gy and Ecology 343 (2007) 1–10www.elsevier.com/locate/jembe

Journal of Experimental Marine Biolo

Effects of diets with distinct protein-to-carbohydrate ratios onnutrient digestibility, growth performance, body composition

and liver intermediary enzyme activities in giltheadsea bream (Sparus aurata, L.) fingerlings

Felipe Fernández a,⁎, Anna G. Miquel a, Marlon Córdoba a, Manuel Varas a,Isidoro Metón b, Anna Caseras b, Isabel V. Baanante b

a Departamento de Ecología, Universidad de Barcelona, Facultad de Biología, Avda. Diagonal 645, E-08028 Barcelona, Spainb Departamento de Bioquímica y Biología Molecular, Universidad de Barcelona, Facultad de Farmacia, Avda. Diagonal 643,

E-08028 Barcelona, Spain

Received 19 June 2006; received in revised form 28 September 2006; accepted 11 October 2006

Abstract

The effect of replacing dietary protein with gelatinized cornstarch (GCS) on apparent digestibility coefficient (ADC), bodycomposition, growth performance and liver enzyme activities involved in control of intermediary metabolism, was studied in Sparusaurata L. Fingerlings of S. aurata were fed 93 days three diets containing 63% protein and 5% gelatinized cornstarch (LC diet), 54%protein and 18%GCS (MC diet) or 47% protein and 26%GCS (HC diet). Diet HC gaveADC values for carbon, nitrogen and drymatterthat were significantly below the corresponding values of the other diets. Fish onMC diet registered higher fresh weight than fish on LCand HC, and higher specific growth rate (SGR) than fish on HC. The lipid body content ranked in the order HCNMCNLC. Highcorrelations between carbohydrate level and liver enzyme activity were found for pyruvate kinase, glucose 6-phosphate dehydrogenase,6-phosphogluconate dehydrogenase and alanine aminotransferase. For cultures of S. aurata, we conclude that carbohydrates like GCScould replace dietary protein, enhance growth rate and reduce nitrogen losses to the ambient waters when used at levels below 20%.© 2006 Elsevier B.V. All rights reserved.

Keywords: Carbohydrate metabolism; Digestibility; Fish culture; Growth; Protein sparing; Sparus aurata

1. Introduction

One of the main objectives of fish nutrition researchis to minimize the amount of protein in diets and coverenergy requirements using carbohydrates or lipids.

⁎ Corresponding author. Departamento de Ecología, Universidad deBarcelona, Facultad de Biología, Avda. Diagonal 645, E-08028Barcelona, Spain. Tel.: +34 934021514; fax: +34 934111438.

E-mail address: ffernandez@ub.edu (F. Fernández).

0022-0981/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jembe.2006.10.057

Excess dietary protein with respect to growth demandsleads to increased amino acid degradation and the loss ofnitrogen to ambient waters, which in turn could result inwater eutrophication (Pillay, 1992; Pearson and Black,2001; Davenport et al., 2003). The protein used in fishdiets is mainly from fish meal, therefore any reduction inits use could also lead to decreased pressure onoverexploited marine fisheries; as in many cases, asignificant part of catches is converted to fish meal(Naylor et al., 2000).

Table 1Composition and proximate analysis of test diets

DIET LC MC HC

(63P5C) (54P18C) (47P26C)

Formulation (%)Fish meal a 81.95 68.40 60.00Fish oil b 7.95 8.73 9.00Corn starch c

Diets without Cr2O3 5.10 17.87 26.00Diets with Cr2O3 4.60 17.37 25.50Mineral mixture d 1.38 1.38 1.38Vitamin mixture e 1.62 1.62 1.62Carrageenan 2.00 2.00 2.00Chromic oxideDiets without Cr2O3 0 0 0Diets with Cr2O3 0.5 0.5 0.5

Chemical analysis f

% fresh weightMoisture 1.31 1.42 1.63

% dry weightProtein 62.9 53.9 46.8Fat 15.5 14.4 14.5Ash 14.0 12.1 11.1Carbohydrates g 7.6 19.6 27.6Phosphorus 2.02 1.93 1.63Calcium 3.06 3.01 2.52

kJ/g dry weightEnergy h 22.44 21.88 21.58a White fish meal (Norse LT-94). Supplied by Sogem Iberica (Rubí,

Barcelona, Spain).b Fish oil with added vitamin A (all-trans retinol: 1000 IU/g) and

vitamin D (cholecalcipherol: 100 IU/g). From Bonafont Quimica(Barcelona, Spain).c Gelatinized corn starch. From Campo Ebro (Zaragoza, Spain).d Mineral mixture (per kg of diet): CaHPO4.H2O, 8.83 g; CaCO3,

2.56 g; KCl, 1.25 g; MgO, 1.0 g; ZnO, 90 mg; FeCO3, 80 mg; MnO2,12.5 mg; CuSO4, 7.5 mg; KI, 1.5 mg; Na2SeO3, 0.28 mg.e Vitamin mixture ( per kg of diet): all-trans retinol, 3 mg;

cholecalcipherol, 25 mg; all-rac-tocopherol acetate, 60 mg; menadione.HNaSO3, 10.6 mg; folic acid, 4.8 mg; nicotinic acid, 360 mg; riboflavin,24 mg; thiamin. HCl, 30 mg; pyridoxine. HCl, 24 mg; cyanocobalamin,0.12 mg; ascorbic acid, 200 mg; calcium pantothenate, 30 mg; biotin,0.42 mg; choline chloride, 3.2 g; myo-inositol, 900 mg.f Chemical analysis performed following standard procedures of the

EU (directives 71/393, for moisture; 93/28, for crude protein (Kjeldahl);71/393 for crude fat (Soxhlet extraction); and 71/250 for ash).g Calculated by difference for diets without Cr2O3.h Calculated from gross composition (protein: 24 kJ/g; lipid: 39 kJ/g;

carbohydrate: 17 kJ/g).

2 F. Fernández et al. / Journal of Experimental Marine Biology and Ecology 343 (2007) 1–10

Carbohydrates can be used to replace proteins in fishdiets. Optimal levels in these diets depend on fish species,carbohydrate type and treatment, and protein and lipidlevels (see reviews by Furuichi and Yone, 1980; Wilson,1994; Shiau, 1997; Hemre et al., 2001). Thus, for a givenfish species, the optimal type and level of carbohydratemust be determined experimentally. Treated starch(gelatinized, dextrinized, extruded) is a suitable dietarycomponent for gilthead sea bream (Fernández et al., 1996,1998; Georgopoulos and Conides, 1999; Venou et al.,2003) and other fish (Bergot, 1979; Bergot and Breque,1983; Jeong et al., 1991; Takeuchi et al., 1994; Wilson,1994; Peres and Oliva-Teles, 2002).

Fish have the enzymes and metabolic pathwaysrequired to regulate the metabolism of carbohydratesingested through diet (Cowey and Walton, 1989;Baanante et al., 1991; Shimeno et al., 1993, 1996;Metón et al., 1999a,b, 2003, 2004; Caseras et al., 2000,2002; Hemre et al., 2001; Dabrowski and Guderley,2002). However, the intermediary metabolism of carniv-orous fish, like rainbow trout, shows only moderateadaptation to high levels of dietary carbohydrate (Coweyand Walton, 1989; Hemre et al., 2001; Moon, 2001).Integrated studies combining enzyme regulation, nutrientdigestibility and growth performance in relation to dietarycarbohydrates are required for cultured species likegilthead sea bream.

The gilthead sea bream is currently the mostextensively cultured fish in the Mediterranean region(FAO, 2006). Recent years have witnessed an increasingnumber of nutritional studies on this species, with the aimto identify the effects of replacing protein with carbohy-drates or lipids (Bonamusa et al., 1992; García-Alcázaret al., 1994; Santinha et al., 1996; Vergara et al., 1996;Fernández et al., 1998; Lupatsch et al., 2001; Companyet al., 1999; Georgopoulos and Conides, 1999; Metónet al., 1999b, 2000, 2004; Venou et al., 2003) or replacingfishmealwith another protein of terrestrial origin (Robainaet al., 1995; Gómez-Requeni et al., 2003; Pereira andOliva-Teles, 2003; Sánchez-Muros et al., 2003).

Here we replaced dietary protein with gelatinizedcornstarch (GCS) and studied the effects of this changeon nutrient digestibility, body composition, growthperformance, nitrogen retention and key liver enzymesin Sparus aurata.

2. Material and methods

2.1. Fish treatments

Gilthead sea bream fingerlings with a mean weight of2.5 g were purchased from a hatchery (Tinamenor,

Cantabria, Spain) and distributed in nine seawateraquariums of 260 l, at a density of 21 fish per aquarium.All aquariums were located in isothermal rooms at 21±0.2 °C and were provided with a closed-circuit watertreatment system consisting of external pump filters andon-line UV lamps. Air pumps and ceramic diffusersaerated the water. Ammonia, salinity (36 g/l) and pH (7–

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8) were checked once a week, and 20% of the seawaterwas replaced weekly. Ammonia was always at traceconcentrations, and salinity and pH were correctedwhen necessary (see Fernández et al., 1996, 1999).

The fish in each three aquariums were hand fed oneof the three experimental diets (Table 1) withoutchromium oxide. The three diets were named LC (lowcarbohydrate), MC (moderate carbohydrate) and HC(high carbohydrate). To facilitate comparisons betweendiets, we also adopted the following conventions: P=%protein, L=% lipid and C=% carbohydrate. Therefore,the LC diet can also be represented as 63P16L5C, as itcontains 63% protein, 16% lipid and 5% carbohydrate(in this case, gelatinized cornstarch, GCS).

During the first week, the fish were fed twice daily tosatiety. Fish were considered satiated when some foodremained uneaten after 15 min of feeding. The fish werethen weighed under anaesthesia (tricaine at 70 mg/l) aftera 24-h fast and three fish from each aquarium were killed,dried in an oven at 70 °C to constant weight and stored at−20 °C until body composition analysis (initial sample).For the remaining fish (18 per aquarium), the ration wasadjusted to 5% of body weight per day, which wasprovided in two meals of 2.5%, one at 9.30 a.m. and theother at 4.30 p.m. This ration was close to satiation for alltreatments and ensured that all the food provided wasconsumed in less than 15 min (usually in less than 5).

The fish were weighed every 7−20 days and the foodration was adjusted to the new weight. As fish grew, thesatiation rations diminished and the ration level waslowered to the same extent for all treatments in order tofall just below satiety. At three months (93 days), fishwere fasted for 24 h and then weighed underanaesthesia; three fish from each aquarium were thenkilled by cervical section, the livers were dissected out,weighed, and immediately frozen in liquid nitrogen andkept at −80 °C until enzyme analysis. Another three fishfrom each aquarium were killed by anaesthesiaoverdose, dried whole in an oven to constant weightin order to obtain dry weights, and stored at −20° untilbody composition analysis.

The remaining live fish (8–12 per aquarium) werefed to satiation (at this point equivalent to a daily rationof about 2% body weight) with the chromium oxideversion of the diets (Table 1) in order to calculatenutrient digestibility. After two weeks, all the fish werekilled under anaesthesia, 6 h after feeding. The gut wasdissected out and divided into three sections aspreviously reported (Fernández et al., 1996, 1999):anterior intestine (2 cm after pyloric caeca), posteriorintestine (2 cm before rectum) and mid-intestine (theportion between the anterior and posterior intestine).

The content of each section was pooled for eachaquarium, dried at 70 °C and kept at −20 °C untilanalysis to calculate nutrient digestibility.

Fish killed at 93 days was analysed for C, N, Ca, P,lipid and ash content. Each fish was defrosted, ground,homogenised in a Potter-Elvehjem homogeniser withthe addition of distilled water, and then dried again.Samples for C and N were analysed with a Carlo ErbaNA 2100 elemental analyser (CE Instruments, Termo-quest Italia). Samples for Ca and P were digested(following procedures described by Furukawa andTsukahara, 1966) and analysed by ICP spectrometry(Polyscan 61E, Thermojarrell Ash Corporation, USA).

Protein was calculated from N content, using a factorof 6.25. Total lipids were measured using a commercialkit from Merck (Merckotest 3321 for Total Lipids). Thesample was previously digested with sulphuric acid at100 °C (Knight et al., 1972). Ash content wasdetermined by combustion in a muffle furnace at550 °C, following AOAC (1984) specifications.

Diets were analysed for C, N, P, Ca, fat and ash by thesame procedures used for fish samples. Protein contentwas also calculated from N content and energy contentwas calculated from diet composition (protein, 24 kJ/g,lipid, 39 kJ/g and carbohydrate, 17 kJ/g, followingBradfield and Llewellyn, 1992). Dietary Cr wasanalysed by ICP spectrometry using the same proce-dures described for P and Ca (the three elements weredetermined simultaneously in the same samples). Gutcontent was also analysed for C, N and Cr following thesame method described above.

2.2. Digestibility and growth parameters

Apparent digestibility coefficient (ADC) of a givennutrient was calculated from the following equation (DeSilva and Anderson, 1995)

ADC ¼ 100−100d% chromium in food% chromium in feces

d% nutrient in feces% nutrient in food

� �:

For dry matter, the equation becomes:

ADC ¼ 100−100d% chromium in food% chromium in feces

� �:

For each aquarium, growth rates were calculated as aspecific growth coefficient (SGC), following the expres-sion: SGR=(ln Wf− ln Wi). 100/t, where Wf is the meanfinal fresh weight of the fish in each aquarium, Wi themean initial fresh weight of the fish in the same aquarium,

Table 2ADC values obtained for Sparus aurata fed HC, MC and LC diets(samples taken from three regions of the intestine)

DIET n Anterior intestine Medium intestine Posterior intestine

CarbonLC 3 73.9±7.9 a 85.5±3.4 b 89.4±3.5 c y

MC 3 76.5±7.6 a 87.5±4.7b 87.4±2.4 b y

HC 3 64.9±21 80.9±9.9 81.7±0.8 x

NitrogenLC 3 82.6±4.7 a 89.2±1.7 b 91.2±1.3 b y

MC 3 81.3±3.7 a 89.5±2.2 b 90.2±1.6 b y

HC 3 72.4±13 84.2±6.6 85.8±0.6 x

Dry matterLC 3 65.2±7.1 a 75.2±1.5 b 82.2±2.1 c y

MC 3 68.4±4.0 a 79.9±3.3 b 80.3±2.0 b y

HC 3 58.0±20 72.6±10 74.2±1.3 x

abc: For a given nutrient and diet, different letters indicate significantADC differences between regions of intestine by Fisher test at 0.05probability level.xyz: For a given nutrient and region of intestine, different lettersindicate significant ADC differences between diets by Fisher test at0.05 probability level.

Table 3Whole body composition of fish at the start of the experiment (initialsamples) and after being fed LC, MC and HC diets for 93 days

Fish group n LC MC HC

Initialg/100 g fresh weight

Dry weight 3 24.8±1.3 x 24.8±1.3 x 24.8±1.3 x

g/100 g dry weightProtein 3 55.6±3.9 55.6±3.9 55.6±3.9Lipid 3 16.8±0.42 x 16.8±0.42 x 16.8±0.42 x

Ash 3 14.9±0.19 14.9±0.19 y 14.9±0.19 y

Carbon 3 46.6±1.3 x 46.6±1.3 x 46.6±1.3 x

Phosphorus 3 2.66±0.14 y 2.66±0.14 y 2.66±0.14 y

Calcium 3 3.70±0.30 3.70±0.30 y 3.70±0.30 y

Fed 93 daysg/100 g fresh weight

Dry weight 3 31.1±2.2 ay 32.8±2.3 by 33.4±2.6 by

LSI 3 1.27±0.17a 1.51±0.39a 1.97±0.22b

g/100 g dry weightProtein 3 56.8±5.8 56.8±3.8 55.2±4.5Lipid 3 19.1±1.9 ay 21.5±2.1 by 23.7±2.2 cy

Ash 3 13.8±1.7 12.3±0.9 x 13.0±1.1 x

Carbon 3 52.7±3.1ay 54.8±2.3 by 53.5±3.1 aby

Phosphorus 3 2.43±0.3 bx 2.19±0.26 ax 2.20±0.24 ax

Calcium 3 3.9±0.6 b 3.3±0.5 ax 3.4±0.5 ax

a, b, c: For a given fish group (initial or fed 93 days) and line (dryweight, LSI, compound or element), different letters indicatesignificant differences between diets by Fisher test ( pb0.05).x, y: For a given diet and element (or compound or dry weight),different letters indicate significant differences between initial andfinal samples by Fisher test ( pb0.05).

4 F. Fernández et al. / Journal of Experimental Marine Biology and Ecology 343 (2007) 1–10

and t is time in days (De Silva andAnderson, 1995). Otherparameters calculated for each aquarium were:

Food Efficiency Ratio (FER) =g weight gain/g feedprovided

Protein Efficiency Ratio (PER) =g weight gain/g feedprotein

Protein retention (PR) =g protein gain 100/g feed proteinCarbon retention (CR) =g carbon gain 100/g feed carbon

Weight gain is defined as the difference between Wfand Wi after 93 days of feeding. Protein and carbon gainwere calculated from the protein and carbon content ofthe fish weighing Wf and Wi.

Using the fish and liver weights at 93 days, we alsocalculated the liver somatic index (LSI) as:

LSI= liver weight 100 /body weight

2.3. Enzyme activities

After 93 days, the livers of fish exposed only to dietswithout Cr, were used to determine enzymes involved incarbohydrate-protein metabolism. Crude extracts wereobtained by homogenisation of the powdered frozenliver (1/5, W/V) in 50 mM Tris–HCl pH 7.5, 4 mMEDTA, 50 mM NaF, 0.5 mM PMSF, 1 mM DTT and250 mM sucrose using a PTA-7 Polytron (position 3,30 s) and centrifugation at 20,000 ×g for 30 min at 4 °C.

The activities of 6-phosphofructo 1-kinase (PFK-1, EC2.7.1.11), pyruvate kinase (PK, EC 2.7.1.40), fructose1,6-bisphosphatase (FBPase-1, EC 3.1.3.11), glucose 6-phosphate dehydrogenase (G6P-DH, EC 1.1.1.49), 6-phosphogluconate dehydrogenase (6PG-DH, EC1.1.1.43), alanine aminotransferase (ALT, EC 2.6.1.2)and aspartate aminotransferase (AST, EC 2.6.1.1) wereassayed in the crude liver extracts using a COBASMIRA S spectrophotometric analyser, as previouslyreported (Metón et al., 1999b). All enzyme assays wereperformed at 30 °C and followed at 340 nm. The totalprotein content in crude liver extracts was also measuredin the COBAS MIRA S at 600 nm and 30 °C, followingthe Bradford (1976) method, using bovine serumalbumin as a standard.

2.4. Statistics

All data were analysed with statistical software. In thecase of ADCs, we use StatView program (SAS InstituteInc., San Francisco, USA) to perform a two factorANOVA (diet and region of intestine) and the FisherLCD post-hoc test to determine significant differences

5F. Fernández et al. / Journal of Experimental Marine Biology and Ecology 343 (2007) 1–10

(p=0.05) between diets or intestinal regions for C, N ordry matter digestibility. As samples from fish in the sameaquarium were pooled for a given region of the intestine,only three values per diet and region (n=3) were used.

Fish composition was analysed by a two factorsANOVA (diet and date) and the Fisher LCD post-hoctest to determine significant differences between diets ordates (initial or 93 days) at p=0.05, using also deStatView program.

The effect of diet on body weight was analysed foreach date on which fish were weighed. For each date, weperformed a nested ANOVA, with diet as the main factorand the factor aquarium nested into the factor diet.Nested design was possible using the Statgraphics Plussoftware (version 5.1, from Statpoint Inc., Herndon,USA). However, the factor aquarium proved to be non-significant, so we change to one factor (diet) ANOVA.Depending on date we had 12–21 fish per aquarium and36–63 fish per diet.

Growth rates in individual fish were not calculated asfish were not tagged. Therefore, the mean weight of fishin each aquarium for a given date was used as the basisto calculate growth rates (n=3), as indicated above. Toestablish differences between diets for a given period,we performed a simple ANOVA, with diet as a singlefactor. Differences between diets were deduced from aFisher post-hoc test, with significance established atp=0.05. We used the same design to analyse growthperformances (FER, PER, PR and CR). In all thesecases, we used the StatView software.

In addition to ANOVA, regression analyses wereused in the case of enzymes, as enzyme activity wasrelated to the level of carbohydrate (and also to thecarbohydrate/protein ratio) of the diet. Linear regression

Fig. 1. Mean fresh weight of Sparus aurata along the experiment. Each columdate. Different letters above deviation bars indicate significant weight differe

gave the best fit in all cases and was used to compare theresponse of different enzymes. In this case, we used theStatView program.

3. Results

3.1. Digestibility

A large part of the absorptive processwas completed inthe anterior intestine, but significant increases occurred inthe medium and posterior intestine (Table 2). We used thelatter to compare overall ADCs.

The ADCs for C, N and dry matter of samples fromthe posterior intestine of fish on the HC diet weresignificantly below the corresponding values of theother diets (MC and LC, Table 2). However, nosignificant differences were found between the MCand LC diets. Therefore, the digestibility of the diets wasaffected when carbohydrate (GCS) reached 26%, but notat 18%.

The samples from the anterior and medium intestineshowed no significant differences in ADC betweendiets, although the values followed a similar trend tothose described for posterior intestine samples.

3.2. Differences in body composition

After 93 days, dry weight, as a percentage of freshweight, was lower in fish on the LC diet than in thoseon the other diets. C content was significantly higher forthe MC group (54.8% of dry weight) than for LC(52.7% of dry weight). No differences betweentreatments were found for protein (or nitrogen) content.The lipid content differed significantly among fish

n represents the mean and standard deviation of fresh weight at a givennces between treatments for each period (Fisher test, p=0.05).

Table 4Growth performances of Sparus aurata fed LC, MC and HC diets for93 days

Diets n LC MC HC

Initial fish weight 63 2.41±0.16 2.65±0.14 2.65±0.12Final fish weight 30–36 29.3±1.4 a 34.5±2.4 b 28.0±1.8 a

SGR 3 2.67±0.02 ab 2.76±0.13 b 2.53±0.09 a

FER 3 0.73±0.05 0.82±0.05 0.74±0.05PER 3 1.17±0.07 a 1.53±0.09 b 1.61±0.11 b

PR (%) 3 21.2±2.2 a 29.1±1.0 b 30.6±2.6 b

CR (%) 3 25.8±2.6 a 31.3±1.1 b 29.0±2.4 ab

SGR: specific growth rate ((ln final wet weight− ln initial wetweight)×100/days); FER: food efficiency ratio (g wet weight gain/gdry diet fed); PER: protein efficiency ratio (g wet weight gain/g proteinfed); PR (%): protein retention efficiency ((final body nitrogen− initialbody nitrogen) ×100/total nitrogen fed); CR (%): carbon retentionefficiency ((final body carbon− initial body carbon)\×100/total carbonfed).a, b: For a given fish weight (initial or final) or index (SGR, FER, PER,PR, CR), different letters indicate significant differences between dietsby Fisher test (5% significance level).

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groups, ranking in the order HCNMCNLC. The P andCa contents of fish on the LC diet were significantlyhigher than those of fish on the MC or HC diets. Therewere no significant differences in ash content betweentreatments.

There was a clear increase in dry weight and lipid andcarbon content and a decrease in ash, phosphorus andcalcium content in the fish fed 93 days compared to theinitial samples (Table 3).

3.3. Growth performance and feed utilization

Partial values of fresh weight obtained during theexperiment are shown in Fig. 1. For the first 25 days, alldiets gave similar values, but at 45 and 60 days fish onthe MC diet registered significantly (Fisher test at 0.05

Table 5Liver enzyme activities of fish fed diets for 93 days with low (LC) intermediafor the relationships between dietary carbohydrate content and enzyme activ

Enzyme LC MC HC

PFK-1 30.01±1.5a 37.94±8.4ab 56.64±PK 214.2±53a 463.1±83 b 740.1±FBPase-1 112.1±16 100.4±11 129.2±G6P-DH 158.2±37 a 176.7±35 a 249.8±6PG-DH 22.0±6.1 a 29.3±2.5 b 40.6±ALT 1404±188 b 959±206 a 719±AST 1067±230 799±131 920±

Enzyme activities are expressed as U/g protein (see Methods).y: enzyme activity; x=% gelatinized corn starch.ab: for a given enzyme, different letters indicate significant differences betw

level) higher values than the HC group. After 74 days,the fresh weight of the MC group was significantlyhigher than the LC and HC groups. The samedifferences were detected after 86 and 93 days.

For the whole period of 93 days (Table 4) fish on theMC diet registered the highest fresh weight and SGRvalues (although SGR differences between LC and MCdiets were not significant), while the lowest values ofPER, PR and CR corresponded to fish on the LC diet.No differences between diets were found for FER.

3.4. Enzyme activities

Our findings indicated that this species adapted theirmetabolism to the level of carbohydrate in the diet(Table 5). For the enzymes involved in glycolysis/gluconeogenesis, the best correlation between carbohy-drate level and enzyme activity was found for PK(r2 =0.90 in Table 5). The activity of this enzymeincreased by more than three fold as carbohydrates inthe diet increased five fold. There was also a goodresponse of PFK-1. However, no significant differencesbetween diets or correlation with dietary starch levelwere observed for FBPase-1 activity.

As the level of dietary GCS increased, there wasalso a significant rise in the activity of regulatoryenzymes of the pentose phosphate pathway, G6P-DHand 6PG-DH. For the former, only the HC diet gavesignificantly higher values than the other diets. For thelatter, clear differences were found between LC, MCand HC (Table 5), and a good correlation was obtainedbetween 6PG-DH activity and dietary starch content(r2 =0.73).

The amino acid-metabolising enzyme ALT showeddependence on dietary composition. ALT activitycorrelated inversely with dietary carbohydrate levels(r2 =0.75) and fish on the HC diet showed half the

te (MC) and high (HC) carbohydrate content and regression equationsity

Regression equation r2 p

22b y=21.74+1.21 x 0.414 0.0274c y=69.93+24.7 x 0.901 0.000131 y=103.2+0.66 x 0.067 0.4222 b y=127.9+4.09 x 0.536 0.0074.9 c y=16.6+0.859 x 0.726 0.000489 a y=18705−493 x 0.749 0.0003266 y=1063−8.26 x 0.107 0.30

een diets at the 0.05 probability level.

7F. Fernández et al. / Journal of Experimental Marine Biology and Ecology 343 (2007) 1–10

activity of those on the LC diet. In contrast, AST did notrespond to the carbohydrate/protein ratio.

4. Discussion

The ADCs obtained for samples of anterior, mediumand posterior intestine from S. aurata corroborate theprevious results of our group (Fernández et al., 1998,1999), indicating that the assimilation of C, N (protein)and dry matter occurs mostly in the stomach and anteriorintestine. However, significant additional absorptiontakes place along the intestine, so samples of posteriorintestine are the most representative source for measur-ing overall digestibility. The discussion that follows isbased upon these samples.

For C, N and dry matter, the HC diet gave ADCs thatwere significantly below the corresponding values forthe other diets. The percentage of ADC reduction forHC compared to the mean obtained for the other twodiets was 7.6% for C, 5.4% for N and 8.7% for drymatter. These figures indicate that, for GCS, a level of26% (HC diet) is probably above the acceptable limit interms of digestibility performance for S. aurata, and that18% (MC) would be near optimal and produce nosignificant effect on ADCs.

Georgopoulos and Conides (1999) fed S. aurata for30 days with diets containing cornstarch and foundprotein ADCs of 92.3, 91.5 and 88.5% for dietscontaining GCS at inclusion levels of 10, 20 and 30%,respectively. The 88.5% ADC is slightly higher than the85.8% reported here for the diet containing 26% GCS,but is consistent with our finding of lower ADCs forprotein when GCS content is around 26%.

Our data on fish composition corroborate those ofother studies that show an increase in dry weight andlipids as fish grow, with the corollary of an inverserelationship between lipid content and water content(see review by Weatherley and Gill, 1987). Dryweight and lipid content were also directly related todietary carbohydrate. Differences in lipid storage canbe associated with differences in the energy/proteinratio of the diets, with higher ratios favouring lipiddeposition.

At the end of the experiment, an increase in the liversomatic index (LSI) in conjunction with the increaseddietary carbohydrate was noted, and the LSI for fish onthe HC diet was significantly higher than that of fish onthe other diets. These results are consistent with otherspreviously found for S. aurata (García de Frutos et al.,1990; Metón et al., 1999b) and other species (Wilson,1994; Hemre et al., 2001) and may be attributed toincreased glycogen deposition (García de Frutos et al.,

1990; Wilson, 1994). However, increased fat depositionin the liver has been observed in other fish like cod(Hemre et al., 1989).

Our SGR values for S. aurata are similar to thosereported by García-Alcázar et al. (1994), who raisedgilthead sea bream fingerlings for 40 days from 2.9 to12.4 g at an ambient temperature of between 21.5 and26.7 °C. In that study, fish received a 49P12L15C diet,which produced an SGR of 3.6 and an FER of 0.81(food conversion factor of 1.23). These figures are verysimilar to values of 3.7 (SGR) and 0.97 (FER) calculatedin this study (but not shown in Table 3) for S. aurataraised from 2.7 to 14.1 g over 45 days on the MC diet(54P14L18C).

Lupatsch et al. (2001) performed a series of trials inEilat (Israel), using gilthead sea bream of 17–32 g,which were grown at 21–24 °C and fed several diets thatdiffered in the proportion of fishmeal, fish oil,cornstarch and cellulose. For the diets containingcornstarch, the weight and protein gain values rankedin the order 43P22L15CN52P18L15CN43P16L29C.This observation corroborates our results, which showbetter growth performances at intermediate carbohy-drate levels. Their results also demonstrated an optimumfor the energy/protein relationship of the diet (either forcarbohydrates or lipids) above which growth depressionoccurs.

Our PER and PR values indicate that high carbohy-drate diets (MC and HC) perform significantly better interms of protein efficiency and protein retentioncompared with the low one (LC). This finding isconsistent with results reported for other fish species andcan be attributed to metabolic control contributing tospare protein when other energy sources are available(Garling and Wilson, 1976; Brauge et al., 1994;Erfanullah, 1998, Stone et al., 2003; see also revisionby Hemre et al., 2001).

Our results indicate MC (54P18C) as the optimaldiet, as it combines high ADCs with elevated SGR,FER, PER, PR and CR, resulting in significantly higherfinal fish weight and moderate lipid content.

Except for the gluconeogenic enzyme FBPase-1and the AST transaminase, our results indicate that keyenzymes involved in glycolysis, the pentose phosphatepathway and amino acid metabolism respond to thelevel of digestible carbohydrates in diets. The resultsfor PK, PFK-1 and ALT are indicative of a proteinsparing effect, with less protein being used to coverenergy demands as more carbohydrate becomesavailable to fuel catabolic pathways. These findingsmatch the higher values of PER and PR found for dietswith a high carbohydrate content. The two-fold

8 F. Fernández et al. / Journal of Experimental Marine Biology and Ecology 343 (2007) 1–10

decrease in ALT activity in the HC group with respectto LC is similar to results reported in carnivorousmammals like the cat. Similar dietary changes in therat (omnivorous) decreased the activity of severalhepatic transaminases 3-to 12-fold (Rogers et al.,1977).

The results obtained for G6P-DH and 6PGDHsuggest that an increase in lipid synthesis occurs ascarbohydrate content increases in the diet. According-ly, the lipid content of the fish body rose as dietarycarbohydrate content increased. In fact, a goodcorrelation (r2 =0.90) was found between final bodylipid and G6P-DH activity. However, this increase canbe considered only as moderate, compared to the fatdeposition found in S. aurata on diets with onlyprotein and fat as energy sources (Lupatsch et al.,2001). In general, these findings are consistent withour previous results on carbohydrate metabolism in S.aurata (Bonamusa et al., 1992; Metón et al., 1999b,2000, 2003) and with results obtained in other species(Walton, 1986; Hemre et al., 2001).

Our results for transaminases in S. aurata are inagreement with those described by Walton (1986) andSanchez-Muros et al. (1998) in rainbow trout. Theformer found that most enzymes that initiate amino acidcatabolism in the trout liver (including AST) wereunaffected by the changes in dietary treatment, whereasALT adapted moderately. Sanchez-Muros et al. (1998)reported that a high-protein diet (61P8L0C) increasedthe liver glutamate dehydrogenase and ALT in rainbowtrout with respect to a control diet (46P8L22C) by 100%and 65%, respectively.

In conclusion, our results combine the study ofnutrient digestibility, growth performance and enzymeactivities and indicate adaptation of S. aurata to dietaryprotein-to-GCS ratio and to the level of GCS in the diet.An optimal level of this starch source is below 26% andprobably above 18%.

Therefore, carbohydrates like GCS can be used toreplace dietary protein and enhance growth rate whenused at 18%. At this level, there was also a clearenhancement of protein retention (29%) respect to thelevel of 5% GCS (21%), with the conclusion that theambient waters would receive less nitrogen and that therisk of eutrophization would be lower when protein arepartially replaced by carbohydrates.

Acknowledgements

We acknowledge Dr. J. Ocaña (Departamento deEstadística, Universidad de Barcelona, Spain) for helpwith statistical treatments. This work was supported by

MCYT (Spain) grants BMC2000-0761 and BIO2003-01098. [SS]

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