the GLM procedure (SPSS;www.spss.com). The t-test was applied after the assumption of normality was satisfied,to two data sets, each characterized by its mean, S.D. and number of data points. Thetest would show whether they represent two distinct sub-populations.

As for the ADC values, the ANOVA were performed on transformed and normal-ized data; any significant differences were tested using the Duncan test at a significanceof P < 0�05. The homogeneity of variance was assessed on statistically significant re-sults using a Kolmogorov–Smirnov goodness-of-fit test. All the data were analysedby one-way ANOVA using the GLM Procedure (SPSS).

RESULTS

HISTOLOGY

The intestinal mucosa (Fig. 1) of P. bogaraveo showed two types of folds:deep, branched folds with prominent central connective stroma [lamina propria

FIG. 1. Intestinal sections of Pagellus bogaraveo fed with the RPC20 diet (see Table I) stained with

haematoxy-esoin–range G. (a) Details of the mucosal folds of the proximal intestine, showing the

rounded, central nuclei of the epithelial cells. Supranuclear and infranuclear absorption vacuoles are

also visible. Note the thin lamina propria (LP). (b) Details of the mucosal folds of the distal intestine,

showing the elongated, basally aligned nuclei of the epithelial cells. Supranuclear absorption

vacuoles are visible. Note the thin LP. Goblet cells are indicated ( ).


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(LP)] and simple folds, with a thin central stroma, lying on and between thecomplex folds. The mucosal epithelium was composed of a single layer ofcolumnar cells lined by microvilli and containing scattered goblet cells. Thenuclei of the epithelial cells were round or ovoid and located more or less inthe centre of the proximal intestine (PI) and middle intestine (MI), while theywere elongated and basal in the distal intestine (DI). The epithelial cells werefinely vacuolated in PI and MI, whereas they contained large supranuclear va-cuoles in DI. A number of lymphocytes were found scattered within the LPand also in the intraepithelial, infranuclear position.The intestinal sections were evaluated following the criteria reported in soy-

bean meal-induced enteritis in Atlantic salmon Salmo salar L. (Baeverfjord &Krogdahl, 1996). The variable evaluated were: the widening and shorteningof the intestinal folds, the enterocytes supranuclear vacuolization extents, thevilli lamina propria widening status and lymphocytes infiltration in the laminapropria and the submucosa.Minor changes in the villi structure were noted in some individuals, irrespective

of the administered diet (Fig. 2). These included different degrees of folding,

FIG. 2. Intestinal sections stained with haematoxy-eosin–orange G. Showing altered intestinal morphol-

ogy in Pagellus bogaraveo. (a) Widening and cellular infiltration of both the lamina propria (LP) and

sub-mucosa (SM) are visible in the photograph of the mid intestine from fish fed RPC0, the control

diet (see Table I). (b) Photograph shows mid intestine from the RPC35 diet. (c) Photograph shows

distal intestine from fish fed RPC0 diet with the indentation of the mucosal lining (*) and the lack of

absorptive vacuoles can be seen in the mucosa cells; the goblet cells are indicated ( ). (d)

Photograph shows distal intestine from fish fed RPC20 diet. These photographs are representative

pictures for all samples which have the same features.


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shortening of folds, irregularly spaced indentations on the villi of the folds andvarying extents of mucosa vacuolization. A slight submucosa widening wasobserved in the MI of four fish fed with RPC0, and two fish fed with RPC20.A total loss of enterocytes vacuolization, accompanied by prominent indentationof the mucosal lining, was observed in one fish fed RPC0. An increased numberof intraepithelial lymphocytes and widening of the LP were displayed in the DI oftwo fish fed with RPC0, two fish fed with RPC20 and one fish fed with RPC35.

ENZYME HISTOCHEMISTRY

Alkaline phosphatase (ALP)ALP showed reactivity in the brush border membrane (BBM) and sometimes in

the villi LP (Fig. 3). A weak reaction was detected at the bottom of the villi, and it

FIG. 3. Alkaline phosphatase (ALP), non-specific esterase (NSE) and 59 nucleotidase (59N) reactivities in

the mid intestine of Pagellus bogaraveo. (a) Section from fish fed with the RPC0 diet, (b) section from

fish fed with the RPC20 diet and (c) section from fish fed with the RPC35 diet (See Table I). The

intense black on the brush-border membrane of the mucosa cells indicates the ALP reactivity. (d), (e)

NSE and (f) 59N histochemical reactivities in the mid intestine. (d) Fish fed with the RPC0 (control

diet), (e) fish fed with the RPC35 diet black areas show NSE reactivity, the lumen (IL) and muscolar

layer (M) are indicated and (f) fish fed with the RPC0 diet [lamina propria (LP), intestinal lumen (IL)

and muscolar layer (M)] are indicated. The grey and dark grey shows 59N reactivity in the villi LP,

which is more intense in the distal half of the villous than in the proximal half.


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increased going towards the tip. No differences appeared in the different tracts inthe fish in the control group, or in any fish fed with the RPC20 or RPC35 diets.

59 Nucleotidase (59N)The villi LP presented a positive reaction to 59N, which is weak at the bottom

and more intense at the top in all the intestinal tracts (Fig. 3). This pattern iscomparable to the ALP reactivity and distribution on the surface of the villimucosa. The mid intestine seemed to show higher reactions than the other tracts.No differences appeared for the 59N in the groups of fish fed with different diets.

Non-specific esterase (NSE)The mucosa layer presented a wide and diffused NSE reaction in the whole

thickness of each intestinal tract (Fig. 3) but did not show a constant distributionor intensity in any of the samples. Slight changes of activity were detected betweenthe fish in the same group and the histochemical detection of this NSE in samplesfrom fish fed with the RPC was similar to the fish fed with the RPC0 diet.

Acid phosphatase (ACP)The ACP reaction showed widespread reactivity spots in the villi LP and the

submucosa and some spots in the mucosa layer in the proximal and the midtracts (Fig. 4). The distal intestine samples also showed ACP activity in thesupranuclear region, unlike the other intestinal tracts that did not highlightany reactivity in the mucosa layer. This enzyme pattern was detected for allthe fish fed the RPC0, or the diets containing diets RPC.

ENZYME ACTIVITIES

Trypsin and chymotrypsinRPC produced a significant increase in tryptic activity in the intestine both at

moderate and high amounts (RPC20 and RPC35 diets; t- test, P < 0�05 andP < 0�01, respectively) (Fig. 5). Unlike trypsin, the RPC20 diet did not produceany significant variations in chymotrypsin, whereas the RPC35 diet causeda stimulating effect on this enzyme activity (t-test, P < 0�05) (Fig. 5).

Carboxypeptidases A and BThe effect produced by RPC on carboxypeptidase A consisted of a significant

increase (t-test, P < 0�01) of this enzyme activity in the intestine of fish fed theRPC35 diet (Fig. 5).RPC caused a significant increase of carboxypeptidase B (t-test, P < 0�05),

but only in the intestine of the fish fed the RPC35 diet, similarly to whatwas found for carboxypeptidase A (Fig. 5).

AmylaseAmylase activity showed a significant progressive increase in response to both

moderate and high RPC concentrations (t-tests both, P < 0�01 for RPC35 andRPC20 diets) (Fig. 5).


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LipaseThe RPC determined a progressive reduction on lipase activity in the intes-

tine at both moderate and high concentrations (t-test, P < 0�05 and P < 0�01,respectively) (Fig. 5).

DIGESTIBILITY

The digestibility results are summarized in Table III, no statistical differencesappeared among the diets for CDM, CCP and CGE. The values recorded for CCP

and CGE for the different diets were c. 90%.

FIG. 4. The acid phosphatase (ACP) histochemical reactivities in the (a), (b) mid and (c) distal intestine.

(a) Fish fed with the RPC0 diet (control diet) and (b) fish fed with the RPC35 diet (see Table I). An

image of the distal intestine from fish fed with the RPC0 is shown in (c). The positive of ACP

reaction in the sub mucosa layer and lamina propria is indicated ( ) in the case of the distal

intestine, the stain reaction for this enzyme is also present on the brush-border membrane mucosa.


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As far as the growth performances, previously reported by Palmegiano et al.(2007), are concerned, all the fish readily accepted the experimental diets; no sta-tistical difference was observed in the feed rate of 1�38 � 0�07% (mean � S.D.)amongst the treatments. The mean biomass gain recorded for diet RPC0 was286�8 � 39�9 g and decreased to 222�0 � 34�1 g for diet RPC35, whereas theEPR ratio showed a similar trend, ranged from 0�83 � 0�05 for diet RPC0 to0�69 � 0�11 in diet RPC35. The recorded G was 0�40, 0�35 and 0�33% day fordiet RPC0, RPC20 and RPC35, respectively. The value of K was 2�70, 2�56and 2�61 for diet RPC0, RPC20 and RPC35, respectively. The proximate compo-sition of fillets did not show differences, the dry matter was 28�1%, and the meanvalues (expressed as percentage on MD) was 70�4, 16�3 and 5�6 for crude protein,ether extract and ash, respectively, and gross energy was 25�8 MJ kg�1 MD.

DISCUSSION

In the present study, an integrated approach was used to link the histologicaland biochemical (digestive enzymes) observations in order to detect the possi-ble changes induced by RPC as a new protein source in P. bogaraveo diets.It is well known that some plant protein inclusion in fish diets may induce

intestinal inflammation, as has been documented for soybean meal-fed rainbowtrout Oncorhynchus mykiss (Walbaum) (Burrells et al., 1999; Ostaszewska et al.,

FIG. 5. (a) Trypsin, (b) chymotripsin, (c) amylase, (d) carboxy peptidase A, (e) carboxy peptidase B and

(f) lipase activity measured in the intestinal tract of the fish fed with experimental diets (see Table I).

Enzyme activities are expressed as units kg�1 body mass (M) (mean � S.E.). *, P < 0�05; ** ¼ P <

0�01 (n ¼ 20).

TABLE III. Diets apparent digestibility coefficients values (Mean � S.D., n ¼ 12) for drymatter (CDM), crude protein (CCP) and gross energy (CGE) (see Table I)

RPC0 RPC0 RPC20 RPC35

ADCs (%)Dry matter 93�0 � 0�0 93�7 � 1�2 92�9 � 0�1Crude protein 91�2 � 0�4 89�1 � 0�2 89�9 � 0�4Gross energy 89�2 � 0�4 88�4 � 0�5 89�0 � 0�3


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2005), S. salar (van den Ingh et al., 1991; Baeverfjord & Krogdahl, 1996; Krogdahlet al., 2003) and Asian sea bass Lates calcarifer (Bloch) (Boonyaratpalin et al.,1998), whereas no significant histological alterations have been reported for lupinkernel meal fed O. mykiss (Glencross et al., 2004). Baeverfjord & Krogdahl (1996)evaluated the inflammatory processes linked to diets, by choosing the wideningand shortening of the intestinal folds, the extent of the enterocytes supranuclearvacuolization, the widening of the villi LP and infiltration of lymphocytes in theLP and submucosa as variable. These variables were utilized and accepted bySanden et al. (2005) and Aslaksen et al. (2007). Krogdahl et al. (2003) reportedthat soybean-containing diets induced an inflammatory reaction, and that the al-terations were related to digestive and absorptive disfunctions linked to the diet.In this study, the minor changes in the intestinal morphology of P. bogaraveosuggest that, according to the above cited variables, the RPC supplementationdid not induce severe inflammatory process. The observed changes can insteadbe considered to be linked to the individual variations, as reported in the previ-ous work on classical intestinal histology of this fish, where the authors exam-ined sections stained by haematoxylin–eosin (Micale et al., 2005).A comparison was carried out between the mucosal enzyme distribution in

fish fed the control diet and the diet with RPC. The results were similar tothose concerning the enzyme reactivity of other fish species (Ribeiro et al.,1999; Bakke-McKellep et al., 2000) and mammal intestines (Landsverk,1980). Bakke-McKellep et al. (2000) reported that reduction in brush-borderenzyme activities (ALP and 59N), observed for the epithelial cell lining of thevillous folds, is probably related to the toxic effects of some diet components.These data have been confirmed through ultrastructural observations of theshortening of microvilli in distal intestinal cells (van den Ingh et al., 1991,1996). In this trial, however, no changes appeared for the different intestinaltracts, except for the ACP in the distal portion of the intestine, where the pos-itive reaction on the supranuclear region suggests that the distal intestinalregion is different from the pyloric caeca region. This supposition could beconfirmed by the similar physiological modifications that have been observedin the distal intestine of S. salar, as suggested by Bakke-McKellep (1999).The same author stated that spots of activity in villi LP and sub mucosa indi-cate the presence of lymphocyte cell lineage, whereas activity in the mucosalayer highlights the presence of active vacuoles such as lysosomes. In the caseof where changes occurred, modifications of the ALP and 59N activities wouldbe seen, as was demonstrated by Bakke-McKellep et al. (2000). However, in thepresence of changes, the ACP activity should increase, either in the number ordistribution of the enzyme spots. The ACP pattern activity observed in boththe distal intestine of P. bogaraveo and S. salar intestine (Bakke-McKellepet al., 2000) was quite similar. These enzyme reactivities and distributions aresimilar to those in mammals. Unlike soybean meal, however, RPC used asa partial substitute of fish meal in a fish diet does not seem to induce modifi-cation of the histochemical pattern of this fish.The digestive enzyme profile made allowed an evaluation to be made of

whether the metabolic functionality of the intestinal tract was modified by thesupplementation of RPC as a dietary protein source. Diversification in enzymepatterns was expected to occur in response to dietary changes; the enzyme levels


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of the examined P. bogaraveo specimens in fact showed a great adaptative capac-ity, which increased significantly after feeding with RPC-supplemented diets,except for lipase. The changes observed in the patterns of proteolytic enzymes,in response to RPC supplementation, could be related to the rice protein (Carusoet al., 2005) and could depend on compensation mechanisms activated in relationto protease inhibitors that are commonly present in plant sources, as reportedfor other fishes (Krogdhal et al., 1994; Haard et al., 1996). In the present study,the in vivo observations are in contrast with those reported by Moyano Lopezet al. (1999), who observed an in vitro protease inhibition on crude digestive ex-tracts using different relative concentrations of plant meals for S. aurata, tilapiaOreochromius niloticus (L.) and African sole Solea senegalensis Kaup species.Moreover, Krogdahl et al. (2003) recorded a decreasing trend of intestinal pro-teolytic enzyme activity in the distal intestine of S. salar fed graded levels of stan-dard soybean meal. Conversely, in P. bogaraveo, RPC seemed to exerta stimulatory effect on intestinal proteolytic activity (Caruso et al., 2005).In this study, amylase activity increased with an increasing RPC inclusion

level; this result is consistent with the higher nitrogen-free extract content inRPC35 than in RPC0 diets. The lipase activity showed a progressive reduction,contrary to the amylase activity, in fish fed with an increasing RPC dietarycontent. The lipase activities are closely correlated to the dietary levels ofthe triglycerides (TG) and phospholipids (PL), as has been demonstrated insea bass Dicentrurchus labrax (L.) larvae fed diets containing different levelsof these lipid fractions (Cahu et al., 2003; Zambonino Infante & Cahu,2007). It is well known that fish meal contains c. 9% of ether extract rich inTG, whereas rice lipids are rich in PL (Choi et al., 2005). In order to clearlyidentify a correlation between TG:PL and lipase activity in P. bogaraveo morespecific analyses are necessary.These results support the hypothesis that fishes can modulate digestive

enzyme activities in response to changes in dietary composition (Fountoulakiet al., 2005).In the present study, the P. bogaraveo have not shown any apparent digest-

ibility coefficient modifications related to the diet. These results are in disagree-ment with a previous work carried out on O. mykiss (Palmegiano et al., 2006)fed with diets containing increasing levels of RPC (20, 35 and 53%) in partialreplacement of fish meal. In this study, the authors reported statistical differ-ences among all the diets with a negative trend to the RPC inclusion level.The possible ADC differences observed in the P. bogaraveo in comparison with

the previous work on O. mykiss could be ascribed to the faeces collecting methods.In fact, it is well known that usually the sedimentation column method overesti-mates ADC due to the leakage of faecal matter in the water column during sam-pling, whereas in the study on O. mykiss, faeces were collected with the Choubertmethod (Choubert et al., 1982), which is more precise than the sedimentation col-umn. The weakness of the sedimentation column method can be supported withperformances obtained in the previous work with O. mykiss (Palmegiano et al.,2006), where a decreasing trend in growth performances were recorded.Knowledge of digestive processes is considered a pre-requisite in the

formulation of diets to fit the nutritional requirements of fishes (Smith, 1989;Genovese et al., 1995).


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Taking into account the results of the present study, it is possible to supposethat RPC can replace as much as 64% of fishmeal without any detrimental ef-fects on the intestinal morphology and physiology.

Research supported by the MIPAF (Ministero delle Politiche Agricole e Forestali) VIProgramma Quadro 2003–2005 Grant. All the authors contributed equally to the workdescribed in this paper.

References

AOAC (1995). Official Methods of Analysis, 15th edn. Washington, DC: Association ofOfficial Analytical Chemist.

Appel, W. (1974). Carboxypeptidases. In Methods of Enzymatic Analysis II (Bergmeyer,H. U., ed.), pp. 996–997. New York, NY: Academic Press.

Aslaksen, M. A., Kraugerud, O. F., Penn, M., Svihus, B., Denstadli, V., Jørgensen, H. Y.,Hillestad, M., Krogdahl, A. & Storebakken, T. (2007). Screening of nutrientdigestibilities and intestinal pathologies in Atlantic salmon, Salmo salar, fed dietswith legumes, oilseeds, or cereals. Aquaculture 272, 541–555.

Baeverfjord, G. & Krogdahl, A. (1996). Development and regression of soybean mealinduced enteritis in Atlantic salmon, Salmo salar L., distal intestine: a comparisonwith the intestines of fasted fish. Journal of Fish Diseases 19, 375–387. doi: 10.1046/j.1365-2761.1996.d01-92.x

Bakke-McKellep, A. M. (1999). Intestinal nutrient absorption in Atlantic salmon (Salmosalar L.) and pathophysiological response of the intestinal mucosa to dietarysoybean meal. PhD Thesis, Norwegian School of Veterinary Science, Oslo, Norway.

Bakke-McKellep, A. M., Press, C. McL., Baeverfjord, G., Krogdahl, A. & Landsverk, T.(2000). Changes in immune and enzyme histochemical phenotypes of cells in theintestinal mucosa of Atlantic salmon, Salmo salar L., with soybean meal-inducedenteritis. Journal of Fish Diseases 23, 115–127. doi: 10.1046/j.1365-2761.2000.00218.x

Bernfeld, P. (1955). Amylase a and b. InMethods in Enzymology (Colowich, S. P., ed.), pp.149–150.New York, NY: Academic Press.

Boonyaratpalin, M., Suraneiranat, P. & Tunpibal, T. (1998). Replacement of fish mealwith various types of soybean products in diets for the Asian seabass, Latescalcarifer. Aquaculture 161, 67–78.

Burrells, C., Williams, P. D., Southgate, P. J. & Crampton, V. O. (1999). Immunological,physiological and pathological responses of rainbow trout (Oncorhynchus mykiss)to increasing dietary concentrations of soybean proteins. Veterinary Immunologyand Immunopathology 72, 277–288. doi: 10.1016/S0165-2427(99)00143-9

Cahu, C. L., Zambonino Infante, J. L. & Barbosa, V. (2003). Effect of dietaryphospholipid level and phospholipid: neutral lipid value on the development ofsea bass (Dicentrarchus labrax) larvae fed a compound diet. British Journal ofNutrition 90, 21–28.

Caruso, G. & Genovese, L. (2000). Osservazioni sulla distribuzione degli enzimi digestiviin differenti specie di Sparidi. Biologia Marina Mediterranea 7, 621–623.

Caruso, G., Genovese, L. & Monticelli, L. (1999). Observations on the enzymaticactivities of some Pagellus bogaraveo (Brunnich 1768) specimens in intensiverearing. Oebalia 25, 31–42.

Caruso, G., Genovese, L., Maimone, G., Manganaro, A., Mancuso, M. & Palmegiano,G. B. (2003). Enzimi digestivi in Pagellus bogaraveo: tre diete sperimentalia confronto. Biologia Marina Mediterranea 10, 430–433.

Caruso, G., Costanzo, M. T., Palmegiano, G. B., Gai, F. & Genovese, L. (2005).Blackspot sea bream (Pagellus bogaraveo) fed on rice protein concentrate meal:effect on digestive enzymes. In Lessons from the Past to Optimise the Future (Howell,B. & Flos, R., eds). EAS Special Publication 35, 158–159.


# 2009 The Authors


Cavallarin, L., Antoniazzi, A., Borreani, G. & Tabacco, E. (2005). Effects of wilting andmechanical conditioning on proteolysis in sainfoin (Onobrychis viciifolia Scop) wiltedherbage and silage. Journal of the Science of Food and Agriculture 85, 831–838.

Cho, C. Y. (1992). Feeding systems for rainbow trout and other salmonids with referenceto current estimates of energy and protein requirements. Aquaculture 100, 107–123.

Choi, S.-K., Takahashi, E., Inatsu, O., Mano, Y. & Ohnishi, M. (2005). Component fattyacids of acidic glycerophospholipids in rice grains: universal order of unsaturationindex in each lipid among varieties. Journal of Oleo Science 54, 369–373.

Choubert, G., De La Noue, J. & Luquet, P. (1982). Digestibility in fish: improved devicefor the automatic collection of faeces. Aquaculture 29, 185–189.

Fernandez, I., Moyano, F. J., Diaz, M. & Martinez, T. (2001). Characterization ofa-amylase activity in five species of Mediterranean sparid fishes (Sparidae, Teleostei).Journal of Experimental Marine Biology and Ecology 262, 1–12.

Fountoulaki, E., Alexis, M. N., Nengas, I. & Venou, B. (2005). Effect of diet compositionon nutrient digestibility and digestive enzyme levels of gilthead sea bream (Sparusaurata L.). Aquaculture Research 36, 1243–1251. doi: 10.1111/j.1365-2109.2005.01232.x

Genovese, L., Patti, F. & Caruso, G. (1995). Studies of the digestive cycle in theamberjack (Seriola dumerilii) (Risso, 1810) in intensive rearing. Oebalia 21, 5–15.

Genovese, L., Maricchiolo, G., Micale, V., Costanzo, M. T., Garaffo, M., Palmegiano,G. B. & Greco, S. (2004). Pagellus bogaraveo: una specie di interesse perl’acquacoltura. Biologia Marina Mediterranea 11, 389–392.

Glencross, B., Evans, D., Hawkins, W. & Jones, B. (2004). Evaluation of dietaryinclusion of yellow lupin (Lupinus luteus) kernel meal on the growth, feedutilisation and tissue histology of rainbow trout (Oncorhynchus mykiss). Aquacul-ture 235, 411–422.

Haard, N. F., Dimes, L. E., Arndt, R. E. & Dong, F. M. (1996). Estimation of proteindigestibility. IV. Digestive proteinases from the pyloric caeca of coho salmon(Oncorhynchus kisutch) fed diets containing soybean meal. Comparative Biochem-istry Physiology B 110, 533–540.

Hummel, B. C. W. (1959). A modified spectrophotometric determination of chymotryp-sin, trypsin and thrombin. Canadian Journal of Biochemistry and Physiology 37,1393–1399.

van den Ingh, T. S. G. A. M., Krogdhal, A., Olli, J. J., Hendricks, H. G. C. J. M. &Koninkx, J. F. J. G. (1991). Effects of soybean containing diets on the proximaland distal intestine in Atlantic salmon (Salmo salar): a morphological study.Aquaculture 94, 297–305.

van den Ingh, T. S. G. A. M., Olli, J. J. & Krogdhal, A. (1996). Alcohol solublecomponents in soybeans causes morphological changes in the distal intestine inAtlantic salmon (Salmo salar): a morphological study. Journal of Fish Diseases 19,47–53.

Kikuchi, K. (1999). Use of defatted soybean meal as a substitute for fish meal in diets ofJapanese flounder (Paralichthys olivaceus). Aquaculture 179, 3–11.

Krogdahl, A. & Bakke-McKellep, A. M. (2005). Fasting and refeeding cause rapidchanges in intestinal tissue mass and digestive capacities of Atlantic salmon (Salmosalar L.). Comparative Biochemistry and Physiology A 141, 450–460.

Krogdahl, A., Lea, T. B. & Olli, J. J. (1994). Soybean protease inhibitors affect intestinaltrypsin activities and amino acids digestibilities in rainbow trout (Oncorhynchusmykiss). Comparative Biochemistry and Physiology A 107, 215–219.

Krogdahl, A., Bakke-McKellep, A. M. & Baeverfjord, G. (2003). Effects of graded levelsof standard soybean meal on intestinal structure, mucosal enzyme activities, andpancreatic response in Atlantic salmon (Salmo salar L.). Aquaculture Nutrition 9,361–371. doi: 10.1046/j.1365-2095.2003.00264.x

Landsverk, T. (1980). Histochemical distribution of enzymes in the small intestine ofyoung milk-fed calves. Acta Veterinaria Scandinavica 21, 402–414.

Maynard, L. A. & Loosly, J. K. (1969). Animal Nutrition. New York, NY: McGraw HillBook Company.


# 2009 The Authors


Micale, V., Genovese, L., Costanzo, M. T., Dapra, F., Gai, F., Muglia, U. & Palmegiano,G. B. (2005). Blackspot seabream (Pagellus bogaraveo) fed on rice proteinconcentrate: intestinal morphology. In Lessons from the Past to Optimise the Future(Howell, B. & Flos, R., eds). EAS Special Publication 35, 328–329.

Moyano Lopez, F. J., Martınez Diaz, I., Diaz Lopez, M. & Alarcon Lopez, F. J. (1999).Inhibition of digestive proteases by vegetable meals in three fish species; seabream(Sparus aurata), tilapia (Oreochromis niloticus) and African sole (Solea senegal-ensis). Comparative Biochemistry and Physiology B 122, 327–332.

NRC (National Research Council) (1993). Nutrient Requirements of Fish, Committee onAnimal Nutrition Board on Agriculture. Washington, DC: National Academy Press.

Opstevd, J., Aknes, A., Hope, B. & Pike, I. H. (2003). Efficiency of feed utilization inAtlantic salmon (Salmo salar L.) fed diets with increasing substitution of fish mealwith vegetable proteins. Aquaculture 221, 365–379.

Ostaszewska, T., Dabrowski, K., Palacios, M. E., Olejniczak, M. & Wieczorek, M.(2005). Growth and morphological changes in the digestive tract of rainbow trout(Oncorhynchus mykiss) and pacu (Piaractus mesopotamicus) due to casein replace-ment with soybean proteins. Aquaculture 245, 273–286.

Palmegiano, G. B., Dapra, F., Forneris, G., Gai, F., Gasco, L., Guo, K., Peiretti, P. G.,Sicuro, B. & Zoccarato, I. (2006). Rice protein concentrate meal as a potentialingredient in practical diets for rainbow trout (Oncorhynchus mykiss). Aquaculture258, 357–367.

Palmegiano, G. B., Costanzo, M. T., Dapra, F., Gai, F., Galletta, M. G., Maricchiolo, G.,Micale, V., Peiretti, P. G. & Genovese, L. (2007). Rice protein concentrate meal aspotential dietary ingredient in practical diets for blackspot sea bream (Pagellusbogaraveo). Journal Animal Physiology and Animal Nutrition 91, 235–239. doi:10.1111/j.1439-0396.2007.00697.x

Ribeiro, L., Sarasquete, C. & Dinis, M. T. (1999). Histological and histochemicaldevelopment of the digestive system of Solea senegalensis (Kaup 1858) larvae.Aquaculture 171, 293–308.

Robaina, L., Corraze, G., Aguirre, P., Blanc, D., Melcion, J. P. & Kaushik, S. (1999).Digestibility, postprandial ammonia excretion and selected plasma metabolites inEuropean sea bass (Dicentrarchus labrax) fed pelleted or extruded diets with orwithout wheat gluten. Aquaculture 179, 45–56.

Sanden, M., Berntssen, M. H. G., Krogdahl, A., Hemre, G.-I. & Bakke-McKellep, A.-M.(2005). An examination of the intestinal tract of Atlantic salmon, Salmo salar L.,parr fed different varieties of soy and maize. Journal of Fish Diseases 28, 317–330.

Smith, L. S. (1989). Digestive functions in teleost fishes. In Fish Nutrition, 2nd edn(Halver, J. E., ed.), pp. 405–407. New York, NY:Academic Press.

Tietz, N. W. & Fiereck, E. A. (1966). A specific method for serum lipase determination.Clinical Chemistry Acta 13, 352–358.

Vergara, J. M., Lopez-calero, G., Robaina, L., Caballero, M. J., Montero, D., Izquierdo,M. S. & Aksnes, A. (1999). Growth, feed utilization and body lipid content ofgilthead seabream (Sparus aurata) fed increasing lipid levels and fish meals ofdifferent quality. Aquaculture 179, 35–44.

Zambonino Infante, J. L. & Cahu, C. L. (2007). Dietary modulation of some digestiveenzymes and metabolic processes in developing marine fish: applications to dietformulation. Aquaculture 268, 98–105.


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