J. Gordon Bell

Recent developments in the essential fatty acid nutrition of fish

Abstract Because of competitive interactions in the metabolism of polyunsaturated fatty acids, tissue and bodily requirements for each of the three dietary essential fatty acids in marine fish, 22:6 n −3, 20:5 n −3 and 20:4 n −6, cannot be meaningfully considered in isolation. Rather, it is necessary to consider requirements in relative as well as absolute amounts, i.e., in terms of the ratio of 22:6 n −3:20:5 n −3:20:4 n −6. This is illustrated by recent research in our laboratories which has suggested that the optimal dietary ratio of 22:6 n −3:20:5 n −3 in sea bass larvae is circa 2:1 with the optimal dietary ratio of 20:5 n −3:20:4 n −6 being circa 1:1. The optimal dietary ratio of 22:6 n −3:20:5 n −3 in turbot and halibut larvae is similarly circa 2:1 but the optimal dietary ratio of 20:5 n −3:20:4 n −6 in these species is 10:1 or greater. In addition, studies with salmon parr point to dietary 18:3 n −3 and 18:2 n −6 being important in determining the optimal tissue ratio of 20:5 n −3:20:4 n −6 for successful parr–smolt transition. We deduce that differences in essential fatty acid requirements for different species of fish reflect different dietary and metabolic adaptations to different habitats and consider how such knowledge can be exploited to develop improved diets for fish, especially in their early stages of development.

Lipid nutrition of marine fish during early development: current status and future directions

Abstract Research on the dietary requirements of marine fish larvae has evolved from considerations of optimal dietary levels of n −3 HUFA to considerations of optimal dietary ratios of the two principal HUFAs, 22:6 n −3 and 20:5 n −3, and more recently to considerations of optimal dietary levels and ratios of all three dietary essential fatty acids, 22:6 n −3, 20:5 n −3 and 20:4 n −6. Our present understanding of the requirements and optimal dietary balance of 22:6 n −3, 20:5 n −3 and 20:4 n −6 is reviewed. Limitations of enriching live feed are considered, particularly from the point of view of achieving an optimal balance between levels of phospholipids and triacylglycerols in enriched live feeds that generate an optimal blend of essential fatty acids and energy-yielding fatty acids. It is concluded that the ideal marine fish larval diet is one containing circa 10% of the dry weight as n −3 HUFA-rich, marine phospholipids with less than 5% triacylglycerols, as exemplified by the lipid compositions of marine fish egg yolk, marine fish larvae themselves and their natural zooplankton prey. Such diets provide 22:6 n −3, 20:5 n −3 and 20:4 n −6 in the desired levels and ratios and simultaneously satisfy known requirements for phospholipids, inositol and choline. Approaches to developing marine fish larval diets more closely resembling this “gold standard” diet are considered.

Arachidonic acid in aquaculture feeds: current status and future opportunities

The importance of arachidonic acid (20:4n6, ARA) in fish nutrition has tended to be overlooked in preference to eicosapentaenoic (20:5n3, EPA) and docosahexaenoic acids (22:6n3, DHA), probably due to the predominance of the latter two HUFA in fish tissues. However, despite the abundance of EPA and DHA in fish tissues, the importance of ARA as the primary eicosanoid precursor has been recognised for some time. Only very recently has the relative importance of ARA been given due acknowledgement and already the potential benefits to fish physiology and biochemistry of optimising the ARA nutrition of fish are becoming clear. In the coming decade, the role of dietary ARA should be the focus of considerably more research activity than has occurred to date. The results will undoubtedly emphasise why this ubiquitous HUFA cannot be underestimated in fish nutrition. D 2003 Elsevier Science B.V. All rights reserved.

Rapeseed oil as an alternative to marine fish oil in diets of post-smolt Atlantic salmon (Salmo salar): changes in flesh fatty acid composition and effectiveness of subsequent fish oil “wash out”

Abstract Duplicate groups of Atlantic salmon post-smolts were fed five practical-type diets in which the added oil 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 16 weeks. After sampling, the remaining fish were switched to a commercial grower diet containing FO as the only added lipid for a further 12 weeks. There were no effects of the inclusion of RO on growth or feed conversion. Fatty acid compositions of flesh total lipid showed significant increases in 18:1n-9, 18:2n-6 and 18:3n-3 with increasing inclusion of RO. The percentages of flesh 20:5n-3 and 22:6n-3 were significantly reduced in fish fed 10%, 25% and 50% RO, compared to fish fed 0% RO, and the percentages in fish fed 100% RO were significantly lower than all other treatments. Following transfer to the FO washout diet, percentages of flesh 20:5n-3 were not significantly different among treatments after 4 weeks while percentages of 22:6n-3 were restored after 12 weeks. However, even after 12 weeks, percentages of flesh 18:2n-6 were still significantly higher in fish previously fed 50% and 100% RO compared to the other treatments, although the final values were reduced by 48% and 65%, respectively, following the 12-week washout period. This study suggests that RO is a potential substitute for FO in Atlantic salmon culture, but that percentages of the n-3 highly unsaturated fatty acid (HUFA), 20:5n-3 and 22:6n-3, are significantly reduced by feeding RO above 50% of added oil. However, percentages of these two HUFA can be restored by feeding a diet containing FO for a period of 12 weeks.

Replacement of dietary fish oil with increasing levels of linseed oil: Modification of flesh fatty acid compositions in Atlantic salmon (Salmo salar) using a fish oil finishing diet

Five groups of salmon, of initial mean weight 127±3 g, were fed increasing levels of dietary linseed oil (LO) in a regression design. The control diet contained capelin oil (FO) only, and the same oil was blended with LO to provide the experimental diets. After an initial period of 40 wk, all groups were switched to a finishing diet containing only FO for a further 24 wk. Growth and flesh lipid contents were not affected by dietary treatment. The FA compositions of flesh total lipids were linearly correlated with dietary FA compositions (r2=0.88–1.00, P<0.0001). LO included at 50% of added dietary lipids reduced flesh DHA and EPA (20∶5n−3) concentrations to 65 and 58%, respectively, of the concentrations in fish fed FO. Feeding 100% LO reduced flesh DHA and EPA concentrations to 38 and 30%, respectively, of the values in fish fed FO. Differences between diet and flesh FA concentrations showed that 16∶0, 18∶1n−9, and especially DHA were preferentially retained in flesh, whereas 18∶2n−6, 18∶3n−3, and 22∶1n−11 were selected against and presumably utilized for energy. In fish previously fed 50 and 100% LO, feeding a finishing diet containing FO for 16 wk restored flesh DHA and EPA concentrations, to ≈80% of the values in fish fed FO throughout. Flesh DHA and EPA concentrations in fish fed up to 50% LO were above recommended intake values for humans for these EFA. This study suggests that LO can be used as a substitute for FO in seawater salmon feeds and that any reductions in DHA and EPA can be largely overcome with a finishing diethigh in FO before harvest.

The effect of dietary lipid on polyunsaturated fatty acid metabolism in Atlantic salmon (Salmo salar) undergoing parr-smolt transformation

The aim of this study was to measure the changes in lipid metabolism which occur during smoltification and seawater transfer in Atlantic salmon (Salmo salar). Duplicate groups of Atlantic salmon parr were fed diets containing either fish oil (FO) or a blend of linseed and rapeseed oils, vegetable oil (VO), from October (week 0) to seawater transfer in May (week 26). From May to August (weeks 26–43), all fish were fed a fish oil-containing diet. Fatty acyl desaturation and elongation activity were followed in isolated hepatocytes incubated with radioactive 18:3n−3 and 18:2n−6. Metabolism of 18:3n−3 was consistently around 5-fold greater than metabolism of 18:2n−6, and total metabolism of both substrate polyunsaturated fatty acids (PUFA) was increased in fish fed both VO and FO up to seawater transfer after which desaturation activities were reduced. Desaturation activities with both 18:3n−3 and 18:2n−6 were significantly greater in fish fed VO, compared to fish fed FO, at 22 and 26 wk. Arachidonic acid (20:4n−6; AA) in liver polar lipids (PL) of fish fed VO increased consistently from weeks 0–22 but varied after seawater transfer. In fish fed FO, AA in liver PL remained constant up to week 17 before increasing at seawater transfer and leveling off thereafter. Eicosapentaenoic acid (20:5n−3; EPA) in liver PL of fish fed VO decreased significantly from week 0–22 before rising at seawater transfer and increasing rapidly posttransfer. EPA in liver PL of fish fed FO showed a similar trend except EPA was always greater in the freshwater phase compared to fish fed VO. Docosahexaenoic acid (DHA) levels in liver PL of fish fed VO remained constant in the freshwater phase before increasing following seawater transfer. In fish fed FO, DHA in liver PL increased from weeks 0–17 reducing and leveling off postseawater transfer. The levels of PGF2α and PGF3α were measured in isolated gill cells stimulated with calcium ionophore A23187. PGF2α production in fish fed VO increased significantly between 0–7 wk before decreasing toward seawater transfer. After transfer, PGF2α production increased to a peak at 35 wk. PGF2α production in fish fed FO was not significantly altered during the trial period. The changes in PGF3α production were broadly similar to those occurring with PGF2α, but the latter was always in excess of the former (2-to 4-fold). Plasma chloride concentrations in fish subjected to seawater challenge at 20 wk were significantly lower in fish fed VO compared to those fed FO. This study has provided new information on the changes in lipid metabolism which accompany parr-smolt transformation and suggests that diets which have a fatty acid composition more similar to that in aquatic invertebrates may be beneficial in effecting successful seawater adaptation.

Highly unsaturated fatty acid synthesis in vertebrates : New insights with the cloning and characterization of a Δ6 desaturase of atlantic salmon

Fish are an important source of the n−3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of HUFA involves enzyme-mediated desaturation, and a Δ5 fatty acyl desaturase cDNA has been cloned from Atlantic salmon (Salmo salar) and functionally characterization of a Δ6 fatty acyl desaturase of Atlantic salmon and describe its genomic structure, tissue expression, and nutritional regulation. A salmon genomic library was screened with a salmon Δ5 desaturase cDNA and positive recombinant phage isolated and subcloned. The full-length cDNA for the putative fatty acyl desaturase was shown to comprise 2106 bp containing an open reading frame of 1365 bp specifying a protein of 454 amino acids (GenBank accession no. AY458652). The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the heme-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. Functional expression showed that this gene possessed predominantly Δ6 desaturase activity. Screening and sequence analysis of the genomic DNA of a single fish revealed that the Δ6 desaturase gene constituted 13 exons in 7965 bp of genomic DNA. Quantitative real-time PCR assay of gene expression in Atlantic salmon showed that both Δ6 and Δ5 fatty acyl desaturase genes, and a fatty acyl elongase gene, were highly expressed in intestine, liver, and brain, and less so in kidney, heart, gill, adipose tissue, muscle, and spleen. Furthermore, expression of both Δ6 and Δ5 fatty acyl desaturase genes in intestine, liver, red muscle, and adipose tissue was higher in salmon fed a diet containing vegetable oil than in fish fed a diet containing fish oil.

Effects of broodstock dietary lipid on fatty acid compositions of eggs from sea bass (Dicentrarchus labrax)

Samples of sea bass (Dicentrarchus labrax) eggs from broodstock which had been fed either a formulated pelleted feed, containing fish and corn oil, or a local trash fish, bogue (Boops boops) were analysed for lipid class compositions, fatty acid compositions of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) and wax ester and fatty alcohol compositions of wax esters. The pelleted feed contained 1.3 mg g−1 of arachidonic acid (20:4n-6; AA) and an AA/eicosapentaenoic acid (20:5n-3; EPA) ratio of 0.1 while the trash fish contained 4.8 mg g−1 AA and an AAEPA ratio of 0.7. Docosahexaenoic acid (22:6n-3; DHA) concentrations were similar for both diets (about 23 mg g−1). The fatty acid compositions of PC, PE and PI from eggs of fish fed trash fish contained significantly more AA, 22:5n-6 and DHA compared to fish fed the pelleted feed. The AAEPA ratios in these phospholipids were around five-fold higher in the trash fish-fed group compared to those fed the fish and corn oil containing diet. In PI, which contains characteristically high levels of AA, the AAEPA ratios were 1.5 and 8.6 for eggs derived from broodstock fed the pelleted diet and the trash fish, respectively. Determination of lipid class compositions of sea bass eggs revealed the presence of high levels of wax esters which were previously unrecorded in this species. The use of broodstock diets containing blends of corn oil and Northern hemisphere fish oils may be undesirable in that they contain high levels of 18:2n-6 and have low ratios of DHAEPA and of AAEPA. In an effort to improve egg quality and larval viability, efforts should be directed towards establishing the best ratio of DHA/EPA/AA in formulated feeds such that requirements for neural function and visual performance are maximised and that production and efficacy of eicosanoids are adequate to permit physiological functions to operate efficiently.

Effect of supplementation with 20:3(n − 6), 20:4(n − 6) and 20:5(n − 3) on the production of prostaglandins E and F of the 1-, 2- and 3-series in turbot (Scophthalmus maximus) brain astroglial cells in primary culture

The production of prostaglandins E and F of the 1-, 2- and 3-series was determined in primary cultures of turbot (Scophthalmus maximus) brain astroglial cells after supplementation with 25 microM dihomo-gamma-linolenic (20:3(n-6)), arachidonic (20:4(n-6)) and eicosapentaenoic (20:5(n-3)) acids. Supplementation by 20:3(n-6), 20:4(n-6) and 20:5(n-3) for 4 days significantly increased the percentages of the respective acids in the total cellular lipid of the turbot astrocytes. The predominant prostaglandins formed by turbot astrocytes in response to stimulation with calcium ionophore A23187 were prostaglandin E2 and prostaglandin F2 alpha under all experimental conditions. The production of prostaglandin E2 was stimulated 2.6-fold, but prostaglandin F2 alpha production was unaffected after supplementation of cultures with 20:4(n-6). However, prostaglandin E2 production in astrocytes was significantly inhibited 3- and 4-fold, and prostaglandin F2 alpha was inhibited 1.6- and 14.6-fold by supplementation with 20:3(n-6) and 20:5(n-3), respectively. Supplementation with 20:3(n-6) also significantly increased the production of prostaglandin E1 (almost 4-fold) and prostaglandin F1 alpha (2.2-fold) whereas supplementation with 20:5(n-3) did not significantly increase the production of prostaglandin E3. Prostaglandin F3 alpha but not prostaglandin E1 were significantly reduced in 20:5(n-3)-supplemented cultures.

Effects of different dietary arachidonic acid : docosahexaenoic acid ratios on phospholipid fatty acid compositions and prostaglandin production in juvenile turbot (Scophthalmus maximus).

Five purified diets containing AA (20:4n-6) at 0.02–0.78% dry weight and DHA (22:6n-3) at 0.93–0.17% dry weight were fed to duplicate groups of juvenile turbot (Scophthalmus maximus) of initial weight 0.87 g for a period of 11 weeks. The dietary DHA:AA ratio ranged from 62 to 0.2. Incorporation of AA into liver phospholipids increased with increasing dietary AA input. Phospholipids from fish fed diets containing 0.02, 0.06 and 0.11% of dry weight as AA generally contained less AA compared to fish fed fish oil while those fed diets containing 0.35 and 0.78% of dry weight as AA had higher AA levels in their phospholipids. The highest levels of AA were found in PI but the greatest percentage increase in AA incorporation was in PE and PC. Brain phospholipid fatty acid compositions were less altered by dietary treatment than those of liver but DHA content of PC and PE in brain was substantially lower in fish fed 0.93% pure DHA compared to those fed fish oil. This suggests that dietary DHA must exceed 1% of dry weight to satisfy the requirements of the developing neural system in juvenile turbot. In both tissues, (20:5n-3) concentration was inversely related to both dietary and tissue PI AA concentration. Similar dietary induced changes in AA, EPA and DHA concentrations occurred in the phospholipids of heart, gill and kidney. PGE2 and 6-ketoPGF1α were measured in homogenates of heart, brain, gill and kidney. In general, fish fed the lowest dietary AA levels had reduced levels of prostaglandins in their tissue homogenates while those fed the highest level of AA had increased prostaglandin levels, compared to fish fed fish oil. In brains, the PGE2 concentration was only significantly increased in fish fed the highest dietary AA.