Vegetable oil blend as alternative lipid resources in dietsfor gilthead seabream, Sparus aurata

Elham A. Wassef Æ Norhan E. Saleh Æ Heyam A. El-Abd El-Hady

Received: 19 May 2008 / Accepted: 26 August 2008� Springer Science+Business Media B.V. 2008

Abstract The recent decreasing worldwide supplies of marine oils have forced the

aquaculture industry to investigate alternative lipid sources for use in marine fish feeds.

The aim of this study was to determine the impact of dietary replacement of fish oil by

vegetable oils on gilthead seabream (Sparus aurata) growth performance, nutritive utili-

zation, body composition, and fatty acid profile as well as feed cost. Two dietary vegetable

oil (VO) mix blends (VO1 and VO2) in which: sunflower (SO), cottonseed (CO) and

linseed (LO) for VO1 or soybean oil (SBO) for VO2, were tested as 60% fish oil (FO)

substitutes versus the 100% FO control or reference diet (FO). Three iso-proteic (46% CP)

and iso-lipidic (18%) experimental diets were hand fed, twice a day, 6 days a week to

apparent visual satiety to triplicate groups of seabream growers (average initial weight,

130.9 ± 3.44 g), until fish reached market size (300–400 g/fish) after 20 weeks at mean

ambient temperature 27.0 ± 1.8�C. All experimental diets were well accepted by seabream

growers regardless of the different lipid sources used, as overall mean feed intake (FI) and

daily intake (DFI) were not significantly different (P [ 0.05) among dietary treatments. In

terms of growth performance, fish fed VO1 diet (with LO) exhibited a relatively lower, but

significant (P \ 0.05), total weight gain (WG) than fish fed all FO diet (FO). However,

mean value of WG of fish fed either vegetable oil-tested diet was nonsignificantly different.

Feeding seabream growers vegetable oil (VO) diets (VO1 or VO2) had no significant effect

on specific growth rate (SGR), daily weight index (DWI), or feed conversion ratio (FCR)

among dietary treatments. Consumption of VO for 20 weeks did not significantly alter the

major nutrient composition of fish, but the muscle fatty acid (FA) profile was significantly

altered compared to the reference FO diet. Comparatively reduced levels of eicosapenta-

enoic acid (EPA) and docosahexaenoic acids (DHA), as well as elevated levels of linoleic

and linolenic acids (LA and LNA) compared with fish fed the FO were noticed. In terms of

economics, 17 or 20% reduction in Kg feed cost was obtained for diets VO1 or VO2,

E. A. Wassef (&) � N. E. SalehFish Nutrition Lab, National Institute of Oceanography and Fisheries (NIOF), Kayed-Bey Street,Anfoushy, Alexandria, Egypte-mail: elamalywa2007@yahoo.com

H. A. El-Abd El-HadyFaculty of Science, Dammam Girls College, King Faisal University, Dammam, Saudi Arabia

123

Aquacult IntDOI 10.1007/s10499-008-9213-7

respectively. In terms of growth performance and cost, VO2 diet showed slight relative

superiority over VO1 diet. However, in terms of liver structure morphology, VO1 diet

(with LO) has resulted in less fat-infiltration and altered hepatic cells than VO2 (with

SBO). As these traits do not affect yield or the price paid for the fish, VO2 diet has

therefore been considered better than VO1 as complementary lipid sources for gilthead

seabream grower diets.

Keywords Fatty acids profile � Gilthead seabream � Growth performance �Sparus aurata � Vegetable oils blend

Introduction

The demand for fish production is constantly increasing due to population growth and

rising consumer demand. Aquaculture is the possible mean for increasing supply of fish

protein for human consumption all over the world. Due to unpredictability and high-cost of

wild-capture fish, the fish culture industry has greatly flourished in Europe in recent years

particularly in the Mediterranean region. On a local basis, the Egyptian aquaculture

industry has not only achieved regular annual growth, but is also continuing to expand at

an outstanding rate (75% increases in production quantity from 2000 to 2006). At present,

aquaculture production accounted for 61% of total fish produced in 2006 and exceeded that

of overall wild fisheries (GAFRD 2008). Marine and brackish water fish farms are cur-

rently running very successfully and contributed 39% of total fish cultured in the same year

(2006), with gilthead seabream, Sparus aurata, European seabass, Dicentrarchus labrax,and mugillids especially Mugil cephalus as the major fish produced.

Gilthead seabream is a commercially important marine fish species cultured in most

countries around the Mediterranean basin, including Egypt. Its production level is rapidly

growing after the development and improvement of technologies used. In Egypt, these

fishes have traditionally also been considered as a high value fishery product (Sadek et al.

2004). Their high marketability, locally and for export, has led to the directing of more

efforts towards the expansion of their culture countrywide. It has been predicted that

cultured seabream will predominate over wild-caught fishes in the near future.

Given the economic importance of feeds and feeding in aquaculture, the need to develop

reliable compounded feeds for seabream with the least cost is evident. In general, aqua-

feeds of marine carnivorous fish species depend heavily upon fish meals and fish oils which

are primarily supplied by wild fisheries. Fish oil has constituted a major dietary ingredient

in compounded fish feeds for carnivorous marine fishes, such as gilthead seabream, due to

its high digestibility and essential fatty acid content, particularly omega-3 highly unsatu-

rated fatty acids (n - 3 HUFA). However, there is increased interest in the inclusion of

vegetable oils in marine fish diets to partially replace and reduce the dependency on fish oil

to ensure a sustainable development of the mariculture industry. Consequently, the issue of

fish oil substitution in aquafeeds, partially or wholly, has come to the forefront only

recently, especially in the light of the forecast that aquafeeds will use 75% (0.96 million

tons) of the world fish oil production by 2010 (Barlow 2000). Therefore, over the past few

years, significant breakthroughs have occurred in the replacement of fish oil by plant oils in

compounded fish feeds in order to reduce dependence on fish oil as well as reduce costs

(Alexis 1997; El-Kerdawy and Salama 1997; Izquierdo et al. 2003; Montero et al. 2003;

Kaushik 2004; Figueiredo-Silva et al. 2005; Saleh 2006; Wassef et al. 2007 and others).

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Thus, replacement of marine fish oils with alternate lipids of plant origin in the feed of

farmed fish should be studied not only to supply lipids at the correct level with the proper

balance of essential fatty acids (EFA) for optimum growth but also to maintain proper

immune function in fish (Montero et al. 2003). As no single raw material will, by itself, be

able to replace fish meal and/or fish oil, the composition must be multi-ingredient. There is

a great need for alternative raw materials for a continued sustainable growth of the

aquaculture industry. Several authors have recommended using a combination of fat

sources in order to have a wide variety of fatty acid profiles, while avoiding extreme diets

(too high in saturates, too low in the n - 3/n - 6 ratio, etc.). The present work was carried

out to investigate the possibility of partial replacement of fish oil in gilthead seabream

grow-out (during their second year of life) diet by two vegetable oil blends in order to

reduce costs and to determine the effect on growth performance, feed utilization, and fatty

acid content of fish. Each vegetable oil blend consisted of three oils chosen for their local

availability and relatively lower price. Moreover, linseed oil is rather exceptional in having

a high amount of omega-3 fatty acids (LNA, 18:3 n - 3 content = 58.6% of total FAs)

amongst oils of plant origin, and was chosen along with soybean oil (containing 8.1%

LNA) with a view to their potential use as the portion source of n - 3 fatty acids in

gilthead seabream aquafeeds. More recently, the effect of feeding gilthead seabream

vegetable oils on their health status and liver structure was investigated (Wassef et al.

2007).

Materials and methods

Fish and experimental conditions

Healthy seabream growers (mean initial weight, 130.9 ± 3.44 g, 1? year old), obtained

from a local marine farm, were randomly distributed in nine cylindrical PVC tanks (1 m3

each) supplied with a flow-through natural seawater system (flow rate of approximately

5 l/min) and provided with continuous aeration and filters. Stocking density was 15 fish per

tank. Since the experiment was conducted during the summer months (June–October),

rearing tanks were partly covered with lids to provide a more shaded environment and

quietness for the experimental fish. Mean water temperature was 27 ± 1.8�C, salinity

ranged between 37 and 38 ppt, dissolved oxygen was above 6 mg/l, and pH was ca. 7

during the whole experimental period (20 weeks). Fish were subjected to a natural

photoperiod of approximately 14 h light/10 h darkness.

Diets

Four crude vegetable oils (VO), namely: sunflower (SO), cottonseed (CO), linseed (LO),

and soybean oil (SBO) were selected because of their local availability, low price, and fatty

acid profile of LO and SBO which makes them likely candidates to partially substitute

imported fish oil (cod liver oil, FO) in compounded fish feeds. CO is the least expensive oil

among them all. A 100% FO reference diet (FO) was firstly formulated, then two iso-

proteic (46% CP) and iso-lipidic (18% L) experimental diets were prepared by substituting

60% of FO of reference diet by two combinations of three vegetable oils (1:1:1, w/w/w):

SO, CO and LO for diet VO1, and SO, CO and SBO for diet VO2. All experimental diets

were produced in the laboratory. Solid dietary ingredients were finely ground to powder

by using a Lab feed mill and then sieved to obtain an almost uniform particle size.

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A ‘‘Biogen’’ powder was further added as a feed attractant. The oils were properly

homogenized before they were mixed to the other powdered ingredients. The dry com-

ponents, except vitamins and minerals premix, were thoroughly mixed in a bench food

mixer, and then the required amount of oil dissolved in premix was added with continuous

mixing for about 10 min to ensure homogeneity of ingredients. An appropriate amount of

water was then gradually added to the mixture until the diet began to clump, forming a

dough-shaped paste. The diet was passed through a commercial meat-mincer to form

spaghetti-like extruded cold diet, then spread to dry in an oven at temperature 40–60�C.

Dry diets were chopped into small stick-shaped pellets of the appropriate size to fish

(2–3 mm diameter for early and late growers). Diets were stored in labeled plastic con-

tainers until use. Formulation and proximate analysis, and fatty acid composition of the

three experimental diets are given in Tables 1 and 2, respectively.

Feeding strategy

Gilthead seabream growers were acclimatized to laboratory conditions for 2 weeks prior to

feeding trial initiation. During the first week, fish were fed a commercial diet (40% crude

protein, CP), and then they were gradually adapted to the FO diet for another week. At end

of the conditioning period, fish in each tank were collectively weighed and the average

initial weight for each dietary treatment was then obtained. Each diet was handfed slowly

Table 1 Ingredients and composition (% DM) of experimental diets fed to gilthead seabream,Sparus aurata, for 20 weeks

Ingredients Diets (g/kg)

FO VO1 VO2

Fish meala 467 467 467

Corn gluten 260 260 260

Wheat flour (local) 158 158 158

Vitamins and minerals premixb 23 23 23

Biogenc 2 2 2

Fish oild 90 36 36

Sunflower oile 0 18 18

Cottonseed oile 0 18 18

Linseed oile 0 18 0

Soybean oile 0 0 18

Proximate analysis (% DM)

Crude protein 46.81 45.98 46.44

Crude lipids 17.89 18.39 18.33

Gross energy (MJ/Kg) 22.42 22.39 22.20

CP/GE (g/MJ) 20.88 20.54 20.92

a Danish 999b According to NRC recommendations (1993)c Biogen (feed attractant) contains Alicin [ 0.247 Mmole/g and Baccillus subtilis Nnatto 6 9 107/g. ChinaWay Corporation, Taiwand Irish producte Local crude vegetable oils

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to meet the appetite of the fish and to minimize loss due to uneaten food. The three test

diets were provided as two meals per day, 6 days a week until apparent visual satiety for

20 weeks until the fish reached a final weight around 300–400 g/fish which is a com-

mercial size for market. The amount of feed consumed for each treatment was then

quantified weekly and at the end of the growth trial. Experimental diets were assigned to

triplicate groups of fish for each treatment.

Analytical procedures

Feed ingredients, experimental diets, and fish samples (before and after the feeding trial)

were analyzed for proximate composition (crude protein, lipids, ash, fiber, and moisture)

according to standard methodology of AOAC (1995). Moisture content was obtained by

weight loss after drying samples in an oven at 105�C until constant weight. Crude protein

was determined as total nitrogen (N) by using a semi-automatic Kjeldahl (VELP

Scientifica, UDK 126) technique and multiplying N by 6.25. Ash content was obtained

from the weight loss after incineration of dried samples at 600�C for about 16 h in a

Muffle Furnace. Crude fiber was determined using sulfuric acid then sodium hydroxide,

12.5% (w/w) for half an hour each, and the final residue was washed with 5% HCl and

water, then filtered, dried, and weighed.

Table 2 Main fatty acid composition (% total) of experimental diets, as fed, to gilthead seabream for20 weeks

Fatty acid Dietary treatments

FO VO1 VO2

14:0 2.39 1.60 1.94

16:0 22.80 20.32 18.38

Total saturates 26.10 a 23.25 b 22.36 c

16:1 4.75 3.29 3.43

18:1 n - 9 28.97 10.91 12.22

22:1 n - 11 4.90 a 3.16 b –

Total monounsaturated 39.09 a 17.94 b 15.65

18:2 n - 6 (LA) 5.59 a 40.87 b 45.27 c

18:3 n - 3 (LNA) 1.24 a 5.30 b 3.35 c

20:4 n - 6 (ARA) 3.88 a 2.62 b 2.79 a

20:5 n - 3 (EPA) 6.63 a 4.37 b 4.17 b

22:6 n - 3 (DHA) 5.37 a 3.97 b 3.40 b

Total PUFA 23.99 a 57.67 b 60.35 c

n - 3 lc PUFA 12.45 a 8.62 b 7.57 b

n - 6 lc PUFA 4.71 a 2.88 b 4.16 a

Total fatty acids 99.18 98.86 98.36

% Lipids 17.89 18.39 18.33

FO 100% fish oil diet, VO1 vegetable oils blend 1 (sunflower:cottonseed:linseed 1:1:1 w/w/w), VO2 vegetableoils blend 2 (sunflower:cottonseed:linseed 1:1:1 w/w/w), Lc PUFA long chain polyunsaturated fatty acids

Values in the same row bearing different letters are significantly different (P [ 0.05)

Totals include some minor components not shown

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Total lipids (L) were extracted from diet and fresh fish muscle samples (three fish from

each dietary treatment) basically according to Folch et al. (1957) method after homoge-

nization in a mixture of chloroform–methanol (2:1, v/v), under icing conditions, using an

Edmond–Buhner HO4 homogenizer. The solvent was removed in a rotary-evaporator and

total lipids were dried in a vacuum desiccator before weighing. The lipid extracts were

redissolved in chloroform–methanol (2:1, v/v) at a final concentration of 100 mg lipid/ml

and were stored at -20�C for fatty acid analysis.

Fatty acid methyl esters (FAME) were prepared from aliquots of total lipids by acid-

catalyzed transmethylation with sulfuric acid in methanol overnight at 50�C (Christie

1982). FAMEs were purified by TLC using hexane/diethyl ether/acetic acid (85:15:15, v/v/v)

as solvent (Tocher and Harvie 1988). Separation of FAMEs was carried out in a Hewlett–

Packard 6890 Gas Liquid Chromatograph system (GLC) equipped with a flame ionization

detector (FID), a crosslinked silica capillary column HP-5 (30 m 9 0.32 mm, i.d.,

0.25 lm film thickness; Hewlett–Packard, CA, USA), on-column injection and using

Helium as carrier gas. The column was programmed for an initial temperature of 150�C

held for 2 min, rising thereafter to 200�C at a rate of 5�C/min to the final temperature

250�C and held for 9 min. Injector and detector temperatures were 220�C and 250�C,

respectively. Helium was used as the carrier gas with flow rate of 0.8 ml/min. The flow

rates of compressed air and hydrogen were 250 ml/min and 20 ml/min, respectively.

Identification and quantification of FAMs were based on the comparison of the sample

retention time with known standards (Sigma Chemicals, St. Louis, USA).

Histological examination of liver

At end of the feeding trial, samples of liver were collected from at least two fish of almost

uniform size for each dietary treatment. Small pieces (about 2 9 2 mm) of liver were fixed

in a 4% glutaraldehyde solution overnight, kept in a 1% osmium tetroxide solution for 1 h,

embedded, sectioned, and stained with uranyl acetate and lead citrate then examined with

Electron Transmission Microscope (ETM) (100 cx, JEOL, Tokyo, Japan). Diameters of

lipid droplets sample were measured and the mean diameter for each dietary treatment was

then calculated.

Data collection and analyses

Growth in weight was monitored at each 4-week interval by weighing all fish of each tank,

before first morning feed provision, then mean weight per tank was obtained. At the end of

the feeding trial (20 weeks), fish were killed by icing and stored frozen at -20�C in labeled

polyethylene bags for final proximate composition and fatty acid analyses.

Growth rate and feed utilization indices were calculated as follows: total weight gain (WG,

g/fish) = Wf-Wi, specific growth rate (SGR, %/day) = 100 9 (ln Wf - ln Wi)/t, daily

growth index (DGI, g/fish/day) = 100 9 (Wf)1/3 - (Wi)

1/3]/t, where Wf and Wi are mean final

and initial weights (g/fish), respectively, and t is time of experiment (days) (Figueiredo-Silva

et al. 2005). Feed conversion ratio (FCR) = dry feed intake (g)/fish live weight gain (g).

Statistical analysis

Mean values and standard deviation for each parameter measured for all treatments were

first calculated. The results were subjected to one-way ANOVA to test the effect of

vegetable oils blend replacement on fish performance. Data were analyzed using SAS,

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version 6.07 (SAS Institute, Cary, North Carolina, USA, 1992). Differences between

means were compared using Duncan’s multiple range test at significance of differences

(P \ 0.05) among dietary treatments.

Results

Growth performance

Feeding gilthead seabream growers either of the tested vegetable oils blend (VO1 or VO2)

for 20 weeks resulted in no significant (P [ 0.05) effect on final weight among treatments

(Table 3). Fish grew to a mean final weight of about 352 g during the experiment. All

experimental diets were well accepted by fish regardless of the different lipid sources

provided. Despite mean feed intake (FI) being relatively higher for VO2 diet than for VO1,

differences in total and daily FI among dietary treatments were nonsignificant (P [ 0.05).

Under our feeding protocol, the average daily feed intake, throughout the growth trial, was

estimated to be 1.65% of mean fish weight. However, total WG tended to be relatively

lower for fish fed vegetable oil blend with LO (VO1 diet) during the trial than that of other

diets. A slight but nonsignificant (P [ 0.05) difference was noticed for WG (g/fish) among

fish fed the two VO-added diet groups. SGR, DWI (1.41 g/fish/day), as well as FCR (1.17)

were almost similar and not significantly different among dietary treatments. Moreover, a

reduction of 17 or 20% per kg of feed cost was gained when the tested diet VO1 or VO2

was used, respectively, as compared to the 100% FO diet (Table 3). These data indicated

that, in terms of performance, both tested VO blends can be used as a 60% dietary FO

substitute, with slight preference for VO2, for gilthead seabream growers without

compromising growth performance or feed utilization.

Table 3 Growth performance indices (mean ± SD) of gilthead seabream fed vegetable oil diets for20 weeks

Indices Dietary treatment

FO VO1 VO2

Initial weight (g/fish) 130.85 ± 2.72 131.08 ± 4.57 130.90 ± 3.44

Final weight (g/fish) 360.5 ± 8.12 340.71 ± 1.48 353.53 ± 9.09

Total weight gain (g/fish) 229.66 ± 6.19 a 209.63 ± 1.75 b 223.22 ± 3.0 ab

Daily growth index (g/day) 1.45 ± 0.02 1.36 ± 0.03 1.43 ± 0.04

Specific growth rate (%/day) 0.85 ± 0.01 0.080 ± 0.02 0.83 ± 0.03

Feed intake (g/fish) 280.5 ± 23.8 244.4 ± 16.40 250.7 ± 22.3

Daily feed intake (fish/day % body weight) 1.79 ± 0.13 1.56 ± 0.07 1.60 ± 0.19

Feed conversion ratio 1.22 ± 0.07 1.19 ± 0.08 1.13 ± 0.09

Price feed/kg (Egyptian Pound) 5.77 4.79 4.64

% Reduction in price 100 17 20

FO 100% fish oil diet, VO1 vegetable oils blend 1 (sunflower:cottonseed:linseed 1:1:1 w/w/w), VO2 vegetableoils blend 2 (sunflower:cottonseed:linseed 1:1:1 w/w/w)

Means corresponding to different dietary treatments bearing different letters are significantly different(P \ 0.05)

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Nutrient composition of fish

Proximate composition of fish following 20 weeks of feeding the experimental diets with

VO blend are shown in Table 4 and indicated no significant differences in crude protein,

lipid, ash, and moisture contents between dietary treatments. However, feeding seabream

growers vegetable oil-supplemented diets had resulted in significant alterations in their

fatty acid (FA) muscle profiles (Table 5). Notably, fatty acid composition of fish clearly

reflected that of the dietary lipids (Tables 2 and 5). Although saturated FA levels were

lower in diets containing VOs than in the FO diet, muscle contents of these FAs (primarily

Table 4 Proximate composition (% wet weight, mean ± SD) of seabream fed vegetable oil diets for20 weeks

Dietary treatment Crude protein Lipids Moisture Ash

Initial 18.68 ± 0.8 8.26 ± 1.5 69.73 ± 0.5 4.60 ± 0.7

Final

FO 17.74 ± 0.88 13.27 ± 0.16 62.01 ± 0.28 4.36 ± 0.25

VO1 16.73 ± 0.09 12.89 ± 1.61 63.63 ± 2.70 3.91 ± 1.29

VO2 17.31 ± 0.10 13.39 ± 0.60 62.53 ± 0.50 4.12 ± 0.60

FO 100% fish oil diet, VO1 vegetable oils blend 1 (sunflower:cottonseed:linseed 1:1:1 w/w/w), VO2 vegetableoils blend 2 (sunflower:cottonseed:linseed 1:1:1 w/w/w)

Table 5 Main fatty acid composition (% total) of gilthead seabream fed vegetable oil diets for 20 weeks

Fatty acid Dietary treatment

FO VO1 VO2

14:0 2.85 a 1.83 b 3.10 a

16:0 18.74 a 21.74 b 22.29 b

Total saturates 23.10 a 24.61 b 26.79 c

16:1 n - 9 7.48 a 5.30 b 5.08 b

18:1 n - 9 39.16 a 7.92 b 8.69 b

22:1 n - 11 3.98 a 2.44 a –

Total monounsaturated 51.12 a 15.96 b 13.88 c

18:2 n - 6 (LOA) 4.18 a 39.91 b 41.12 c

18:3 n - 3 (LNA) 0.41 a 4.34 b 1.34 c

20:4 n - 6 (ARA) 3.44 a 2.21 b 3.06 a

20:5 n - 3 (EPA) 6.64 a 4.31 b 5.97 a

22:6 n - 3 (DHA) 5.70 a 4.12 b 4.73 b

Total PUFA 23.54 a 56.02 b 57.46 c

n - 3 lc PUFA 12.83 a 8.94 a 11.15 c

n - 6 lc PUFA 6.12 a 2.83 b 3.85 c

Total fatty acids 97.76 96.59 98.13

FO 100% fish oil diet, VO1 vegetable oils blend 1 (sunflower:cottonseed:linseed 1:1:1 w/w/w), VO2 vegetableoils blend 2 (sunflower:cottonseed:linseed 1:1:1 w/w/w)

Values in the same row bearing different letters are significantly different (P \ 0.05)

Totals include some minor components not shown

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palmitic acid, 16:0), were not significantly reduced at the end of the feeding trial. Total

monounsaturated FA in fish groups fed either VO blend were very close to each other, but

were significantly (P \ 0.05) reduced according to that in the diet. Fish fed the FO diet had

an elevated level of oleic acid (18:1 n - 9) compared to other fish groups. Meanwhile, the

value of FA 22:1 content was not significantly (P [ 0.05) different among treatments.

Total polyunsaturated fatty acids (PUFA) content were remarkably high in both fish groups

fed the VO, due largely to elevated levels of linolenic (LOA, 18:3 n - 3) or linoleic acid

(LNA, 18:2 n - 6), compared with fish fed the sole FO diet.

Levels of n - 3 long chain PUFA (HUFA) in muscle were also reduced to 30 or 13%

for fish fed VO1 and VO2 diets, respectively, compared to levels in FO fish muscle

(Table 5).

As expected, fish fed the 100% FO diet were characterized by their highest total highly

unsaturated fatty acids (HUFA) and total omega-3 FA, mainly eicosapentaeonic (EPA,

20:5 n - 3) and docosahexaeonic acid (DHA, 22:6 n - 3), among all treatments. DHA

flesh contents were always higher than in corresponding diet content (Table 2) indicating

its preferential retention among other FAs. Concentration of both LOA and LNA acids in

muscles of fish fed VO were significantly higher than in fish fed sole FO diet, but this

elevation was proportional to the levels found in their diets (Table 2).

Ultra-structure morphology of liver

Examination of liver tissue from gilthead seabream fed sole FO diet showed normal

hepatocytes hexagonal in shape with large, round, and centrally located nuclei and slight

lipid droplets (mean diameter 2.85 ± 2.12 l) in the cytoplasm (Fig. 1). Fish fed VO1 diet

revealed hepatocytes with relatively larger-size lipid droplets (4.2 ± 3.9 l), normal nuclei

but displaced, with cell-organelles, towards cell-periphery (Fig. 2). Hepatic cells in fish fed

VO2 diet showed shrinkage and pycnotic nuclei, condensed chromatin materials, and the

highest number but relatively smaller (3.85 ± 4.2 l) lipid droplets within the cytoplasm

Fig. 1 ETM photograph of liver from gilthead seabream fed 100% FO diet showing central andhomogenous-shaped nucleus (N), slight lipid droplets (L), and normal distribution of chromatin materials(12,0009)

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(sign of fat-infiltration) (Fig. 3). Comparative morphology of liver cells between fish

groups fed vegetable oils indicated slight preference for diet VO1, which showed less fat

deposition and hepatocytes alteration than VO1, relative to reference FO diet.

Fig. 2 ETM photograph of liver from gilthead seabream fed VO1-diet, reveals larger lipid droplets,nucleus displacement, and reduced chromatin materials (12,0009)

Fig. 3 ETM photograph of liver from gilthead seabream fed VO2-diet illustrates numerous smaller lipiddroplets (great fat-infiltration), shrinkage, and nucleus picnosis, and condensation of chromatin materials(12,0009)

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Discussion

Several studies have been carried out to investigate certain vegetable oils as possible

sustainable partial substitutes for fish oils in gilthead seabream compounded aquafeeds

(Kalogeropoulos et al. 1992; Alexis 1997; El-Kerdawy and Salama 1997; Izquierdo et al.

2003; Montero et al. 2003; Caballero et al. 2004; Saleh 2006; Wassef et al. 2007).

These studies have used linseed, soybean, sunflower, rapeseed, and olive oil either

solely or in combination in feed formulation for the species with levels of dietary lipids

between 10 and 12% (Alexis 1997; El-Kerdawy and Salama 1997), 18% (Saleh 2006) and

25% (Izquierdo et al. 2003; Montero et al. 2003; Caballero et al. 2004). These authors had

suggested a 60% fish oil replacement level at most in order not to compromise growth

performance or feed utilization efficiency of fish. Higher levels of dietary fish oil substi-

tution up to 70% (Saleh 2006) or 80% (Montero et al. 2003) had resulted in remarkably

reduced growth performance, feed utilization efficiency, and altered liver structure

(Caballero et al. 2004; Saleh 2006) as well as the fish immune system (Montero et al.

2003). However, among these earlier studies, experiments with cottonseed oil either singly

or in combination are just those of Saleh (2006) and Wassef et al. (2007), respectively. The

first study had used seabream fingerlings (IW, 4.2 g/fish), whereas the latter have examined

the effect on health status and liver structure of the grow-out stage. In addition to its local

availability and lowest price amongst all tested vegetable oils, cottonseed oil is rich in

tocopherols (natural anti-oxidants) which have variable vitamin E activity which contribute

to its stability giving the product that contains it a long shelf-life (NPCA 2003).

The present study has shown that it is possible to substitute 60% of dietary fish oil of

gilthead seabream growers by mixtures of three vegetable oils, namely sunflower, cot-

tonseed and either linseed oil or soybean oil, without a negative effect on growth, feed

utilization, or main nutrient composition of the fish. These results are in agreement with

earlier findings when vegetable and marine oils were mixed in diets for juvenile/young

gilthead seabream (Izquierdo et al. 2003; Saleh 2006) and European seabass (Dicentrar-chus labrax) (Mourente et al. 2005).

Under our feeding protocol, the tested VO-supplemented diets (VO1 and VO2) were

well accepted by gilthead seabream growers with an average daily feed intake of 1.65% of

fish body weight (130–400 g/fish) at a mean temperature of 27�C throughout the growth

trial. This value was very close to that obtained for the species of 100–200 g (1.81% of

biomass) grown at 23�C (Lupatsch et al. 2003), but relatively lower than the corresponding

value (2.26% body weight) for smaller juvenile fish (IW, 10 g) fed a mixture of vegetable

oils as a partial substitute of fish oil at 20–24�C (Izquierdo et al. 2003). For commercial

semi-intensive gilthead seabream culture, a similar daily feed consumption of about 2% of

biomass was previously recorded in earthen ponds in Egypt (Sadek et al. 2004). These

slight variations of feed intake rate are mainly related to initial fish size, prevailing tem-

perature, and other environmental conditions. In general, daily growth indices as well as

feed conversion ratios obtained in the present work were considered good, and even better

than the corresponding values recorded earlier for the species fed VO-supplemented diets

(Alexis 1997; El-Kerdawy and Salama 1997; Saleh 2006). The overall mean feed con-

version ratio obtained in the present study (1.17), was very close to 1.0, which is excellent

for the species under the specified experimental conditions. Similar values have been

reported separately: 1.02 by Izquierdo et al. (2003), 1.47 by Lupatsch et al. (2003), and

1.77 by Saleh (2006). The last author had investigated linseed, soybean, or cottonseed oils

individually as a partial substitute for fish oil, at three levels, 50, 60 and 70%, in diets for

gilthead seabream fingerlings (IW, 4.2 g). She recorded, for the 60% level, significantly

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(P \ 0.05) lower protein efficiency ratios and protein productive values for fish fed LO60

diet compared to those of fish fed the 100% fish oil diet. In the meantime, the present study

indicated that using a mixture of those three vegetable oils (SO, CO and LO) had no

apparent negative impact on protein utilization of gilthead seabream growers (Table 4).

Saleh (2006) had also reported significant elevation in hepato- and viscera-somatic indices

(HSI and VSI) in fish groups fed vegetable oil-supplemented diets compared to those of

fish fed all fish oil diet, and suggested relatively higher fat deposition both within liver and

viscera in fish that had been primed with diets containing VOs. In general, HSI increased as

the percent of fish oil substitution level increased.

The present work has also shown that consumption of vegetable oils in seabream grower

diets for 20 weeks did not significantly alter their major nutrients composition (Table 4),

but had modified the body fatty acids profile. Most vegetable oils are rich in unsaturated

18C fatty acids (oleic, 18:1 n - 9; linoleic 18:2 n - 6; linolenic acid 18:3 n - 3) but are

poor sources of n - 3 HUFAs. In general, marine fish species have high requirements for

n - 3 HUFAs, particularly EPA and DHA, as they are not capable of bioconversion C18

PUFAs to C20 and C22 HUFAs. Therefore, this must be taken in consideration when

vegetable oils are used in their diets (Sargent et al. 2002).

The fatty acid compositions of gilthead seabream flesh lipids in the present study were

closely related to dietary fatty acid input. Izquierdo et al. (2003) observed that fatty acid

profile of gilthead seabream muscle fed vegetable oils mixed diet (linseed, soybean and

rapeseed as 60% fish oil replacement) more closely resembled the general profile of fish

fed the fish oil diet. However, while dietary fatty acids influence flesh fatty acids, specific

fatty acids are selectively retained or utilized in different tissues. In the present study, DHA

was selectively deposited and retained, as flesh DHA levels were always higher than diet

levels (Tables 2 and 5). This has also been observed in European seabass (Mourente et al.

2005) among other fish species. The mechanism of selective FA deposition may include

the high specifity of fatty acyl transferase for DHA and the relative resistance of DHA to

beta-oxidation because of the complex catabolic pathway required for this fatty acid (Bell

et al. 2001). The elevated levels of some PUFAs in gilthead seabream muscles despite their

relatively lower dietary levels suggested the stimulation of delta-6 and delta-5 desaturase

activity in marine fish (Izquierdo et al. 2003). The latter authors stated that it could be

possible, under certain circumstances, to stimulate the delta-6 and delta-5 desaturase

activity in marine fish. In the present study, consumption of vegetable oils reduced the

muscle content of arachidonic acid (20:4 n - 6) to a lower degree than their corresponding

reductions in the diet after fish oil replacement, which highlights its importance to the

species. The relatively lower concentrations of linoleic and linolenic acid in flesh of fish

fed the VO-tested diets as compared to their levels in the corresponding diet (particularly

for VO2 diet) may suggest that gilthead bream utilize these fatty acids for oxidation. This

result supports similar data reported for European seabass (Mourente et al. 2005), and

contrasts with those for red seabream (Pagrus auratus) (Glencross et al. 2003a) and sharp

snout seabream (Diplodus puntazzoas) (Piedecausa et al. 2007). Total replacement of

dietary fish oil by either soybean or linseed oils in sharp snout seabream, a closely related

sparid fish, was feasible and did not affect growth or feed utilization after 3 months feeding

(Piedecausa et al. 2007). Fillet percentages were significantly lower in fish that had been

fed on a fish oil diet. Similarly, both refined canola and soybean oils have been considered

as potential as total fish oil-substitutes in diets for juvenile red seabream (Pagrus auratus)

(Glencross et al. 2003b). However, a significant reduction in growth and feed utilization

was observed when crude canola oil was used as a complete (100%) fish oil-alternative.

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As regards economics, the present study findings, that the SBO diet was the least

expensive diet when compared to either fish oil or linseed oil diet and had the best

economic feed conversion ratio (bottom section of Table 3), are in agreement with those of

Piedecausa et al. (2007) for sharp snout seabream.

It may be worth mentioning that consumption of food rich in n - 3 HUFA-like fish has a

positive effect on human health, such as prevention of cardiovascular pathologies (Herold and

Kinsella 1986), and auto-immune diseases (Hwang 1989). Thus, the beneficial effects of fish

as human food can be maintained even when the farmed fish have been fed diets with high

inclusions of vegetable oils (Izquierdo et al. 2003). Almost all studies undertaken in different

fish species thus clearly point out that replacement of fish oil by plant oils modifies flesh fatty

acid composition in a significant manner. The degree of changes in fatty acid profiles and the

consequences in terms of nutritional value depends upon several factors: dietary fat level,

level of substitution, duration of feeding, size, and growth rate of fish (Kaushik 2004). Since

there is a relationship between dietary fatty acid composition and flesh fatty acid composition,

it follows that any possible effects of dietary vegetable oils can be reversed by re-feeding fish

with diets containing adequate amounts of fish oil. Once fish are grown with vegetable oils

over the major part of life cycle, a finishing diet based on fish oil as the major source can be

used to tailor the final flesh fatty acid composition to a desired level for human nutrition and

dietetics (Glencross et al. 2003a; Mourente et al. 2005).

Given the importance of fish fats, the relative ease with which the fatty acid composition

of fish body oils can be tailored by altering the fatty acid composition of their feeds is

indeed a very positive aspect of aquaculture (Kaushik 2004). Therefore, one way to boost

the n - 3 HUFA concentration of farmed fish may be to use ‘‘finishing’’ feeds containing

fish oil. The fatty acid composition of the fish flesh could thus be altered to meet the

consumer expectation of a product that is rich in n - 3 HUFAs and low in n - 6 fatty

acids (Jobling et al. 2002; Glencross et al. 2003b). The lipid accumulation in hepatocytes

of gilthead seabream growers of the present study (Figs. 1–3) may reflect the storage of

lipidic reserve without pathological consequences. Hemoglobin and hematocrit contents,

erythrocyte, leukocyte, and thrombocyte count as indicators of health status of gilthead

seabream growers remained significantly unaffected after feeding VO-supplemented diets

for 5 months (Wassef et al. 2007). The fat-infiltration and alterations found in the livers of

gilthead seabream fed VO diets for 6 months are reversible when the fish are re-fed with a

balanced diet (100% fish oil), denoting the non-pathological character of these liver

alterations (Caballero et al. 2004).

In conclusion, results of the present study indicated that it is feasible to replace 60% of

dietary fish oil in gilthead seabream diets with either of the tested vegetable oil blends

(VO1 or VO2) without any negative effect on performance, feed utilization, and major

nutrient composition of fish. In terms of growth and economics, VO2 blend was considered

better than VO1, regardless of the relatively higher fat-infiltration and liver cell structure,

as these traits do not affect yield or the price paid for the fish and are considered to be a

reversible and not a pathological character.

Acknowledgment The authors are grateful to Dr. O. Malak for her help in preparation of liver sections.

References

Alexis MN (1997) Fish meal and fish oil replacers in Mediterranean marine fish diets. In: Tacon A, BasurcoB (eds) Proc. workshop ‘‘Feeding tomorrow’s fish,’’ Mazarron, Spain, Cahier Options Mediterr, Vol22, June 24–26, 1996, pp 183–204

Aquacult Int

123

AOAC (Association of Official Analytical Chemists) (1995) International official methods of analysis, 16thedn. Arlington, Virginia, USA

Barlow S (2000) Fish meal and fish oil: sustainable feed ingredients for aquafeeds. Glob Aquac Advocate4:85–88

Bell JG, McEvoy J, Tocher DR, McGhee F, Campbell PJ, Sargent JR (2001) Replacement of fish oil withrapeseed oil in diets of Atlantic salmon (Salmo salar) affects tissue lipid composition and hepatocytefatty acid metabolism. J Nutr 131:222–230

Caballero MJ, Izquierdo MS, Kjorsvik E, Fernandez AJ, Rosenlund G (2004) Histological alterations in theliver of seabream Sparus aurata, caused by short- or long-term feeding with vegetable oils: recovery ofnormal morphology after feeding fish oil as the sole lipid source. J Fish Dis 27:531–541. doi:10.1111/j.1365-2761.2004.00572.x

Christie WW (1982) Lipid analysis, 2nd edn. Pergamon Press, OxfordEl-Kerdawy A, Salama A (1997) Effect of dietary lipid sources on the growth and fatty acid composition of

gilthead bream Sparus aurata. In: Tacon A, Basurco B (eds) Proc. Workshop ‘‘Feeding tomorrow’sfish,’’ Mazarron, Spain, Cahier Options Medit., Vol 22, June 24–26, 1996, pp 235–241

Figueiredo-Silva A, Rocha E, Dias J, Silva P, Rema P, Gomes E et al (2005) Partial replacement of fish oilby soybean oil on lipid distribution and liver histology in European seabass (Dicentrarchus labrax) andrainbow trout (Oncorhynchus mykiss) juveniles. Aquacult Nutr 11:147–155. doi:10.1111/j.1365-2095.2004.00337.x

Folch J, Lees M, Sloan-Stanley GH (1957) A simple method for the isolation and purification of total lipidsfrom animal tissues. J Biol Chem 226:497–509

General Authority for Fish Resources Development (GAFRD) (2008) Annual fishery statistical report of fishproduction in Egypt during 2006 (in Arabic)

Glencross BD, Hawkins W, Curnow J (2003a) Evaluation of Canola oils as alternative lipid resources indiets for juvenile red seabream, Pagrus auratus. Aquacult Nutr 9:305–315. doi:10.1046/j.1365-2095.2003.00257.x

Glencross BD, Hawkins W, Curnow J (2003b) Restoration of fatty acid composition of red seabream(Pagrus auratus) using a fish oil finishing diet after grow-out on plant oil based diets. Aquacult Nutr9:409–418. doi:10.1046/j.1365-2095.2003.00272.x

Herold PM, Kinsella JE (1986) Fish oil consumption and decreased risk of cardiovascular disease, acomparison of findings from animal and human feeding trials. Am J Clin Nutr 43:566–598

Hwang D (1989) Essential fatty acids and immune responses. FASEB J 3:2052–2061Izquierdo MS, 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. Aquacult Nutr9:397–407. doi:10.1046/j.1365-2095.2003.00270.x

Jobling M, Larsen AV, Andreseasen B, Olsen RI, Sigholt T (2002) Influence of dietary shift on temporalchanges in fat deposition and fatty acid composition of Atlantic salmon post-smolt during the earlyphase of seawater rearing. Aquacult Res 33:875–889. doi:10.1046/j.1365-2109.2002.00727.x

Kalogeropoulos N, Alexis MN, Henderson RJ (1992) Effects of dietary soybean and cod-liver oil levels ongrowth and body composition of gilthead seabream (Sparus aurata). Aquaculture 104:203–308. doi:10.1016/0044-8486(92)90211-3

Kaushik SJ (2004) Fish oil replacement in aquafeeds. Aquafeeds Formulation Beyond Mag 1:3–6Lupatsch I, Kissil GW, Sklan D (2003) Defining energy and protein requirements of gilthead seabream

(Sparus aurata). Israeli J Aquacult Bamidgeh 55:243–257Montero D, Kalinowski T, Obach A, Robaina L, Tort L, Caballero MJ et al (2003) Vegetable lipid sources

for gilthead seabream (Sparus aurata): effects on fish health. Aquaculture 225:353–370. doi:10.1016/S0044-8486(03)00301-6

Mourente G, Good JE, Bell JG (2005) Partial substitution of fish oil with rapeseed, linseed and olive oils indiets for European seabass (Dicentrarchus labrax L.): effects on flesh fatty acid composition, plasmaprostaglandins E2 and E2a immune function and effectiveness of a fish oil finishing diet. AquacultNutr 11:25–40. doi:10.1111/j.1365-2095.2004.00320.x

National Cottonseed Products Association (NPCA) (2003) www.cottonseedcom/publicationsPiedecausa MA, Mazon MJ, Garcia BG, Hernandez MD (2007) Effects of total replacement of fish oil by

vegetable oils in the diets of sharp snout seabream (Diplodus puntazzo). Aquaculture 263:211–219.doi:10.1016/j.aquaculture.2006.09.039

Sadek SS, Osman MF, Mansour MA (2004) Growth, survival and feed conversion rates of seabream(Sparus aurata) cultured in earthen brackish water ponds fed different feed types. Aquacult Int 1:1–13

Saleh NE (2006) Evaluation of some plant lipid sources in gilthead seabream Sparus aurata diets. PhDthesis, Alexandria University

Aquacult Int

123

Sargent JR, Tocher DR, Bell JG (2002) The lipids. In: Halver JE, Hardy RW (eds) Fish nutrition, 3rd edn.Elsevier, USA, pp 181–257

Tocher DR, Harvie DG (1988) Fatty scid compositions of the major phosphoglycerides from fish neuraltissues: (n - 3) and (n - 6) polyunsaturated fatty acids in rainbow trout (Salmo gairdneri L.) and cod(Gadus morhua) brains and retinas. Fish Physiol Biochem 5:229–239. doi:10.1007/BF01874800

Wassef EA, Wahby OM, Sakr EM (2007) Effect of dietary vegetable oils on health and liver histology ofgilthead seabream (Sparus aurata) growers. Aquacult Res 38:852–861. doi:10.1111/j.1365-2109.2007.01738.x

Aquacult Int

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