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Articles

Replacement of Fish Oil with Rapeseed Oil in Diets of Atlantic Salmon (Salmo salar) Affects Tissue Lipid Compositions and Hepatocyte Fatty Acid Metabolism¹

Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA, Scotland, UK and ^* BioMar Limited, Grangemouth, FK3 8UL, Scotland, UK

²To whom correspondence should be addressed. E-mail: gjb1{at}stir.ac.uk.

	ABSTRACT

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Duplicate groups of Atlantic salmon post-smolts were fed five practical-type diets in which the added lipid was 100% fish oil [FO; 0% rapeseed oil (0% RO)], 90% FO + 10% RO (10% RO), 75% FO + 25% RO (25% RO), 50% FO + 50% RO (50% RO) or 100% RO, for a period of 17 wk. There were no effects of diet on growth rate or feed conversion nor were any histopathological lesions found in liver, heart, muscle or kidney. The greatest accumulation of muscle lipid was in fish fed 0% RO, which corresponded to significantly lower muscle protein in this group. The highest lipid levels in liver were found in fish fed 100% RO. Fatty acid compositions of muscle lipid correlated with RO inclusion in that the proportions of 18:1(n-9), 18:2(n-6) and 18:3(n-3) all increased with increasing dietary RO (r = 0.98–1.00, P < 0.013). The concentrations of eicosapentaenoic acid [20:5(n-3)] and docosahexaenoic acid [22:6(n-3)] in muscle lipid were significantly reduced (P < 0.05), along with total saturated fatty acids, with increasing dietary RO. Diet-induced changes in liver fatty acid compositions were broadly similar to those in muscle. Hepatic fatty acid desaturation and elongation activities, measured using [1-¹⁴C] 18:3(n-3), were increased with increasing dietary RO. Limited supplies of marine fish oils require that substitutes be found if growth in aquaculture is to be maintained such that fish health and product quality are not compromised. Thus, RO can be used successfully as a substitute for fish oil in the culture of Atlantic salmon in sea water although at levels of RO >50% of dietary lipid, substantial reductions occur in muscle 20:5(n-3), 22:6(n-3) and the (n-3)/(n-6) polyunsaturated fatty acid (PUFA) ratio, which will result in reduced availability of the (n-3) highly unsaturated fatty acids that are beneficial for human health.

KEY WORDS: • Atlantic salmon • rapeseed • canola • polyunsaturated fatty acid

	INTRODUCTION

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The beneficial effects to humans of consuming fish, particularly oily fish such as salmon, herring and mackerel with a high content of the (n-3) highly unsaturated fatty acids (HUFA),⁴ eicosapentaenoic acid [EPA; 20:5(n-3)] and docosahexaenoic acid [DHA; 22:6(n-3], have been well documented (1 2 3 4 5) . However, global capture fisheries are a finite resource. As a result of overfishing and subsequently tighter regulation, future demand for wild-caught fisheries products will exceed supply (6) .

Fish produced by aquaculture currently represent the fastest growing food sector, which is projected to increase at 10%/y for at least the next 10 y (7) . Aquaculture has traditionally utilized products from industrial fisheries, namely, fish meal and oil, to convert relatively cheap protein and oil into high value products, a practice that is sound both scientifically and commercially. However, of the global fish oil production in 1996 of 1.4 million metric tonnes (mt), 576 kilo tonnes (kt) were used for salmon and trout production with the predicted use of fish oil for aquaculture estimated to rise to 85% of the total available by 2010 (8 ,9) . In addition, competition for fish oil for inclusion in human nutritional supplements and agricultural feeds other than for aquaculture will soon make fish oil a highly prized commodity. Clearly, if aquaculture is to continue to expand, alternatives to its current dependence on fish oil must be developed.

Many freshwater fish, including salmonids, can convert 18:3(n-3) to 20:5(n-3) and then to 22:6(n-3) (10 11 12) ; by the same enzymatic pathways of desaturation and elongation, they can convert 18:2(n-6) to 20:4(n-6). In fresh water, Atlantic salmon parr consume substantial quantities of invertebrates that contain an abundance of 18:2(n-6) and 18:3(n-3) with lower amounts of 20:5(n-3) and almost no 22:6(n-3) (13) . It is not surprising, therefore, that salmon parr have been grown successfully to smoltification (sea water transfer) with diets containing rapeseed and linseed oil (14 ,15) . After smoltification, salmon enter the marine environment in which their natural zooplanktonic crustacean and piscine prey are rich in 20:5(n-3) and 22:6(n-3). Therefore, it might be expected that the genes encoding the desaturase and elongase enzymes responsible for the conversion of 18:3(n-3) to 22:6(n-3) are downregulated, possibly irreversibly, soon after salmon migrate to sea. However, a number of studies have suggested that salmonids can utilize vegetable oils in seawater provided the diets contain enough 18:3(n-3) to satisfy essential fatty acid (EFA) requirements (16 17 18) . More recent evidence suggests that there is no "switch off" of fatty acid–metabolizing enzymes in salmon post-smolts and that fish fed vegetable oils showed increased conversion of 18:3(n-3) to 22:6(n-3) [and 18:2(n-6) to 20:4(n-6)] compared with those fed fish oil (FO) (19) .

At the present time, the product of the salmon aquaculture industry is of high nutritional quality with an abundance of (n-3) HUFA and a high (n-3)/(n-6) polunsaturated fatty acid (PUFA) ratio (20) . Clearly, producers and consumers of salmon will want to minimize any perceived reduction in quality arising from the inclusion of vegetable oils, both in terms of growth and health of the fish and in the healthy image of salmon as part of the human diet. The high growth rates of cultured salmon are due to the use of high energy (oil) diets, which are rich in the C-20 and C-22 monoenoic fatty acids characteristic of northern hemisphere marine oils. Thus, any substitute for fish oil in salmon feeds should meet the following criteria: 1) avoid excessive deposition of 18:2(n-6); 2) enhance conversion of 18:3(n-3) to 20:5(n-3) and 22:6(n-3); and 3) provide sufficient energy in the form of monoenoic fatty acids to maintain high growth rates. In this regard, rapeseed oil (RO) is a potential candidate in that it has moderate levels of 18:2(n-6) and 18:3(n-3), in a ratio of 2:1, and an abundance of 18:1(n-9). The ratio of 18:2(n-6)/18:3(n-3) in rapeseed oil makes it of benefit to human health, as well as fish health (21) . In this study, duplicate groups of Atlantic salmon post-smolts were fed diets containing RO at 10, 25, 50 and 100% of the added oil, in comparison to FO (0% RO), for 17 wk. Growth and feed utilization were measured along with muscle and liver total lipid and fatty acid composition, muscle proximate composition, muscle carotenoid content [astaxanthin (Ax)] and hepatocyte fatty acid desaturation and elongation activities.

	MATERIALS AND METHODS

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Fish, husbandry and experimental diets.

Atlantic salmon S1 smolts (n = 350; initial mean weight, 80 g) were obtained from the F.R.S. Marine Research Unit, Aultbea, Wester Ross, Scotland. The smolts were randomly distributed into 10 1-m diameter tanks of 500-L capacity supplied with nonrecirculated sea water at 10 L/min at the F.R.S. Marine Research Unit. The tanks were subjected to a photoperiod regime of 12 h light:12 h dark and the temperature over the experimental period (August–December 1999) ranged from 7.9 to 14.2°C with an average temperature of 11.7 ± 1.5°C. The diets were supplied by automatic feeders, which were activated for a few seconds every 15 min during daylight hours and adjusted to provide 20 g/kg biomass each day. Fish were weighed individually at the start and finish of the experiment and bulk weighed every 28 d, with the ration adjusted accordingly. An initial sample was taken at the start of the trial, after 9 wk, and final sampling was performed after 17 wk of the feeding experiment. The experiment was conducted in accordance with the British Home Office guidelines regarding research on experimental animals.

Five practical-type commercial extruded diets were formulated (BioMar, Brande, Denmark) to provide 47% crude protein and 25% lipid and differed only in their content of rapeseed oil which was added at 0, 10, 25, 50 and 100% of the total added oil, the remainder of which was FO (Table 1). The diets were designed to satisfy the nutritional requirements of salmonid fish (22) . All diets supplied sufficient (n-3) PUFA to meet requirements for normal growth and development. In diets with high levels of RO, the (n-3) PUFA requirement was met by PUFA derived from dietary fish meal. The proximate composition of the experimental diets including total carotenoid and Ax contents is shown in Table 2 and the dietary fatty acid compositions are shown in Table 3 .

An initial sample of six fish was taken at the start of the experiment to determine baseline levels of lipid and fatty acid composition in liver and white muscle, and carotenoid content in muscle. A similar sample of 12 fish per dietary treatment was taken after 9 wk. After 17 wk, each analytical measurement was performed on at least 10 fish, selected at random, from each tank. Fish were killed by a blow to the head, and samples of liver were dissected and immediately placed in liquid nitrogen. A muscle sample, representative of the edible portion, was obtained by cutting a steak from the "Norwegian Quality Cut" region, between the dorsal and ventral fins. This section was then skinned, deboned, the red muscle trimmed off and the remaining white muscle homogenized, after removal of the dorsal fat body, in a Waring Blender (Fisher Scientific, Loughbourgh, UK). The homogenate was frozen in liquid nitrogen and all samples were stored at -40°C before analysis.

Proximate analysis.

The nutrient composition of the five experimental diets and muscle samples was determined by proximate analysis. Moisture was determined by thermal drying to constant weight in an oven at 110°C for 24 h. Sample weight was recorded before drying and after removal from the oven, after cooling in a desiccator. Moisture was expressed as a percentage of wet weight. Crude protein was determined by combustion using the Dumas process (23) . Crude lipid in diets was determined by acid hydrolysis followed by Soxhlet extraction (24) . Ash content (g/100 g dry weight) was determined by dry ashing in porcelain crucibles in a muffle furnace at 600°C overnight (24) .

Lipid extraction and fatty acid analysis.

Total lipid was extracted from liver and muscle by homogenizing in 20 volumes of chloroform/methanol (2:1, v/v) in an Ultra-Turrax tissue disrupter (Fisher Scientific) and measured gravimetrically according to the method of Folch et al. (25) . Fatty acid methyl esters (FAME) were prepared by acid-catalyzed transesterification of total lipid according to the method of Christie (26) . Extraction and purification of FAME were performed as described by Ghioni et al. (27) . FAME were separated and quantified by gas-liquid chromatography (Carlo Erba Vega 6000, Milan, Italy) using a 30 m x 0.32 mm i.d. capillary column (CP Wax 52CB, Chrompak, London, UK). Hydrogen was used as carrier gas and temperature programming was from 50 to 150°C at 40°C/min and then to 230°C at 2.0°C/min. Individual methyl esters were identified by comparison with known standards and by reference to published data (28) . The gas chromatography method used does not separate 22:1(n-13) from 22:1(n-11) although in the text these combined fatty acids are referred to as 22:1.

Ax measurement.

Total carotenoid was extracted from salmon muscle largely by the method of Barua et al. (29) . Tissue samples were homogenized in 5 mL of absolute ethanol and 5 mL of ethyl acetate using an Ultra-Turrax tissue disrupter (Fisher Scientific). The homogenate was centrifuged (1000 x g, 5 min) and the supernatant removed to a stoppered glass tube. The pellet was rehomogenized in 5 mL of ethyl acetate and recentrifuged, and the supernatant was combined with the first supernatant. Finally, the pellet was rehomogenized in 10 mL of hexane and recentrifuged, and the supernatant combined with the pooled supernatant. The pooled supernatant was dried under a stream of nitrogen and vacuum-desiccated for 2 h before redissolving the residue in 2 mL of hexane containing 0.2g/L BHT. Total carotenoid was measured spectrophotometrically at 470 nm using the E_1% (wt/v) of 2100. Measurement of Ax was carried out using a 5-µm Luna ODS2 column (4.6 x 150 mm, Phenomenex, Macclesfield, UK). The chromatographic system was equipped with a Waters Model 501 pump and Ax was detected at 470 nm using a Waters 490E multiwavelength UV/vis detector [Millipore (UK), Watford, UK]. An isocratic solvent system was used containing ethyl acetate/methanol/water (20:72:8, v/v/v) at a flow rate of 1 mL/min. Ax was detected at 470 nm and quantified using an external standard of Ax obtained from Roche (Heanor, UK). Ax in diets was extracted and measured similarly after an enzymatic digestion of the ground extruded pellets with Maxatase (International Biosynthetics, Rijswijk, Netherlands). Portions of ground diet (1 g) were mixed with 10 mL water and 10 mg of Maxatase in a 50-mL stoppered glass tube followed by incubation in a water bath at 50°C for 30 min.

Preparation of isolated hepatocytes.

The gall bladder and main blood vessels were carefully dissected from the liver. The liver was perfused via the hepatic vein with solution A [calcium- and magnesium-free Hank’s balanced salt solution (HBSS) + 10 mmol/L HEPES + 1 mmol/L EDTA] to clear blood from the tissue. The liver was chopped finely with scissors and incubated with 20 mL of solution A containing 1 g/L collagenase in a 25 mL "Reacti-flask" in a shaking water bath at 20°C for 45 min. The digested liver was filtered through 100-µm nylon gauze and the cells collected by centrifugation at 1000 x g for 5 min. The cell pellet was washed with 20 mL of solution A containing fatty acid–free bovine serum albumin (FAF-BSA, 10 g/L) and recentrifuged. The hepatocytes were resuspended in 10 mL of Medium 199 containing 10 mmol/L HEPES, 2 mmol/L glutamine, 1 x 10⁵ U/L penicillin and 0.1 g/L streptomycin. Cell suspensions (100 µL) were mixed with 400 µL of trypan blue; hepatocytes were counted and their viability assessed using a hemocytometer. Cell suspensions (100 µL) were also retained for protein determination as described below.

Assay of hepatocyte fatty acyl desaturation/elongation activities.

Fatty acyl desaturation and elongation activities were determined in isolated hepatocytes as described by Buzzi (30) . Hepatocyte suspensions (5 mL) were dispensed into two 25-cm² tissue culture flasks. Hepatocytes were incubated with 9.25 KBq of [1-¹⁴C] 18:3(n-3) added as a complex with FAF-BSA. Briefly, 925 KBq of fatty acid (0.5 µmol) in ethanol was placed in a reaction vial, solvent evaporated under a stream of nitrogen and 100 µL of 0.1mol/L KOH added. The mixture was stirred for 10 min at room temperature before the addition of 5 mL of 50 g/L FAF-BSA in HBSS containing 10 mmol/L HEPES buffer; the mixture was stirred for 45 min at 20°C. After isotope addition, the flasks were incubated at 20°C for 2 h. After incubation, the cell layer was dislodged by gentle rocking, the cell suspension transferred to glass conical test tubes and the flasks washed with 1 mL of ice-cold HBSS containing 10 g/L FAF-BSA. The cell suspensions were centrifuged at 300 x g for 2 min, the supernatants discarded and the cell pellets washed with 5 mL of ice-cold HBSS/FAF-BSA and recentrifuged. The supernatants were again discarded and the tubes placed upside down on paper towels to blot for 15 s before extraction of total lipid using ice-cold chloroform/methanol (2:1, v/v) containing 0.1 g/L BHT essentially according to Folch et al. (25) and as described in detail previously (31) .

Total lipid was transmethylated and FAME prepared as described by Tocher et al. (31) . The methyl esters were redissolved in 100 µL hexane containing 0.1 g/L BHT and applied as 2.5-cm streaks to TLC plates impregnated by spraying with 2 g silver nitrate in 20 mL acetonitrile and preactivated at 110°C for 30 min. Plates were fully developed in toluene/acetonitrile (95:5, v/v) (32) . Autoradiography was performed with Kodak MR2 film (Sigma, Poole, UK) for 4–7 d at room temperature. Silica corresponding to individual PUFA was scraped into scintillation mini-vials containing 2.5 mL of scintillation fluid (Ecoscint A. National Diagnostics, Atlanta, GA) and radioactivity determined in a TRI-CARB 2000CA scintillation counter (United Technologies Packard, Pangbourne, UK). Results were corrected for counting efficiency and quenching of ¹⁴C under exactly these conditions.

Protein determination.

Protein concentration in hepatocytes was determined according to the method of Lowry et al. (33) after incubation with 0.25 mL of 2.5 g/L SDS, 1 mol/L NaOH for 45 min at 60°C.

Histology.

Samples of liver, heart, kidney and white muscle were fixed in 200 mL/L buffered formol saline and embedded in paraffin wax. Sections (5 µm) were cut and stained with hematoxylin and eosin for histological analysis. Pathological assessment was carried out on coded, randomized slides to eliminate bias in interpretation. Examination of slides was performed using a Reichert (Microstar IV) compound microscope at an objective magnification of X40 or X100. All organs were examined for general abnormalities such as tumors, infections, inflammation, loss or replacement of tissues.

Statistical analysis.

Significance of difference (P < 0.05) between dietary treatments was determined by one-way ANOVA. Differences between means were determined by Tukey’s test. Data identified as nonhomogeneous, using Bartlett’s test, were subjected to log or arcsin square-root transformation before applying the ANOVA. ANOVA was performed using a Graphpad Prism (version 2.0) statistical package (Graphpad Software, San Diego, CA).

	RESULTS

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There were no significant differences in initial weights of the fish at the start of the trial (80.3–85.3g). Similarly, the final weights and lengths were not significantly different (320–364g and 29.5–30.9 cm, respectively), either between dietary treatments or between replicate tanks from the same treatment. Mortalities over the experimental period were <3% for all groups. Fish increased in weight by around fourfold and specific growth rates (SGR) varied between 1.20 and 1.27. Feed efficiency ratios (g feed/g weight gain; FER) were very similar for all treatments, ranging from 0.75 to 0.78. Although not significantly different (P = 0.19), fish fed the 100% RO diet had the lowest final mean weights and SGR. No histopathologies were evident in liver, heart, muscle or kidney samples taken from fish fed the five experimental diets.

The proximate composition of white muscle (Table 4) showed no significant differences in moisture content, but lipid content was significantly greater, albeit only modestly, in fish fed 0% RO (FO) compared with those fed 50% RO. Conversely, the protein content of muscle was significantly lower, again only modestly so, in fish fed 0% RO compared with all other treatments. Ash content was greatest in fish fed 10 and 50% RO followed by 0 and 25% RO and lowest in fish fed 100% RO. Liver lipid content (Table 4) was significantly higher in fish fed 100% RO compared to fed fish fed 0, 10 and 25% RO. There were no significant differences in total carotenoid pigment or Ax deposition in muscle in fish fed the different experimental diets.

View larger version (34K): [in this window] [in a new window]   Figure 1. Relationship between dietary fatty acid concentrations and muscle fatty acid concentrations of 18:2(n-6), 18:3(n-3), 18:1(n-9) and 22:6(n-3) in total lipids of Atlantic salmon fed either 0% rapeseed oil (RO), 10, 25, 50 or 100% RO.

View this table: [in this window] [in a new window]   Table 6. Correlation coefficients (r) and slopes from plots of dietary fatty acid concentrations vs. fatty acid concentrations in muscle including the difference () between diet and muscle fatty acid values for 0% rapeseed oil (RO) and 100% RO treatments1

View larger version (37K): [in this window] [in a new window]   Figure 2. Relationship between dietary fatty acid concentrations and liver fatty acid concentrations of 18:2(n-6), 18:3(n-3), 18:1(n-9) and 22:6(n-3) in total lipids of Atlantic salmon fed either 0% rapeseed oil (RO), 10, 25, 50 or 100% RO.

View this table: [in this window] [in a new window]   Table 7. Correlation coefficients (r) and slopes from plots of dietary fatty acid concentrations vs. fatty acid concentrations in liver including the difference () between diet and liver fatty acid values for 0% rapeseed oil (RO) and 100% RO treatments1

View larger version (22K): [in this window] [in a new window]   Figure 3. Total amount of [1-14C] 18:3(n-3) desaturated and elongated/(h · mg protein) by isolated hepatocytes from Atlantic salmon post-smolts fed diets containing 0, 10, 25, 50 and 100% supplementary dietary lipid as rapeseed oil (RO). Each column represents the mean ± SD for four separate hepatocyte preparations. Means without a common letter differ, P < 0.05.

View larger version (32K): [in this window] [in a new window]   Figure 4. Amount of [1-14C] 18:3(n-3) desaturated and elongated to18:4(n-3), 20:4(n-3), 20:5(n-3) and 22:6(n-3) by isolated hepatocytes from Atlantic salmon post-smolts fed diets containing 0, 10, 25, 50 and 100% supplementary dietary lipid as rapeseed oil (RO). The 20:5(n-3) column also contains a small amount of 22:5(n-3), which represents <10% of the total activity in the 20:5(n-3) column. Each column represents the mean ± SD for four separate hepatocyte preparations. Means without a common letter differ, P < 0.05.

	DISCUSSION

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The percentage of lipid in the muscle was significantly greater in fish fed 0% RO compared with fish fed 50% RO although the differences between the dietary treatments were small. Conversely, the muscle protein content was significantly lower in fish fed 0% RO compared with all other treatments, and the second highest muscle lipid level, found in fish fed 100% RO, was correlated with the second lowest protein content. This relationship between muscle protein and lipid content has been observed in previous studies with salmonids (36 ,37) . Variation in the type and quantity of dietary PUFA can influence uptake and deposition of carotenoid pigments in salmon flesh (38) . In the present study, there were no significant differences in total muscle carotenoid and Ax concentrations among the five dietary treatments. Flesh pigmentation is an important factor in perception of flesh quality in salmonids (20 ,39) . A previous study observed reduced pigment deposition in salmon when fish oil was replaced with soybean oil or beef tallow (40) .

The fatty acid compositions of tissue lipids of Atlantic salmon are readily influenced by the fatty acid composition of dietary lipid (35 ,41) . This is amply confirmed in the present study, which clearly establishes linear correlations, with different slopes and intercepts, between the percentage of individual fatty acids in dietary lipids and in muscle total lipid, consisting of 85% triacylglycerols (TAG) and 10% phospholipids (PL), and in liver total lipid, consisting of 30% TAG and 50% PL (20 ,42) .

Therefore, although the fatty acid composition of the total lipid is a combination of the TAG and PL compositions, the former predominate in salmon muscle and are thereby the primary influence on the composition of total muscle lipid. In this study, we chose to concentrate on total lipid rather than consider the different attributes of TAG and PL because total lipid reflects the fatty acids available to the consumers of fish. The correlations described above are of practical use for predicting outcomes of feeding different blends of a given substituting oil with fish oil. They also reveal how different fatty acids in dietary lipid are selected for or against relative to tissue lipids. Thus, the data in Tables 5 and 6 and Figure 1 establish that 22:6(n-3) is selectively deposited in muscle lipids. Possible mechanisms underlying this selective deposition include high specificity of fatty acyl transferases for 22:6(n-3) and/or relative resistance of 22:6(n-3) to ß-oxidation stemming from the complex catabolic pathway for this fatty acid. It is of interest that complete replacement of fish oil in the diet with rapeseed oil reduced the percentage of 22:6(n-3) in dietary lipid by more than fourfold but in muscle lipid by only twofold. This residual dietary 22:6(n-3) reflects the presence of fish meal in the diet, and its associated 22:6(n-3)–rich PL and TAG.

In contrast to 22:6(n-3), the monoenes 22:1 and 18:1(n-9) are discriminated against in muscle lipids relative to dietary lipids when present at high concentrations, most clearly seen in the 0 and 100% RO diets, respectively. This is readily accounted for by the ease of ß-oxidation of these monoenes (43 ,44) . The high oil diets used currently in salmon culture and in this study are of course designed to generate energy through extensive fatty acid oxidation, thereby sparing amino acid oxidation and enhancing the growth rate of the fish. It is interesting that when 22:1 and 18:1(n-9) are present in dietary lipid at similar percentages, as in the 0% RO diet, 22:1 appears to be catabolized in preference to 18:1(n-9) (Table 6) . Nevertheless, the data here are consistent with18:1(n-9) being readily oxidized at high concentrations, as in the 100% RO diet, as is the case also for 18:2(n-6) and 18:3(n-3).

The foregoing considerations for muscle total lipid hold also for liver total lipid, except that the trends in liver lipid are quantitatively greater than in muscle lipid. This reflects the predominance in liver of PL, rich in 20:5(n-3) and especially 22:6(n-3), containing a modest percentage of 18:2(n-6) and 18:3(n-3) and negligible 22:1, and whose composition is not so readily influenced by dietary input as muscle lipid, which is rich in TAG (20 ,42) .

Increases in hepatic desaturation and elongation activities have been observed in previous studies in which salmon were fed vegetable oils or vegetable oil blends (14 ,15 ,19) . In the present study, hepatic desaturation and elongation of 18:3(n-3) were clearly enhanced progressively by replacing dietary FO with RO. However, only very small amounts of end product 22:6(n-3) were formed in the short term radiolabeling assay using hepatocytes; the major product was the intermediate 20:4(n-3). This intermediate was not detected in large amounts in fatty acid analyses of hepatic lipids, nor was there any alteration in the ratio of 20:5(n-3) to 22:6(n-3) in hepatic lipids. This suggests that although substituting FO in the diet with RO clearly activates hepatic desaturation of 18:3(n-3) to 18:4(n-3) and its further elongation to 20:4(n-3), the reactions may proceed too slowly to influence significantly the dietary-induced changes in fatty acid compositions of tissue lipids.

Salmon production worldwide in 1996 was 0.64 million mt and production for 2000 is expected to be 0.8 million mt with a 1 million mt harvest predicted 2002. With global fish oil supplies static, or even in decline, the expansion of aquaculture production in general, and salmon production in particular, will occur only if other oil sources are utilized. A number of studies have investigated the suitability of different vegetable oils as replacements for fish oil in a range of fish species including, rainbow trout (Oncorhynchus mykiss) (45 ,46) , brown trout (Salmo trutta) (47) , arctic charr (Salvelinus alpinus) (48) , brook charr (Salvelinus fontinalis) (18) , Atlantic salmon (16 ,17 ,40) and chinook salmon (Oncohynchus tshawytscha) (34) . A notable recent study is that of Torstensen et al. (35) investigating the effects of capelin oil, palm oil and oleic acid–enriched sunflower as dietary lipid sources for Atlantic salmon. Only the last-mentioned study used an oil-rich diet formulation similar to the high energy feeds currently favored for salmonid culture.

Given present concerns about the imbalance of (n-6) and (n-3) PUFA in the diets of developed nations and the encouragement to consume oily fish, such as mackerel, sardines, salmon and trout, it is important that cultured salmon maintain a high level of essential (n-3) HUFA in the edible flesh (3 4 5) . Therefore, successful substitution of fish oil with vegetable oils should minimize depletion of 20:5(n-3) and 22:6(n-3), minimize deposition of 18:2(n-6) and maximize desaturation and elongation of 18:3(n-3) to 20:5(n-3) and 22:6(n-3) while permitting growth rates equivalent to those presently achieved with fish oils. The present study has investigated the potential of rapeseed oil, fed at different inclusion levels, to satisfy these criteria in Atlantic salmon post-smolts in sea water.

In our previous studies with Atlantic salmon fed vegetable oils, including corn, sunflower, grape seed and safflower oils, there was a significant increase in concentrations of arachidonic acid [20:4(n-6)] in fish tissues, resulting in increased eicosanoid production and the appearance of cardiac histopathologies (17 , 49 50 51) . All of the oils mentioned above contain high levels of 18:2(n-6) and only trace levels of 18:3(n-3). Elevated percentages of 20:4(n-6) were not observed in tissue lipids in the present study, nor were cardiac histopathologies observed, consistent with negligible conversion of 18:2(n-6) to 20:4(n-6) resulting from replacement of dietary FO with RO. The presence of substantial levels of 18:3(n-3) in rapeseed oil, despite an excess of 18:2(n-6), can account for the lack of a significant conversion of 18:2(n-6) to 20:4(n-6), due to competitive interactions between (n-3) and (n-6) PUFA in hepatic desaturation and elongation pathways (52 53 54) . However, fully understanding how simultaneously varying dietary levels of precursor 18:2(n-6) and 18:3(n-3), and end product 20:4(n-6), 20:5(n-3) and 22:6(n-3) affects hepatic elongation-desaturation pathways requires further study. Nonetheless, the finding here that substituting dietary FO with RO activates the pathways at least in part offers hope that conditions can be identified whereby at least significant conversion of 18:3(n-3) in the substituting oil to 22:6(n-3) occurs to offset in part the decrease in dietary 22:6(n-3) caused by reduction of dietary FO.

In summary, the present study suggests that rapeseed oil is an effective substitute for fish oil in Atlantic salmon in terms of permitting similar growth rates and feed efficiency, and having no apparent ill effects on fish health. However, inclusion of RO at levels in excess of 50% of supplementary lipid results in significant decreases in the (n-3)/(n-6) PUFA ratio and the EPA and DHA concentrations in fish flesh such that the nutritional benefits of the fish to the human consumer would be considerably reduced. This does not preclude the use of higher levels of rapeseed oil in dietary formulations for Atlantic salmon because such diets could be used for the majority of the growth cycle providing, at an appropriate time before marketing, the fish were returned to a fish oil-containing diet thereby restoring the 18:2(n-6), EPA and DHA concentrations to their "normal" values. These, and other aspects of fish oil substitution, are the subject of ongoing research activity in our laboratory.

	ACKNOWLEDGMENTS

 
We would like to thank Philip MacGlaughlin and the staff of the Fisheries Research Services Marine Research Unit for their assistance with fish husbandry.

	FOOTNOTES

 
¹ Supported by a grant award from the Home Grown Cereals Authority. The study was also supported by the "Aquagene" initiative, funded jointly by the Natural Environment Research Council and the Scottish Higher Education Funding Council.

³ Present address; North Atlantic Fisheries College, Port Arthur, Scalloway, Shetland ZE1 0UN, Scotland, UK.

⁴ Abbreviations used: Ax, astaxanthin; BSA, bovine serum albumin; DHA, docosahexaenoic acid; EFA, essential fatty acid; EPA, eicosapentaenoic acid; FAF, fatty acid–free; FAME, fatty acid methyl esters; FER, feed efficiency ratio; FO, fish oil; HBSS, Hank’s balanced salt solution; HUFA, highly unsaturated fatty acids; kt, kilo tonnes; mt, metric tonnes; PL, phospholipid; PUFA, polyunsaturated fatty acids; RO, rapeseed oil; SGR, specific growth rate; TAG, triacylglycerol.

Manuscript received October 30, 2000. Initial review completed December 4, 2000. Revision accepted February 14, 2001.

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