Available online at www.sciencedirect.com

008) 154–161www.elsevier.com/locate/aqua-online

Aquaculture 276 (2

Effects of replacement of dietary fish oil by soybean oil on growthperformance and liver biochemical composition in juvenile

black seabream, Acanthopagrus schlegeli

Shiming Peng a, Liqiao Chen a,⁎, Jian G. Qin b, Junli Hou a, Na Yu a,Zhangqiang Long a, Jinyun Ye c, Xinjin Sun a

a School of Life Science, East China Normal University, Shanghai, 200062, Chinab School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia

c Zhejiang Institute of Freshwater Fisheries, Huzhou, Zhejiang, 313001, China

Received 20 November 2007; received in revised form 23 January 2008; accepted 24 January 2008

Abstract

A 9-week feeding experiment was carried out on juvenile black seabream Acanthopagrus schlegeli to evaluate the effects of dietaryreplacement of fish oil by soybean oil on fish growth and liver biochemical composition. Fish in triplicate were fed four diets, in which 0% (FO ascontrol), 60% (60SO), 80% (80SO) and 100% (100SO) of fish oil was replaced by soybean oil. The weight gain of fish fed 60SO or 80SO diet wassimilar to that of fish fed the control diet, but a total replacement of fish oil by soybean oil significantly reduced fish growth. Although theinclusion of soybean oil resulted in an increase in the crude lipid content of the liver, the level of fish oil replacement did not significantly alter thehepatosomatic index, feed conversion ratio, condition factor and liver proximate composition. The inclusion of soybean oil in seabream dietsincreased hepatic α-tocopherol concentrations, but reduced thiobarbituric acid-reactive substances and plasma cholesterol. Linoleic acid andlinolenic acid significantly increased in fish fed soybean oil diets, but docosahexaenoic acid, eicosapentaenoic acid and the ratio n−3/n−6 weresignificantly reduced by the inclusion of dietary soybean oil (Pb0.05). Our results indicated that the inclusion of soybean oil increased the hepaticα-tocopherol content and reduced lipid peroxidation in fish. However, complete substitution of fish oil with soybean oil reduced growth efficiency.Thus, 60–80% replacement of fish oil by soybean oil is recommended in diet formulation for black seabream.© 2008 Elsevier B.V. All rights reserved.

Keywords: Black seabream; Acanthopagrus schlegeli; Fish oil replacement; Soybean oil; Growth; Biochemical composition

1. Introduction

Dietary lipids play an important role as a source of energy forfish growth and as carriers for fat soluble vitamins. Fish oilcontains high quantities of n−3 HUFA and other essential fattyacids (EFA) necessary for marine fish (Sargent and Tacon,1999). They serve as a functional element maintaining metab-olism and contain attractants that enhance diet palatability. Thedemand for fish oils in aquafeeds has dramatically increased in

⁎ Corresponding author. School of Life Science, East China NormalUniversity, Shanghai, 200062, China. Tel./fax: +86 21 62233637.

E-mail address: lqchen@bio.ecnu.edu.cn (L. Chen).

0044-8486/$ - see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.aquaculture.2008.01.035

the last decade (Barlow, 2000) and has placed unsustainablepressure on this finite resource (Tacon, 2004). Thus, the partialreplacement of fish oils with vegetable oils in artificial feeds hasgained increasing interest from aquaculturists (Caddy, 1999;Valdimarsson and James, 2001).

Aquaculture has continued to grow more rapidly than allother animal food-producing sectors with a global averagegrowth rate of 8.8% per year since 1970 (FAO). Current pro-jections anticipate that in a few years, global fish oil productionmay not be enough to supply the increasing demand of animalfeed. On the contrary, production of global vegetable oil hassteadily increased in recent years, reaching a volume of 100times more than fish oil (Bimbo, 1990). Therefore, replacement

Table 1Ingredient and proximate composition of the experimental diets

Experimental diets

FO a 60SO 80SO 100SO

Ingredients (g/kg)Fish meal 350 350 350 350Soybean meal 400 400 400 400Wheat flour 129.3 129.3 129.3 129.3L-methionine 2 2 2 2L-lysine 8 8 8 8Anchovy oil 90 36 18 0Soybean oil 0 54 72 90Vitamin premix b 10 10 10 10L-ascorbic acid b 0.5 0.5 0.5 0.5DL-α-tocopherol acetate b 0.2 0.2 0.2 0.2Mineral premix c 10 10 10 10

Proximate compositionCrude protein (% DM) 45.26 45.29 45.37 45.94Crude fat (% DM) 14.95 15.36 15.16 15.22Ash (% DM) 11.74 11.97 11.92 11.03α-tocopherol (mg/kg) 107.41 106.03 105.59 105.27

a FO = 100% fish oil; 60SO = 60% soybean oil; 80SO = 80% soybean oil;100SO = 100% soybean oil.b Supplied (mg kg−1 diet): myo-inositol, 400; nicotinic acid, 150; calcium

pantothenate, 44; riboflavin, 20; pyridoxine hydrochloride, 12; menadione, 10;thiamine hydrochloride, 10; retinyl acetate, 7.3; folic acid, 5; biotin, 1;cholecalciferol, 0.06; cyanocobalamin, 0.02. L-ascorbic acid: 93% AA activity;DL-α-tocopherol acetate: 50% vitamin E activity.c Supplied (kg−1 diet): KH2PO4, 22 g; FeSO4·7H2O, 1.0 g; ZnSO4·7H2O,

0.13 g; MnSO4·4H2O, 52.8 mg; CuSO4·5H2O, 12 mg; CoSO4·7H2O, 2 mg; KI,2 mg.

155S. Peng et al. / Aquaculture 276 (2008) 154–161

of fish oil with vegetable oils appears to be a viable option giventheir availability, low cost and absence of dioxins and pollutants(Caballero et al., 2002; Izquierdo et al., 2003).

A key requirement for the replacement of fish oil in aqua-feeds is to supply equivalent energy with balanced essentialfatty acids. This is necessary in order to sustain high growth andsurvival, feed conversion efficiency, immune competence, dis-ease resistance, and flesh quality. However, the lack of balancedfatty acid profiles (Sargent et al., 2002), low palatability(Guillou et al., 1995) and digestibility (Caballero et al., 2002)may hinder the practice of fish oil replacement by vegetable oilsin fish diets. Despite this drawback, a total replacement of fishoil by vegetable oil has been successfully achieved in turbotPsetta maxima (Regost et al., 2003) and partial replacementhas been reported in numerous fish species. For instance, up to60% fish oil was successfully replaced by vegetable oil injuvenile European sea bass Dicentrarchus labrax L. (Monteroet al., 2005; Mourente et al., 2005a) and gilthead sea breamSparus aurata (Izquierdo et al., 2005). One side effect of in-cluding vegetable oils is that it alters the n−3/n−6 fatty acidratio and interferes with eicosanoid synthesis (Fracalossi et al.,1994; Sargent et al., 2002). This can lead to decreased growthperformance and has been observed as a result of 80% fish oilsubstitution in European sea bass (Montero et al., 2005) andgilthead sea bream diets (Montero et al., 2003; Izquierdo et al.,2005).

In the past, most studies on fish oil replacement have focusedon the growth response to the addition of vegetable oil in variousfreshwater fish species such as rainbow trout Oncorhynchusmykiss (Fonseca-Madrigal et al., 2005) and Atlantic salmonSalmo salar L. (Bell et al., 2001; Torstensen et al., 2004), andmarine fish species such as gilthead sea bream S. aurata(Caballero et al., 2003; Montero et al., 2003; Izquierdo et al.,2003, 2005), European sea bassD. labrax (Montero et al., 2005;Mourente et al., 2005a), grouper Epinephelus malabaricus (Linand Shiau, 2007), sharpsnout seabream Diplodus puntazzo(Piedecausa et al., 2007) and turbot P. maxima (Bell et al., 1995;Regost et al., 2003). It is therefore necessary to investigate thefunctional response of fish to the supplement of dietary lipids.

The liver plays a central role in lipid metabolism includingfatty acid synthesis and degradation through enzyme regula-tions, and it is also a sensitive organ reflecting dietary lipidchange in fish (Kiessling and Kiessling, 1993; Henderson,1996). The functional response of red drum, Sciaenops ocellatusto imbalanced dietary fatty acids has been detected throughmonitoring the change of biochemical composition in the liver(Craig et al., 1999). The liver cells can be functionally damagedby lipid peroxidation when the diet contains a high level ofunsaturated animal fatty acids (Kanazawa, 1993; Mates et al.,1999). In contrast, the use of vegetable oils has been reported toreduce lipid peroxidation in mammals (Lopez-Bote et al., 1997)and fish (Stephan et al., 1995; Alvarez et al., 1998). For instance,50% fish oil replacement by corn oil in grouper E. malabaricusdiets reduced peroxidation in the liver (Lin and Shiau, 2007).Therefore, the response of fat deposition and peroxidation in theliver should be considered a physiological indicator whenevaluating fish oil replacement by other lipid alternatives.

Black seabream Acanthopagrus schlegeli is a valuable com-mercial species for aquaculture in many parts of Asia (Changand Yueh, 1990). The objective of this study was to investigatethe response of black seabream to partial and total replacementof dietary fish oil with a soybean oil alternative. In this study,fish performance was evaluated through conventional variablessuch as growth, survival and food conversion ratio. In addition,the response of proximate body composition of juvenile blackseabream to dietary lipid manipulation was also examined.More importantly, we further measured the responses of thio-barbituric acid-reactive substances (TBARS), an index of lipidperoxidation and oxidative stress, α-tocopherol, an antioxidant,in liver cells, and triglycerides and cholesterol in blood plasmato evaluate the possible oxidative and physiological stress offish subjected to various levels of vegetable oil in the diet. Thisstudy will provide evidence and explanation for fish adaptationto dietary lipid manipulation.

2. Materials and methods

2.1. Experimental diets

Four iso-nitrogenous, iso-energetic, iso-lipidic experimental diets were for-mulated with 15% lipid derived from the following combinations: (1) 100% fishoil as control (FO), (2) 60% soybean oil and 40% fish oil (60SO), (3) 80%soybean oil and 20% fish oil (80SO), (4) 100% soybean oil (100SO) (Table 1).The fatty acid compositions of experimental diets were given in Table 2.

Table 2Fatty acid profiles of experimental diets (% of total fatty acids)

Fatty acids Experimental diets

FO 60SO 80SO 100SO

14:0 6.51 3.65 2.52 1.6716:0 24.27 18.56 16.75 15.07Saturates 41.28 30.06 26.09 22.8116:1 7.43 3.65 2.50 1.3618:1n9 16.63 17.63 17.88 17.89MUFA 26.32 22.63 20.62 19.4318:2n6 7.41 30.87 39.50 46.2818:3n6 0.51 0.46 0.45 0.4320:3n6 3.24 1.69 1.14 0.70n−6 PUFA 11.16 33.06 41.09 47.4118:3n3 2.23 4.47 5.34 6.0520:5n3 7.69 3.98 2.87 1.8622:6n3 10.97 5.56 3.91 2.37n−3 PUFA 20.89 14.01 12.12 10.28n−3/n−6 1.87 0.42 0.29 0.22n−3 HUFA 18.67 9.55 6.78 4.23

Table 3Growth performance of black seabream following feeding on experimental diets(mean±SE, n=3)

Dietarytreatments

FO 60SO 80SO 100SO

Weightgain (%) a

307.54±11.93 b 313.49±8.92 b 270.90±9.78 a b 220.97±7.35 a

DGI b 2.63±0.10 b 2.61±0.05 b 2.34±0.06 a b 2.03±0.05 a

FCRc 1.09±0.03 1.11±0.05 1.12±0.06 1.32±0.14HSI (%) d 3.02±0.07 3.09±0.19 3.13±0.41 3.20±0.15CF (%) e 3.01±0.08 2.93±0.05 2.87±0.06 2.90±0.06Survival(%) f

87.5±1.79 87.14±2.85 85.71±3.57 86.14±2.00

Different superscript letters within each row represent significant differences(Pb0.05).a Weight gain=100×(final weight− initial weight) / (initial weight).b DGI, daily growth index=100×((final body weight)1/3− (initial body

weight)1/3) / days.c FCR, feed conversion ratio=dry feed weight/wet weight gain.d HSI, hepatosomatic index=100×(liver weight) / (total fish weight).e CF, condition factor=100×final weight (g) / (fork length (cm))3.f Survival=100×(initial fish number−dead fish number) / (initial fish number).

156 S. Peng et al. / Aquaculture 276 (2008) 154–161

2.2. Fish maintenance and experimental design

Black seabream (A. schlegeli) juveniles were obtained from a local fish farm.Prior to the experiment, all fish were acclimatized to the environmentalcondition by feeding the FO diet for 2 weeks. A total of 360 fish (mean weight±S.E., 20.26±0.22 g) were randomly assigned to twelve 800-l fiberglass tankswith 30 fish each and the four experimental diets were randomly assigned to thetanks with three replicates. Fish were fed twice daily at approximately 08:00 and16:00 h to apparent satiation and the experiment was conducted over 9 weeks.Photoperiod was set at 12 h light/12 h dark and experimental tanks weresupplied with seawater (26‰ salinity) at a flow through rate of 10 l/min withaeration. Throughout the experiment, water temperature and dissolved oxygenwere maintained between 26.0–28.0 °C and 6.1–7.3 mg/l respectively whileammonia and nitrate remained below 0.1 mg/l.

2.3. Fish performance and sample collection

At the completion of the 9-week trial, feeding was stopped 24 h beforeharvesting. Fish were anaesthetized with MS 222 (100 μg/ml) before weightsand lengths were determined. Body length was measured to the nearest 0.1 mm.Weights of fish and liver were determined by blotting dry on filter paper beforeweighing. Hepatosomatic index (HSI) was calculated by dividing the liverweight by the total fish weight. Mortality was estimated from the number of fishstocked and harvested.

Blood samples were taken from the caudal vessel with heparinized syringesfrom five randomly selected fish in each tank and the liver and white muscleswere separately extracted from these fish after blood was taken. Fish from eachtank were pooled for further analyses. The remaining fish in each tank wereground and pooled for determining the whole body proximate composition. Theextracted livers, white muscle and the fish were stored at −70 °C until analysis.Plasma was drawn after centrifugation at 3000×g for 20 min at 4 °C, and storedat −70 °C until further analyses.

2.4. Chemical analysis

2.4.1. Moisture, ash, protein and lipid determinationsThe analyses of proximate composition on feed ingredients, experimental

diets and fish tissues were performed by the standard methods of AOAC (1995).Samples of white muscle, liver and whole body were dried to a constant weightat 105 °C to determine moisture. Protein was determined by measuring nitrogen(N×6.25) using the Kjeldahl method. Lipids were extracted by ether usingSoxhlet, and ash was combusted at 550 °C.

2.4.2. Plasma protein, triacylglycerol and cholesterol contentsProtein and triacylglycerol (TAG) in plasma and total cholesterol were

analyzed on the Technicon RA-1000 clinical analyser system (Bayer, Germany)according to the standard Technicon methods (Sandnes et al., 1988).

2.4.3. Hepatic α-tocopherol and thiobarbituric acid-reactive substances(TBARS)

Vitamin E concentrations (α-tocopherol) were measured in livers usingreverse phase high performance liquid chromatography (Lewis and Lall, 2007).Thiobarbituric acid-reactive substances (TBARS) were measured using themethod of Rueda-Jasso et al. (2004) and the results were expressed as nmol ofMDA/mg protein. The protein of liver homogenates was determined by themethod of Lowry et al. (1951).

2.4.4. Lipid extraction and fatty acid analysisTotal lipid was extracted using chloroform: methanol (2:1, v/v) according to

the method of Folch et al. (1957). The capillary gas chromatography (GC)method was employed to determine the fatty acid profile. The HP6890 (FIDdetector) and SPTM-2380 column (30 m×0.25 mm×0.20 ìm) were used on theGC machine. Separation was carried out with nitrogen as the carrier gas. Thecolumn temperature was programmed from 140 to 240 °C at 4 °C /min, held for5 min at 140 °C and 10 min at 240 °C, with a detector at 260 °C. A split injector(50:1) at 260 °C was used. Fatty acids were identified by the comparison of theirretention time to a chromatographic Sigma standard. Peak areas were deter-mined using Varian software.

2.5. Statistical analysis

Data were analyzed using one-way ANOVA (SPSS Inc., Chicago, IL, USA).Tukey test was applied as a multiple sample comparison when significant maineffect was detected. The level of significant difference was set at Pb0.05.

3. Results

3.1. Growth performance

All diets were well accepted by black seabream and thus no sig-nificant differences were found in feed intake during the experimentalperiod. The use of soybean oil in black seabream diets did not affectfish survival (Table 3). The inclusion of 60% or 80% soybean oil indiets did not significantly change weight gain compared with the FO

Table 4Composition of whole body, muscle and liver (in % of wet weight) in black seabream after 9 weeks (mean±SE, n=3)

Dietary treatments

Initial FO 60SO 80SO 100SO

Whole bodyMoisture 74.85±0.35 73.72±0.84 73.41±0.22 71.26±1.89 72.23±2.23Crude protein 14.62±0.20 15.77±0.65 15.33±0.11 15.32±1.02 15.13±0.13Crude lipid 3.23±0.11 3.95±0.18 4.01±0.01 4.51±0.21 4.68±0.35Ash 5.54±0.19 4.90±0.32 5.10±0.26 5.44±0.38 5.30±0.47

MuscleMoisture 79.38±0.45 78.32±0.61 78.38±0.37 78.53±0.08 79.47±0.66Crude protein 15.51±0.32 16.39±0.44 16.17±0.16 16.49±0.13 16.13±0.13Crude lipid 0.95±0.01 0.98±0.06a 1.13±0.00b 1.18±0.03b 1.16±0.03b

Ash 1.30±0.04 1.24±0.00 1.24±0.01 1.25±0.00 1.19±0.08

LiverMoisture 60.01±0.56 59.03±0.96 60.89±0.89 62.63±0.46 62.29±1.39Crude lipid 20.02±0.43 21.19±0.12 21.42±0.82 22.48±0.78 23.82±0.81

Different superscript letters within each row represent significant differences (Pb0.05). Statistics not performed on the initial sample.

157S. Peng et al. / Aquaculture 276 (2008) 154–161

diet (Table 3). Lowest weight gain was observed in fish fed the 100SOdiet, and was significantly lower than that of fish fed FO diet. Dailygrowth index (DGI) followed similar trends as weight gain. No sig-nificant differences were found in hepatosomatic index (HSI), con-dition factor (CF) and feed conversion ratio (FCR) betweenexperimental diets (Table 3), although there was an increasing trendof HSI and FCR with more soybean oil inclusion.

3.2. Whole body and muscle proximate compositions

Proximate composition of the whole body did not change signifi-cantly due to soybean oil inclusion (Table 4). No significant differenceswere found in muscle moisture, crude protein and ash contents betweendiets. However, muscle lipid was increased by the inclusion of soybeanoil in the diets (Table 4). The muscle lipid contents of fish fed 60SO,80SO and 100SO diets were significantly higher than those of fish fedFO diet (Pb0.05), but there was no significant difference in musclelipid contents between 60SO, 80SO and 100SO diets.

3.3. Plasma biochemical composition

Plasma protein was relatively stable throughout the experimentalperiod (Table 5). Plasma cholesterol showed a declining trend withincreasing levels of soybean oil. Total cholesterol of plasma in fish fedthe 100SO diet (5.08 mM) was significantly lower than that of fish fedFO diet (7.09 mM) (Pb0.05). Levels of triacylglycerol (TAG) inplasma did not differ significantly between diets (Table 5).

Table 5Total protein, total cholesterol and triacylglycerol in black seabream plasma after9-week trial (mean±SE, n=3)

Dietary treatments Protein (g/l) Cholesterol (mM) Triacylglycerol (mM)

Initial 24.87±1.98 7.26±0.51 8.78±0.36FO 25.00±2.31 7.09±0.45b 9.58±0.7060SO 26.33±3.18 6.44±0.42ab 8.91±0.4280SO 27.00±3.51 6.00±0.39ab 9.31±0.76100SO 25.67±0.88 5.08±0.40a 9.29±0.22

Different superscript letters within each column represent significant differences(Pb0.05). Statistics not performed on the initial sample.

3.4. Liver biochemical composition

Lipid and moisture content in the liver were not significantly af-fected by dietary treatments, although there was an increasing trend oftheir contents with more fish oil replacement (Table 4). The α-tocopherol in the liver was significantly increased by the inclusion ofsoybean oil in the diets (Pb0.05) (Table 6). However, the α-tocopherolconcentrations between the soybean oil diets (60SO, 80SO and 100SO)were not significantly different. The inclusion of soybean oil in thediets reduced the hepatic TBARS values (Table 6). The hepatic TBARSvalues of fish fed 80% and 100% soybean oil diets were significantlylower than that of fish fed FO diet (Pb0.05).

The fatty acid profile in the liver differed between dietary lipids(Table 7). After 9 weeks, total lipids in the liver of fish fed the control dietcontained 42.72% saturates, 43.03% monounsaturated fatty acids(MUFA) and 13.50% polyunsaturated fatty acids (PUFA) with n−3PUFA being the major component (7.98%) (Table 7). Saturated fattyacid andMUFAwere highest in fish fed the FO diet, whereas PUFAwerehigher in fish fed soybean oil diets than that of fish fed the FO diet. Thecontents of docosahexaenoic acid (22:6n3, DHA) and, eicosapentaenoicacid (20:5n3, EPA), and the n−3/n−6 ratio were significantly reduced bythe inclusion of dietary soybean oil (Pb0.05). MUFA and oleic acid(18:1n−9, OA) significantly reduced in the liver of fish fed soybean oildiets (Pb0.05). Linoleic acid (18:2n6, LA) and linolenic acid (18:3n3,LNA) significantly increased in fish fed the soybean oil diets (Pb0.05).The LA content in the liver of fish fed 80SO and 100SO diets weresignificantly higher than that in fish fed either FO or 60SO diets

Table 6Hepatic α-tocopherol and thiobarbituric acid-reactive substances (TBARS)values of black seabream after 9-week trial (mean±SE, n =3)

Dietarytreatments

Hepatic α-tocopherol (μg/gwet tissue)

Hepatic TBARS values (nmol/mg prot)

FO 52.00±3.36a 5.77±0.63c

60SO 62.13±5.19ab 5.36±0.99bc

80SO 66.40±1.53b 2.86±0.18a

100SO 64.05±2.41b 3.06±0.50ab

Different superscript letters within each column represent significant differences(Pb0.05).

Table 7Fatty acid composition of liver in black seabream fed different diets for 9 weeks(g FA/100g total FA) (mean±SE, n=3)

Fatty acids Dietary treatments

FO 60SO 80SO 100SO

Saturate 42.72±0.52b 41.68±0.61ab 37.59±1.72a 39.71±1.22ab

18:1n9 33.66±0.50b 29.63±0.08a 28.76±1.12a 28.67±0.28a

20:1n9 1.04±0.01b 0.89±0.06b 0.77±0.06ab 0.55±0.05a

MUFA 43.03±0.73c 35.41±0.29b 33.09±1.28ab 31.90±0.31a

18:2n6 3.93±0.31a 12.63±1.50b 18.20±1.76c 18.06±1.63c

18:3n6 0.90±0.04a 3.66±0.29b 5.55±1.08c 5.92±0.22c

20:4n6 0.70±0.24 0.67±0.04 0.52±0.00 0.56±0.31n−6PUFA 5.53±0.11a 16.95±1.17b 24.26±2.83c 24.54±1.10c

18:3n3 0.53±0.06a 1.65±0.03b 1.71±0.21b 1.43±0.18b

20:5n3 2.36±0.19d 1.35±0.10c 1.02±0.01b 0.54±0.06a

22:6n3 5.10±0.42d 2.24±0.05c 1.55±0.03b 0.93±0.00a

n−3PUFA 7.98±0.67c 5.23±0.18b 4.28±0.24ab 2.89±0.24a

PUFA 13.50±0.77a 22.18±1.00b 28.54±3.07c 27.43±0.86bc

n−3HUFA 7.46±0.61d 3.58±0.15c 2.57±0.04b 1.47±0.07a

n−3/n−6 1.45±0.10c 0.31±0.03b 0.18±0.01ab 0.12±0.02a

Different superscript letters within each row represent significant differences(Pb0.05).

158 S. Peng et al. / Aquaculture 276 (2008) 154–161

(Pb0.05). The inclusion of soybean oil in diets significantly increased thecontent of n−6 polyunsaturated fatty acids (Pb0.05), whereas thecontent of n−3 polyunsaturated fatty acids in fish fed FO diet wassignificantly lower than that in fish fed the soybean oil diet at any level(Pb0.05). The content of n−3 HUFA in liver of fish fed the FO diet wassignificantly higher than that of any other experimental diets (Pb0.05).Increased levels of soybean oil in fish diets significantly reduced the n−3HUFA content in the liver (Pb0.05).

4. Discussion

In our study, weight gain in fish fed the diet containing only40% fish oil was similar to fish fed the diet containing 100% fishoil. This finding is consistent with previous studies showingsuccessful partial replacement of dietary fish oil with vegetableoils in other marine fish species without showing negative signsof growth (Mourente et al., 2005a,b; Lin and Shiau, 2007;Piedecausa et al., 2007). For example, it is possible to replace upto 60% of fish oil with vegetable oils in European sea bass duringgrow out (Izquierdo et al., 2003; Montero et al., 2005; Mourenteet al., 2005a). Piedecausa et al. (2007) indicated that fish oil canbe successfully replaced with soybean or linseed oil in sharps-nout seabream D. puntazzo diets for a period of 92 days withoutreducing growth. The present study also showed that 60%replacement did not reduce fish growth compared to the FO diet.A decline in weight gain was noticed with 80% fish oil replace-ment although this difference was not significant different to thecontrol.

Subhadra et al. (2006) suggested that fish growth efficacycan be affected by diet composition and feeding trial duration.For instance, Llorens et al. (2007) showed that the dietarysoybean oil levels (0%, 24%, 48% and 72%) did not influencethe growth performance of gilthead sea bream until 211 days inthe trial. However, at the end of the trial (day 309), fish fed the72% soybean oil diet weighed the lowest. Similarly, the 8 month

study by Montero et al. (2005), using rapeseed oil at 60%substitution resulted in reduced growth of European sea bass,whereas the 89 day study by Izquierdo et al. (2003) includingrapeseed oil at the same level of substitution did not observe anyreduction in growth rate in the same species. It is likely that fishfed 80% fish oil replacement in our study might exhibit inferiorgrowth if the present experiment had lasted longer.

The declining trend of fish growth with further inclusion ofsoybean oil in this study may be related to the limited capacityof black seabream to elongate and desaturate fatty acids asreported in juvenile sea bream S. aurata (Mourente and Tocher,1993). Although symptoms of EFA-deficient such as fin erosion(Castell et al., 1972) were not observed in any of the dietarytreatments, weight gain of black seabream fed 100SO diet wassignificantly low than that fish fed either FO or 60SO diet. Then−3 HUFA concentration in the 100SO diet was 4.23% of thetotal fatty acids (Table 2), which is equivalent to 0.64% ofthe diet by weight. However, the n−3 HUFA concentrationsin 80SO diet accounted for 1.03% of the diet, which did notsignificantly reduce the growth of black seabream compared tofish fed the FO diet in 9 weeks, suggesting that the dietary n−3HUFA requirement for juvenile black seabream should be morethan 1%. This value is similar to gilthead bream that require atleast 1% of n−3 HUFA in dry diet to maintain normal growth(Ibeas et al., 1996). Our study indicated that 80% fish oilreplacement by soybean oil could sustain the growth of blackseabream for a period of 9 weeks, but the declining trend ingrowth suggests that this level of replacement may not be ap-plicable for a longer term grow out.

No effect of dietary soybean oil was observed in the wholebody or tissue composition of black seabream, which is con-sistent with the results in brown trout Salmo trutta (Arzel et al.,1994), turbot P. maxima (Regost et al., 2003) and gilthead seabream (Benedito-Palos et al., 2007). However, fish fed soybeanoil diets showed a slight increase of hepatic lipid content, whichis a characteristic of dietary and hormonal imbalances, such asnon-alcoholic steatohepatitis (McClain et al., 2004) andmetabolic syndrome (Avramoglu et al., 2006). Meanwhile, then−3/n−6 ratio dropped markedly with increasing vegetableoil inclusion in diets. The imbalance of n−3 and n−6 fattyacids in liver could leads to lipid deposits in the liver (Takeuchiet al., 1979). This supports our observation that fish fed highpercentage soybean oil diets tended to deposit more liver lipidthan those fed a FO diet, and is consistent with a previous studyin sharpsnout seabream (Piedecausa et al., 2007). Benedito-Palos et al. (2007) also reported that gilthead sea bream fed avegetable oil diet showed a high level of fat deposition in liver.High lipid deposits in the liver may be functionally importantand limit the possibility of including more vegetable oils in fishdiets but this hypothesis requires future test.

Our study showed that experimental diets with soybean oildecreased plasma cholesterol, showing a hypocholesterolemiceffect. Similar results have been reported in other fish. Forinstance, Richard et al. (2006a) showed that diets containingvegetable oil blends decreased the total plasma cholesterol inEuropean sea bass and in rainbow troutO. mykiss (Richard et al.,2006b). This is possibly because diets containing vegetable oils

159S. Peng et al. / Aquaculture 276 (2008) 154–161

are rich in oleic acid (OA), linoleic acid (LA) and linolenic acid(LNA) and these acids are known to reduce cholesterol(Dietschy, 1998; Fernandez and West, 2005). Another explana-tion could be the presence of phytosterols in vegetable oils whichcan affect cholesterol absorption (Gilman et al., 2003).

The present study also demonstrated an increase in vitamin E(i.e., α-tocopherol) and a reduction of TBARS in the liver ofblack seabream fed soybean oil diets. This suggests thatvegetable oil could reduce lipid peroxidation in fish. Fish oil isrich in n−3 PUFA such as DHA and EPA, but fish tissue fattyacids may be subject to oxidation by increasing the level ofdietary fish oil (Stephan et al., 1995). Hemre and Sandnes (1999)reported a reduction of vitamin E in Atlantic salmon musclewhen fish were fed fish oil containing high PUFA. Additionally,previous reports have also indicated that α-tocopherol could berapidly utilized in tissues of fish challenged by the oxidativestress caused by feeding rancid diets. For instance, the tissue α-tocopherol levels in rainbow trout (Hung et al., 1980), sea bass(Hung et al., 1981), European sea bass (Obach et al., 1993) andcatfish (Baker and Davies, 1997) were negatively relatedto the content of oxidized oil in diet. A tenfold reduction of α-tocopherol was observed in the hepatic tissue of fish fed oxidizedlipids as compared with the fish fed an equivalent amount ofa fresh diet (Baker and Davies, 1997). The reason for high vita-min E depletion in the fish tissue exposed to lipid oxidative stressis probably because vitamin E was used to counteract lipid per-oxidation in tissues. The coupled relationship between enhancedvitamin E in the liver and the amount of fish oil replacement byvegetable oil in fish diets suggests that soybean oil could reducelipid peroxidation in fish tissue since vitamin E is a biologicalantioxidant for maintaining flesh quality of seafood (Stephanet al., 1995; Tocher et al., 2002). Although the mechanism forescalating levels of liver vitamin E in fish fed soybean oil re-quires further investigation, the inclusion of soybean oil in fishdiet did reduce lipid peroxidation as indicated by the low level ofTBARS in liver.

The level of TBARS is one of the most popular and com-monly used methods to determine tissue peroxidation (Rosminiet al., 1996). In the present study, the hepatic TBARS value washigher in fish fed FO diet than fish fed vegetable oil diets, whichcorrespond with earlier observations in turbot (Stephan et al.,1995), grouper (Lin and Shiau, 2007) and mammals (Lopez-Bote et al., 1997), where the substitution of fish oil by vegetableoils resulted in a decrease in the levels of TBARS and indicateda reduction in susceptibility to lipid peroxidation. In addition,Alvarez et al. (1998) also reported significantly higher suscep-tibility to muscle oxidation in trout and sea bass fed on diets richin fish oil compared with fish fed on low fish oil diets. In thepresent study, the highest hepatic α-tocopherol content andlowest hepatic TBARS values in liver both occurred in fish fed80% soybean oil diet, suggesting that such a level of fish oilreplacement in diet could reduce the possibility of lipid perox-idation and oxidative stress in black seabream.

The modification of vegetable oil supplements in artificialfish diets could alter the FA profiles in a fish's body. The impactof dietary lipid composition on flesh quality is a major concernin fish because marine species have limited enzyme activity

enabling them to elongate and desaturate polyunsaturated FAswith 18 carbon atoms to form longer chained PUFA (Sargentand Tacon, 1999). The liver is a sensitive organ and responds tochanges in the FA composition of the diet (Arzel et al., 1994;Bell et al., 1995; Guillou et al., 1995). The present study showedthat the total MUFA and the total PUFAwere influenced by thedietary soybean oil at a significant level. Replacement of fish oilwith the soybean oil resulted in a significant increase in lino-leic acid (18:2n6) and total PUFA in fish liver cells. However,the n−3 PUFA in the liver, particularly EPA (C20:5n−3) andDHA (C22:6n−3), significantly reduced in fish fed soybean oildiets which is similar to reports in Atlantic salmon (Bell et al.,2003) and rainbow trout (Drew et al., 2007). Furthermore,selective retainment of fatty acid in the liver was observed inthis study. For instance, oleic acid (C18:1n−9) in liver cells wasmuch higher than that in the diets provided. The oleic acid ac-cumulation in liver was also found in rainbow trout (Skonberget al., 1994) and European sea bass (Montero et al., 2005), sug-gesting that diet lipid could be selectively absorbed by the liver.This may explain the cause of fat liver disease in fish fed a dietwith a high fat content.

In conclusion, this study shows the feasibility of fish oilreplacement by soybean oil at 60% and the possibility of re-placing up to 80% fish oil based on observed fish growthperformance. Biochemical evidence suggests that the inclusionof soybean oil increased the hepatic α-tocopherol content andreduced lipid peroxidation in fish. Dietary soybean oil modifiedthe fish liver fatty acid profile with the most pronounced impactbeing observed in the EPA and DHA contents. This functionalresponse by the liver may limit the inclusion of vegetable oil atlevels above 80% in black seabream diets.

Acknowledgements

The study was supported by the Program of Shanghai Sub-ject Chief Scientist (05XD14005) to L. Chen, the ‘Shu Guang’project of Shanghai Municipal Education Commission, Shang-hai Education Development Foundation (06GG06) and theZhejiang Key Technologies Program (Grant No 2002C12015,2006C12005), China. Thanks are also due to Xiaowei Duan,Wei Wang, Yunkai Li, Ping Wu, Chao Liu, Qian Yao, WeiZhang, Xinjin Sun, Yue Wang and Hao Zhang for theirassistance in the study. We much appreciate the comments fromtwo anonymous reviewers to improve the clarity and focus ofthis manuscript. Craig Meakin has kindly edited the English textto improve readability.

References

Alvarez, M.J., Lopez-Bote, C.J., Diez, A., Corraze, G., Arzel, J., Kaushik, S.J.,Bautista, J.M., 1998. Dietary fish oil and digestible protein modifysusceptibility to lipid peroxidation in the muscle of rainbow trout(Oncorhynchus mykiss) and sea bass (Dicentrarchus labrax). Br. J. Nutr. 80,281–289.

Arzel, J., Martinez-Lopez, F.X., Metallier, R., Stephan, G., Viau, M., Gandemer,G., Guillaume, J., 1994. Effect of dietary lipid ongrowth performance and bodycomposition of brown trout (Salmo trutta) raised in seawater. Aquaculture 123,361–375.

160 S. Peng et al. / Aquaculture 276 (2008) 154–161

Association of Official Analytical Chemists (AOAC), 1995. Official methods ofanalysis of Official Analytical Chemists International. 16th ed. Associationof Official Analytical Chemists, Arlington, VA, USA.

Avramoglu, R.K., Basciano, H., Adeli, K., 2006. Lipid and lipoproteindysregulation in insulin resistant states. Clin. Chim. Acta 368, 1–19.

Baker, R.T.M., Davies, S.J., 1997. Modulation of tissue α-tocopherol in Africancatfish, Clarias gariepinus (Burchell), fed oxidized oils, and thecompensatory effect of supplemental dietary vitamin E. Aquac. Nutr. 3,91–97.

Barlow, S., 2000. Fishmeal and oil: sustainable feed ingredients for aquafeeds.Glob. Aquac. Advocate 4, 85–88.

Bell, J.G., Tocher, D.R., MacDonald, F.M., Sargent, J.R., 1995. Effects ofdietary borage oil [enriched in γ-linoleic acid, 18:3(n−6)] or marine fish oil[enriched in eicosapentaenoic acid, 20:5(n−3)] on growth, mortalities, liverhistopatology and lipid composition of juvenile turbot (Scophthalmusmaximus). Fish Physiol. Biochem. 14, 373–383.

Bell, J.G., McEvoy, J., Tocher, D.R., McGhee, F., Campbell, P.J., Sargent, J.R.,2001. Replacement of fish oil with rapeseed oil in diets of Atlantic salmon(Salmo salar) affects tissue lipid compositions and hepatocyte fatty acidmetabolism. J. Nutr. 131, 1535–1543.

Bell, J.G., McGhee, F., Campbell, P.J., Sargent, J.R., 2003. Rapeseed oil as analternative to marine fish oil in diets of post-smolt Atlantic salmon (Salmosalar): changes in flesh fatty acid composition and effectiveness of sub-sequent fish oil “wash out”. Aquaculture 218, 515–528.

Benedito-Palos, L., Saera-Vila, A., Calduch-Giner, J.A., Kaushik, S., Pérez-Sánchez, J., 2007. Combined replacement of fish meal and oil in practicaldiets for fast growing juveniles of gilthead sea bream (Sparus aurata L.):networking of systemic and local components of GH/IGF axis. Aquaculture267, 199–212.

Bimbo, A.P., 1990. Production of fish oil. Ch6 in fish oils nutrition. In: Stansby,M.E. (Ed.), Van Nostrand Reinhold, New York, pp. 141–180.

Caballero, M.J., Obach, A., Rosenlund, G., Montero, D., Gisvold, M.,Izquierdo, M.S., 2002. Impact of different dietary lipid sources on growth,lipid digestibility, tissue fatty acid composition and histology of rainbowtrout, Oncorhynchus mykiss. Aquaculture 214, 253–271.

Caballero, M.J., Izquierdo, M.S., Kjørsvik, E., Montero, D., Socorro, J.,Fernandez, A.J., Rosenlund, G., 2003. Morphological aspects of intestinalcells from gilthead sea bream (Sparus aurata) fed diets containing differentlipid sources. Aquaculture 225, 325–340.

Caddy, J.F., 1999. Fisheries management in the twenty-first century: will newparadigms apply? Rev. Fish Biol. Fish. 9, 1–43.

Castell, J.D., Sinnhuber, R.O., Wales, J.H., Lee, J.D., 1972. Essential fatty acidsin the diet of rainbow trout (Salmo gairdneri): growth, feed conversion andsome gross deficiency symptoms. J. Nutr. 102, 77–86.

Chang, C.F., Yueh, W.S., 1990. Annual cycle of gonadal histology and steroidprofiles in the juvenile males and adult females of the protandrous blackporgy, Acanthopagrus schlegeli. Aquaculture 91, 176–196.

Craig, S.R., Washburn, B.S., Gatlin, D.M., 1999. Effects of dietary lipids onbody composition and liver function in juvenile red drum, Sciaenopsocellatus. Fish Physiol. Biochem. 21, 249–255.

Dietschy, J.M., 1998. Dietary fatty acids and the regulation of plasma lowdensity lipoprotein cholesterol concentrations. J. Nutr. 128, 444S–448S.

Drew, M.D., Ogunkoya, A.E., Janz, D.M., Van Kessel, A.G., 2007. Dietaryinfluence of replacing fish meal and oil with canola protein concentrate andvegetable oils on growth performance, fatty acid composition and organo-chlorine residues in rainbow trout (Oncorhynchus mykiss). Aquaculture 267,260–268.

Fernandez, M.L., West, K.L., 2005. Mechanisms by which dietary fatty acidsmodulate plasma lipids. J. Nutr. 135, 2075–2078.

Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for theisolation and purification of total lipids from animal tissues. J. Biol. Chem.226, 497–509.

Fonseca-Madrigal, J., Karalazos, V., Campbell, P.J., Bell, J.G., Tocher, D.R.,2005. Influence of dietary palm oil on growth, tissue fatty acid compositions,and fatty acid metabolism in liver and intestine in rainbow trout(Oncorhynchus mykiss). Aquac. Nutr. 11, 241–250.

Fracalossi, D.M., Craig-Schmidt, M.C., Lovell, R.T., 1994. Effect of dietarylipid sources on production of leukotriene B by head kidney of channel

catfish held at different water temperatures. J. Aquat. Anim. Health 6,242–250.

Gilman, C.I., Leusch, F.D.L., Breckenridge, W.C., MacLathcy, D.L., 2003.Effects of a phytosterol mixture on male fish plasma lipoprotein fractionsand testis P450scc activity. Gen. Comp. Endocrinol. 130, 172–184.

Guillou, A., Soucy, P., Khalil, M., Abambounou, L., 1995. Effects of dietaryvegetable and marine lipid on growth, muscle fatty acid composition andorganoleptic quality of flesh of brook charr (Salvelinus fontinalis).Aquaculture 136, 351–362.

Hemre, G.I., Sandnes, K., 1999. Effect of dietary lipid level on musclecomposition in Atlantic salmon Salmo salar. Aquacult. Nutr. 5, 9–16.

Henderson, R.J., 1996. Fatty acid metabolism in freshwater fish with particularreference to polyunsaturated fatty acids. Arch. Tierernahr. 49, 5–22.

Hung, S.S.O., Cho, C.Y., Slinger, S.J., 1980. Measurement of oxidation in fishoil and its effect on vitamin E nutrition of rainbow trout (Salmo gairdneri).Can. J. Fish. Aquat. Sci. 37, 1248–1253.

Hung, S.S.O., Cho, C.Y., Slinger, S.J., 1981. Effect of oxidized fish oil, DL-α-tocopheryl acetate and ethoxyquin supplementation on the vitamin Enutrition of rainbow trout (Salmo gairdneri) fed practical diets. J. Nutr. 111,648–654.

Ibeas, C., Cejas, J., Gomez, T., Jerez, S., Lorenzo, A., 1996. Influence of dietaryn−3 highly unsaturated fatty acids levels on juvenile gilthead seabream(Sparus aurata) growth and tissue fatty acid composition. Aquaculture 142,221–235.

Izquierdo, M.S., Obach, A., Arantzamendi, L., Montero, D., Robaina, L.,Rosenlund, G., 2003. Dietary lipid sources for seabream and seabass:growth performance, tissue composition and flesh quality. Aquac. Nutr. 9,397–407.

Izquierdo, M.S., Montero, D., Robaina, L., Caballero, M.J., Rosenlund, G.,Gines, R., 2005. Alterations in fillet fatty acid profile and flesh quality ingilthead seabream (Sparus aurata) fed vegetable oils for a long term period.Recovery of fatty acid profiles by fish oil feeding. Aquaculture 250,431–444.

Kanazawa, K., 1993. Tissue injury induced by dietary products of lipid per-oxidation. In: Corongiu, F. (Ed.), Free Radicals and Antioxidants in Nutrition.Richelieu Press, London, pp. 383–399.

Kiessling, K.-H., Kiessling, A., 1993. Selective utilization of fatty acids inrainbow trout (Onchorhychus mykiss Walbaum) red muscle mitochondria.Can. J. Zool. 71, 248–251.

Lewis, L.M., Lall, S.P., 2007. Effects of moderately oxidized dietary lipid andthe role of vitamin E on the development of skeletal abnormalities injuvenile Atlantic halibut (Hippoglossus hippoglossus). Aquaculture 262,142–155.

Lin, Y.H., Shiau, S.Y., 2007. Effects of dietary blend of fish oil with corn oil ongrowth and non-specific immune responses of grouper, Epinephelusmalabaricus. Aquac. Nutr. 13, 137–144.

Llorens, S.M., Vidal, A.T., Monino, A.V., Torres, M.P., Cerda, M.J., 2007.Effects of dietary soybean oil concentration on growth, nutrient utilizationand muscle fatty acid composition of gilthead sea bream (Sparus aurata L.).Aquac. Res. 38, 76–81.

Lopez-Bote, C.J., Rey, A.I., Sanz, M., Gray, J.I., Buckley, D.J., 1997. Dietaryvegetable oils and α-tocopherol reduce lipid oxidation in rabbit muscle.J. Nutr. 127, 1176–1182.

Lowry, O.H., Rosebrough, N.L., Randall, R.I., 1951. Protein measurement withFolin phenol reagent. J. Biol. Chem. 193, 265–275.

Mates, J.M., Perez-Gomez, C., De Castro, I.N., 1999. Antioxidant enzymes andhuman diseases. Clin. Biochem. 32, 595–603.

McClain, C.J., Mokshagundam, S.P., Barve, S.S., Song, Z., Hill, D.B., Chen, T.,Deaciuc, I., 2004. Mechanisms of non-alcoholic steatohepatitis. Alcohol 34,67–79.

Montero, D., Kalinowski, T., Obach, A., Robaina, L., Tort, L., Caballero, M.J.,Izquierdo, M.S., 2003. Vegetable lipid sources for gilthead seabream(Sparus aurata): effects on fish health. Aquaculture 225, 353–370.

Montero, D., Robaina, L., Caballero, M.J., Ginés, R., Izquierdo, M.S., 2005.Growth, feed utilization and flesh quality of European sea bass(Dicentrarchus labrax) fed diets containing vegetable oils: a time-coursestudy on the effect of a re-feeding period with a 100% fish oil diet.Aquaculture 248, 121–134.

161S. Peng et al. / Aquaculture 276 (2008) 154–161

Mourente, G., Tocher, D.R., 1993. Incorporation and metabolism of 14C-labelledpolyunsaturated fatty acids in juvenile sea bream Sparus aurata L. in vivo.Fish Physiol. Biochem. 21, 443–453.

Mourente, G., Dick, J.R., Bell, J.G., Tocher, D.R., 2005a. Effect of partialsubstitution of dietary fish oil by vegetable oils on desaturation and β-oxidation of [1-14C] 18:3n−3 (LNA) and [1-14C] 20:5n−3 (EPA) inhepatocytes and enterocytes of European sea bass (Dicentrarchus labrax L.).Aquaculture 248, 173–186.

Mourente, G., Good, J.E., Bell, J.G., 2005b. Partial substitution of fish oil withrapeseed, linseed and olive oils in diets for European sea bass(Dicentrarchus labrax L.): effects on flesh fatty acid composition, plasmaprostaglandins E2 and F2a, immune function and effectiveness of a fish oilfinishing diet. Aquac. Nutr. 11, 25–40.

Obach, A., Quentel, C., Laurencin, F.B., 1993. Effects of alpha-tocopherol anddietary oxidized fish oil on the immune response of sea bass Dicentrarchuslabrax. Dis. Aquat. Org. 15, 175–185.

Piedecausa, M.A., Mazón, M.J., García, B.G., Hernández, M.D., 2007. Effectsof total replacement of fish oil by vegetable oils in the diets of sharpsnoutseabream (Diplodus puntazzo). Aquaculture 263, 211–219.

Regost, C., Arzel, J., Robin, J., Rosenlund, G., Kaushik, S.J., 2003. Totalreplacement of fish oil by soybean or linseed oil with a return to fish oil inturbot (Psetta maxima): 1. Growth performance, flesh fatty acid profile, andlipid metabolism. Aquaculture 217, 465–482.

Richard, N.,Mourente, G., Kaushik, S., Corraze, G., 2006a. Replacement of a largeportion of fish oil by vegetable oils does not affect lipogenesis, lipid transportand tissue lipid uptake in European seabass (Dicentrarchus labrax L.).Aquaculture 261, 1077–1087.

Richard, N., Kaushik, S., Larroquet, L., Panserat, S., Corraze, G., 2006b. Replacingdietary fish oil by vegetable oils has little effects on lipogenesis, lipid transportand tissue lipid uptake in rainbow trout (Oncorhynchus mykiss). Br. J. Nutr. 96,299–309.

Rosmini, M.R., Perlo, F., Perez-Alvarez, J.A., Pagan-Moreno, M.J., Gago-Gago, A., Lopez-Santovena, F., Aranda-Catala, V., 1996. TBA test by anextractive method applied to ‘Pate’. Meat Sci. 42, 103–110.

Rueda-Jasso, R., Conceição, L.E.C., Dias, J., De Coen,W., Gomes, E., Rees, J.F.,Soares, F., Dinis, M.T., Sorgeloos, P., 2004. Effect of dietary non-protein

energy levels on condition and oxidative status of Senegalese sole (Soleasenegalensis) juveniles. Aquaculture 231, 417–433.

Sandnes, K., Lie, Ø., Waagbø, R., 1988. Normal ranges of some blood chemistryparameters in adult farmed Atlantic salmon, Salmo salar. J. Fish Biol. 32,129–136.

Sargent, J.R., Tacon, A.G.J., 1999. Development of farmed fish: a nutritionallynecessary alternative to meat. Proc. Nutr. Soc. 58, 377–383.

Sargent, J., Tocher, D.R., Bell, J.G., 2002. The lipids, In: Halver, J.E., Hardy, R.W.(Eds.), Fish Nutrition, 3rd ed. Academic Press, San Diego, pp. 181–257.

Skonberg, D.I., Rasco, B.A., Dong, F.M., 1994. Fatty acid composition ofsalmonid muscle changes in response to a high oleic acid diet. J. Nutr. 124,1628–1638.

Stephan, G., Guillaume, J., Lamour, F., 1995. Lipid peroxidation in turbot(Scophthalmus maximus) tissue: effect of dietary vitamin E and dietary n−6or n−3 polyunsaturated fatty acids. Aquaculture 130, 251–268.

Subhadra, B., Lochmann, R., Rawles, S., Chen, R., 2006. Effect of dietary lipidsource on the growth, tissue composition and hematological parameters oflargemouth bass (Micropterus salmoides). Aquaculture 255, 210–222.

Tacon, A.G.J., 2004. Use of fish meal and fish oil in aquaculture: a globalperspective. Aquat. Resour., Cult. Dev. 1, 3–14.

Takeuchi, L., Takeda, H., Watanabe, T., 1979. Availability of dietary phosphorousin carp and rainbow trout. Bull. Jpn. Soc. Sci. Fish. 45, 1527–1532.

Tocher, D.R., Mourente, G., Van dereecken, A., Evjemo, J.O., Dlaz, E., Bell, J.G.,Geurden, I., Lavens, P., Olsen, Y., 2002. Effects of dietary vitamin E onantioxidant defencemechanisms of juvenile turbot (ScophthalmusmaximusL.),halibut (Hippoglossus hippoglossus L.) and sea bream (Sparus aurata L.).Aquac. Nutr. 8, 195–207.

Torstensen, B.E., Frøyland, L., Lie, Ø., 2004. Replacing dietary fish oil withincreasing levels of rapeseed oil and olive oil — effects on Atlantic salmon(Salmo salar) tissue and lipoprotein composition and lipogenic enzymeactivities. Aquac. Nutr. 10, 175–192.

Valdimarsson, G., James, D., 2001. World fisheries—utilisation of catches. OceanCoast. Manag. 44, 619–633.