Comparative Biochemistry and Physiology, Part A 151 (2008) 4550

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Comparative Biochemistry and Physiology, Part A

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a

Effect of water temperature and dietary starch on growth and metabolic utilization of
diets in gilthead sea bream (Sparus aurata) juveniles
A. Couto a,b, P. Enes a,b, H. Peres b, A. Oliva-Teles a,b,
a
Departamento de Zoologia e Antropologia, Faculdade de Cincias, Universidade do Porto, 4099-002 Porto, Portugal
b
CIMAR/CIIMAR-Centro Interdisciplinar de Investigao Marinha e Ambiental, Universidade do Porto, 4050-123 Porto, Portugal

A R T I C L E I N F O A B S T R A C T

Article history: We evaluated the effect of dietary starch level on growth performance, feed utilization, whole-body
Received 8 January 2008 composition and activity of selected key enzymes of intermediary metabolism in gilthead sea bream juveniles
Received in revised form 23 May 2008 reared at 18 and 25 C. A diet was formulated to contain 48% crude protein, 12% lipids and 30% gelatinized maize
Accepted 24 May 2008
starch (diet 30GS). Two other diets were formulated to include the same level of ingredients as diet 30GS except
Available online 30 June 2008
for the gelatinized starch, which was included at 20% (diet 20GS) or 10% (diet 10GS). No adjustment to diet
Keywords:
composition was otherwise made. Each diet was fed to triplicate groups of gilthead sea bream (30 g initial mass)
Carbohydrate for 8 weeks, on a pair-feeding scheme. The higher temperature improved growth performance but the opposite
Enzyme activity was true for feed efciency and protein efciency ratio. Independently of temperature, growth performance,
Feed utilization feed efciency and protein efciency ratio were lower in sh fed diet 30GS. No effect of temperature or dietary
Gilthead sea bream starch level on whole-body composition was noticed. Hepatosomatic index and liver glycogen were higher at
Growth performance 18 C and, within each temperature, in sh fed diet 30GS. Glycemia was not affected by temperature, but was
Water temperature lower in sh fed diet 10GS. Data on enzyme activities showed that increasing water temperature enhances liver
Whole-body composition
glucokinase (GK) and pyruvate kinase (PK) activities, suggesting that gilthead sea bream is more apt to use
dietary starch at higher temperatures. No effect of temperature was noticed on hexokinase (HK), fructose-1,6-
bisphosphatase (FBPase), glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GDH)
activities. Dietary starch enhanced PK and FBPase activities while depressed GDH activity, suggesting a lack of
signicant regulation of hepatic glucose utilization and production in this species. HK, GK and G6PD activities
were unaffected by dietary composition. Irrespectively of water temperature, gelatinized starch may be
included up to 20% in diets for gilthead sea bream juveniles; at higher dietary levels, growth and efciency of
feed utilization are depressed.
2008 Elsevier Inc. All rights reserved.

1. Introduction 2002). Thus, it is a priority to optimise the protein to energy ratio of the
diets and improve the protein sparing by non-protein energy sources
Gilthead sea bream (Sparus aurata) is one of the most important such as lipids and carbohydrates (Wilson, 1994; Hemre et al., 2002).
marine sh species reared in the Mediterranean region. Gilthead sea Carbohydrate is the least expensive form of dietary energy; how-
bream is a carnivorous species with high protein requirement (Oliva- ever its utilization by sh depends of several factors, such as sh
Teles, 2000). The protein used in sh diets is mainly obtained from sh species, dietary carbohydrate level, origin, molecular complexity and
meal, an overexploited resource which is becoming scarce (Watanabe, physical state (Wilson, 1994; Stone, 2003). As sh are poikilothermic
2002); thus, protein stands amongst the most expensive components animals, environmental temperature also affects their feed intake,
of sh diets. Optimization of protein utilization for growth by reduc- growth and metabolic responses (Peres and Oliva-Teles, 1999).
tion of its use for energy purposes is therefore a research priority not Although few studies are available regarding the utilization of
only from an economical but also from an environmental perspective, carbohydrates at different temperatures, such studies indicate that
since protein catabolism by-products account for the major source of temperature affects carbohydrate utilization (Mdale et al., 1991, 1999;
nitrogen loading to the surrounding waters (Cowey, 1995; Watanabe, Shikata et al., 1995; Papoutsoglou and Lyndon, 2005; Enes et al., 2006b,
2008b; Moreira et al., 2008). Technological processing including ap-
plication of moisture and heat, which promotes gelatinization,
improves starch utilization (Kaushik and Oliva-Teles, 1985; Krogdahl
Corresponding author. Departamento de Zoologia e Antropologia, Faculdade de
Cincias, Universidade do Porto, 4099-002 Porto, Portugal. Tel.: +351 22 340 1507;
et al., 2005). Gelatinization breaks down the starch granules increasing
fax: +351 22 340 1511. its surface area, which renders starch more susceptible to enzymatic
E-mail address: [email protected] (A. Oliva-Teles). attack (Bergot and Brque, 1983; Stone, 2003). The higher digestibility

1095-6433/$ see front matter 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpa.2008.05.013
46 A. Couto et al. / Comparative Biochemistry and Physiology, Part A 151 (2008) 4550

of gelatinized starch compared to raw starch has been shown in several The experimental facilities consisted of two thermo-regulated re-
species (Bergot and Brque, 1983; Gouveia et al., 1995; Peres and Oliva- circulation water systems each one equipped with 9 berglass
Teles, 2002; Stone et al., 2003), including gilthead sea bream (Venou cylindrical tanks of 250 L water capacity, supplied with a continuous
et al., 2003). Treated starch was previously shown to improve per- ow of ltered seawater. During the trial, water temperature was
formance and provide some protein sparing in gilthead sea bream, maintained at 25 0.5 C in one system and at 18 0.5 C in the other
when used up to 20% (Fernandez et al., 2007; Enes et al., 2008a). system, and salinity averaged 35 1. After transport to the experi-
One difculty in interpreting nutrient utilization is that modifying a mental facilities, sh were kept in quarantine for 2 weeks and fed a
dietary nutrient inclusion level generally implies a concomitant mod- commercial diet. Then they were transferred to the experimental sys-
ication of other nutrient levels, therefore confounding the inter- tems and allowed to adapt to the experimental conditions for 5 days.
pretation of effect of the nutrient under study. With the present study Thereafter, groups of 25 sh with an initial mean body mass of 30 g
we aimed, by pair-feeding the animals diets identical in all nutrients were randomly distributed to each tank. Diets were randomly assigned
but carbohydrate, to gain further information on the effect of car- to triplicate groups of these sh. During the trial, sh were fed by hand
bohydrate level (gelatinized maize starch) on growth performance, two times a day, 6 days a week, on a pair-feeding scheme as follows:
body composition and activity of key enzymes of intermediary metab- the group receiving diet 30GS was fed to apparent visual satiation,
olism in gilthead sea bream juveniles. This study also aims to evaluate while the other groups received 90% (diet 20GS) and 80% (diet 10GS) of
the effect of water temperature on carbohydrate utilization, this way the ration given to group 30GS, so that every group was fed the same
contributing to further understanding the potential use of carbohy- amount of all nutrients except carbohydrate. Utmost care was taken to
drates as energy sources in gilthead sea bream reared at different assure that all feed supplied was consumed. The trial lasted 8 weeks;
temperatures. during this period two sh from different tanks died from unidentied
reasons. During the trial, the sh were bulk weighed every 2 weeks,
2. Materials and methods after 1 day of feed deprivation. Ten sh from the initial stock pop-
ulation and six sh from each tank at the end of the trial were sampled
The experiment was carried out at the Marine Zoology Station and pooled for whole-body composition analysis. Whole sh, viscera
of Faculty of Sciences, at Porto University, with gilthead sea bream and liver mass of these sh were recorded for determination of
(S. aurata) juveniles obtained from a commercial hatchery. hepatosomatic and visceral indices. To minimize manipulation stress,
A sh meal based diet was formulated to contain 48% crude protein, the remaining sh continued to be fed for three more days, after which
12% crude lipids and 30% gelatinized starch (diet 30GS); two other diets blood from three sh and liver from six sh per tank were sampled 6 h
were formulated to include the same levels of all ingredients as diet after the morning meal. Blood was collected from the caudal vein with
30GS except for the gelatinized starch, which was included at 20% (diet a heparinised syringe, immediately centrifuged and the plasma frozen
20GS) or 10% (diet 10GS). No adjustment to diet composition was for analysis. Livers were frozen in liquid nitrogen and stored at 80 C.
otherwise made. All dietary ingredients were nely ground, well Chemical composition of diets and whole sh were analyzed using
mixed and dry pelleted in a laboratory pellet mill (CPM) through 3 mm the following procedures: dry matter after drying in an oven at 105 C
dye. Ingredients and proximate composition of the experimental diets until constant weight; ash by incineration in a mufe furnace at 450 C
are presented in Table 1. for 16 h; protein content (N 6.25) by the Kjeldahl method after acid
digestion using Kjeltec digestion and distillation units; lipid by
petroleum ether extraction (SoxTec HT System); starch according
Table 1 to Thivend et al. (1972); gross energy by direct combustion in an
Ingredient composition and proximate analysis of the experimental diets adiabatic bomb calorimeter (PARR model 1261). Hepatic glycogen was
Diets determined as described by Plummer (1987) and plasma glucose was
determined according to the enzymaticcolorimetric method from
30GS 20GS 10GS
Spinreact (glucose kit, cod. 1001191).
Ingredients (% dry weight) For measurement of hexoquinase (HK; EC 2.7.1.1)/glucokinase (GK;
Fish meala 59.8 64.5 74.6
EC 2.7.1.2) and L-type pyruvate kinase (PK; EC 2.7.1.40) activities, a frozen
Soluble sh protein concentrateb 1.0 1.1 1.3
Cod liver oil 5.7 6.3 7.1 sample of liver (200 mg) was homogenized (dilution 1/10) in ice-cold
Gelatinized starchc 30.0 22.2 12.5 buffer (80 mM Tris; 5 mM EDTA; 2 mM DTT; 1 mM benzamidine; 1 mM
Vitamin premixd 1.0 1.1 1.3 4-(2-aminoethyl) benzenesulfonyl uoride, pH 7.6). After centrifugation
Mineral premixe 1.0 1.1 1.3
(900 g for 10 min), the resultant supernatant was separated for HK/GK
Choline chloride (50%) 0.5 0.6 0.6
Binderf 1.0 1.1 1.3 and PK activity measurements. The HK (low Km HKs) and GK (high Km
HK or HK IV) activities were measured at 37 C using 0.5 mM and
Proximate analysis (% dry weight) 100 mM of glucose, respectively, by coupling ribulose-5-phosphate
Dry matter (DM) 95.10 92.91 92.82 formation from glucose-6-phosphate to the reduction of -NADP using
Crude protein 47.17 52.76 58.34
puried glucose-6-phosphate dehydrogenase (Sigma) and 6-phospho-
Crude fat 11.68 13.37 14.73
Starch 25.5 18.5 8.0 gluconate dehydrogenase (Sigma) as coupling enzymes (Tranulis et al.,
Ash 11.83 13.25 14.90 1996; Panserat et al., 2000). The method for measuring GK activity on
Gross energy (kJ g 1 DM) 20.40 20.90 21.39 frozen samples needed correction by measuring glucose dehydrogenase
a
Vacuum Dried LT. Pesquera Diamante, S. A. Peru. (EC 1.1.1.47) activity as described by Tranulis et al. (1996). To measure PK
b
G-Special. Soropche, France. activity the previously separated supernatant was centrifuged at
c
C-Gel Instant12016, Cerestar, Mechelen, Belgium. 10 000 g for 20 min, for isolation of the cytosolic fraction. The
d
Vitamins (mg kg 1 diet): retinol, 18,000 (IU kg 1 diet); calciferol, 2000 (IU kg 1
diet); alpha tocopherol, 35; menadion sodium bis., 10; thiamin, 15; riboavin, 25; Ca
consumption of -NADH was monitored at 340 nm and 37 C, using
pantothenate, 50; nicotinic acid, 200; pyridoxine, 5; folic acid, 10; cyanocobalamin, lactate dehydrogenase (Sigma) in excess as coupling enzyme (Foster and
0.02; biotin, 1.5; ascorbyl monophosphate, 50; inositol, 400. Moon, 1985).
e
Minerals (mg kg 1 diet): cobalt sulphate, 1.91; copper sulphate, 19.6; iron sulphate, The activity of fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11)
200; sodium uoride, 2.21; potassium iodide, 0.78; magnesium oxide, 830; manganese
was measured on a frozen sample of liver (200 mg), homogenized
oxide, 26; sodium selenite, 0.66; zinc oxide, 37.5; potassium chloride, 1.15 (g kg 1 diet);
sodium chloride, 0.40 (g kg 1 diet); dibasic calcium phosphate, 5.9 (g kg 1 diet). (dilution 1/10) in ice-cold buffer (20 mM Tris; 5 mM EDTA; 2 mM DTT;
f
Aquacube (Guar gum, polymethyl carbamide, Manioc starch blend, hydrate calcium 0.24 M sucrose, pH 8). The homogenate was centrifuged at 900 g
sulphate). Agil, England. for 10 min. Enzyme assay was performed on the cytosolic fraction by
 A. Couto et al. / Comparative Biochemistry and Physiology, Part A 151 (2008) 4550 47

Table 2 a standard. Enzymatic activities were expressed as mU per mg of

Growth performance, feed, nitrogen and energy utilization of sea bream fed the hepatic soluble protein (specic activity). One unit of enzyme activity
experimental diets
(U) was dened as the amount of enzyme catalysing the hydrolysis of
Temperature 25 C 18 C 1 mol of substrate per min under standard conditions (37 C).
Diets 30GS 20GS 10GS SEM 30GS 20GS 10GS SEM Statistical analysis of data was done by two-way analysis of variance
using the signicance level of 0.05 for rejection of the null hypothesis.
Initial body mass (g) 30.66 30.72 30.64 0.03 30.68 30.72 30.73 0.01
Final body mass (g) 64.44 67.16 69.49 0.83 46.71 50.65 51.21 0.81 Whenever statistical differences were found, a Turkey's multiple range
Mass gain 10.10 10.42 10.86 0.13 6.40 7.46 7.59 0.21 test was performed to identify differences among means. All statistical
(g kg ABW 1 day 1) analysis was done using a SPSS 15.0 software package.
Feed intake 19.45 16.69 14.77 0.69 12.17 10.26 9.25 0.45
(g kg ABW 1 day 1)
Protein intake 0.54 0.55 0.55 0.00 0.25 0.26 0.26 0.00
3. Results
(g sh 1 day 1)
Starch intake 0.29 0.19 0.07 0.03 0.14 0.09 0.03 0.01 Final body mass and mass gain were higher in sh reared at 25 than
(g sh 1 day 1) at 18 C (Table 2). Within each water temperature these parameters
Feed efciencya 0.52 0.62 0.74 0.03 0.52 0.73 0.82 0.04
were lower in sh fed diet 30GS than the other diets. Feed intake was
Protein efciency ratiob 1.10 1.18 1.26 0.03 1.11 1.38 1.41 0.05
Nitrogen retention 17.57 19.11 21.45 0.70 18.29 22.31 24.01 0.98 lower at the lowest temperature and differences in feed intake among
(% N intake) c groups were due to the pair-feeding scheme applied. Differences were
Energy Retention 21.59 24.16 29.65 1.28 20.84 28.61 32.48 1.77 found in FE and PER between rearing temperatures, with higher values
(% E intake) d at 18 than at 25 C, except for sh fed diet 30GS (Table 2). FE increased
Two-way ANOVA
with the decrease of dietary starch level, while PER was only lower in
the 30GS group comparatively to the 20GS and 10GS groups. Retention
Variation source Temperature Diet Interaction Diete
of nitrogen and energy (in % of intake) were higher at 18 than at 25 C
30GS 20GS 10GS and increased with the reduction of dietary starch level (Table 2).
Initial body mass (g) ns ns ns At the end of the trial, sh exhibited similar whole-body com-
Final body mass (g) ns a b b position (Table 3). Visceral (VI) and hepatosomatic (HSI) indices were
Mass gain ns a b b higher at 18 than at 25 C. VI of sh fed diet 30GS was higher than that
(g kg ABW 1 day 1)
of sh fed diet 10GS, while HSI of sh fed diet 30GS was higher than
Feed intake a b c
(g kg ABW 1 day 1) that of groups fed diets 20GS and 10GS, which were similar amongst
Protein intake ns ns them. Liver glycogen was also higher at 18 than at 25 C and decreased
(g sh 1) with the decrease of dietary starch level (Table 3). Glycemia was
Starch intake a b c
(g sh 1)
Feed efciency a b c
Table 3
Protein efciency a b b
Whole-body composition (% fresh weight), visceral and hepatosomatic indices and
ratio
plasmatic glucose of sea bream fed the experimental diets
N Retention ns a b b
(% N intake) Temperature 25 C 18 C
E Retention ns a b c
(% N intake) Diets 30GS 20GS 10GS SEM 30GS 20GS 10GS SEM

SEM: pooled standard error of the mean. Dry matter 31.28 30.48 30.36 0.21 30.83 30.70 30.92 0.30
Nsnon signicant; *P b 0.05; **P b 0.01; ***P b 0.001. Crude protein 15.65 15.77 16.26 0.15 15.65 15.67 16.02 0.16

Average body mass (initial body mass + nal body mass)/2. Crude lipids 12.10 11.55 11.40 0.18 12.27 11.78 12.10 0.20
a
FE: wet mass gain/dry feed intake. Ash 3.69 4.00 3.57 0.08 3.92 3.70 3.72 0.09
b
PER: wet mass gain/crude protein intake. Gross energy (kJg 1) 8.30 7.99 8.24 0.06 8.24 8.20 8.28 0.05
c
N retention (%): (N gain/N intake) 100. VIa 6.47 5.96 5.95 0.11 8.06 7.76 7.35 0.16
d
Energy retention (%): (energy gain/energy intake) 100. HSIb 1.54 1.13 1.16 0.04 3.06 2.60 2.16 0.10
e
Different letters in the same row stand for statistical differences between diets Liver glycogen 5.17 3.45 2.07 0.35 12.05 7.77 6.04 0.59
P b 0.05. (g 100 g 1 liver)
Plasma glucose (mmol L 1) 6.71 6.10 3.44 0.37 6.87 6.53 4.19 0.37

monitoring -NADPH formation, using puried glucose-6-phosphate

dehydrogenase (Sigma) and 6-phosphogluconate dehydrogenase Two-way ANOVA
(Sigma) as coupling enzymes (Tranulis et al., 1996). Variation source Temperature Diet Interaction Dietc
To measure glucose-6-phosphate dehydrogenase (G6PD; EC 30GS 20GS 10GS
1.1.1.49) activity, a frozen sample of liver (200 mg) was homogenized
Dry matter ns ns ns
(dilution 1/5) in ice-cold buffer (0.02 M Tris; 0.25 M sucrose; 2 mM
Crude protein ns ns ns
EDTA; 0.1 M NaF; 0.5 mM PMSF; 0.01 M -mercapto-ethanol, pH 7.4). Crude lipids ns ns ns
Homogenates were centrifuged at 30 000 g for 20 min, and the Ash ns ns ns
formation of -NADPH was monitored at 37 C and 340 nm (Bautista Crude energy ns ns ns
et al., 1988). (kJ g 1)
VI ns a ab b
For measurement of glutamate dehydrogenase (GDH; EC 1.4.1.2) HSI a b b
activity, a frozen sample of liver (200 mg) was homogenized (dilution Liver glycogen a b c
1/10) in ice-cold buffer (30 mM HEPES, 0.25 mM sucrose, 0.5 mM EDTA, (g 100 g 1 liver)
5 mM K2HPO4, 1 mM DTT, pH 7.4). After centrifugation (900 g for Plasma glucose ns ns a a b
(mmol L 1)
10 min), the resultant supernatant was sonicated for 1 min (pulse 1 s,
amplitude 50) and centrifuged again at 15 000 g for 20 min. The GDH SEM: pooled standard error of the mean; nsnon signicant; *P b 0.05; **P b 0.01;
***P b 0.001.
activity was measured using 10 mM of L-glutamate, at 37 C and a
Visceral index: (viscera mass/body mass) 100.
followed at 340 nm (Bergemeyer, 1974). b
Hepatosomatic index: (liver mass/body mass) 100.
Protein concentration was determined according to Bradford c
Different letters in the same row stand for statistical differences between diets
(1976) using Sigma protein assay kit with bovine serum albumin as P b 0.05.
48 A. Couto et al. / Comparative Biochemistry and Physiology, Part A 151 (2008) 4550

Table 4
Selected hepatic glycolytic, gluconeogenic, lipogenic and amino acid catabolic enzyme activities (mU mg 1 protein) in sea bream fed the experimental diets

Temperature 25 C 18 C

Diets 30GS 20GS 10GS SEM 30GS 20GS 10GS SEM

Glycolysis
Hexokinase 0.29 0.27 0.27 0.03 0.23 0.31 0.16 0.03
Glucokinase 13.83 15.39 9.44 1.26 9.75 10.49 7.51 0.82
Pyruvate kinase 79.01 45.29 31.64 4.81 32.24 34.70 34.07 1.64

Gluconeogenesis
Fructose-1,6-bisphosphatase 44.82 32.49 24.61 2.81 38.87 29.47 24.65 2.68

Lipogenesis
Glucose-6-phosphate dehydrogenase 155.78 128.78 121.81 6.43 119.42 143.51 127.65 4.88

Aminoacid metabolism
Glutamate dehydrogenase 59.83 79.15 136.22 9.52 64.03 87.15 140.33 8.84

Two-way ANOVA

Variation source Temperature Diet Interaction Diet1

30GS 20GS 10GS

Hexokinase ns ns ns
Glucokinase ns ns
Pyruvate kinase a b b
Fructose-1,6-bisphosphatase ns ns a b b
Glucose-6-phosphate dehydrogenase ns ns
Glutamate dehydrogenase ns ns a a b

SEM: pooled standard error of the mean; nsnon signicant; *P b 0.05; **P b 0.01; ***P b 0.001.
1
Different letters in the same row stand for statistical differences between diets P b 0.05.

unaffected by temperature, but it was lower in sh fed diet 10GS than different carbohydrate levels at different temperatures, which differs
the other diets (Table 3). from the temperature-dependent response observed in Atlantic salm-
Hexokinase (HK) and glucokinase (GK) activities (Table 4) showed no on Salmo salar by Hemre et al. (1995). In that study, the authors
differences among dietary treatments. GK activity was higher at 25 than reported that sh had problems adapting to medium and high dietary
at 18 C, but temperature had no effect on HK activity. Pyruvate kinase starch levels at low (winter) temperatures, but not at higher (summer)
(PK) activity was also higher at 25 than at 18 C. At the higher temperatures. Indeed, differences in the response to dietary nutrient
temperature, but not at the lower, PK activity increased with the dietary level at different temperatures seem to be species specic. Accord-
starch level. The gluconeogenic enzyme fructose-1,6-bisphosphatase ingly, Yamamoto et al. (2001) observed that while macronutrient self-
(FBPase) activity was not affected by temperature and was higher in sh selection in rainbow trout is not inuenced by temperature, in com-
fed diet 30GS than the other diets. Glutamate dehydrogenase (GDH) mon carp there is a temperature related change in the most appro-
activity was also not affected by water temperature and was higher priate macronutrient ratios.
in sh fed diet 10GS than the other diets, while glucose-6-phosphate Regardless of temperature, in the present study FE decreased with
dehydrogenase (G6PD) activity was not affected either by diet com- increasing dietary starch level. Although our results contradict those of
position or water temperature. Fernandez et al. (2007) and Enes et al. (2008a) in the same species, they
are in agreement with those reported by Moreira et al. (2008) in
4. Discussion European sea bass fed similar diets and following an identical feeding
scheme. Discrepancies in the different studies may therefore be related
As expected, growth rate was higher in sh reared at 25 than at to an adjustment of feed intake in sh fed diets with different pro-
18 C since the higher temperature is within the optimum range for portions of protein:carbohydrate.
growth of gilthead sea bream, while 18 C is clearly below the Data on PER indicate that gilthead sea bream used 10% as efciently
optimum. Within each temperature, growth was higher with diets as 20% GS for protein sparing, while higher dietary starch levels
including 10 or 20% gelatinized starch (GS) than 30% GS. Similarly, Enes depressed protein utilization. Similar results were also observed in
et al. (2008a) found no differences in growth of gilthead sea bream gilthead sea bream with dietary starch levels up to 20% (Enes et al.,
juveniles fed either a carbohydrate-free diet or diets including 10 or 2008a). Although sh grow faster at high temperature, feed, protein
20% of either native or waxy maize starch and Fernandez et al. (2007) and energy utilization were more efcient at low temperature.
observed higher growth of juveniles fed a diet with 18% GS than with Similarly, Fountoulaki et al. (2005) in the same species found higher
26% GS. Overall, results of these studies seem to indicate that gilthead FE at low (winter) than at high (summer) temperatures, whereas Enes
sea bream juveniles tolerate up to 20% carbohydrates, while growth et al. (2008b) observed higher FE and PER at 25 than at 18 C in gilthead
depression occurs at higher dietary inclusion levels. This agrees with sea bream fed diets including either glucose or waxy maize starch.
the recommendation advanced by Wilson (1994) that diets for Although a positive correlation between dietary carbohydrate level
carnivorous sh should include no more than 20% carbohydrate. and body lipid content was previously found in gilthead sea bream
However, this should be carefully conrmed for each species as for (Fernandez et al., 2007) and in other sh species (Kaushik and Oliva-
instance in European sea bass Dicentrarchus labrax juveniles, by Teles, 1985; Shimeno et al., 1996; Lanari et al., 1999), such an effect was
applying a methodological approach identical to that of the present not observed in the present trial. Not many data are available on the
study, Moreira et al. (2008) observed no growth differences in sh fed effect of temperature on sh whole-body composition. In European sea
diets including up to 30% GS. bass, Peres and Oliva-Teles (1999) and Moreira et al. (2008) observed
Both in this study with gilthead sea bream and in that of Moreira signicant differences in whole-body composition due to temperature,
et al. (2008) with European sea bass there was a lack of adaptation to although trends differed in both studies. In carp Cyprinus carpio, higher
 A. Couto et al. / Comparative Biochemistry and Physiology, Part A 151 (2008) 4550 49

body fat content was observed in sh reared at 18 than at 25 C (Mdale However, very recently, Enes et al. (2008c) conrmed in European
et al., 1999). Although no effect of water temperature on whole-body sea bass and gilthead sea bream that at least for GK such effect was
composition was observed in our study, both lower temperature and indeed do to temperature. In contrast to what was previously ob-
high dietary starch level promoted higher visceral (VI) and hepatoso- served in gilthead sea bream (Enes et al., 2008b), present data
matic (HSI) indices and liver glycogen deposition. Similar effects of showed no effect of rearing temperature on HK activity. Thus, the
temperature (Enes et al., 2008b) and dietary carbohydrate level effect of temperature on the regulation of HK activity requires to be
(Fernandez et al., 2007) were previously observed in this species. The further claried.
increase in VI cannot be linked to a deposition of excess dietary energy Although Panserat et al. (2002) showed that in gilthead sea bream
as lipids in the viscera since whole-body lipid content does not support hepatic FBPase gene expression was depressed by feeding a 20%
this argument. Instead, VI increase seems to be strongly correlated to carbohydrate diet comparatively to a carbohydrate-free diet, our data
liver glycogen deposition. The higher glycogen content and thus HSI indicate that high levels of dietary starch (30%) promoted an increase
observed at lower water temperature may be a consequence of lower of FBPase activity. Furthermore, no differences in FBPase activity were
metabolic rates at this temperature. observed in gilthead sea bream fed either a carbohydrate-free diet or
This study showed that glycemia was not affected by water diets including 10 or 20% of either native or waxy maize starch (Enes
temperature even though feed intake was higher at the highest tem- et al., 2008a). Overall, these results sustain the hypothesis of a lack of
perature. This agrees with the results of Enes et al. (2008b) also in this signicant regulation of hepatic glucose utilization and production,
species. Overall, these results suggest that temperature does not affect which may contribute to explain the relatively low efciency of
glucose regulation in gilthead sea bream juveniles. Fish generally have dietary glucose utilization by gilthead sea bream juveniles.
a limited ability to metabolize glucose, and high digestible carbohy- Excess hepatic dietary glucose is directed towards glycogen
drate intake results in post-prandial hyperglycemia (Wilson, 1994; synthesis or lipogenesis. In the present study it seems that it was
Peres et al., 1999). Accordingly, results of the present study showed a mainly directed towards glycogen synthesis as the lipogenic enzyme
higher glycemia in sh fed 30 and 20% GS than 10% GS. Indeed, 6 h after G6PD activity was unaffected by dietary carbohydrate level. Moreover,
feeding, plasma glucose in the 10GS group was already near the basal data from whole-body composition showed no differences in whole-
glucose levels previously described for this species (Peres et al., 1999). body lipid content among groups. On the contrary, other studies
Our results cannot be explained by differences in starch digest- performed also with gilthead sea bream reported an increase of G6PD
ibility. Although data are not presented, apparent digestibility of starch activity as well as whole-body lipid content with increasing dietary
was very high in all diets (N99%) and it was not affected by water carbohydrate levels (Metn et al., 1999; Fernandez et al., 2007; Enes
temperature. Results on GS digestibility are similar to those of Enes et al., 2008a). Irrespective of temperature, amino acid catabolism
et al. (2008a) in the same species in sh fed up to 20% of raw or waxy showed a clear reduction with increasing dietary starch levels, as
starch. These results indicate that gilthead sea bream adapts, at the evidenced by GDH activities, suggesting a protein sparing by dietary
digestibility level, to high levels of dietary starch, independently of carbohydrates. However, these results conict with those of whole-
temperature. This lack of temperature effect on starch digestibility may body N retention. Other studies performed on this very same species
be explained either by the lower feed intake, and hence lower starch further observed a decrease of alanine aminotransferase (ALAT) ac-
intake at low temperatures, or by an adaptation of the carbohydrase tivity with increasing dietary carbohydrate (Metn et al., 1999;
activity or expression at different temperatures; this needs to be Fernandez et al., 2007; Enes et al., 2008a).
elucidated in further studies. In common carp, Shikata et al. (1995) observed that hepatopan-
At 25 C, the enhanced PK activity levels due to the higher starch creatic enzyme activities were higher in cold-acclimated sh than in
intake correlates to the glycemia levels observed in these groups. Such warm-acclimated sh, suggesting a thermal compensation for low
inducible enzymatic response may also contribute to explain the acclimation temperature. The lack of differences due to temperature in
increased liver glycogen levels and HSI observed in sh fed the higher activities of FBPase, G6PD and GDH observed in the present study also
starch level. Fernandez et al. (2007) in gilthead sea bream also seems to indicate an acclimation to low temperature in the activity of
reported higher PK activity in sh fed with high carbohydrate diets these enzymes, as also suggested by Ibarz et al. (2007). This however
and related these ndings to a protein sparing effect by carbohydrates. did not occur with the glycolytic enzymes, GK and PK, which were
However, this is not corroborated by the present data, as N retention more active at the higher temperature.
did not improve with an increase of dietary carbohydrate level. On the Overall, results of the present study indicate that increasing water
contrary, at high dietary starch level, there was even a decrease in N temperature enhances liver glycolytic capacities, suggesting that
retention. Contrary to PK, HK activity was unaffected by dietary starch gilthead sea bream is more apt to use dietary starch at higher rearing
level conrming that this enzyme is not under nutritional regulation temperatures. Furthermore, dietary starch enhanced PK activities
as already observed in this species (Enes et al., 2008a,b) as well as in while depressed protein catabolism. Gluconeogenesis was enhanced
other species (Panserat et al., 2000; Enes et al., 2006a,b). Somehow by high levels of dietary starch (30%) suggesting a lack of signicant
unexpectedly, dietary starch level did not affect GK activity. Indeed, regulation of hepatic glucose synthesis in this species. Our data
previous studies in sh, including gilthead sea bream (Panserat et al., corroborate previous results indicating that diets for gilthead sea
2000; Caseras et al., 2002; Enes et al., 2008a) showed an increase of bream should not include more than 20% carbohydrate.
hepatic GK activity with the increase of dietary carbohydrates
(Tranulis et al., 1996; Borrebaek and Christophersen, 2000; Panserat Acknowledgements
et al., 2000; Enes et al., 2006a).
Regarding temperature effect, GK and PK activities were higher in This work was partially supported by the Fundao para a Cincia e
the warm-acclimated sh, suggesting an enhanced glycolytic activity a Tecnologia, Portugal (project POCI-CVT-57695-2004). The second
at a higher temperature. A reection of these ndings is the lower author was supported by a grant (BD/11414/2002) from Fundao para
glycogen deposition in liver of sh reared at the higher temperature. a Cincia e a Tecnologia, Portugal. We would like to express our thanks
This is in line with the results of Hemre et al. (1995) in Atlantic to Mr. P. Correia for the assistance during the growth trial.
salmon who found a temperature-dependent glucose tolerance: sh
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