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*
Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA, Scotland, UK and
BioMar Ltd., North Shore Road, Grangemouth Docks, Grangemouth, FK3 8UL, Scotland, UK
2To whom correspondence should be addressed. E-mail: gjb1{at}stir.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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10-fold greater in fish fed 100% PO than in those fed 0% PO. This study suggests that PO can be used successfully as a substitute for FO in the culture of Atlantic salmon in sea water. However, at levels of PO inclusion above 50% of dietary lipid, significant reductions in muscle 20:5(n-3), 22:6(n-3) and the (n-3):(n-6) PUFA ratio occur, resulting in reduced availability of these essential (n-3) highly unsaturated fatty acids to the consumer.
KEY WORDS: • Atlantic salmon • palm oil • PUFA • lipid metabolism • fish oil
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
). However, global capture of wild fish is currently in decline due to over fishing and environmental changes such that any increased demand for fish products will have to be met by aquaculture (4
).
Aquaculture production has grown by >10% per year since 1984 and is projected to at least double over the next decade and beyond (4
,5
). Production of Atlantic salmon was 480,000 metric tons (mt) in 1996 and is expected to reach 1 million mt by 2005. Aquaculture has traditionally used 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 (FO) production in 1996 of 1.4 million mt, 576,000 mt were used for salmon and trout production and the predicted use of FO for aquaculture is estimated to rise to 85% of the total available by 2010 (6
,7
). Meanwhile, reduced supply of FO and fish meal due to stagnation in capture fisheries and events such as El Nino has led to the investigation of alternative lipid sources for use in salmonid production. Commercial salmon feeds are generally rich in oil, constituting between 25% and 35% of the diet at different stages of the seawater growth phase.
Dietary lipids provide essential PUFA for normal growth and development of cells and tissues (8
) and are also a major source of energy in Atlantic salmon diets (9
,10
). Studies investigating mitochondrial ß-oxidation have suggested that saturated and monounsaturated fatty acids are preferred over PUFA for energy production in fish (11
). In rainbow trout (Oncorhynchus mykiss) liver, 22:1(n-11) and 16:0 were the best substrates for mitochondrial ß-oxidation in vitro while in red muscle 16:0, 16:1, 18:1(n-9) and 18:2(n-6) were the preferred substrates (12
,13
). However, digestion and absorption of saturated and monounsaturated fatty acids is inferior to PUFA in fish, probably due to melting point effects and the tendency for long-chain saturated and monounsaturated fatty acids to form insoluble soaps with divalent cations in the gut (14
,15
). Therefore, when selecting potential vegetable oils for substituting FO in diets of fish, particularly coldwater species, energy availability as well as PUFA content must be considered.
Farmed Atlantic salmon is currently of high nutritional quality with an abundance of (n-3) HUFA and a high (n-3):(n-6) PUFA ratio (16
,17
). Clearly, producers and consumers of salmon will want to minimize any reduction in quality, in terms of the health of the farmed fish, its organoleptic qualities and its health benefits in the human diet, arising from the substitution of FO with vegetable oils. The high growth rates of cultured salmon are due to the use of high energy diets, i.e., diets rich in Northern hemisphere marine FO with high concentrations of C20 and C22 monoenoic fatty acids. Thus, potential substitutes for FO in salmon feeds should avoid excessive deposition of 18:2(n-6), retain high levels of 20:5(n-3) and 22:6(n-3) and provide sufficient energy in the form of saturated and monounsaturated fatty acids to maintain high growth rates. Palm oil (PO) is currently second behind soybean oil in world seed oil production tonnage (18
) and it is rich in 16:0 and 18:1(n-9) fatty acids, but has relatively low levels of 18:2(n-6), as well as carotenes, tocopherols and tocotrienols (19
). Crude PO is, therefore, a potential candidate for FO substitution in salmonid fish. In this study, duplicate groups of Atlantic salmon postsmolts were fed diets containing PO at 25%, 50% and 100% of their added oil compared with FO (0% PO), for 30 wk. Growth and feed utilization were measured along with muscle and liver total lipid and fatty acid compositions, muscle proximate composition, muscle carotenoid content (astaxanthin; Ax) and hepatocyte fatty acid desaturation and elongation activities.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Two hundred forty Atlantic salmon S1/2 smolts (salmon moved to sea water in <1 y; initial mean weight, 55 g) were obtained from Marine Harvest (Loch Garry, Invernesshire, Scotland). The smolts were randomly distributed into eight 1-m diameter tanks of 500-L capacity supplied with nonrecirculated sea water at 10 L/min at the Fisheries Research Services, 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 (February to September 2000) ranged from 5.9°C to 14.7°C with an average temperature of 11.0°C ± 2.3°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 and the ration adjusted accordingly. An initial sample was taken at the start of the trial, after 12 wk, and final sampling was performed after 30 wk of the feeding experiment. The experiment was conducted in accordance with the British Home Office guidelines regarding research on experimental animals.
Four practical-type commercial extruded diets were formulated (BioMar, Brande, Denmark) to provide 47% crude protein and 28% lipid. These diets differed only in their content of crude PO, which was added at 0, 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 including (n-3) essential fatty acids, which are provided by the inclusion of dietary fishmeal (20
). 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
.
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An initial sample of six fish was taken at the start of the experiment to determine baseline levels of lipid and fatty acid compositions in liver and white muscle and carotenoid content in muscle. A similar sample of six fish per dietary treatment was taken after 12 wk of the trial. After 30 wk, each analytical measurement was performed on at least eight 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 and the muscle homogenized, after removing the dorsal fat body in a Waring Blendor (Fisher Scientific, Loughborough, U.K.). The homogenate was frozen in liquid nitrogen and all samples were stored at -40°C before analysis.
Proximate analysis.
The nutrient composition of the four 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 the percentage of wet weight. Crude protein was determined by combustion using the Dumas process (21
). Crude lipid in diets was determined by acid hydrolysis followed by Soxhlet extraction (22
). Ash content (percentage of dry weight) was determined by dry ashing in porcelain crucibles in a muffle furnace at 600°C overnight (22
).
Lipid extraction and fatty acid analysis.
Total lipid was extracted from liver and muscle by homogenising in 20 volumes of chloroform/methanol (2:1, v/v) in an Ultra-Turrax tissue disrupter (Fisher Scientific, Loughborough, UK). Total lipid was prepared according to the method of Folch et al. (23
) and nonlipid impurities removed by washing with 88 g/L KCl. The weight of lipid was determined gravimetrically after evaporation of solvent and overnight desiccation in vacuo. Fatty acid methyl esters (FAME) were prepared by acid-catalyzed transesterification of total lipid according to the method of Christie (24
). Extraction and purification of FAME were performed as described by Ghioni et al. (25
). FAME were separated and quantified by gas-liquid chromatography (Carlo Erba Vega 8160; 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°C to 150°C at 40°C/min and then to 225°C at 2.0°C/min. Individual methyl esters were identified by comparison with known standards and by reference to published data (26
). Data were collected and processed using the Chromcard for Windows, Version 1.19 computer package (Thermoquest Italia S.p.A., Milan, Italy).
Ax measurement.
Total carotenoid was extracted from salmon muscle primarily by the method of Barua et al. (27
). Tissue samples were homogenized in 5 mL of absolute ethanol and 5 mL of ethyl acetate using an Ultra-Turrax tissue disrupter. The homogenate was centrifuged (1000 x g, 5 min) and the supernatant removed to a stoppered glass tube. The pellet was rehomogenised 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 butylated hydroxy toluene (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). One gram portions of ground diet 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 + 1mmol/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 (10 g/L, FAF-BSA) 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 suspension (100 µL) was mixed with 400 µL of trypan blue, and hepatocytes were counted and their viability assessed using a hemocytometer. Cell suspension (100 µL) was retained for protein determination as described below.
Assay of hepatocyte fatty acyl desaturation/elongation activities.
Five milliliters of each hepatocyte suspension were dispensed into two 25-cm2 tissue culture flasks. Hepatocytes were incubated with 0.25 µCi of [1-14C] 18:3(n-3) added as a complex with FAF-BSA. Briefly, 25 µCi 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 0.1 mol/L KOH added. The mixture was stirred for 10 min at room temperature before 5 mL of 50 g/L FAF-BSA in HBSS containing 10 mmol/L HEPES buffer were added and the mixture stirred for 45 min at 20°C. After addition of isotope 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. (23
) and as described in detail previously (28
).
Total lipid was transmethylated and FAME prepared as described by Tocher et al. (28
). 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) (29
). Autoradiography was performed with Kodak MR2 film (Sigma, Poole, UK) for 4–7 d at room temperature. Silica zones corresponding to individual PUFA were 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. (30
) 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 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 non-homogeneous, 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).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Final weights and lengths were not different, either, among dietary treatments or between replicate tanks from the same treatment. Mortalities over the experimental period were <9% for all treatments. All groups of fish increased in weight by around sevenfold and specific growth rates (SGR) varied between 0.93 and 0.98. Feed conversion ratios (FCR) were similar for all treatments ranging from 1.47 to 1.55. No histopathologies were evident in liver, heart or muscle samples taken from fish fed the four experimental diets.
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). Conversely, muscle lipid levels were higher in fish fed 0% PO compared with fish fed 50% and 100% PO. Muscle ash levels were greater in fish fed 100% PO compared with fish fed 0 and 50% PO. Muscle protein content was greater in fish fed the 50% and 100% PO diets compared with fish fed the 0% PO diet. Liver lipid and muscle total carotenoid pigment and Ax concentrations (Table 5)
were not affected by dietary treatment.
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. Plots of concentration (g/100 g) of fatty acids in flesh lipid (Table 6)
against their concentration (g/100 g) in dietary lipid (Table 3)
yielded straight lines, typical examples of which are shown in Figure 1
. Correlation coefficients and slopes of the lines for individual fatty acids are shown in Table 7
, which establishes that different fatty acids have different slopes. For those fatty acids whose concentration in flesh lipids increased as their concentration in dietary lipid increased (due to increasing levels of PO), i.e., 16:0, 18:0, 18:1(n-9) and 18:2(n-6), a slope greater than unity means the concentration in flesh lipid was higher than in dietary lipid. This applied to 18:1(n-9) and 18:2(n-6) with slopes of 1.33 and 1.23, respectively. However, 16:0 and 18:0 with slopes of 0.23 and 0.50, respectively, were present in flesh lipid in lower concentrations than in dietary lipid. Thus, 18:1(n-9) and 18:2(n-6) were preferentially deposited in flesh lipid relative to dietary lipid, whereas 16:0 and 18:0 were preferentially discriminated against in flesh lipid relative to dietary lipid. Equally for those fatty acids whose concentration in flesh lipids decreased due to their absence from or presence in very low concentrations in dietary lipid, e.g., 18:3(n-3), a slope greater than unity means they were being preferentially mobilized as their concentration in dietary lipid decreased (with increasing levels of PO in the diet) and a slope less than unity, as for 18:3(n-3) (Table 7)
, means they were being preferentially retained. Thus, 18:3(n-3), 20:1(n-9), 22:1(n-11), 20:5(n-3) and 22:6(n-3) were all preferentially retained in flesh lipid because their concentrations in dietary lipid decreased with increasing levels of PO in the diet. 20:1(n-9) was the most strongly retained and 18:3(n-3) the least strongly retained.
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and Figure 1
is that the concentration of 22:6(n-3) in flesh lipid decreased only slightly, from 16:3 g/100 g to 14.6 g/100 g, as dietary FO was replaced progressively with PO up to 50% of each. However, the ratio of (n-3):(n-6) PUFA in flesh lipid is approximately halved from 6.2 to 3.3 after substitution of one-half of the dietary FO with PO (Table 6)
. Complete replacement of FO with palm (100% PO diet) caused a further halving of the concentration of 22:6(n-3) in flesh lipid from 14.6 g/100 g to 7.7 g/100 g and a further, now drastic, reduction of the (n-3):(n-6) PUFA ratio from 3.3 to 0.9 (Table 6)
.
Hepatocyte fatty acid desaturation and elongation activities were significantly affected by dietary PO inclusion (Figs. 2
-4). Total (summed) desaturation products (Fig. 2)
of [1-14C] 18:3(n-3) were significantly elevated, by more than fourfold, in hepatocytes from fish fed 100% PO compared with the other three dietary treatments. The summed products of 
6-desaturation, C18–20 elongation, 5-desaturation and 22:6(n-3) production from [1-14C] 18:3(n-3) are shown in Figure 3
. In fish fed 100% PO there was significant stimulation of all steps in the hepatic pathway of 22:6(n-3) production from 18:3(n-3). The individual desaturation products from [1-14C] 18:3(n-3) are shown in Figure 4
. The main product of 18:3(n-3) metabolism was 20:4(n-3) followed by 20:5(n-3), 18:4(n-3), 22:5(n-3) and 22:6(n-3) and the hepatic production of all five radiolabeled fatty acids was stimulated by feeding 100% PO.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), hold promise that blends of dietary oils can be developed that allow substantial replacement of marine FO with vegetable oil alternatives. Both rapeseed and PO can meet the energy requirements of the fish by providing easily oxidized fatty acids and, simultaneously, generate flesh fatty acid compositions that are beneficial to the consumer both by maximizing retention of desirable fatty acids such as 22:6(n-3) and by minimizing deposition of undesirable fatty acids such as 18:2(n-6).
This study demonstrated that feeding diets containing PO, ranging from 25% to 100% of added oil, had no significant effect on growth rate or feed conversion ratio, compared with fish fed marine FO (0% PO). The SGR and FCR values were somewhat poorer than would normally be expected for salmon but may be due to the following. The experiment was begun in February with S1/2 smolts when feed intake is low due to both physiological adaptation to sea water and low water temperatures. In the period from May to August, SGR and FCR values improved markedly but there was a downturn in the final month due to the tanks having exceeded their optimum biomass. In comparison with the recent study of Torstensen et al. (32
), where PO was used in Atlantic salmon diets, the growth and feed conversion data in this were favorable, although the study of Torstensen et al. (32
) used considerably larger fish.
Although PO has been little studied as a lipid source for salmonids, a number of studies have investigated dietary PO in warm water species. Viegas and Contreras (33
) found that dietary PO gave a similar growth and feed conversion to soybean oil in fingerling tambaqui (Colossoma macropomum) and a similar result was observed with Nile tilapia (Oreochromis niloticus L.) (34
). African catfish (Heterobranchus longifilis) showed highest growth when given PO or copra oil when compared with peanut, cotton seed or cod liver oil (35
), although Ng et al. (36
) found that PO gave similar growth to cod liver oil, corn oil and soybean oil in a tropical bagrid catfish (Mystus nemurus). However, all these warm water fish species are fed diets with low lipid concentration (circa. 10%) and none would normally encounter much (n-3) HUFA in their natural diets.
Nonetheless, a number of other vegetable oils have been used experimentally as replacements for FO in the culture of salmonid fish, including rainbow trout (Oncorhynchus mykiss) (37
,38
), brown trout (Salmo trutta) (39
), arctic charr (Salvelinus alpinus) (40
), brook charr (Salvelinus fontinalis) (41
), Atlantic salmon (42
–44
) and chinook salmon (Oncohynchus tshawytscha) (45
).
In this study, muscle lipid levels were significantly higher in fish fed 0% PO (100% FO) compared with fish fed 50% and 100% PO. Conversely, the muscle protein content was significantly lower in fish fed 0% PO, compared with fish fed 50% and 100% PO, and the second highest muscle lipid level, found in fish fed 25% PO, was correlated with the second lowest protein content. This relationship between muscle protein and lipid content has been observed in previous studies with salmonids (46
,47
). This is similar to a previous study in which FO was replaced by rapeseed oil although in the latter study the lowest lipid levels were in fish fed 50% rapeseed oil and the fish fed 100% rapeseed oil were not different from the FO-fed fish (31
). This suggests that different vegetable oils may have different effects with respect to adiposity. It should be stressed, however, that in both these studies fish were not grown to market size so that the full extent of differences in adiposity due to the different dietary oils cannot be fully assessed. Lipid deposition in the liver was not affected by dietary treatment in the present study. However, in salmon fed 100% rapeseed oil lipid deposition in the liver was significantly increased compared with fish fed FO (31
). Alterations in dietary oils have been shown to influence uptake and deposition of carotenoid pigments in salmon muscle (48
). In this study there were no significant differences in total muscle carotenoid or Ax concentrations among the four dietary treatments. An earlier study observed reduced pigment deposition in salmon fed diets containing beef tallow or soybean oil (42
). Flesh carotenoid pigmentation is an important factor in retailer and consumer perception of flesh quality in salmonids (16
,49
) and carotenoids are also important cellular antioxidants (50
).
The fatty acid compositions of tissue lipids in Atlantic salmon are well known to be readily influenced by the fatty acid composition of dietary lipids (31
,32
,43
,51
). This is clearly the case in this study also, where linear correlations existed between the concentrations of given fatty acids in flesh total lipid and their concentrations in dietary lipid. Linear correlations also existed in trials similar to the present one, with salmon, where FO was progressively replaced with rapeseed oil (31
). Such linear correlations can be used to predict how flesh fatty acid composition changes with changing dietary levels of a given oil. However, they cannot presently be used to predict how flesh fatty acid composition changes with different oils. Thus, this study with increasing dietary PO shows that 18:1(n-9) and 18:2(n-6) are preferentially deposited in flesh lipid, whereas 20:1(n-9) and 22:1(n-11) are selectively retained. Our previous study with increasing dietary rapeseed oil showed that 20:1(n-9) and 22:1(n-11) were selectively mobilized from flesh lipid, whereas 18:1(n-9) and 18:2(n-6), although accumulating in flesh lipid, were discriminated against in flesh lipid relative to the diet (31
). Such differences presumably reflect different rates of assimilation of dietary fatty acids together with different rates of catabolism of fatty acids once assimilated. Moreover, there seem to be interactions between different fatty acids in determining the final composition of flesh lipids. Thus, in the present PO trial 20:1(n-9) and 22:1(n-11) were selectively retained in flesh lipid relative to dietary lipid, i.e., they do not appear to be favored for mobilization and catabolism. In the rapeseed oil trial 20:1(n-9) and 22:1(n-11) were selectively depleted in flesh lipid relative to dietary lipid, i.e., they appeared to be favored for mobilization and catabolism. This presumably reflects differences in the ease of assimilation and catabolism of associated fatty acids present in different amounts in the two oils tested, principally 18:1(n-9) and 16:0 in the palm oil and 18:1(n-9), 18:2(n-6) and 18:3(n-3) in the rapeseed oil. The underlying mechanisms determining flesh fatty acid composition remain unknown. However, it is already clear from the present, and our previous study (31
), that up to 50% of dietary FO can be replaced with vegetable oil with only modest decreases in flesh 22:6(n-3).
Several studies with salmon have recorded increased activities of hepatic desaturation and elongation when vegetable oils have replaced FO in dietary formulations (31
,52
–54
). In this study, hepatic desaturation and elongation of radiolabeled 18:3(n-3) was increased progressively with added dietary PO, although only fish fed 100% PO were significantly different from the other three treatments with a10-fold higher activity than fish fed 0% PO. This stimulatory effect by PO was considerably greater than in previous studies using blends of rapeseed and linseed oils (31
,52
,53
). Although 20:4(n-3) was the major product of 18:3(n-3) metabolism, all steps in the metabolic conversion of 18:3(n-3) to 22:6(n-3) were stimulated, particularly in fish fed 100% PO. PO contains negligible amounts of (n-3) PUFA and a moderate amount of 18:2(n-6). The increased flux through the 6-desaturation step was confirmed by the increase in 20:3(n-6) (10-fold) in livers from fish fed 100% PO (0.2 g/100g in 0% PO to 2.8 g/100g in 100% PO), although there was no increase in 20:4(n-6) in the total lipid fraction. The stimulation of hepatic desaturation and elongation activity by vegetable oils presumably reflects increased substrate availability [18:2(n-6) and/or 18:3(n-3)] coupled with reduced end product concentration [20:5(n-3) and 22:6(n-3)], with high levels of the latter inhibiting the pathway (55
,56
).
In previous studies with Atlantic salmon fed 18:2(n-6)-rich vegetable oils, including corn, sunflower, grape seed and safflower oils, there was a significant increase in tissue 20:4(n-6) deposition, increased eicosanoid production and the appearance of cardiac histopathologies (44
,57
–59
). Although the above oils contain more 18:2(n-6) than PO, the oil level in the present study was 28% compared with 15% in the earlier studies. Nonetheless, elevated levels of 20:4(n-6) were not seen in this study, nor were any cardiac histopathologies observed.
In conclusion, this study suggests that PO can be used as an effective substitute for FO in Atlantic salmon in that it allows similar growth and feed conversion while having no apparent negative effects on fish health. Despite evidence to suggest that, at lower water temperatures, the digestibility of saturated and monounsaturated fatty acids present in PO may be impaired (32
), this was not reflected in reduced growth rates in this study. Inclusion of PO at levels above 50% resulted in significant reductions in (n-3) HUFA in the flesh such that the nutritional benefit to the consumer would be reduced. Higher levels of PO could, in principle, be used in feed formulations for salmon provided that, at an appropriate time before harvest, fish were placed on a FO diet so that flesh levels of 18:2(n-6), 20:5(n-3) and 22:6(n-3) were normalized (16
,17
). These and other aspects of FO substitution are subject to ongoing research activity in our laboratory.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: Ax, astaxanthin; BHT, butylated hydroxytoluene; BSA, bovine serum albumin; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FAF, fatty acid-free; FAME, fatty acid methyl esters; FCR, feed conversion ratio; FO, fish oil; HBSS, Hank’s balanced salt solution; HUFA, highly unsaturated fatty acids; mt, metric tonnes; PO, palm oil; SGR, specific growth rate; TLC, thin-layer chromatography.
Manuscript received 4 June 2001. Initial review completed 26 July 2001. Revision accepted 16 November 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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