Journal of Experimental Marine Biology and Ecology 343 (2007) 1 – 10

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Effects of diets with distinct protein-to-carbohydrate ratios on

nutrient digestibility, growth performance, body composition
and liver intermediary enzyme activities in gilthead
sea 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, Spain
b
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), body
composition, growth performance and liver enzyme activities involved in control of intermediary metabolism, was studied in Sparus
aurata 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 gave ADC values for carbon, nitrogen and dry matter
that were significantly below the corresponding values of the other diets. Fish on MC diet registered higher fresh weight than fish on LC
and HC, and higher specific growth rate (SGR) than fish on HC. The lipid body content ranked in the order HC N MC N LC. High
correlations 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 GCS
could 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 Excess dietary protein with respect to growth demands

One of the main objectives of fish nutrition research
is to minimize the amount of protein in diets and cover
energy requirements using carbohydrates or lipids.
⁎ Corresponding author. Departamento de Ecología, Universidad de
Barcelona, Facultad de Biología, Avda. Diagonal 645, E-08028
Barcelona, 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
leads to increased amino acid degradation and the loss of
nitrogen to ambient waters, which in turn could result in
water eutrophication (Pillay, 1992; Pearson and Black,
2001; Davenport et al., 2003). The protein used in fish
diets is mainly from fish meal, therefore any reduction in
its use could also lead to decreased pressure on
overexploited marine fisheries; as in many cases, a
significant part of catches is converted to fish meal
(Naylor et al., 2000).
2 F. Fernández et al. / Journal of Experimental Marine Biology and Ecology 343 (2007) 1–10

Carbohydrates can be used to replace proteins in fish Cantabria, Spain) and distributed in nine seawater
diets. Optimal levels in these diets depend on fish species, aquariums of 260 l, at a density of 21 fish per aquarium.
carbohydrate type and treatment, and protein and lipid All aquariums were located in isothermal rooms at 21 ±
levels (see reviews by Furuichi and Yone, 1980; Wilson, 0.2 °C and were provided with a closed-circuit water
1994; Shiau, 1997; Hemre et al., 2001). Thus, for a given treatment system consisting of external pump filters and
fish species, the optimal type and level of carbohydrate on-line UV lamps. Air pumps and ceramic diffusers
must be determined experimentally. Treated starch aerated the water. Ammonia, salinity (36 g/l) and pH (7–
(gelatinized, dextrinized, extruded) is a suitable dietary
component for gilthead sea bream (Fernández et al., 1996,
1998; Georgopoulos and Conides, 1999; Venou et al., Table 1
Composition and proximate analysis of test diets
2003) and other fish (Bergot, 1979; Bergot and Breque,
1983; Jeong et al., 1991; Takeuchi et al., 1994; Wilson, DIET LC MC HC
1994; Peres and Oliva-Teles, 2002). (63P5C) (54P18C) (47P26C)
Fish have the enzymes and metabolic pathways Formulation (%)
required to regulate the metabolism of carbohydrates Fish meal a 81.95 68.40 60.00
ingested through diet (Cowey and Walton, 1989; Fish oil b 7.95 8.73 9.00
Corn starch c
Baanante et al., 1991; Shimeno et al., 1993, 1996;
Diets without Cr2O3 5.10 17.87 26.00
Metón et al., 1999a,b, 2003, 2004; Caseras et al., 2000, Diets with Cr2O3 4.60 17.37 25.50
2002; Hemre et al., 2001; Dabrowski and Guderley, Mineral mixture d 1.38 1.38 1.38
2002). However, the intermediary metabolism of carniv- Vitamin mixture e 1.62 1.62 1.62
orous fish, like rainbow trout, shows only moderate Carrageenan 2.00 2.00 2.00
Chromic oxide
adaptation to high levels of dietary carbohydrate (Cowey
Diets without Cr2O3 0 0 0
and Walton, 1989; Hemre et al., 2001; Moon, 2001). Diets with Cr2O3 0.5 0.5 0.5
Integrated studies combining enzyme regulation, nutrient
digestibility and growth performance in relation to dietary Chemical analysis f
carbohydrates are required for cultured species like % fresh weight
Moisture 1.31 1.42 1.63
gilthead sea bream.
% dry weight
The gilthead sea bream is currently the most Protein 62.9 53.9 46.8
extensively cultured fish in the Mediterranean region Fat 15.5 14.4 14.5
(FAO, 2006). Recent years have witnessed an increasing Ash 14.0 12.1 11.1
number of nutritional studies on this species, with the aim Carbohydrates g 7.6 19.6 27.6
Phosphorus 2.02 1.93 1.63
to identify the effects of replacing protein with carbohy-
Calcium 3.06 3.01 2.52
drates or lipids (Bonamusa et al., 1992; García-Alcázar kJ/g dry weight
et al., 1994; Santinha et al., 1996; Vergara et al., 1996; Energy h 22.44 21.88 21.58
Fernández et al., 1998; Lupatsch et al., 2001; Company a
White fish meal (Norse LT-94). Supplied by Sogem Iberica (Rubí,
et al., 1999; Georgopoulos and Conides, 1999; Metón Barcelona, Spain).
b
et al., 1999b, 2000, 2004; Venou et al., 2003) or replacing Fish oil with added vitamin A (all-trans retinol: 1000 IU/g) and
fish meal with another protein of terrestrial origin (Robaina vitamin D (cholecalcipherol: 100 IU/g). From Bonafont Quimica
(Barcelona, Spain).
et al., 1995; Gómez-Requeni et al., 2003; Pereira and c
Gelatinized corn starch. From Campo Ebro (Zaragoza, Spain).
Oliva-Teles, 2003; Sánchez-Muros et al., 2003). d
Mineral mixture ( per kg of diet): CaHPO4.H2O, 8.83 g; CaCO3,
Here we replaced dietary protein with gelatinized 2.56 g; KCl, 1.25 g; MgO, 1.0 g; ZnO, 90 mg; FeCO3, 80 mg; MnO2,
cornstarch (GCS) and studied the effects of this change 12.5 mg; CuSO4, 7.5 mg; KI, 1.5 mg; Na2SeO3, 0.28 mg.
e
on nutrient digestibility, body composition, growth Vitamin mixture ( per kg of diet): all-trans retinol, 3 mg;
performance, nitrogen retention and key liver enzymes cholecalcipherol, 25 mg; all-rac-tocopherol acetate, 60 mg; menadione.
HNaSO3, 10.6 mg; folic acid, 4.8 mg; nicotinic acid, 360 mg; riboflavin,
in Sparus aurata. 24 mg; thiamin. HCl, 30 mg; pyridoxine. HCl, 24 mg; cyanocobalamin,
0.12 mg; ascorbic acid, 200 mg; calcium pantothenate, 30 mg; biotin,
2. Material and methods 0.42 mg; choline chloride, 3.2 g; myo-inositol, 900 mg.
f
Chemical analysis performed following standard procedures of the
2.1. Fish treatments 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.
Gilthead sea bream fingerlings with a mean weight of h
Calculated from gross composition ( protein: 24 kJ/g; lipid: 39 kJ/g;
2.5 g were purchased from a hatchery (Tinamenor, carbohydrate: 17 kJ/g).
 F. Fernández et al. / Journal of Experimental Marine Biology and Ecology 343 (2007) 1–10
8) were checked once a week, and 20% of the seawater
was replaced weekly. Ammonia was always at trace
concentrations, and salinity and pH were corrected
when necessary (see Fernández et al., 1996, 1999).
The fish in each three aquariums were hand fed one
of the three experimental diets (Table 1) without
chromium oxide. The three diets were named LC (low
carbohydrate), MC (moderate carbohydrate) and HC
(high carbohydrate). To facilitate comparisons between
diets, we also adopted the following conventions: P = %
protein, L = % lipid and C = % carbohydrate. Therefore,
the LC diet can also be represented as 63P16L5C, as it
contains 63% protein, 16% lipid and 5% carbohydrate
(in this case, gelatinized cornstarch, GCS).
During the first week, the fish were fed twice daily to
satiety. Fish were considered satiated when some food
remained uneaten after 15 min of feeding. The fish were
then weighed under anaesthesia (tricaine at 70 mg/l) after
a 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 was
adjusted to 5% of body weight per day, which was
provided in two meals of 2.5%, one at 9.30 a.m. and the
other at 4.30 p.m. This ration was close to satiation for all
treatments and ensured that all the food provided was
consumed in less than 15 min (usually in less than 5).
The fish were weighed every 7−20 days and the food
ration was adjusted to the new weight. As fish grew, the
satiation rations diminished and the ration level was
lowered to the same extent for all treatments in order to
fall just below satiety. At three months (93 days), fish
were fasted for 24 h and then weighed under
anaesthesia; three fish from each aquarium were then
killed by cervical section, the livers were dissected out,
weighed, and immediately frozen in liquid nitrogen and
kept at −80 °C until enzyme analysis. Another three fish
from each aquarium were killed by anaesthesia
overdose, dried whole in an oven to constant weight
in order to obtain dry weights, and stored at − 20° until
body composition analysis.
The remaining live fish (8–12 per aquarium) were
fed to satiation (at this point equivalent to a daily ration
of about 2% body weight) with the chromium oxide
version of the diets (Table 1) in order to calculate
nutrient digestibility. After two weeks, all the fish were
killed under anaesthesia, 6 h after feeding. The gut was
dissected out and divided into three sections as
previously reported (Fernández et al., 1996, 1999):
anterior intestine (2 cm after pyloric caeca), posterior
intestine (2 cm before rectum) and mid-intestine (the
portion between the anterior and posterior intestine).
3
The content of each section was pooled for each
aquarium, dried at 70 °C and kept at − 20 °C until
analysis 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 with
the addition of distilled water, and then dried again.
Samples for C and N were analysed with a Carlo Erba
NA 2100 elemental analyser (CE Instruments, Termo-
quest Italia). Samples for Ca and P were digested
(following procedures described by Furukawa and
Tsukahara, 1966) and analysed by ICP spectrometry
(Polyscan 61E, Thermojarrell Ash Corporation, USA).
Protein was calculated from N content, using a factor
of 6.25. Total lipids were measured using a commercial
kit from Merck (Merckotest 3321 for Total Lipids). The
sample was previously digested with sulphuric acid at
100 °C (Knight et al., 1972). Ash content was
determined by combustion in a muffle furnace at
550 °C, following AOAC (1984) specifications.
Diets were analysed for C, N, P, Ca, fat and ash by the
same procedures used for fish samples. Protein content
was also calculated from N content and energy content
was calculated from diet composition (protein, 24 kJ/g,
lipid, 39 kJ/g and carbohydrate, 17 kJ/g, following
Bradfield and Llewellyn, 1992). Dietary Cr was
analysed by ICP spectrometry using the same proce-
dures described for P and Ca (the three elements were
determined simultaneously in the same samples). Gut
content was also analysed for C, N and Cr following the
same method described above.
2.2. Digestibility and growth parameters
Apparent digestibility coefficient (ADC) of a given
nutrient was calculated from the following equation (De
Silva and Anderson, 1995)
 
% chromium in food % nutrient in feces
ADC ¼ 100−100d d :
% chromium in feces % nutrient in food
For dry matter, the equation becomes:
 
% chromium in food
ADC ¼ 100−100d :
% chromium in feces
For each aquarium, growth rates were calculated as a
specific growth coefficient (SGC), following the expres-
sion: SGR = (ln Wf − ln Wi). 100/t, where Wf is the mean
final fresh weight of the fish in each aquarium, Wi the
mean initial fresh weight of the fish in the same aquarium,
4 F. Fernández et al. / Journal of Experimental Marine Biology and Ecology 343 (2007) 1–10

Table 2 The activities of 6-phosphofructo 1-kinase (PFK-1, EC

ADC values obtained for Sparus aurata fed HC, MC and LC diets 2.7.1.11), pyruvate kinase (PK, EC 2.7.1.40), fructose
(samples taken from three regions of the intestine)
1,6-bisphosphatase (FBPase-1, EC 3.1.3.11), glucose 6-
DIET n Anterior intestine Medium intestine Posterior intestine phosphate dehydrogenase (G6P-DH, EC 1.1.1.49), 6-
Carbon phosphogluconate dehydrogenase (6PG-DH, EC
LC 3 73.9 ± 7.9 a 85.5 ± 3.4 b 89.4 ± 3.5 c y 1.1.1.43), alanine aminotransferase (ALT, EC 2.6.1.2)
MC 3 76.5 ± 7.6 a 87.5 ± 4.7b 87.4 ± 2.4 b y
and aspartate aminotransferase (AST, EC 2.6.1.1) were
HC 3 64.9 ± 21 80.9 ± 9.9 81.7 ± 0.8 x
assayed in the crude liver extracts using a COBAS
Nitrogen MIRA S spectrophotometric analyser, as previously
LC 3 82.6 ± 4.7 a 89.2 ± 1.7 b 91.2 ± 1.3 b y
reported (Metón et al., 1999b). All enzyme assays were
MC 3 81.3 ± 3.7 a 89.5 ± 2.2 b 90.2 ± 1.6 b y
performed at 30 °C and followed at 340 nm. The total
HC 3 72.4 ± 13 84.2 ± 6.6 85.8 ± 0.6 x
protein content in crude liver extracts was also measured
Dry matter in the COBAS MIRA S at 600 nm and 30 °C, following
LC 3 65.2 ± 7.1 a 75.2 ± 1.5 b 82.2 ± 2.1 c y the Bradford (1976) method, using bovine serum
MC 3 68.4 ± 4.0 a 79.9 ± 3.3 b 80.3 ± 2.0 b y albumin as a standard.
HC 3 58.0 ± 20 72.6 ± 10 74.2 ± 1.3 x
abc: For a given nutrient and diet, different letters indicate significant 2.4. Statistics
ADC differences between regions of intestine by Fisher test at 0.05
probability level.
All data were analysed with statistical software. In the
xyz: For a given nutrient and region of intestine, different letters
indicate significant ADC differences between diets by Fisher test at case of ADCs, we use StatView program (SAS Institute
0.05 probability level. Inc., San Francisco, USA) to perform a two factor
ANOVA (diet and region of intestine) and the Fisher
LCD post-hoc test to determine significant differences
and t is time in days (De Silva and Anderson, 1995). Other
parameters calculated for each aquarium were: Table 3
Whole body composition of fish at the start of the experiment (initial
Food Efficiency Ratio (FER) = g weight gain/g feed samples) and after being fed LC, MC and HC diets for 93 days
provided Fish group n LC MC HC
Protein Efficiency Ratio (PER) = g weight gain/g feed Initial
protein g/100 g fresh weight
Protein retention (PR) = g protein gain 100/g feed protein Dry weight 3 24.8 ± 1.3 x 24.8 ± 1.3 x 24.8 ± 1.3 x
g/100 g dry weight
Carbon retention (CR) = g carbon gain 100/g feed carbon
Protein 3 55.6 ± 3.9 55.6 ± 3.9 55.6 ± 3.9
Lipid 3 16.8 ± 0.42 x 16.8 ± 0.42 x 16.8 ± 0.42 x
Weight gain is defined as the difference between Wf Ash 3 14.9 ± 0.19 14.9 ± 0.19 y 14.9 ± 0.19 y
and Wi after 93 days of feeding. Protein and carbon gain Carbon 3 46.6 ± 1.3 x 46.6 ± 1.3 x 46.6 ± 1.3 x
were calculated from the protein and carbon content of 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
the fish weighing Wf and Wi.
Using the fish and liver weights at 93 days, we also Fed 93 days
calculated the liver somatic index (LSI) as: g/100 g fresh weight
Dry weight 3 31.1 ± 2.2 ay 32.8 ± 2.3 by 33.4 ± 2.6 by
LSI = liver weight 100 / body weight LSI 3 1.27 ± 0.17a 1.51 ± 0.39a 1.97 ± 0.22b
g/100 g dry weight
Protein 3 56.8 ± 5.8 56.8 ± 3.8 55.2 ± 4.5
2.3. Enzyme activities Lipid 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
After 93 days, the livers of fish exposed only to diets Carbon 3 52.7 ± 3.1ay 54.8 ± 2.3 by 53.5 ± 3.1 aby
without Cr, were used to determine enzymes involved in 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
carbohydrate-protein metabolism. Crude extracts were
obtained by homogenisation of the powdered frozen a, b, c: For a given fish group (initial or fed 93 days) and line (dry
liver (1/5, W/V) in 50 mM Tris–HCl pH 7.5, 4 mM weight, LSI, compound or element), different letters indicate
significant differences between diets by Fisher test ( p b 0.05).
EDTA, 50 mM NaF, 0.5 mM PMSF, 1 mM DTT and x, y: For a given diet and element (or compound or dry weight),
250 mM sucrose using a PTA-7 Polytron (position 3, different letters indicate significant differences between initial and
30 s) and centrifugation at 20,000 ×g for 30 min at 4 °C. final samples by Fisher test ( p b 0.05).
 F. 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 or
dry matter digestibility. As samples from fish in the same
aquarium 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 factors
ANOVA (diet and date) and the Fisher LCD post-hoc
test to determine significant differences between diets or
dates (initial or 93 days) at p = 0.05, using also de
StatView program.
The effect of diet on body weight was analysed for
each date on which fish were weighed. For each date, we
performed a nested ANOVA, with diet as the main factor
and the factor aquarium nested into the factor diet.
Nested design was possible using the Statgraphics Plus
software (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 and
36–63 fish per diet.
Growth rates in individual fish were not calculated as
fish were not tagged. Therefore, the mean weight of fish
in each aquarium for a given date was used as the basis
to calculate growth rates (n = 3), as indicated above. To
establish differences between diets for a given period,
we performed a simple ANOVA, with diet as a single
factor. Differences between diets were deduced from a
Fisher post-hoc test, with significance established at
p = 0.05. We used the same design to analyse growth
performances (FER, PER, PR and CR). In all these
cases, we used the StatView software.
In addition to ANOVA, regression analyses were
used in the case of enzymes, as enzyme activity was
related to the level of carbohydrate (and also to the
carbohydrate/protein ratio) of the diet. Linear regression
Fig. 1. Mean fresh weight of Sparus aurata along the experiment. Each column represents the mean and standard deviation of fresh weight at a given
date. Different letters above deviation bars indicate significant weight differences between treatments for each period (Fisher test, p = 0.05).
5
gave the best fit in all cases and was used to compare the
response of different enzymes. In this case, we used the
StatView program.
3. Results
3.1. Digestibility
A large part of the absorptive process was completed in
the anterior intestine, but significant increases occurred in
the medium and posterior intestine (Table 2). We used the
latter to compare overall ADCs.
The ADCs for C, N and dry matter of samples from
the posterior intestine of fish on the HC diet were
significantly below the corresponding values of the
other diets (MC and LC, Table 2). However, no
significant differences were found between the MC
and LC diets. Therefore, the digestibility of the diets was
affected when carbohydrate (GCS) reached 26%, but not
at 18%.
The samples from the anterior and medium intestine
showed no significant differences in ADC between
diets, although the values followed a similar trend to
those described for posterior intestine samples.
3.2. Differences in body composition
After 93 days, dry weight, as a percentage of fresh
weight, was lower in fish on the LC diet than in those
on the other diets. C content was significantly higher for
the MC group (54.8% of dry weight) than for LC
(52.7% of dry weight). No differences between
treatments were found for protein (or nitrogen) content.
The lipid content differed significantly among fish
6 F. Fernández et al. / Journal of Experimental Marine Biology and Ecology 343 (2007) 1–10

Table 4 level) higher values than the HC group. After 74 days,

Growth performances of Sparus aurata fed LC, MC and HC diets for the fresh weight of the MC group was significantly
93 days
higher than the LC and HC groups. The same
Diets n LC MC HC differences were detected after 86 and 93 days.
Initial fish weight 63 2.41 ± 0.16 2.65 ± 0.14 2.65 ± 0.12 For the whole period of 93 days (Table 4) fish on the
Final fish weight 30–36 29.3 ± 1.4 a 34.5 ± 2.4 b 28.0 ± 1.8 a MC diet registered the highest fresh weight and SGR
SGR 3 2.67 ± 0.02 ab 2.76 ± 0.13 b 2.53 ± 0.09 a
values (although SGR differences between LC and MC
FER 3 0.73 ± 0.05 0.82 ± 0.05 0.74 ± 0.05
PER 3 1.17 ± 0.07 a 1.53 ± 0.09 b 1.61 ± 0.11 b diets were not significant), while the lowest values of
PR (%) 3 21.2 ± 2.2 a 29.1 ± 1.0 b 30.6 ± 2.6 b PER, PR and CR corresponded to fish on the LC diet.
CR (%) 3 25.8 ± 2.6 a 31.3 ± 1.1 b 29.0 ± 2.4 ab No differences between diets were found for FER.
SGR: specific growth rate ((ln final wet weight − ln initial wet
weight) × 100/days); FER: food efficiency ratio (g wet weight gain/g 3.4. Enzyme activities
dry diet fed); PER: protein efficiency ratio (g wet weight gain/g protein
fed); PR (%): protein retention efficiency ((final body nitrogen − initial
Our findings indicated that this species adapted their
body nitrogen) × 100/total nitrogen fed); CR (%): carbon retention
efficiency ((final body carbon − initial body carbon)\ × 100/total carbon metabolism to the level of carbohydrate in the diet
fed). (Table 5). For the enzymes involved in glycolysis/
a, b: For a given fish weight (initial or final) or index (SGR, FER, PER, gluconeogenesis, the best correlation between carbohy-
PR, CR), different letters indicate significant differences between diets drate level and enzyme activity was found for PK
by Fisher test (5% significance level).
(r2 = 0.90 in Table 5). The activity of this enzyme
increased by more than three fold as carbohydrates in
the diet increased five fold. There was also a good
groups, ranking in the order HC N MC N LC. The P and response of PFK-1. However, no significant differences
Ca contents of fish on the LC diet were significantly between diets or correlation with dietary starch level
higher than those of fish on the MC or HC diets. There were observed for FBPase-1 activity.
were no significant differences in ash content between As the level of dietary GCS increased, there was
treatments. also a significant rise in the activity of regulatory
There was a clear increase in dry weight and lipid and enzymes of the pentose phosphate pathway, G6P-DH
carbon content and a decrease in ash, phosphorus and and 6PG-DH. For the former, only the HC diet gave
calcium content in the fish fed 93 days compared to the significantly higher values than the other diets. For the
initial samples (Table 3). latter, clear differences were found between LC, MC
and HC (Table 5), and a good correlation was obtained
3.3. Growth performance and feed utilization between 6PG-DH activity and dietary starch content
(r2 = 0.73).
Partial values of fresh weight obtained during the The amino acid-metabolising enzyme ALT showed
experiment are shown in Fig. 1. For the first 25 days, all dependence on dietary composition. ALT activity
diets gave similar values, but at 45 and 60 days fish on correlated inversely with dietary carbohydrate levels
the MC diet registered significantly (Fisher test at 0.05 (r2 = 0.75) and fish on the HC diet showed half the

Table 5
Liver enzyme activities of fish fed diets for 93 days with low (LC) intermediate (MC) and high (HC) carbohydrate content and regression equations
for the relationships between dietary carbohydrate content and enzyme activity
Enzyme LC MC HC Regression equation r2 p
a ab b
PFK-1 30.01 ± 1.5 37.94 ± 8.4 56.64 ± 22 y = 21.74 + 1.21 x 0.414 0.02
PK 214.2 ± 53a 463.1 ± 83 b 740.1 ± 74c y = 69.93 + 24.7 x 0.901 0.0001
FBPase-1 112.1 ± 16 100.4 ± 11 129.2 ± 31 y = 103.2 + 0.66 x 0.067 0.42
G6P-DH 158.2 ± 37 a 176.7 ± 35 a 249.8 ± 22 b y = 127.9 + 4.09 x 0.536 0.007
6PG-DH 22.0 ± 6.1 a 29.3 ± 2.5 b 40.6 ± 4.9 c y = 16.6 + 0.859 x 0.726 0.0004
ALT 1404 ± 188 b 959 ± 206 a 719 ± 89 a y = 18705 − 493 x 0.749 0.0003
AST 1067 ± 230 799 ± 131 920 ± 266 y = 1063 − 8.26 x 0.107 0.30
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 between diets at the 0.05 probability level.
 F. 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 not
respond to the carbohydrate/protein ratio.
4. Discussion
The ADCs obtained for samples of anterior, medium
and posterior intestine from S. aurata corroborate the
previous 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 anterior
intestine. However, significant additional absorption
takes place along the intestine, so samples of posterior
intestine are the most representative source for measur-
ing overall digestibility. The discussion that follows is
based upon these samples.
For C, N and dry matter, the HC diet gave ADCs that
were significantly below the corresponding values for
the other diets. The percentage of ADC reduction for
HC compared to the mean obtained for the other two
diets was 7.6% for C, 5.4% for N and 8.7% for dry
matter. These figures indicate that, for GCS, a level of
26% (HC diet) is probably above the acceptable limit in
terms of digestibility performance for S. aurata, and that
18% (MC) would be near optimal and produce no
significant effect on ADCs.
Georgopoulos and Conides (1999) fed S. aurata for
30 days with diets containing cornstarch and found
protein ADCs of 92.3, 91.5 and 88.5% for diets
containing GCS at inclusion levels of 10, 20 and 30%,
respectively. The 88.5% ADC is slightly higher than the
85.8% reported here for the diet containing 26% GCS,
but is consistent with our finding of lower ADCs for
protein when GCS content is around 26%.
Our data on fish composition corroborate those of
other studies that show an increase in dry weight and
lipids as fish grow, with the corollary of an inverse
relationship between lipid content and water content
(see review by Weatherley and Gill, 1987). Dry
weight and lipid content were also directly related to
dietary carbohydrate. Differences in lipid storage can
be associated with differences in the energy/protein
ratio of the diets, with higher ratios favouring lipid
deposition.
At the end of the experiment, an increase in the liver
somatic index (LSI) in conjunction with the increased
dietary carbohydrate was noted, and the LSI for fish on
the HC diet was significantly higher than that of fish on
the other diets. These results are consistent with others
previously 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 to
increased glycogen deposition (García de Frutos et al.,
7
1990; Wilson, 1994). However, increased fat deposition
in the liver has been observed in other fish like cod
(Hemre et al., 1989).
Our SGR values for S. aurata are similar to those
reported by García-Alcázar et al. (1994), who raised
gilthead sea bream fingerlings for 40 days from 2.9 to
12.4 g at an ambient temperature of between 21.5 and
26.7 °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 very
similar to values of 3.7 (SGR) and 0.97 (FER) calculated
in this study (but not shown in Table 3) for S. aurata
raised from 2.7 to 14.1 g over 45 days on the MC diet
(54P14L18C).
Lupatsch et al. (2001) performed a series of trials in
Eilat (Israel), using gilthead sea bream of 17–32 g,
which were grown at 21–24 °C and fed several diets that
differed in the proportion of fishmeal, fish oil,
cornstarch and cellulose. For the diets containing
cornstarch, the weight and protein gain values ranked
in the order 43P22L15C N 52P18L15C N 43P16L29C.
This observation corroborates our results, which show
better growth performances at intermediate carbohy-
drate levels. Their results also demonstrated an optimum
for the energy/protein relationship of the diet (either for
carbohydrates or lipids) above which growth depression
occurs.
Our PER and PR values indicate that high carbohy-
drate diets (MC and HC) perform significantly better in
terms of protein efficiency and protein retention
compared with the low one (LC). This finding is
consistent with results reported for other fish species and
can be attributed to metabolic control contributing to
spare protein when other energy sources are available
(Garling and Wilson, 1976; Brauge et al., 1994;
Erfanullah, 1998, Stone et al., 2003; see also revision
by Hemre et al., 2001).
Our results indicate MC (54P18C) as the optimal
diet, as it combines high ADCs with elevated SGR,
FER, PER, PR and CR, resulting in significantly higher
final fish weight and moderate lipid content.
Except for the gluconeogenic enzyme FBPase-1
and the AST transaminase, our results indicate that key
enzymes involved in glycolysis, the pentose phosphate
pathway and amino acid metabolism respond to the
level of digestible carbohydrates in diets. The results
for PK, PFK-1 and ALT are indicative of a protein
sparing effect, with less protein being used to cover
energy demands as more carbohydrate becomes
available to fuel catabolic pathways. These findings
match the higher values of PER and PR found for diets
with 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 respect
to LC is similar to results reported in carnivorous
mammals like the cat. Similar dietary changes in the
rat (omnivorous) decreased the activity of several
hepatic transaminases 3-to 12-fold (Rogers et al.,
1977).
The results obtained for G6P-DH and 6PGDH
suggest that an increase in lipid synthesis occurs as
carbohydrate content increases in the diet. According-
ly, the lipid content of the fish body rose as dietary
carbohydrate content increased. In fact, a good
correlation (r 2 = 0.90) was found between final body
lipid and G6P-DH activity. However, this increase can
be considered only as moderate, compared to the fat
deposition found in S. aurata on diets with only
protein and fat as energy sources (Lupatsch et al.,
2001). In general, these findings are consistent with
our 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 in
agreement with those described by Walton (1986) and
Sanchez-Muros et al. (1998) in rainbow trout. The
former found that most enzymes that initiate amino acid
catabolism in the trout liver (including AST) were
unaffected by the changes in dietary treatment, whereas
ALT adapted moderately. Sanchez-Muros et al. (1998)
reported that a high-protein diet (61P8L0C) increased
the liver glutamate dehydrogenase and ALT in rainbow
trout with respect to a control diet (46P8L22C) by 100%
and 65%, respectively.
In conclusion, our results combine the study of
nutrient digestibility, growth performance and enzyme
activities and indicate adaptation of S. aurata to dietary
protein-to-GCS ratio and to the level of GCS in the diet.
An optimal level of this starch source is below 26% and
probably above 18%.
Therefore, carbohydrates like GCS can be used to
replace dietary protein and enhance growth rate when
used at 18%. At this level, there was also a clear
enhancement of protein retention (29%) respect to the
level of 5% GCS (21%), with the conclusion that the
ambient waters would receive less nitrogen and that the
risk of eutrophization would be lower when protein are
partially replaced by carbohydrates.
Acknowledgements
We acknowledge Dr. J. Ocaña (Departamento de
Estadística, Universidad de Barcelona, Spain) for help
with statistical treatments. This work was supported by
MCYT (Spain) grants BMC2000-0761 and BIO2003-
01098. [SS]
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