Effects of dietary organic chromium on gilthead seabream (Sparus aurata L.) performances and liver microsomal metabolism

Effects of dietary organic chromium on gilthead

seabream (Sparus aurata L.) performances and liver

microsomal metabolism

P P Gatta1, A Piva1, M Paolini2, S Testi1, A Bonaldo1, A Antelli2 & A Mordenti1

1Dipartimento di Morfo®siologia Veterinaria e Produzioni Animali, UniversitaÁ degli Studi di Bologna, Via Tolara di

Sopra, 50-40064 Ozzano Emilia, Bologna, Italy2Dipartimento di Farmacologia, UniversitaÁ degli Studi di Bologna, Via Irnerio, 48-40126 Bologna, Italy

Correspondence: P P Gatta, Dipartimento di Morfo®siologia Veterinaria e Produzioni Animali, UniversitaÁ degli Studi di Bologna, Via Tolara

di Sopra, 50-40064 Ozzano Emilia, Bologna, Italy. Tel: (+39) 051792995, Fax: (+39) 051792869, e-mail: [email protected]

Abstract

The effect of yeast and chromium yeast on gilthead

seabream (Sparus auratus L) performance, carcass

indices and body composition was studied. Whether

supplementation affected liver microsomal mixed

function oxidases using either multibioprobes (test-

osterone) or highly speci®c substrates to cytochrome

P450 (CYP) isoforms was also investigated.

Seabream juveniles (35±37 g initial weight) were

allocated into 12 800 L tanks of 50 ®sh each for

87 days and fed pelleted experimental diets, i.e.

control, yeast supplemented (1.6%) and chromium

yeast supplemented at both low (800 p.p.b.) and

high chromium level (53 810 p.p.b.). At the end of

the experiment, growth, feed conversion ratio,

thermal-unit growth coef®cient, carcass yield,

hepatosomatic index, and carcass and ®llet

proximate compositions were similar among

treatments and only condition factor was statistic-

ally different. Organic chromium at both doses

affected CYP-catalysed drug reactions slightly, as

shown by the modest effect on the regio- and

stereoselective hydroxylations of testosterone, as

well as the metabolism of the selected probes.

Overall, we found that chromium yeast did not

change performance substantially, nor carcass

indices, carcass and ®llet chemical compositions,

or hepatic xenobiotic metabolizing enzymes of

gilthead seabream.

Keywords: chromium, gilthead seabream,

nutrition, toxicology

Introduction

Over the last few decades, chromium has been

recognized as a new and important micronutrient,

essential in human (Jeejebhoy et al. 1977) and

animal nutrition (Shwartz & Mertz 1959), in¯uenc-

ing many aspects of metabolism (Mordenti, Piva &

Piva 1997). In particular, it has been shown to have

a positive in¯uence on the growth, reproductive

ef®ciency and carcass composition of pigs and cattle.

Furthermore, there is evidence that dietary chro-

mium supplementation can exert some bene®cial

effects on both the nonspeci®c and speci®c immune

systems of cattle (Mallard & Borgs 1997).

So far, several studies dealing with the effect of

chromium in ®sh have been related to its role in

carbohydrate utilization. Shiau & Lin (1993) found

a signi®cant improvement in weight gain, energy

deposition and liver glycogen content when tilapia

(Oreochromis niloticus L. 3 O. aureus L) juveniles

were fed a glucose-based diet supplemented with

chromium chloride. In the same experiment, no

differences were recorded when diets contained

starch instead of glucose as a carbohydrate source.

Another study (Shiau & Chen 1993), designed to

60 ã 2001 Blackwell Science Ltd

Aquaculture Research, 2001, 32 (Suppl. 1), 60±69

compare the in¯uence of different chromium

sources on tilapia (O. niloticus 3 O. aureus) carbo-

hydrate utilization, demonstrated a greater weight

gain in ®sh fed glucose diets supplemented with

chromium than those with an unsupplemented diet.

Hertz et al. (1989) were able to demonstrate that

chromium salts improved glucose utilization and

inhibited gluconeogenesis in common carp

(Cyprinus carpio L) juveniles. The ef®cacy of chro-

mium as a growth enhancer at certain dosages was

also reported by Tacon & Beveridge (1982) in trout

(Oncorhynchus mykiss Walbaum) and by Jain et al.

(1994) in Indian major carp (Labeo rohita

Hamilton).

On the other hand, con¯icting results have been

found when dietary chromium added as chromic

oxide was tested in ®sh. On the basis of an

experiment in tilapia (O. niloticus 3 O. aureus),

Shiau & Liang (1995) concluded that chromic

oxide alters glucose utilization and nutrient digest-

ibility in this ®sh, while Ng & Wilson (1997)

reported the opposite response in channel cat®sh

(Ictalurus punctatus Ra®nesque) fed diets containing

graded levels of supplemental chromic oxide.

Fernandez et al. (1999) came to the same conclu-

sion in their study of gilthead seabream (Sparus

aurata), but only about the in¯uence of chromic

oxide on organic compound utilization.

Most of the experiments carried out so far have

provided clear evidence that chromium exerts some

effect on the metabolism of ®sh, even though feeding

doses, regimes, chromium forms and ®sh species

differed among trials, and hence direct comparisons

among studies is dif®cult.

The form of the trace mineral used for dietary

supplementation is of particular concern. Recently,

Paripatananont & Lovell (1997) compared some

dietary trace elements fed both as organic and

inorganic forms, and found the former to be

absorbed more readily than the latter. Usually, the

source of trace mineral for feed supplementation is

the mineral salt and rarely as organic form where a

speci®c mineral is bound to an organic compound.

A typical group of organically bound is the yeast-

trace mineral. Because the yeast alone is considered

an ingredient with several nutritional effects in ®sh

(Rumsey et al. 1991; Sanderson & Jolly 1994;

Gatesoupe 1999), this is a further variable to

consider in conducting studies with yeast-trace

mineral.

Chromium can be found in several oxidation

states, some of them (exavalent chromium) with

possible toxic implications and hence any toxico-

logical effect in ®sh as a consequence of dietary

supplementation shall be ruled out. Recent studies

considered this heavy metal as a water pollutant

(Calamari & SolbeÂ 1994) instead of a dangerous

nutrient when overdosed.

As no data are currently available in the literature

about the in¯uence of organic chromium on the

metabolism in marine ®sh, the aim of the present

study was to assess whether dietary supplementation

with chromium yeast could modify performance,

carcass and ®llet composition, as well as some

biochemical markers related to liver microsomal

metabolism in juvenile gilthead seabream.

Materials and methods

Fish

Seabream juveniles (Table 1) supplied by Novagriter

Farm (Campomarino, Campobasso, Italy) were

allocated into 12 tanks of 50 ®sh each. A sample

of 35 ®sh from the initial group was anaesthetized,

killed and refrigerated for body measurements and

carcass proximate composition. Every group was

assigned randomly to a 800-L quadrangular tank

supplied with sea water in a ¯ow-through system

having a ¯ow rate of 60 L min±1. Water tempera-

ture (Fig. 1) ranged from 22.0 to 27.4 °C, except

the last week before the end of the experiment it

decreased to 18.8 °C. Dissolved oxygen ranged from

5.5 to 6.5 p.p.m. and water salinity from 35% to

36%. Water parameters, such as temperature and

dissolved oxygen, were monitored daily while

salinity was measured weekly. Each tank was

cleaned twice a week. After 1 week of adaptation,

®sh were fed experimental diets until the end of the

trial, which lasted 87 days. Each group of ®sh was

weighed three times during the experiment: at the

beginning, after 5 weeks and at the end. Each

treatment was fed to triplicate tanks of ®sh.

Diets and feeding regime

Experimental diets (Table 2) were produced using a

pelletizer machine (General Dies, Colognola ai Colli,

Verona, Italy) at the facilities of Dipartimento di

Morfo®siologia Veterinaria e Produzioni Animali

(DIMORFIPA, University of Bologna). Pellet dia-

meter was 3.0 mm. Except for control, experimental

diets were supplemented with 1.6% of yeast-based

products, such as Diamond XP (Y) supplied by

ã 2001 Blackwell Science Ltd, Aquaculture Research, 32 (Suppl. 1), 60±69 61

Aquaculture Research, 2001, 32 (Suppl. 1), 60±69 Dietary organic chromium and gilthead seabream P P Gatta et al.

Diamond V Mills (Cedar Rapids, Iowa, USA) or

chromium yeast supplied by Diamond V Mills and

Alltech to provide two dietary chromium levels, i.e.

800 mg kg±1 (low chromium yeast) and

53 810 mg kg±1 (high chromium yeast). Five

weeks after the beginning of the experiment, diet

formula was changed to slightly reduce protein

percentage (Table 1). Feeds were fed by hand four

times a day at a rate of 3 and 2.2% of biomass for

the ®rst (5 weeks) and the second period (7 weeks),

respectively. In case of ®sh satiation in one or more

tanks, the feeder stopped feeding ®sh and the

remaining feed was weighed in order to record

only the amount of eaten feed per tank.

Sampling and storage conditions

At the middle (5 weeks from the beginning) and the

end of the experiment, all ®sh per tank were

anaesthetized, caught and immediately weighed.

Fish observed 24 h of starvation before weighing. At

the end of the experiment, 20 ®sh per tank were

sampled randomly, packed into a polystyrene box

with a ¯ake-ice and transported to the DIMORFIPA,

where they were put into a refrigerator (1 °C). The

next day, each ®sh was handled to measure total

length, body weight, viscera weight and liver

weight. Five ®sh per tank were cut into small

pieces, pooled and homogenized with a La

Moulinette food processor (Moulinex, Milan, Italy)

twice for 10 s, while other ®ve ®sh per tank were

®lleted, skinned and the muscle from the right and

left sides of each ®sh were homogenized together.

All homogenized samples were stored at ±20 °C

until chemical analyses. Only whole-body pooled

samples were freeze-dried before analysis. Livers

from six ®sh per treatment were removed rapidly

and processed separately and the S9 fraction

Watertemperature

(¡C)

16.0

18.0

20.0

22.0

24.0

26.0

28.0

30.0

1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85

Day

Figure 1 Water temperature (°C) during the feeding trial.

Table 1 Performance and carcass indices of gilthead seabream fed the experimental diets for 87 days (mean 6 SD)

Control Yeast only

Low

chromium yeast

High

chromium yeast

Initial weight (g) 35.4 6 2.05 36.8 6 1.51 36.8 6 1.51 37.1 6 0.61

Final weight (g) 148.3 6 6.66 147.7 6 8.50 144.5 6 2.12 152.0 6 2.65

Intermediate growth (g)1 38.5 6 2.78 38.4 6 3.14 38.1 6 2.00 37.7 6 160

Final growth (g) 112.9 6 5.04 110.9 6 8.24 107.7 6 0.83 114.9 6 3.10

Intermediate FCR1 1.09 6 0.04 1.14 6 0.06 1.14 6 0.06 1.13 6 002

Final FCR 1.40 6 0.05 1.47 6 0.05 1.40 6 0.02 1.38 6 0.02

Intermediate TGC1 0.099 6 0.003 0.097 6 0.005 0.097 6 0.004 0.095 6 0.003

Final TGC 0.097 6 0.001 0.095 6 0.004 0.092 6 0001 0.097 6 0.002

CF (%) 1.81a 6 0.04 1.81a 6 0.03 1.74b 6 0.00 1.83a 6 0.02

CY (%) 92.86 6 0.84 92.84 6 1.07 92.66 6 0.76 92.90 6 0.67

HSI (%) 2.16 6 0.29 2.09 6 0.22 2.21 6 0.31 2.28 6 0.24

1Data obtained after 5 weeks.

Means within a row with different letters differ (P < 0.05). FCR, feed conversion ratio; TGC, thermal-unit growth

coef®cient; CF, condition factor; CY, carcass yield; HSI, hepatosomatic index. See Statistical analyses and calculations for

formulae.

62 ã 2001 Blackwell Science Ltd, Aquaculture Research, 32 (Suppl. 1), 60±69

Dietary organic chromium and gilthead seabream P P Gatta et al. Aquaculture Research, 2001, 32 (Suppl. 1), 60±69

(9000 g) was then prepared (Paolini et al. 1999a).

The postmitochondrial supernatant was then centri-

fuged for 60 min at 105 000 g, pellet resuspended

in 0.1 M K2P2O7, 1 mM EDTA (pH 7.4) and

centrifuged again for 60 min at 105 000 g to give

the ®nal fraction. Washed microsomes were then

resuspended with a hand-driven Potter Elvehjem

homogenizer in a 10-mM Tris-HCl buffer (pH 7.4)

containing 1 mM EDTA and 20% (v/v) glycerol;

fractions were immediately frozen in liquid nitrogen

and stored at ±80 °C prior to use.

Chemical analyses

Moisture, protein and ash were determined accord-

ing to AOAC methods (1990).

Total lipids were extracted by means of a solution

of chloroform/methanol 2 : 1 (v/v) (Folch, Lees &

Stanley 1957). Brie¯y, »5 g of sample was weighed

in a 300-mL Erlenmeyer conical ¯ask containing

100 mL of a chloroform/methanol solution and left

in a stirrer for 30 min. The content was then ®ltered

through a paper ®lter and the sample placed into

the ¯ask with 70 mL of a chloroform/methanol

solution and the procedure described previously was

followed. This operation was repeated once more

and the total amount of solvent was collected into a

spherical separator funnel. After the addition of

80 mL of a 2% NaCl water solution and the

separation into two phases, the lower phase was

collected into a glass ¯ask and evaporated in a

rotatory evaporator. Lipid content was determined

gravimetrically.

Crude ®bre was measured according to Martillotti

et al. (1987). Starch was determined spectro-

photometrically after enzymatic digestion into

glucose.

The concentration of total chromium in feed

samples was determined by inductively coupled

plasma atomic emission spectrometry after wet

digestion with nitric acid.

Gross energy of diets was determined using a

bomb calorimeter (Parr Instrument, Moline, IL,

USA).

Table 2 Diet chemical composition and chromium yeast content (I, ®rst period; II, second period)

Control Yeast only

Low

chromium yeast

High

chromium yeast

I II I II I II I II

Ingredients

Fish meal (%) 52.0 50.0 52.0 50.0 52.0 50.0 52.0 50.0

Soybean meal (%) 18.0 18.0 18.0 18.0 18.0 18.0 18.0 18.0

Fish oil (%) 13.0 13.0 13.0 13.0 13.0 13.0 13.0 13.0

Gelatinized starch (%) 11.9 13.9 10.3 12.3 10.3 12.3 10.3 12.3

Starch (%) 4.0 4.0 4.0 40 4.0 4.0 4.0 4.0

Yeast or chromium yeast (%) 0 0 1.6 1.6 1.6 1.6 1.6 1.6

Vitamin and mineral premix1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1

Analysed composition (dry matter basis)

Moisture (%) 3.26 5.49 4.96 6.00 2.53 6.33 2.40 5.51

Crude protein (%) 51.00 48.71 50.83 49.33 50.93 48.91 51.39 49.68

Crude lipid (%) 17.63 17.55 17.57 18.41 16.83 18.67 17.13 18.10

Crude ®bre (%) 1.61 ND 1.79 ND 1.82 ND 1.67 ND

Starch (%) 13.23 ND 13.44 ND 12.47 ND 12.28 ND

Ash (%) 8.37 7.89 8.35 8.15 8.61 8.06 7.77 8.05

Gross energy2 (kJ g±1) 22 447 ND 22 846 ND 22 534 ND 22 553 ND

Cr from Cr-yeast (mg kg±1) 0 ND 0 ND 800 ND 53 810 ND

1Provides per kg of premix: vitamin A, 1 600 000 IU; vitamin D3, 160 000 IU; vitamin E, 22 000 mg; vitamin K3,

368 mg; vitamin B1, 1280 mg; vitamin B2, 1280 mg; vitamin B6, 2450 mg; vitamin B12, 1.2 mg; ascorbic acid, 30 g;

vitamin PP, 7850 mg; inositol, 6000 mg; D-calcium pantothenate, 13720 mg; folic acid, 368 mg; biotin, 20 mg; zinc

sulphate, 11 375 mg; manganese sulphate, 4800 mg; copper sulphate, 500 mg; potassium iodide, 272 mg.

ND, not determined.2Value obtained using a bomb calorimeter.



Aminopyrine N-demethylase activity

Activity was determined by quanti®cation of CH2O

release, according to Mazel (1971). The total

incubation volume was 3 mL, composed of 0.5 mL

of a water solution of 50 mM aminopyrine and

25 mM MgCl2, 1.48 mL of a 0.60-mM NADP+,

3.33 mM G6P in 50 mM Tris-HCl buffer (pH 7.4),

0.02 mL G6PDH (0.93 U mL±1) and 0.125 mL of

sample (0.5 mg of protein). After 5 min of incuba-

tion at 37 °C, the yellow colour developed by the

reaction of the released CH2O with the Nash reagent

was read at 412 nm, and the molar absorption of

8000 used for calculation (Nash 1953).

p-Nitrophenol hydroxylase activity

Activity was determined in a ®nal volume of

2 mL : 2 mM p-nitrophenol in 50 mM Tris-HCl

buffer (pH 7.4), 5 mM MgCl2 and a NADPH-

generating system consisting of 0.4 mM NADP+,

30 mM isocitrate, 0.2 U of isocitrate dehydrogenase

and 1.5 mg of proteins. After 10 min at 37 °C, the

reaction was terminated by addition 0.5 mL of a

0.6 N perchloric acid. Precipitated proteins were

removed by centrifugation and 1 mL of resultant

supernatant mixed with 1 mL 10 N NaOH.

Absorbance at 546 nm was immediately measured

and 4-nitrocatechol determined (e = 10.28 mM±1

cm±1) (Reinke & Mayer 1985).

Pentoxyresoru®n O-dealkylase,

ethoxyresoru®n O-deethylase

Reaction mixture consisted of 0.025 mM MgCl2,

200 mM pentoxyresoru®n, 0.32 mg of proteins and

130 mM NADPH in 2.0 mL 0.05 M Tris-HCl buffer

(pH 7.4). Resoru®n formation at 37 °C was calcu-

lated by comparing the rate of increase in relative

¯uorescence to the ¯uorescence of known amounts

of resoru®n (excitation at 562 nm, emission at

586 nm) (Lubet et al. 1985). Ethoxyresoru®n O-

deethylase was measured in the same manner

described for the pentoxyresoru®n assay, except

that substrate concentration was 1.7 mM ethoxyr-

esoru®n (Burke et al. 1985).

Ethoxycoumarin O-deethylase activity

Activity was determined by quanti®cation of

umbelliferone formation, according to Aitio

(1978). Incubation mixture consisted of 2.6 mL,

composed of 1 mM ethoxycoumarin, 5 mM MgCl2,

NADPH-generating system (see aminopyrine assay)

and 25 mL of sample (0.1 mg of proteins). After

5 min of incubation at 37 °C, reaction was stopped

with 85 mL of 0.31-M Trichloroacetic acid (TCA).

The pH of the mixture was brought to about 10 by

adding 0.65 mL of 1.6 M NaOH-glycine buffer

(pH 10.3). The amount of umbelliferone was

measured ¯uorimetrically (excitation at 390 nm;

emission at 440 nm).

Testosterone hydroxylase activity

Incubations contained liver microsomes (equivalent

to 1±2 mg protein), 0.6 mM NADP+, 8 mM glucose

6-phosphate, 1.4 U glucose 6-phosphate dehydro-

genase and 1 mM MgCl2, in a ®nal volume of 2 mL

of 0.1-M phosphate Na+/K+ buffer (pH 7.4). The

mixture was preincubated for 5 min at 37 °C. The

reaction was performed at 37 °C by shaking and

started by the addition of 80 mM testosterone

(dissolved in methanol). After 10 min, the reaction

was stopped with 5 mL ice-cold dichloromethane

and 12 nmol corticosterone (internal standard) in

methanol. After 1 min of vortexing, phases were

separated by centrifugation at 2000 g for 10 min

and the aqueous phase was extracted once more

with 2 mL dichloromethane. The organic phase was

extracted with 2 mL 0.02 M NaOH to remove lipid

constituents, dried over anhydrous sodium sulphate

and transferred to a small tube. Dichloromethane

was evaporated at 37 °C under nitrogen and the

dried samples stored at ±20 °C. The samples were

dissolved in 100 mL methanol and analysed by

HPLC (Platt et al. 1989).

HPLC chromatographic separations were per-

formed using a system consisting of a high-pressure

pump (model 600E, multisolvent delivery system;

Waters, Milford, MA, USA), a sample injection valve

(Rheodyne Model 7121, Cotati, CA, USA) with a 20-

mL sample loop and an ultraviolet detector (254 nm;

model 486, tunable absorbance detector, Waters)

connected to an integrator (chromatography man-

ager; Millennium 2010, Milford, MA, USA). For

reversed-phase separation of metabolites, a NOVA-

PAK C18 analytical column (60 AÊ , 4 mm,

3.9 3 150 mm, Waters) was used as stationary

phase. The mobile phase consisted of a mixture of

solvent A (7.5% v/v tetrahydrofuran in water) and

solvent B (7.5% v/v tetrahydrofuran and 60% v/v

methanol in water) at a 1-mL min±1 ¯ow rate.

Metabolite separation was performed by a gradient



from 30 to 100% (v/v) of solvent B over 30 min The

eluent was monitored at 254 nm and the area under

the absorption band was integrated. The concentra-

tion of metabolites was determined by the ratio

between respective metabolite peak areas and

corticosterone (internal standard), and the calibra-

tion curves obtained with synthetic testosterone

derivatives (van Der Hoeven 1984; Paolini et al.

1997).

Protein concentration

Protein concentration was determined according to

the method described by Lowry et al. (1951) and

revised by Bailey (1967), using bovine serum

albumin as a standard and diluting samples 200

times to provide a suitable protein concentration.

Statistical analyses and calculations

All results on growth performance, body and carcass

measurement and proximate analyses were analysed

statistically using ANOVA, while toxicological effects

were assessed by means of Wilcoxon's rank methods,

as reported by Box & Hunter (1967). The software

used was Sigma Plot 5.0 (London, UK). Signi®cance

level was P < 0.05.

The following formulae were used:

FCR (feed conversion ratio) = feed per ®sh/

weight gain per ®sh

CF (condition factor) = (body weight/

total length3) 3 100

CY (carcass yield) = degutted ®sh 3 100/

body weight

HSI (hepatosomatic index) = liver weight 3

100/body weight

Thermal-unit growth coef®cient (TGC) =

100 3 (®nal body weight1/3 ± initial body

weight1/3)/S(temperature [°C] 3 days)

Results

Regardless of the source and dose of dietary

chromium as well as of yeast supplementation, no

differences were detected among treatments on

seabream growth, FCR and TGC (Table 1). The

same pattern resulted for carcass indices, i.e. carcass

yield and hepatosomatic index, while only condition

factor was statistically different among treatments

(Table 1), suggesting differences in the chemical

composition of ®sh. However, carcass and ®llet

proximate composition (Tables 3 and 4) did not

con®rm this hypothesis and were very similar

between groups without statistical differences.

Table 5 shows the effect of 800 or 53 810 p.p.b.

chromium yeast in the feed on liver microsomal

cytochrome P450 (CYP) machinery of gilthead

seabream. Yeast alone did not affect at any of the

selected monooxygenases. While the N-demethyl-

ation of aminopyrine and the hydroxylation of

p-nitrophenol were unchanged by dietary treat-

ment, a slight decrease (21 to 36%, at higher and

lower doses, respectively) in the deethylation of

ethoxycoumarin was seen at both doses tested. A

signi®cant (P < 0.01) but modest increase in the

dealkylation of pentoxyresoru®n (up to 45%) and a

decrease (up to 63%) in the deethylation of

ethoxyresoru®n were measured.

In Table 6, the effects of chromium yeast on

testosterone metabolism in gilthead seabream hepa-

tic subcellular preparations is shown. No differences

between the control (untreated) and yeast-treated

groups were seen. On the contrary, with the

exception of testosterone 7a- and 6b-hydroxylase

activities, which were substantially unaffected by

either lower or higher organic chromium dose,

some appreciable differences in the hydroxylation of

the other testosterone positions were observed

among various experimental conditions. For

example, an inactivating effect was recorded for

the testosterone 6a- (up to 31% loss, lower dose),

16a- (29 and 26% loss, lower and higher doses,

Table 3 Fillet proximate composition (mean 6 SD)

Control Yeast only

Low

chromium yeast

High

chromium yeast

Moisture (%) 70.13 6 1.20 70.08 6 0.82 69.59 6 1.35 70.04 6 0.95

Protein (%) 20.17 6 0.46 20.55 6 0.64 20.60 6 0.37 19.74 6 0.56

Lipid (%) 6.80 6 1.16 6.38 6 1.09 7.16 6 1.27 7.28 6 0.99

Ash (%) 1.54 6 0.06 1.59 6 0.05 1.53 6 0.10 1.54 6 0.07



Table 5 Effect of dietary treatments on gilthead seabream (Sparus aurata L) liver in microsomal mixed function

monooxygenase. Each value represents the mean 6 SD of six independent experiments

Control Yeast only

Low

chromium yeast

High

chromium yeast

Monooxygenases

Aminopyrine N-demethylase (nmol mg±1 min±1) 1.28 6 0.13 1.36 6 0.18 1.04 6 0.27c 0.90 6 0.08d

p-Nitrophenol hydroxylase (nmol mg±1 min±1) 0.16 6 0.02 0.14 6 0.01 0.11 6 0.01 0.16 6 002c

Ethoxycoumarin O-deethylase (nmol mg±1 min±1) 0.14 6 0.02 0.15 6 0.03 0.09 6 0.01b,d 0.11 6 0.01a,c

Ethoxyresoru®n O-deethylase (pmol mg±1 min±1) 1.00 6 0.11 1.03 6 0.09 0.37 6 0.03b,d 0.37 6 0.02b,d

Pentoxyresoru®n O-dealkylase (pmol mg±1 min±1) 0.22 6 0.02 0.21 6 0.03 0.32 6 0.02b 0.47 6 0.05b,d

1Signi®cant differences between treated groups and their respected controls (untreated) using Wilcoxon's rank method

(P < 0.05).2Signi®cant differences between treated groups and their respected controls (untreated) using Wilcoxon's rank method

(P < 0.01).3Signi®cant differences between treated groups and their respected controls (yeast) using Wilcoxon's rank method

(P < 0.05).4Signi®cant differences between treated groups and their respected controls (yeast) using Wilcoxon's rank method

(P < 0.01).

Table 4 Carcass proximate composition (percentage dry weight) (mean 6 SD)

Control Yeast only

Low

chromium yeast

High

chromium yeast

Protein (%) 45.87 6 0.97 46.54 6 2.07 46.03 6 1.87 46.55 6 0.80

Lipid (%) 38.77 6 1.00 37.81 6 2.07 38.36 6 1.35 38.29 6 0.89

Ash (%) 9.67 6 0.46 9.88 6 0.71 9.81 6 0.82 9.40 6 0.40

Table 6 Testosterone hydroxlase in gilthead seabream (Sparus aurata) liver microsomes). Each value represents the

mean 6 SD of six independent experiments

Control Yeast only

Low

chromium yeast

High

chromium yeast

Linked monooxygenases

6a-Hydroxytestosterone (pmol mg±1 min±1) 5.92 6 0.24 5.13 6 0.56 4.10 6 0.482,3 4.31 6 0.512,3

7a-Hydroxytestosterone (pmol mg±1 min±1) 2.07 6 0.12 1.97 6 0.08 1.73 6 0.154 2.06 6 0.20

6b-Hydroxytestosterone (pmol mg±1 min±1) 13.47 6 0.77 14.39 6 1.68 10.47 6 1.711,2 13.27 6 089


16b-Hydroxytestosterone (pmol mg±1 min±1) 1.71 6 0.21 1.58 6 0.14 1.01 6 0.172,3 0.86 6 0.092,4


2b-Hydroxytestosterone (pmol mg±1 min±1) 17.33 6 2.11 15.25 6 0.94 152 6 0.092,4 1.27 6 0.052

Androst-4-ene-3,17-dione (nmol mg±1 min±1) 1.19 6 0.03 1.09 6 0.14 0.79 6 0.042,4 0.89 6 0.083

1Signi®cant differences between treated groups and their respected controls using Wilcoxon's rank method (P < 0.05).2Signi®cant differences between treated groups and their respected controls using Wilcoxon's rank method (P < 0.01).3Signi®cant differences between treated groups and their respected controls using Wilcoxon's rank method (P < 0.05).4Signi®cant differences between treated groups and their respected controls using Wilcoxon's rank method (P < 0.01).



respectively), 16b- (41 and 50% loss, lower and

higher doses, respectively), 2a- (65% loss, lower

dose) and 2b- (93 and 91% loss, higher and lower

doses, respectively) -hydroxylase activities. Androst-

4-ene-3,17-dione-linked monooxygenase activity

was also reduced by chromium (25 and 34% loss,

higher and lower doses, respectively).

Discussion

Growth, feed conversion ratio and thermal-unit

growth coef®cient exhibited good responses to diets,

despite the fact that they contained a considerable

amount of plant protein and a medium lipid level

(17±18%). Gilthead seabream is a marine species

well suited to utilize dietary plant proteins, at least

to a certain level without any remarkable reduction

in performance (Robaina et al. 1995).

The inclusion of yeast in the diets yielded little

effect on feed performance. A previous study

reported a positive in¯uence on growth and feed

conversion ratio in trout fed semipuri®ed diets

containing brewer's dried yeast (Rumsey et al.

1991) at the rate of 25% of the diet, a level well

beyond the 1.6% used in this experiment. Therefore

higher yeast inclusion into the diet may be

necessary to affect gilthead seabream performance.

Assuming that chromium is a component of the

glucose tolerance factors increasing insulin activity

as in mammals (Mordenti et al. 1997), its role in ®sh

nutrition could be related to the actions of this

hormone. Duguay & Mommsen (1994) describe

insulin as the major anabolic hormone in ®sh,

which stimulates the uptake of glucose and amino

acids by skeletal muscle and liver, and increases the

rate of protein synthesis in these tissues. Jobling

(1994) differentiates the insulin activity in mam-

mals and in ®sh where it appears to be more

important in protein than in carbohydrate metabo-

lism. Those ®ndings could explain the growth-

promoting effect found in ®sh fed diets supplemen-

ted with chromium, even though little is known on

how it acts on ®sh metabolism. Most of the

experiments showing an effect of dietary chromium

involved herbivorous or omnivorous ®sh species

where carbohydrate digestion is more important

than in carnivorous ®sh. Tacon & Beveridge (1982)

studied dietary trivalent chromium (chromium

chloride) in trout feeds, but those data should be

used carefully because a single group of ®sh per

dietary treatment was employed and also they fed

semipuri®ed diets containing a low lipid level (12%).

A slightly but not signi®cant increase in growth and

a reduced feed conversion ratio was achieved in ®sh

fed 1 mg chromium kg diet±1. Only Fernandez et al.

(1999) reported an experiment carried out in

gilthead seabream; they supplemented experimental

diets with chromic oxide and found this form to be

neutral in relation to the utilization of organic

compounds, but able to modify the utilization of

mineral salts. In this case it should be noted that

dietary lipid level was quite low and hence the

protein/lipid ratio and the whole energy content

were not as high as usually found in commercial

diets. Chromic oxide was shown to be ineffective, as

it was not absorbed from the gut. When other

chromium forms are included in diets, assuming

their higher availability, the ef®cacy of chromium

on ®sh performances may be related to the dietary

nutrient concentration, having a greater effect

when higher protein and lipid contents are present.

In this scenario, we could speculate that all diets

tested in the present study may not be concentrated

suf®ciently for chromium to have measurable effect.

Concerning the in¯uence of chromium on hepatic

microsomal CYP biochemistry, here we show that

chromium treatments did not substantially change

CYP-linked monooxygenases in gilthead seabream

hepatic subcellular preparations. Indeed, with the

exception of the androst-4-ene-3,17-dione-asso-

ciated mixed function oxidases, which were reduced

to about 34% at the lower dose tested, the selected

CYP-dependent oxidases were affected only slightly

by chromium yeast. This is particularly true if we

remember that the observed changes were indepen-

dent of the dose employed. More precisely, even

though some alterations in CYP-catalysed drug

reactions found in the present investigation were

statistically signi®cant, they should not be of great

biological relevance. It should be taken into account

that the difference of the microsomal monooxygen-

ase units would be considered in evaluating the

biological outcomes linked to CYP modulation by

xenobiotics; while a twofold induction, for example,

in the N-demethylation of aminopyrine from 5 to

10 nmol mg±1 min±1 represents a `net' increase of

5000 pmol mg protein±1 and time unit, the

corresponding (twofold) induction of activities,

such as the O-deethylation of ethoxyresoru®n from

5 to 10 pmol mg±1 min±1, represents instead a net

increase of 5 pmol mg±1 min±1 only (Paolini et al.

1999b).

Taken as a whole, dietary yeast or chromium

yeast did not improve gilthead seabream perform-



ance, carcass indices, or carcass and ®llet chemical

compositions, at least at the levels used in this

study. Some hepatic xenobiotic metabolizing

enzymes were modi®ed slightly by chromium

treatments regardless of dose supplemented.

Further studies are necessary to clarify if different

dosages and longer-term feeding could affect gilt-

head seabream performances and microsomal

metabolism.

Acknowledgments

The authors would like to thank Giorgia Bignami,

Margherita Drudi and the DIMORFIPA laboratory

staff for their work, as well as Signor Agostino De

Fenza (Novagriter Fish Farm). The study was

supported by a joint grant from Ministero

dell'UniversitaÁ e Ricerca Scienti®ca e Tecnologica

(providing 40% and 60% of funds) and the

University of Bologna.

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Effects of dietary organic chromium on gilthead seabream (Sparus aurata L.) performances and liver microsomal metabolism