Influence of dietary n-3 fatty acids on the desaturation and elongation of [1-14C] 18:2 n-6 and [1-14C] 18:3 n-3 in Atlantic salmon hepatocytes

Atlantic salmon (Salmo salar) were fed diets containing fish oil supplemented with 22:6n-3 (FO diet) or linseed oil supplemented with 20:5n-3 (LO diet) for 6 months. The effects of these diets, both containing about 36% n-3 fatty acids, on the esterification, desaturation and elongation of [1-¹⁴C] 18:2n-6 and [1-¹⁴C] 18:3n-3 were investigated in isolated hepatocytes. The percentages of radioactivity which was esterified from [1-¹⁴C] 18:2n-6 or [1-¹⁴C]18:3n-3 into total lipids, were approximately 20% lower in hepatocytes from fish fed the FO diet than in hepatocytes from fish fed the LO diet. The percentages of radioactivity esterified in both groups were further reduced when 0.1 mM unlabelled 22:6n-3 was added to the incubation. The percentage of desaturation and elongation products formed from [1-¹⁴C] 18:2n-6 was twice as high in hepatocytes from salmon fed the FO diet as it was in hepatocytes from fish fed the LO diet. The ratio of 18:2n-6 to 18:3n-3 was five times higher in the FO diet, and this probably promoted the conversion of 18:2n-6 to longer chain n-6 fatty acids. When 0.1mM unlabelled 22:6n-3 was added to the incubation medium, the percentages of desaturation and elongation products formed were unchanged. Thus, a high level of 22:6n-3 in the diet is apparently not inhibiting the conversion of 18:2n-6 to 20:4n-6, as long as the amount of 18:2n-6 present is substantially higher than that of 18:3n-3. No desaturation and elongation products were recovered from the phospholipids of hepatocytes incubated with [1-¹⁴C] 18:3n-3 in any of the groups. However, the `dead end' elongation product 20:3n-3 was found in the triacylglycerol fraction, and the percentage of this fatty acid increased when 22:6n-3 was added to the incubation medium.     

Polyunsaturated fatty acid metabolism in Atlantic salmon (Salmo salar) undergoing parr-smolt transformation and the effects of dietary linseed and rapeseed oils

Duplicate groups of Atlantic salmon parr were fed diets containing either fish oil (FO), rapeseed oil (RO), linseed oil (LO) or linseed oil supplemented with arachidonic acid (20:4n-6; AA) (LOA) from October (week 0) to seawater transfer in March (week 19). From March to July (weeks 20–34) all fish were fed a fish oil-containing diet. Fatty acyl desaturation and elongation activity in isolated hepatocytes incubated with [1-¹⁴C]18:3n-3 increased in all dietary groups, peaking in early March about one month prior to seawater transfer. Desaturation activities at their peak were significantly greater in fish fed the vegetable oils, particularly RO, compared to fish fed FO. Docosahexaenoic acid (22:6n-3:DHA) and AA in liver and gill polar lipids (PL) increased in all dietary groups during the freshwater phase whereas eicosapentaenoic acid (20:5n-3; EPA) increased greatly in all groups after seawater transfer. The AA/EPA ratio in tissue PL increased up to seawater transfer and then decreased after transfer. AA levels and the AA/EPA ratio in gill PL were generally higher in the LOA group. The levels of 18:3n-3 in muscle total lipid were increased significantly in the LO, LOA and, to a lesser extent, RO groups prior to transfer but were reduced to initial levels by the termination of the experiment (week 34). In contrast, 18:2n-6 in muscle total lipid was significantly increased after 18 weeks in fish fed the diets supplemented with RO and LO, and was significantly greater in the FO and RO groups at the termination of the experiment. Gill PGF production showed a large peak about two months after transfer to seawater. The production of total PGF post-transfer was significantly lower in fish previously fed the LOA diet. However, plasma chloride concentrations in fish subjected to a seawater challenge at 18 weeks were all lower in fish fed the diets with vegetable oils. This effect was significant in the case of fish receiving the diet with LOA, compared to those fed the diet containing FO. The present study showed that during parr-smolt transformation in Atlantic salmon there is a pre-adaptive increase in hepatocyte fatty acyl desaturation/elongation activities that is controlled primarily by environmental factors such as photoperiod and temperature but that can also be significantly modulated by diet. Feeding salmon parr diets supplemented with rapeseed or linseed oils prevented inhibition of the desaturase activities that is induced by feeding parr diets with fish oils and thus influenced the smoltification process by altering tissue PL fatty acid compositions and eicosanoid production. These effects, in turn, had a beneficial effect on the ability of the fish to osmoregulate and thus adapt to salinity changes.     

Nutritional regulation of hepatocyte fatty acid desaturation and polyunsaturated fatty acid composition in zebrafish (Danio rerio) and tilapia (Oreochromis niloticus)

The desaturation and elongation of [1-¹⁴C]18:3n-3 was investigated in hepatocytes of the tropical warm freshwater species, zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus). The hepatocyte fatty acid desaturation/elongation pathway was assayed before and after the fish were fed two experimental diets, a control diet containing fish oil (FO) and a diet containing vegetable oil (VO; a blend of olive, linseed and high oleic acid sunflower oils) for 10 weeks. The VO diet was formulated to provide 1% each of 18:2n-6 and 18:3n-3, and so satisfy the possible EFA requirements of zebrafish and tilapia. At the end of the dietary trial, the lipid and fatty acid composition was determined in whole zebrafish, and liver, white muscle and brain of tilapia. Both zebrafish and tilapia expressed a hepatocyte fatty acid desaturation/elongation pattern consistent with them being freshwater and planktonivorous fish. The data also showed that hepatic fatty acid desaturation/elongation was nutritionally regulated with the activities being higher in fish fed the VO diet compared to fish fed the FO diet. In zebrafish, the main effect of the VO diet was increased fatty acid Δ6 desaturase activity resulting in the production of significantly more 18:4n-3 compared to fish fed the FO diet. In tilapia, all activities in the pathway were greater in fish fed the VO diet resulting in increased amounts of all fatty acids in the pathway, but primarily eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). However, the fatty acid compositional data indicated that despite increased activity, desaturation of 18:3n-3 was insufficient to maintain tissue proportions of EPA and DHA in fish fed the VO diet at the same level as in fish fed the FO diet. Practically, these results indicate that manipulation of tilapia diets in commercial culture in response to the declining global fish oil market would have important consequences for fish fatty acid composition and the health of consumers. Scientifically, zebrafish and tilapia, both the subject of active genome mapping projects, could be useful models for studies of lipid and fatty acid metabolism at a molecular biological and genetic level. This revised version was published online in August 2006 with corrections to the Cover Date.     

Partial substitution of fish oil with rapeseed, linseed and olive oils in diets for European sea bass (Dicentrarchus labrax L.): effects on flesh fatty acid composition, plasma prostaglandins E2 and F2α, immune function and effectiveness of a fish oil finishing diet

Triplicate groups of European sea bass (Dicentrarchus labrax L.), of initial weight 90 g, were fed four practical‐type diets in which the added oil was 1000 g kg^?1 fish oil (FO) (control diet), 600 g kg^?1 rapeseed oil (RO) and 400 g kg^?1 FO, 600 g kg^?1 linseed oil (LO) and 400 g kg^?1 FO, and 600 g kg^?1 olive oil (OO) and 400 g kg^?1 FO for 34 weeks. After sampling, the remaining fish were switched to the 1000 g kg^?1 FO diet for a further 14 weeks. Fatty acid composition of flesh total lipid was influenced by dietary fatty acid input but specific fatty acids were selectively retained or utilized. There was selective deposition and retention of docosahexaenoic acid (DHA; 22:6n‐3). Eicosapentaenoic acid (EPA; 20:5n‐3) and DHA were significantly reduced and linolenic (LNA; 18:3n‐3), linoleic (LA; 18:2n‐6) and oleic (OA; 18:1n‐9) acids significantly increased in flesh lipids following the inclusion of 600 g kg^?1 RO, LO and OO in the diets. No significant differences were found among different treatments on plasma concentrations of prostaglandin E₂ and prostaglandin F_2α. Evaluation of non‐specific immune function, showed that the number of circulating leucocytes was significantly affected (P < 0.001), as was macrophage respiratory burst activity (P < 0.006) in fish fed vegetable oil diets. Accumulation of large amounts of lipid droplets were observed within the hepatocytes in relation to decreased levels of dietary n‐3 HUFA, although no signs of cellular necrosis was evident. After feeding a FO finishing diet for 14 weeks, DHA and total n‐3 HUFA levels were restored to values in control fish although EPA remained 18% higher in control than in the other treatments. This study suggests that vegetable oils such as RO, LO and OO can potentially be used as partial substitutes for dietary FO in European sea bass culture, during the grow out phase, without compromising growth rates but may alter some immune parameters.     

Effects of diets containing linseed oil on fatty acid desaturation and oxidation in hepatocytes and intestinal enterocytes in Atlantic salmon (Salmo salar)

We hypothesized that replacing fish oil with 18:3n-3-rich linseed oil may enable salmon to maintain the levels of tissue n-3HUFA levels through a combination of increased desaturation activity and increased substrate fatty acid provision. To this end we investigated desaturation/elongation of [1-¹⁴C18:3n-3 in hepatocytes and intestinal enterocytes, and determined the extent to which 18:3n-3 was oxidized and desaturated by measuring both simultaneously in a combined assay. Salmon smolts were stocked randomly into five seawater pens and fed for 40 weeks on diets in which the fish oil was replaced in a graded manner by linseed oil. At the end of the trial, fatty acyl desaturation/elongation and oxidation activities were determined in isolated hepatocytes and intestinal enterocytes using [1-¹⁴C]18:3n-3 as substrate, and samples of liver and intestinal tissue were collected for analysis of lipid and fatty acid composition. The results showed that, despite increased desaturation of [1-¹⁴C]18:3n-3 in hepatocytes, provision of dietary 18:3n-3 did not prevent the decrease in tissue n-3HUFA in fish fed linseed oil. Intestinal enterocytes were a site of significant fatty acid desaturation but, in contrast to hepatocytes, the activity was not increased by feeding linseed oil and was generally lower in fish fed linseed oil compared to fish fed only fish oil. In contrast, oxidation of [1-¹⁴C]18:3n-3 in enterocytes was generally increased in fish fed linseed oil compared to fish fed the diet containing only fish oil. However, oxidation of [1-¹⁴C]18:3n-3 in hepatocytes was 4- to 8-fold lower than in enterocytes and was not affected by diet. Furthermore, oxidation of [1-¹⁴C]18:3n-3 in enterocytes exceeded desaturation irrespective of dietary treatment, whereas similar amounts of [1-¹⁴C]18:3n-3 were desaturated and oxidized in hepatocytes from fish fed only fish oil and desaturation exceeded oxidation by 3-fold in fish fed the diet containing 100% linseed oil. The molecular mechanisms underpinning these results were discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of essential fatty acid-deficient diets on growth, mortality, tissue histopathology and fatty acid compositions in juvenile turbot (Scophthalmus maximus)

Three diets in which the lipid component was supplied either as fish oil (FO), linseed oil (LO) or olive oil (OO) were fed to duplicate groups of juvenile turbot (Scophthalmus maximus) of initial weight 1.2 g for a period of up to 12 weeks. The latter two diets resulted in a significant reduction in specific growth rate and an increased mortality compared to the FO (control) fed fish. A liver histopathology was evident in around half of the fish fed the LO and OO diets but was absent in fish fed FO. The lesion showed indications of cellular alterations consisting of foci of densely basophilic cells but without evidence of inflammatory activity. The total lipid fatty acid composition of the carcass from fish fed LO had increased percentages of 18:2n-6 and 18:3n-3, but decreased percentages of all other polyunsaturated fatty acids (PUFA) including the physiologically important 20:4n-6, 20:5n-3 and 22:6n-3, compared to fish fed FO. Almost 2/3 of the total fatty acids in the carcass of OO-fed fish were monounsaturated while the percentages of total saturated fatty acids and all other PUFA, except 18:2n-6, were significantly reduced compared to fish fed FO. Broadly similar effects on total lipid fatty acid composition were observed in liver. In the liver glycerophospholipid classes of fish fed LO, percentages of 18:2n-6, 18:3n-3 and 20:3n-3 were significantly increased whereas all C20 and C22 PUFA, with the exception of 20:5n-3 in PI, were significantly reduced compared to fish fed FO. The liver glycerophospholipids of fish fed OO all showed significantly increased total monounsaturates, 18:2n-6, 20:2n-6, 18:2n-9 and 20:2n-9 as well as reduced percentages of 20:4n-6 and 22:6n-3, compared to fish fed FO. The brain glycerophospholipids showed broadly similar changes in response to dietary treatment although the magnitude of fatty acid alterations was less than those observed in liver. The greater mortalities in the OO-fed fish compared to the LO-fed fish suggests that incorporation of 18:3n-3 into tissue phospholipids can offset losses of long-chain PUFA more effectively than incorporation of 18:1n-9. However, levels of dietary long-chain PUFA must be optimised to allow normal growth and development. We conclude that the very low flux through the fatty acid desaturase/elongase pathways in turbot is not up-regulated by diets deficient in 20:5n-3 and 22:6n-3.     

Effects of dietary lipid level and vegetable oil on fatty acid metabolism in Atlantic salmon (Salmo salar L.) over the whole production cycle

Changes in fatty acid metabolism in Atlantic salmon (Salmo salar) induced by vegetable oil (VO) replacement of fish oil (FO) and high dietary oil in aquaculture diets can have negative impacts on the nutritional quality of the product for the human consumer, including altered flesh fatty acid composition and lipid content. A dietary trial was designed to investigate the twin problems of FO replacement and high energy diets in salmon throughout the entire production cycle. Salmon were grown from first feeding to around 2 kg on diets in which FO was completely replaced by a 1:1 blend of linseed and rapeseed oils at low (14–17%) and high (25–35%) dietary oil levels. This paper reports specifically on the influence of diet on various aspects of fatty acid metabolism. Fatty acid compositions of liver, intestinal tissue and gill were altered by the diets with increased proportions of C₁₈ polyunsaturated fatty acids and decreased proportions of n-3 highly unsaturated fatty acids (HUFA) in fish fed VO compared to fish fed FO. HUFA synthesis in hepatocytes and enterocytes was significantly higher in fish fed VO, whereas β-oxidation was unaltered by either dietary oil content or type. Over the entire production cycle, HUFA synthesis in hepatocytes showed a decreasing trend with age interrupted by a large peak in activity at seawater transfer. Gill cell prostaglandin (PG) production showed a possible seasonal trend, with peak activities in winter and low activities in summer and at seawater transfer. PG production in seawater was lower in fish fed the high oil diets with the lowest PG production generally observed in fish fed high VO. The changes in fatty acid metabolism induced by high dietary oil and VO replacement contribute to altered flesh lipid content and fatty acid compositions, and so merit continued investigation to minimize any negative impacts that sustainable, environmentally-friendly and cost-effective aquaculture diets could have in the future. Abbreviations: FO - fish oil; HUFA - highly unsaturated fatty acids acids (carbon chain length ≥C ₂₀ with ≥3 double bonds); LO - linseed oil; RO - rapeseed oil; VO - vegetable oil. This revised version was published online in July 2006 with corrections to the Cover Date.     

Histological alterations in the liver of sea bream, Sparus aurata L., caused by short- or long-term feeding with vegetable oils. Recovery of normal morphology after feeding fish oil as the sole lipid source

This study evaluated the effects of fish oil (FO) replacement by vegetable oils [soybean oil (SO), rapeseed oil (RO), linseed oil (LO)] and subsequent feeding with FO on the liver morphology of sea bream. A short-term trial (3 months) and long-term trial (6 months) were carried out feeding sea bream with the following experimental diets: FO100%; SO60% + FO40%; RO60% +FO40%; LO60% + FO40%; SO + RO +LO60% + FO40%. Finally, all groups from the long-term trial were fed with FO100% for 95 days (washout period). Liver samples were taken for histological and biochemical studies. In both the short- and long-term trials, livers of sea bream fed LO60% and SO + RO + LO60% showed a similar hepatic morphology to that observed in fish fed FO100%. In contrast, sea bream fed SO60% showed an intense steatosis, with foci of swollen hepatocytes containing numerous lipid vacuoles. After the washout period, a considerable reduction of the cytoplasmic vacuolation and the lipid vacuole accumulation were observed in the livers of fish fed the different experimental diets. The results of this study suggested that the type of non-essential fatty acid, characteristic of vegetable oils, induces the appearance of steatosis in the following order: linoleic acid > linolenic acid > oleic acid. However, the liver alterations found during the experimental periods with vegetable oils are reversible when the fish are re-fed with a balanced diet (FO100%), indicating the non-pathological character of these histological changes.     

Effects of diets rich in linoleic (18:2n - 6) and α-linolenic (18:3n - 3) acids on the growth,lipid class and fatty acid compositions and eicosanoid production in juvenile turbot (Scophthalmus maximus L.)

Three practical-type diets utilizing fishmeal and casein as the protein sources and containing fish oil (FO), safflower oil (SO) or linseed oil (LO) were fed to duplicate groups of juvenile turbot (Scophthalmus maximus) of initial weight 1.2 g for a period of 12 weeks. No differences in final weight, mortality or development of pathological lesions were evident either between duplicate tanks or between dietary treatments over this period. Fish fed diets containing SO and LO contained significantly greater amounts of liver triacylglycerol compared to fish fed FO. The major C₁₈ polyunsaturated fatty acids (PUFA) in SO and LO diets, 18:2(n-6) and 18:3(n-3) respectively, were readily incorporated into both total lipid and individual phospholipids of turbot tissues. There was no accumulation of the Δ6-desaturation products of these fatty acids, namely 18:3(n-6) and 18:4(n-3), in any of the tissues examined. The products of elongation of 18:2(n-6) and and 18:3(n-3), 20:2(n-6) and 20:3(n-3) respectively, accumulated in both total lipid and phospholipids with the highest levels of 20:2(n-6) in liver PC and 20:3(n-3) in liver PE. Eicosapentaenoic acid [EPA, 20:5(n-3)] levels exceeded those of arachidonic acid [AA, 20:4(n-6)] in phosphatidylinositol (PI) from liver and gill of fish fed LO. EPA levels in liver PI from fish fed LO were 3-fold and 2-fold greater than SO-fed and FO-fed fish, respectively. Fish fed diets containing SO and LO had significantly reduced levels of AA in liver and muscle total lipid and lower AA in individual phospholipid classes of liver and gill compared to FO-fed fish. The concentration of thromboxane B₂ was significantly reduced in plasma and isolated gill cells stimulated with calcium ionophore A23187 of fish fed SO and LO compared to those fed FO. Prostaglandin E produced by isolated gill cells stimulated with A23187 was significantly reduced in fish fed both SO and LO compared to fish fed FO.     

Effects of different dietary lipid sources in the diet for Pangasius nasutus (Bleeker, 1863) juveniles on growth performance,feed efficiency,body indices and muscle and liver fatty acid compositions

Four isonitrogenous (300 g kg^?1 crude protein), isoenergetic (21 kJ g^?1) experimental diets were formulated to contain fish oil (FO), soybean oil (SBO), crude palm oil (CPO) and linseed oil (LO), respectively, as the lipid sources, added at 120 g kg^?1 of crude lipid each. The diets were fed by hand to triplicate groups of Pangasius nasutus (Bleeker, 1863) juveniles (mean weight 10.66 ± 0.04 g), to apparent satiation twice daily for 12 weeks. Fish survival rate was 100% among all the treatments. Growth performance (DGR) was similar among fish fed the SBO, CPO and LO diets, but was significantly (P < 0.05) higher in the CPO compared to fish fed the control (FO) diet. Fish fed SBO and CPO diets also recorded significantly (P < 0.05) higher intraperitoneal fat compared to fish fed the control, whereas fish fed the LO diet did not significantly differ from the other treatments. Muscle and liver fatty acid profile of fish from all the treatments generally mirrored the composition of the diets fed and the major fatty acids recorded were 18:3n‐3 and 18:2n‐6 in the tissues of fish fed the LO and SBO treatments, respectively. Results of this study suggests that P. nasutus fed diets containing vegetable oils (especially CPO and SBO) produce better growth performance, without compromising fish survival and feed efficiency compared with those fed a diet containing only FO.