Effects of total replacement of fish oil by vegetable oils in the diets of sharpsnout seabream (Diplodus puntazzo)

Aquaculture 263 (2007) 211 – 219 www.elsevier.com/locate/aqua-online

Effects of total replacement of fish oil by vegetable oils in the diets of sharpsnout seabream (Diplodus puntazzo) M.A. Piedecausa, M.J. Mazón, B. García García, M.D. Hernández ⁎ IMIDA-Acuicultura. Consejería de Agricultura y Agua de la Región de Murcia. Apdo. 65. 30740, San Pedro del Pinatar. Murcia, Spain Received 13 June 2006; received in revised form 28 September 2006; accepted 28 September 2006

Abstract The aim of this study was to determine the impact of dietary replacement of fish oil by vegetable oils on sharpsnout seabream growth, nutritive utilization, somatic parameters, body composition, feed digestibility, and muscle fatty acid profile, as well as to make an estimate of its economic repercussions. To this end, three isonitrogenous (48% crude protein) and isoenergetic (23 MJ/kg) experimental diets were formulated, using three different lipid sources: fish oil (FO), soybean oil (SO) and linseed oil (LO). These diets were fed to triplicate groups of 30 sharpsnout seabream with an initial average weight of 14.9 g, three times a day to apparent satiation, over 92 days at 24.6 ± 1.1 °C. Our results show that the replacement of fish oil with soybean or linseed oil in sharpsnout seabream diets does not affect growth or feed utilization after three months of feeding. Fish on an SO diet exhibited higher hepatosomatic indices, whereas fillet percentages were significantly lower in fish that had been fed an FO diet. Apparent digestibility coefficients for dry matter, crude protein and crude lipid were significantly lower in fish that had consumed an LO diet. The muscle fatty acid composition reflected that of the diet. Consumption of vegetable oils reduced the muscle content of ARA (arachidonic acid), EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) to a lower degree than their corresponding reductions in the diet after fish oil replacement, which highlights their importance. Vegetable oils also increased the muscle content of linoleic and linolenic acids. In terms of economic performance, the SO diet was the least expensive diet, and had the best economic conversion ratio. © 2006 Elsevier B.V. All rights reserved. Keywords: Sharpsnout seabream; Diplodus puntazzo; Fish oil replacement; Soybean oil; Linseed oil; Alternative lipid sources

1. Introduction Sharpsnout seabream is a promising fish with many qualities that make it an excellent new species for culture (Franicevic, 1989; García Gómez and Ortega Ros, 1993; Caggiano et al., 1993; Abellán et al., 1994). This is a sparid fish, having more omnivorous feeding habits and a quality very similar to that of gilthead seabream. Pilot pre-growout and growout studies have been conducted ⁎ Corresponding author. Tel./fax: +34 968 184518. E-mail address: [email protected] (M.D. Hernández). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.09.039

with highly promising results, under both intensive and extensive culture conditions (Bermúdez et al., 1989; Kentouri et al., 1992; Divanach et al., 1993; Gatland, 1995). Feeding costs account for around 35–50% (García García et al., 2001; Vielma et al., 2000) of the total expenses at intensive aquaculture facilities. For those trying to culture new species, achieving a competitive economic performance is a high priority. In just a few decades, fish farming has developed into a highly efficient industry producing animal protein for human consumption. Besides good growing conditions, having a reliable supply of effective feeds is a prerequisite

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Table 1 Experimental diet composition (g kg− 1diet)

2. Materials and methods

Ingredients

Diet

2.1. Animals and housing

FO

SO

LO

Fish meal Wheat meal Wheat gluten Cod liver oil Soybean oil Linseed oil Vitamin–mineral premix a

460 220 150 160 0 0 10

460 220 150 0 160 0 10

460 220 150 0 0 160 10

a Vitamin and mineral premix, according to NRC (1993) recommendations for fish.

for productive and sustainable fish farming. In general, fish feed is made using fish oil as the main source of lipids, both because it has been readily available and because it has a high content of n-3 HUFA (highly unsaturated fatty acids), which are considered essential fatty acids for marine fish (Sargent and Tacon, 1999). However, even the most optimistic projections anticipate that in a few years, global fish oil production may not be enough to cover the increasing demand for animal feed. On the contrary, global vegetable oil production has increased in recent years, reaching volumes 100 times that of fish oil (Bimbo, 1990). As a result, prices for vegetable oils have been more stable and have even decreased in some markets, with some vegetable oils becoming even less expensive than fish oil. Some vegetable oils, such as soybean and linseed oil, are considered good alternative lipid sources for salmonids and freshwater fish (Bell et al., 2001; Rosenlund et al., 2001; Caballero et al., 2002). However, the use of vegetable oils as the only lipid source in marine fish feed is limited by their low ability to convert the linoleic and linolenic acids (abundant in many vegetable oils) into arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, which are essential for marine fish and are found in high concentrations in fish oil. Therefore, successful replacement of fish oil with vegetable oils would reduce both the absolute dependence on this ingredient and associated costs. Fishes with more omnivorous feeding habits, such as sharpsnout seabream (Sala and Ballesteros, 1997), may be able to use dietary vegetable oils in a more efficient manner. Therefore, the aim of this study was to assess the effects of complete replacement of dietary fish oil by soybean or linseed oil on sharpsnout seabream growth, its nutritive utilization, somatic parameters, body composition, feed digestibility, and muscle fatty acid profile, as well as to estimate the economic impact of such a replacement.

Sharpsnout seabream (with an initial average weight of 14.3 g) were obtained from the Valle Ca Zuliani Societa Agricola S.R.L. (Pila di Porto Tolle, Italy) hatchery and kept at the IMIDA aquaculture facilities (San Pedro del Pinatar, Murcia, Spain). Fish were allowed to acclimatize in raceway-type, 5500-l opencircuit seawater tanks, feeding on a commercially available feed for gilthead seabream. Afterwards, the fish were distributed between nine 360-l cylindroconical tanks supplied with running seawater (salinity: 37 g/l; NO − 2 :b 0.1 m g/l; NO − 3 :b0.1 mg/l; NH3:b0.5 mg/l; pH: 7.7). Testing conditions included 30 fish per tank, with each diet being experimentally tested in triplicate. The tanks were part of a recirculating system fitted with biological filtration, an ultraviolet lamp, and a thermostat that controlled the experimental temperature (Cerezo and García García, 2004). The water flow was constantly regulated to maintain dissolved oxygen at 70% of the saturation level. Animals were kept under natural photoperiod (37°50′N, 0°46′W) conditions at constant temperature (24.6 ± 1.1 °C), and allowed to feed to satiety with experimental diets (Table 1) three times a day, 7 days a week. The experiment lasted 92 days (from June to September). 2.2. Experimental diets Based on previous works (Hernández et al., 2001, 2003), three isonitrogenous and isoenergetic diets were formulated with a lipid content of about 20%. Cod liver oil was the only lipid source added to the fish oil (FO) diet, which served as a control. For the other two diets, cod liver oil was completely replaced by either soybean oil (SO) or linseed oil (LO). The ingredients were mixed Table 2 Proximate composition analysis of experimental diets (as % of dry matter) Components

Diet FO

SO

LO

Dry matter Crude protein Crude fat Ash NFE a Fiber Gross energy (MJ/kg of feed)

85.6 48.7 21.5 10.3 4.4 0.6 21.6

88.4 49.2 21.6 8.0 8.9 0.7 22.8

89.5 48.6 21.1 7.9 11.2 0.7 22.6

a

Nitrogen free extract.

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and the diets prepared by cooking-extrusion, using a semi-industrial extruder (E 19/25 D BRABENDER). The processing conditions selected were: 150 rpm screw speed, 90/95/105 °C temperature, and 2 mm diameter pellets. Samples of all diets were subjected to proximate composition analysis, the results of which are presented in Table 2.

digestibility coefficients (ADC) were calculated using the formula:

2.3. Sampling procedures and growth parameter evaluation

2.5. Analytical methods

Fish were weighed at the beginning of the experiment, and then on a monthly basis. Feed intake was monitored for each experimental group, in order to measure their daily intake rate (DIR). The effects on growth were determined by evaluating a number of growth and nutrient utilization indexes, including weight gain, specific growth rate (SGR), feed conversion ratio (FCR), and protein productive value (PPV), as well as the survival percentage. At the beginning and end of the experimental period, six animals from each tank were sacrificed in order to collect data on fish length and total weight, as well as liver, intraperitoneal fat, and digestive and fillet weights. These data were used to calculate the animals' condition factor (CF), hepatosomatic index (HSI), digestosomatic index (DSI), intraperitoneal fat index (IPF) and percentage of fillet (PF) using the following formulas:

ADCð%Þ ¼ 100−½100ð% I diet=% I faecesÞ  ð% N faeces=% N dietÞ where I is the inert marker, and N is the nutrient.

Proximate analyses of diet ingredients, diets, faeces and whole bodies of fish were based on the procedures from the AOAC (1997). Once measurements had been taken, all fish samples were frozen at − 20 °C until analyzed. The body composition was determined for six fish in each tank. After homogenization of individual fish, crude protein (Kjeldahl method, with a 6.25 nitrogen-to-protein conversion factor), crude fat (ethyl–ether extraction using a SOXTEC System HT6 extractor), moisture (drying at 105 ± 1 °C to constant weight) and total ash (incineration at 450 ± 2 °C to constant weight) contents were determined. Diet ingredients and diets were also tested for crude fibre (Weende method) and gross energy (IKA Adiabatic Calorimeter C-5000). AIA was determined by the method of Atkinson et al. (1984). All analyses were performed in triplicate. 2.6. Fatty acids

DIR SGR FCR PPV CF HSI DSI IPF PF

([feed intake / mean weight] / no. days) × 100 100 × (ln[final body weight] − ln[initial body weight]) / No. days Feed intake / weight gain 100 × (retained protein / ingested protein) 100 × (body weight / total length3) 100 × (liver weight / body weight) 100 × (digestive weight / body weight) 100 × (mesenteric fat weight / body weight) 100 × (fillet weight / body weight)

2.4. Digestibility measurements The digestibility study was carried out during the last month of the experiment (from August to September). Groups of sharpsnout seabream were fed experimental diets (FO, SO and LO) containing 0.5 g·kg− 1 of Celite (diatomaceous earth). After a seven-day adaptation period, faeces were collected for 30 days, 6 days a week, centrifuged (4 °C, 4000 rpm, 15 min), freezedried and used to analyze the natural marker AIA (acid insoluble ash) and nutrients (see below). Apparent

After 92 days of feeding, muscle samples were collected from 9 fish per diet, which were immediately frozen and kept at − 85 °C for further analysis. The fatty acid profile was determined after methanolysis in a gas chromatograph (Varian 3900) equipped with a FID detector, and using a 50-m/0.25 mm ID CP-Sil-88 column with helium as the carrier gas. The injector and detector temperatures were both kept at 250 °C. The column temperature was programmed for an initial temperature of 110 °C for 4 min, 10 °C/min to 160 °C, 4 °C/min to 210 °C and held for 4 min, and then to a final temperature of 240 °C at 4 °C/min, and held for another 4 min. Methyl esters were extracted from methanol with hexane, and automatically injected on the gas chromatograph. Fatty acid methyl esters were identified by comparison to external standards (SIGMA). 2.7. Economic estimates Based on the price of each raw material and the amounts that were required to make the different diets, we calculated the cost for one kilogram of each diet. The

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Table 3 Effects of dietary fish oil replacement with soybean oil and linseed oil on sharpsnout seabream growth and nutritive utilization

Initial weight (g) Final weight (g) SGR DIR FCR PPV Survival %

FO

SO

LO

14.83 ± 0.10 72.95 ± 3.40 1.73 ± 0.05 1.66 ± 0.03 1.15 ± 0.03 33.72 ± 1.23 86.67 ± 6.67b

14.97 ± 0.20 70.64 ± 4.30 1.69 ± 0.06 1.63 ± 0.05 1.15 ± 0.05 33.65 ± 1.37 85.56 ± 8.39b

15.11 ± 0.20 64.90 ± 9.60 1.58 ± 0.14 1.62 ± 017 1.21 ± 0.21 32.44 ± 5.92 34.44 ± 26.94a

Data represent the mean ± standard deviation of three replicates. Values on the same line and different superscripts are significantly different (P b 0.05).

raw material prices used were the average prices in 2006 (Official FOB prices, Mercados Agroalimentarios), due to the fact that they may experience significant changes throughout the year. Table 9 shows estimated prices for all the diets used in these experiments. The economic conversion ratio (ECR) was determined using the following equation: ECR ¼ Cost of Diet  Feed Conversion Ratio ðFCRÞ

2.8. Statistical analysis In order to detect statistically significant differences, experimental values were compared using a one-way analysis of variance (ANOVA), and differences between means were tested for significance using Tukey's multiple range test. The significance level was set at p b 0.05. Statistical analyses were performed with the software package Statistica® Version 6.0. Values throughout the text are expressed as mean ± standard deviation. 3. Results Fish survival rates over 85% were recorded with fish oil- and soybean oil-containing diets, but fish fed linseed Table 4 Effects of dietary fish oil replacement with soybean oil and linseed oil on sharpsnout seabream somatic parameters

Condition factor Hepatosomatic index Digestosomatic index Intraperitoneal fat % Fillet %

FO

SO

LO

2.0 ± 0.1 1.6 ± 0.4a 2.6 ± 0.4 2.0 ± 0.7 49.1 ± 3.6a

2.0 ± 0.1 2.0 ± 0.4b 2.5 ± 0.5 1.6 ± 0.4 52.3 ± 3.8b

2.0 ± 0.1 1.5 ± 0.3a 2.4 ± 0.4 1.7 ± 0.5 52.5 ± 4.1b

Data are expressed as mean ± standard deviation. Values on the same line and different superscripts are significantly different (P b 0.05).

Table 5 Effects of dietary fish oil replacement with soybean oil and linseed oil on sharpsnout seabream body composition Initial body composition Moisture 68.7 ± 0.40 Crude 16.1 ± 0.20 protein Crude fat 11.0 ± 0.55 Ash 4.0 ± 0.40

FO

SO

LO

65.2 ± 0.99 16.6 ± 0.42

65.2 ± 1.96 16.9 ± 0.29

65.2 ± 1.74 16.8 ± 0.45

14.2 ± 1.31 14.0 ± 1.60 13.5 ± 1.72 4.1 ± 0.29a 3.6 ± 0.29b 3.6 ± 0.42b

Values are presented as a percentage of wet substance (mean ± standard deviations). Values on the same line and with different superscripts are significantly different (P b 0.05).

oil displayed a high mortality rate after July's sampling. Among the dead fish, 88% died suddenly two days after July's sampling. The subjects showed signs of ulcers and external hemorrhages stemming from acute opportunistic bacterial infections associated with handling the animals in high temperatures. Experimental diets were well accepted by the animals, all of which fed in an active manner throughout the entire experiment. Although feed intake during the trial tended to be somewhat lower for the LO diet, the overall average daily feed intake was not significantly different from that of the other diets. Average fish weight increased continuously throughout the experiment (Table 3). Average SGR values decreased from 2.43 at the beginning of the trial to 1.20 at the end. Fish growth and feed utilization values are shown in Table 3. Dietary lipid sources did not significantly affect SGR, which averaged 1.66 for the whole period. Still, there was a small but clear tendency for linseed oil-fed fish to grow at a slower rate. With regards to nutritional utilization of the diet (Table 3), we obtained an average FCR value of 1.1, which is quite good; however, fish that were fed an LO diet tended to have a slightly higher FCR, and the inverse tendency was seen for PPV. Somatic parameters for sharpsnout seabream across the different diets are presented in Table 4. Fillet percentage was significantly lower in fish that had been fed an FO diet, whereas those that ate an SO diet displayed a Table 6 Apparent digestibility coefficients (ADCs) of experimental diet components ADCs (%)

FO

SO

LO

Dry matter Protein Lipid

87.4 ± 0.39a 96.9 ± 0.10a 97.6 ± 0.12a

87.5 ± 0.34a 97.3 ± 0.17a 97.2 ± 0.23a

78.3 ± 0.91b 94.0 ± 0.45b 94.1 ± 0.26b

Data are expressed as mean ± standard deviation. Values on the same line and with different superscripts are significantly different (P b 0.05).

M.A. Piedecausa et al. / Aquaculture 263 (2007) 211–219 Table 7 Fatty acid profiles of experimental diets (% of total fatty acids) Fatty acid a

FO

SO

LO

14:0 15:0 16:0 17:0 18:0 20:0 22:0 24:0 Σ Saturated 16:1 17:1 18:1n-9 18:1n-7 20:1n-9 22:1n-11 24:1n-9 Σ Monounsaturated 18:2n-6 18:3n-3 18:3n-6 18:4n-3 20:2 20:3n-3 20:4n-6 20:5n-3 22:5n-3 22:6n-3 Σ Polyunsaturated Σn-3 Σn-6 n-3/n-6 EPA/DHA

3.8 0.3 16.0 0.2 2.8 0.1 0.2 0.1 23.6 5.6 0.4 16.3 4.2 0.9 7.6 0.7 35.8 4.3 10.0 0.1 2.1 0.4 0.8 0.6 9.3 1.2 13.0 42.1 36.5 5.8 7.1 0.7

0.8 0.1 8.0 0.1 2.8 0.3 0.4 0.2 12.8 0.9 0.1 19.9 1.7 0.2 1.7 0.2 24.7 46.8 26.4 0.6 0.3 0.1 0.3 0.1 1.5 0.1 2.3 78.7 31.0 47.6 0.6 0.6

0.8 0.1 7.7 0.1 3.2 0.1 0.2 0.1 12.3 0.9 0.1 18.0 0.9 0.7 1.7 0.3 22.6 18.8 43.6 0.7 0.3 0.1 0.3 0.1 1.5 0.2 2.4 68.2 48.3 19.7 2.4 0.6

a

Some minor fatty acids (b0.1 g/100 g of fatty acids) are not shown.

higher hepatosomatic index. With regards to body composition, no significant moisture, crude protein or crude lipid content differences were found among the dietary groups tested (Table 5). Slight (albeit significant) fluctuations were found in ash content. The type of oil used affected the diet's digestibility (Table 6). Apparent digestibility coefficients for dry matter, crude protein and crude lipids were significantly lower in fish fed an LO diet. Fatty acid analysis of the total dietary lipids reflected the addition of vegetable oils. Table 7 shows the fatty acid profiles for the experimental diets expressed as percentages of total fatty acids. Total saturated fatty acids ranged from 12.3% in the LO diet to 23.6% in the FO diet. Total monounsaturated fatty acids (mainly oleic acid, 18:1 n-9) ranged from 22.6% in the LO diet to 35.8% in the FO diet. Linoleic acid (18:2 n-6) increased after the addition of vegetable oils, an effect that was particularly pronounced after replacing fish oil with

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soybean oil (from 4.3% in the FO diet to 46.8% in the SO diet). Vegetable oils also increased the percentage of linolenic acid (18:3 n-3), but in this case, much more so when fish oil was replaced by linseed oil (from 10.0% in the FO diet to 43.6% in the LO diet). EPA percentages ranged from 9.3% in the FO diet to 1.5% in the SO diet, while DHA values ranged from 13.0% in the FO diet to 2.3% in the SO diet. Compared to their respective values in the FO diet, the use of vegetable oils reduced the contents of ARA, EPA and DHA by 80, 84, and 82%, respectively. The muscle fatty acid composition clearly reflected the lipid composition of the diet (Table 8). Replacement of fish oil with vegetable oils reduced the diet's saturated and monounsaturated fatty acid content and Table 8 Fatty acid composition percentages of sharpsnout seabream muscle under the different experimental diets Fatty acida 14:0 15:0 16:0 17:0 18:0 20:0 22:0 24:0 ΣSaturated 16:1 17:1 18:1n-7 18:1n-9 20:1n-9 22:1n-11 24:1n-9 ΣMonounsaturated 18:2n-6 18:3n-3 18:3n-6 18:4n-3 20:2 20:3n-3 20:3n-6 20:4n-6 20:5n-3 22:5n-3 22:6n-3 ΣPolyunsaturated Σn-3 Σn-6 n-3/n-6 EPA/DHA

FO

SO a

4.6 ± 0.12 0.4 ± 0.01a 18.1 ± 0.43a 0.2 ± 0.01a 3.6 ± 0.16b 0.1 ± 0.01b 0.1 ± 0.01b 0.1 ± 0.01 27.3 ± 0.51a 7.2 ± 0.15a 0.4 ± 0.01a 4.4 ± 0.07a 22.1 ± 0.85 1.1 ± 0.69a 2.8 ± 0.15a 0.3 ± 0.02a 38.5 ± 1.44a 5.4 ± 0.34b 6.9 ± 0.77b 0.1 ± 0.046b 1.5 ± 0.02a 0.4 ± 0.01b 0.4 ± 0.03b 0.1 ± 0.01b 0.5 ± 0.04a 7.0 ± 0.35a 2.8 ± 0.16a 11.9 ± 0.99a 37.1 ± 1.82b 30.5 ± 1.77c 6.2 ± 0.35b 4.9 ± 0.39a 0.6 ± 0.02a

LO b

1.5 ± 0.13 0.2 ± 0.01b 13.6 ± 0.35c 0.2 ± 0.01c 2.1 ± 0.18a 0.2 ± 0.01a 0.1 ± 0.01a 0.1 ± 0.02 18.0 ± 0.49b 3.3 ± 0.17b 0.2 ± 0.03b 2.1 ± 0.07c 22.7 ± 0.75 0.1 ± 0.06b 0.7 ± 0.01b 0.2 ± 0.02b 29.4 ± 0.73b 42.1 ± 1.06a 8.3 ± 0.91c 0.7 ± 0.13c 0.8 ± 0.07b 1.0 ± 0.01a 0.4 ± 0.03b 0.7 ± 0.12a 0.2 ± 0.03b 2.2 ± 0.26b 1.1 ± 0.11b 4.1 ± 0.53b 61.8 ± 1.91c 17.0 ± 0.55b 43.8 ± 1.20a 0.4 ± 0.02b 0.5 ± 0.03b

1.6 ± 0.11b 0.2 ± 0.01b 12.9 ± 0.54b 0.1 ± 0.01b 2.9 ± 0.12c 0.1 ± 0.01c 0.1 ± 0.02b 0.1 ± 0.05 18.1 ± 0.67c 2.2 ± 0.19b 0.2 ± 0.02b 1.8 ± 0.05b 21.9 ± 0.53 1.0 ± 0.06c 0.8 ± 0.06b 0.2 ± 0.02b 28.3 ± 0.59b 13.5 ± 0.51c 29.5 ± 1.11a 0.7 ± 0.28a 0.9 ± 0.98c 0.5 ± 0.04c 1.3 ± 0.11a 0.2 ± 0.02b 0.2 ± 0.03b 2.10.25b 0.9 ± 0.11b 3.8 ± 0.48b 53.6 ± 1.14a 38.5 ± 0.80a 14.6 ± 0.45c 2.6 ± 0.06c 0.5 ± 0.02b

Data represent the mean ± standard deviation of three replicates. Values on the same line and with different superscripts are significantly different (P b 0.05). a Some minor fatty acids (b0.1 g/100 g fatty acids) are not shown.

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Table 9 Economic parameter results for experimental diets

FCR Feed cost (€/kg) ECR (€/kg)

FO

SO

LO

1.15 0.61 0.71

1.15 0.59 0.68

1.21 0.66 0.80

also, albeit to a lesser extent, their proportions in the animals' flesh. Both vegetable oils also increased the muscle content of linoleic and linolenic acids. This increase was particularly pronounced in the case of linoleic acid with the SO diet and for linolenic acid with the LO diet. Fish oil replacement reduced the muscle content of ARA, EPA and DHA in the same proportion; reduction in the diet, however, was more pronounced for EPA followed by DHA. Regarding the economic estimates (Table 9), the SO diet was the least expensive, and the one having the best economic conversion ratio (ECR). For an average fish farm with a production of 800 tons per year, feeding costs would amount to 566,131 € with fish oil, 541,843 € with soybean oil, and 639,809 € with linseed oil. Using the less expensive soybean oil would reduced feeding costs by 24,288 €, which means a saving of 4.3% with respect to fish oil. The use of linseed oil, on the other hand, would increase feeding costs by 73,678 €, i.e., an increment of 13% with respect to fish oil. 4. Discussion This experiment yielded quite acceptable growth results, with SGR values similar to those previously described for this time of the year (Faranda et al., 1983; Bermúdez et al., 1989; Divanach et al., 1993; Hernández et al., 2001, 2007). Our results indicate that fish oil can be successfully replaced with soybean or linseed oil in sharpsnout seabream diets for a period of time of 92 days without affecting fish growth or the nutritive utilization of the diet. These results are consistent with previous studies showing the viability of partially replacing dietary fish oil with soybean oil (Reinitz and Yu, 1981; Hardy et al., 1987; Greene and Selivonchick, 1990; Guillou et al., 1995; Rosenlund et al., 2001; Caballero et al., 2002) and linseed oil (Mugrditchian et al., 1981; Greene and Selivonchick, 1990; Rosenlund et al., 2001) in salmonids, without a negative impact on animals' growth or feed nutritive utilization. In like manner, Regost et al. (2001, 2003) replaced fish oil with soybean or linseed oil in turbot diets for 91 days without compromising the animals' growth. Lower growth rate values were found by Izquierdo et al. (2005) after six

months of feeding gilthead seabream diets with an 80% soybean or linseed oil content, although no significant differences were observed with 60% replacement levels. El-Kerdawy and Salama (1997) reported that 50% replacement of fish oil with soybean oil did not affect gilthead seabream growth, whereas a similar replacement with linseed or rapeseed oil impaired the animals' growth. In this study, we found high survival rates in fish that had been fed FO and SO experimental diets, but somewhat lower rates for LO. In our experience, fish fed linseed oil appear to be more sensitive to stress conditions (such as handling during sampling) than those fed fish oil and soybean oil. A number of studies have reported effects on the immune system of animals raised on diets with vegetable lipid sources. Lower survival rates due to infection by Edwarsiella ictaluri, as well as a reduced ability to produce antibodies, were reported by Fracalossi and Lovell (1994) after feeding catfish a diet with 7% linseed oil. Furthermore, n-3 HUFA deficiency in trout diets reduced the animals' resistance to the IHN virus (Kiron et al., 1995); and more recently, Montero et al. (2003) concluded that long-term feeding of gilthead seabream with 60% soybean oil may lead to immunosuppression, whereas linseed oil could alter the response to stress. Fish eating a diet containing soybean oil displayed a significantly higher hepatosomatic index than those fed fish oil or linseed oil. By contrast, previous studies have not demonstrated significant HSI differences on other fish species, including Atlantic salmon (Rosenlund et al., 2001; Menoyo et al., 2003; Bendiksen et al., 2003; Ng et al., 2004), turbot (Regost et al., 2003), and European sea bass (Mourente et al., 2005). At the morphological level, the livers from fish on the soybean oil diet had a less consistent texture and were easier to break up than those from fish fed the other diets. Likewise, Caballero et al. (2004) observed histological alterations in the liver of sea bream after 60% replacement of dietary fish oil with soybean or rapeseed oil, but not when they used linseed oil or a mix of vegetable oils at similar replacement levels. According to these authors, linseed oil does not alter hepatic morphology because its high proportion of 18:3n3 would promote hepatic lipid oxidation, yielding hepatosomatic indices similar to those of fish fed fish oil-containing diet, whereas the high percentage of 18:2n6 in soybean oil would promote lipid accumulation. Kalogeropoulos et al. (1992) demonstrated a high accumulation of linoleic acid in gilthead sea bream liver, even when they were fed a diet with a low content of this fatty acid. Moreover, in a study evaluating the activity of selected enzymes (Menoyo et al., 2004), hepatic lipid oxidation in gilthead sea bream fed soybean

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oil was reduced by 21% and 11% when compared to fish on fish oil and linseed oil, respectively. All three experimental diets had similarly high apparent digestibility coefficients, with only a slightly (albeit significant) lower value in the case of linseed oil. This may be due to a higher affinity of digestive lipases for some fatty acids and/or to absorption rate and efficiency differences between fatty acids leading to a different utilization of individual fatty acids (Gunasekera et al., 2002). In this sense, interspecific differences appear to exist that may merit further investigation. The essential fatty acid requirements for sharpsnout seabream are not known, but our results suggest that they may be low. Since we did not observe any symptoms of deficiency in sharpsnout seabream fed soybean oil and linseed oil, it would appear that the percentage of fish meal contained in all diets provides a sufficient amount of essential fatty acids. The muscle fatty acid profile reflects that of the diet. Similar results have been previously reported for other fish species such as rainbow trout (Yu et al., 1977; Reinitz and Yu, 1981) and catfish (Yingst and Stickney, 1979). Inclusion of vegetable oils in the diet affects the fish fatty acid profile, an effect that is more obvious in marine species because of their limited ability to convert C18 into polyunsaturated fatty acids (Watanabe, 1982). Particularly important among these changes is the marked muscle incorporation of 18:2n-6 and of 18:3n-3 in fish fed soybean and linseed oil diets, respectively. Vegetable oils, particularly soybean oil, greatly reduced the n3/n6 fatty acid ratio of the diet. It has been established that n-3 HUFA are essential for marine fish in many species (Sargent et al., 1989), and that the use of vegetable oils reduces the content of these fatty acids in the diet. The fish n3/n6 lipid ratio is strongly influenced by the diet's n3/n6 ratio. It has been shown that the n-3 and n-6 series fatty acids can act as substrates for a number of enzymes involved in lipid metabolism in fish, hence the importance of balancing the n3/n6 PUFA ratio in diets for growout marine fish (Sargent et al., 1989). The n-3/n-6 ratio dropped markedly in fish fed soybean oil. One of the main signs of n-3 and n-6 fatty acid imbalance are increased lipid deposits in the liver (Takeuchi et al., 1979). This supports our observation that the fish on soybean oil displayed higher hepatosomatic indices than those from the other groups, which is probably due to higher lipid deposition. Feeding on vegetable oils lowered the muscle content of EPA, DHA and ARA, but not as much as might have been expected, based on their low proportion in the diet. This highlights their importance as essential fatty acids. A similar effect has also been reported for Atlantic salmon (Bell et al., 2001, 2002, 2003), trout (Caballero et al., 2002), African

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catfish (Ng et al., 2003), turbot (Regost et al., 2003), sea bass (Mourente et al., 2005), and sea bream (Izquierdo et al., 2005). A deficiency in these essential fatty acids increases hepatic lipid deposits in many species (Castell et al., 1972; Takeuchi et al., 1990; Lemaire et al., 1991). DHA levels were higher than those for EPA in all diets. In fish fed a diet containing fish oil, the muscle content of DHA doubled that of EPA, and was not affected by replacement with vegetable oils. Saturated fatty acids are a major muscle component, which may be the reason why they increase in fish muscle. On the other hand, since oleic acid is well represented in both the diet and the muscle, it would appear that the muscle can make good use of it, mainly as an energy source (Léger et al., 1981). In conclusion, this study shows that fish oil can be replaced by soybean oil or linseed oil in sharpsnout seabream diets for a period of 92 days without negatively affecting fish performance. Vegetable oil replacement modified the fish muscle fatty acid profile, reducing the levels of EPA, DHA y ARA. It would be interesting to analyze the effects of incorporating these oils in sharpsnout seabream diets for longer periods of time. Acknowledgments This research was supported by grants from the ‘Planes Nacionales de Acuicultura (JACUMAR)’. References Abellán, E., García-Alcázar, A., Ortega, A., García-Alcázar, S., Martín, P., 1994. Cultivo de nuevas especies de espáridos mediterráneos: experiencias de preengorde y engorde del sargo común (Diplodus sargus sargus, L. 1758) y del sargo picudo (Diplodus puntazzo, Cetti, 1777). Inf. Téc. Inst. Esp. Oceanogr. 148, 1−ll. AOAC, 1997. Oficial Methods of Análisis, 16th ed. Association of Official Analytical Chemists, Washington. Atkinson, J.L., Milton, J.W., Slinger, S.J., 1984. Evaluation of acidinsoluble ash as an indicator of feed digestibility in rainbow trout (Salmo gairdneri). Can. J. Fish. Aquat. Sci. 41, 1384–1386. Bell, J.G., McEvoy, J., Tocher, D.R., McGhee, F., Campbell, P.J., Sargent, J.R., 2001. Replacement of fish oil with rapessed oil in diets of Atlantic salmon (Salmo salar) affects tissue lipid composition and hepatocyte fatty acid metabolism. J. Nutr. 131, 1535–1543. Bell, J.G., Henderson, R.J., Tocher, D.R., McGhee, F., Dick, J.R., Porter, A., Smullen, R., Sargent, J.R., 2002. Substituting fish oil with crude palm oil in the diet of Atlantic salmon (Salmo salar) affects tissue fatty acid compositions and hepatic fatty acid metabolism. J. Nutr. 132, 222–230. Bell, J.G., Tocher, D.R., Henderson, R.J., Dick, J.R., Crampton, V.O., 2003. Altered fatty acid compositions in Atlantic salmon (Salmo salar) fed diets containing linseed and rapessed oils can be partially restored by a subsequent fish oil finfishing diet. J. Nutr. 133, 2793–2801.

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