J.G. Bell

Requirements, presentation and sources of polyunsaturated fatty acids in marine fish larval feeds

Abstract Current procedures for optimising the presentation of dietary polyunsaturated fatty acid (PUFA) to marine fish larvae are reviewed in relation to the advantages and disadvantages of using (a) single-cell eukaryotic organisms or, (b) purified oils, as primary sources of these essential nutrients in larval production systems. For the former option (a), phototrophic and heterotrophic organisms can both be used to advantage and current knowledge of the origins and functions of PUFA in marine organisms can help identify suitable organisms. For the latter option (b), control of PUFA peroxidation by various antioxidant systems is essential. Progress in this field requires definition of the optimal dietary ratio of 22:6( n −3): 20:5( n −3): 20:4( n −6); the significance of phospholipid vs. neutral lipid in larval diets, and larval requirements for antioxidant vitamins.

Effects of purified diets containing different combinations of arachidonic and docosahexaenoic acid on survival, growth and fatty acid composition of juvenile turbot (Scophthalmus maximus)

The objective of this study was to determine the relative essential fatty acid (EFA) growth-promoting activities of pure arachidonic (AA, 20:4n−6) and docosahexaenoic (DHA, 22:6n−3) acids and various concentrations of these two acids in the diet of juvenile turbot (Scophthalmus maximus). Casein-based, semi-purified diets containing 15% fish oil or 14% hydrogenated coconut oil/oleic acid (1:1) supplemented with 1% 20:4n−6, 1% 22:6n−3 or 1% of various combinations of these two acids were fed to duplicate groups of 26 juvenile turbot for 11 weeks. In this trial, feeding the diet containing 20:4n−6 as the only highly unsaturated fatty acid (HUFA) resulted in higher growth and survival than any of the mixtures of the two fatty acids or 22:6n−3 alone. The diet containing 22:6n−3 as the sole HUFA resulted in the lowest growth and survival of all dietary treatments. The control diet with 15% fish oil resulted in a greater growth rate than any of the pure HUFA-supplemented diets. There was a significant effect of dietary lipid on the somatic index of the brain but not heart, kidney or liver. The percentage of lipid in the liver, but not of heart, brain, eyes, gills or kidney, was influenced by dietary lipid, with the highest percentage in fish supplemented with DHA alone. After 11 weeks, the 20:4n−6 and 22:6n−3 levels in whole-body total lipids were strongly influenced by the content of these fatty acids in the diets. The relative effect of dietary levels of these two fatty acids on their content in fish lipids varied considerably among the various organs and tissues of the fish that were analyzed. Brain and eye lipids were generally highest in 22:6n−3 while gill and kidney lipids were consistently higher in 20:4n−6 than the other organs analyzed. The effect of dietary 20:4n−6 on the content of that HUFA in organ lipid was greatest in gill and liver. The greatest impact of dietary 22:6n−3 level on content of that acid in organ lipid was seen in gill and kidney. There were also significant effects of dietary HUFA content on organ lipid levels of saturated, mono-unsaturated fatty acids and other members of the n−3 and n−6 PUFA, and HUFA series. The present study suggests that the EFA growth-promoting activity of arachidonic acid provides strong support for the contention that dietary 20:4n−6 is essential for juvenile turbot.

Optimising lipid nutrition in first-feeding flatfish larvae

Although global production of flatfish has increased in recent years, both in terms of numbers of fish and diversification into new species, problems still remain with low survival rates and difficulties with metamorphosis. This short review highlights some advances made in optimising lipid nutrition in an attempt to overcome some of these problems. Copepod nauplii are the best live prey for first-feeding flatfish larvae. Rotifers can provide a useful method of essential fatty acid delivery but Artemia are poorer in this regard, especially at first feeding. Copepods are nutritionally beneficial due to their naturally high levels of the essential highly unsaturated fatty acids (HUFA), 20:5n-3 (eicosapentaenoic acid; EPA) and 22:6n-3 (docosahexaenoic acid; DHA), which are predominantly in the form of phospholipids. Rotifers can be enriched with fish oil emulsions to provide compositions similar to copepods, while enriched Artemia are difficult to enrich with high levels of DHA and the HUFA tend to be located in triglycerides rather than phospholipids. There is considerable evidence that the superior efficacy of copepods and rotifers is largely due to the digestibility and availability of HUFA supplied as pre-formed phospholipids. In addition to the essentiality of EPA and DHA, the requirement for 20:4n-6 (arachidonic acid; ARA) should also be considered. Improvements in dorsal pigmentation in turbot and halibut can be achieved by providing ratios of DHA/EPA of >2:1 but, perhaps more importantly, an EPA/ARA ratio of >5:1. This suggests that eicosanoids are involved in the control of pigmentation and this is further supported by the use of enrichments containing 18:3n-6, the elongation product of which (20:3n-6), is a potent inhibitor of ARA-derived eicosanoid formation, as is EPA.

Growth, survival, lipid composition and pigmentation of turbot (Scophthalmus maximus) larvae fed live-prey enriched in Arachidonic and Eicosapentaenoic acids

Abstract Turbot larvae were fed live-prey enriched with different levels of arachidonic (ARA) and eicosapentaenoic (EPA) acids to study the effects of these fatty acids on body composition and pigmentation success. Significantly reduced pigmentation was obtained in those fish fed medium and high ARA diets for 43 days. Growth and survival were the same for all groups. The incorporation of ARA and EPA in fish eyes, brains, livers and carcasses reflected the percentage of these fatty acids in the diets. ARA accumulation was similar in all tissues, but brain accumulated EPA was less efficient than the other tissues examined. A highly significant, negative correlation was found between the %ARA in turbot juvenile brain total lipids and pigmentation success. A weaker, positive correlation was found between brain EPA and pigmentation. Increasing dietary ARA affected the fatty acid composition of turbot brain phosphoglycerides more than increasing dietary EPA, especially in phosphatidylinositol (PI) and phosphatidylethanolamine (PE). A negative relationship was found between percentage normal pigmentation and ARA levels in brain phosphatidylcholine (PC), PE and phosphatidylserine (PS). Elevated levels of ARA in PI also resulted in malpigmented juveniles, but EPA:ARA ratios ≥1 in PI were associated with normal pigmentation. We conclude that, given a sufficiency of dietary docosahexaenoic acid (DHA), the optimum dietary level of EPA is not a function of DHA, but of dietary ARA.

Increased activities of hepatic antioxidant defence enzymes in juvenile gilthead sea bream (Sparus aurata L.) fed dietary oxidised oil: attenuation by dietary vitamin E

Abstract Previously, we had shown that altering the highly unsaturated fatty acid (HUFA)/vitamin E ratios in gilthead sea bream livers significantly affected their peroxidation status, with fish fed a diet rich in HUFA and low in vitamin E showing significantly higher values of lipid peroxidation products, without, however, significant effects on liver antioxidant defence enzyme activities. The aim of the present trial was to further characterise the biochemical indicators of peroxidative stress in juvenile gilthead sea bream. A high pro-oxidative stress was induced by feeding diets containing around 7% of the dry weight as n −3 HUFA. The potential peroxidative stress was increased by oxidising the oil, increasing the peroxide value of the oil some 10-fold. These oils were fed without or with supplemental vitamin E (α-tocopheryl acetate at 200 mg kg −1 dry diet) giving four diets in total. Fish were sampled after 30 and 60 days of feeding the experimental diets. None of the diets had any serious deleterious effects on growth and mortality of the fish during the trial. Similarly, there were few significant effects due to dietary oxidised oil or supplementary vitamin E on liver lipid and fatty acid profiles and, in particular, the proportions of HUFA were not decreased by dietary oxidised oil. The vitamin E content of the liver reflected the vitamin E content of the diets but was also affected by dietary oxidised oil being reduced by oxidised oil in fish fed diets without supplemental vitamin E but, unexpectedly, increased by oxidised oil in fish fed diets supplemented with vitamin E. Liver thiobarbituric acid reactive substances (TBARS) levels were significantly lower in fish fed diets supplemented with vitamin E whereas dietary oxidised oil had no major effect on lipid peroxidation products. Catalase (CAT) and superoxide dismutase (SOD) activities were both increased in fish fed dietary oxidised oil and reduced by supplementary vitamin E after 30 days feeding. In contrast, glutathione peroxidase (GPX) was less affected by the diets, and the activities of glutathione-S-transferase (GST) and glutathione reductase (GR) were only reduced by dietary vitamin E after 60 days of feeding. However, all the enzyme activities were significantly affected by the duration of feeding, but the number of interactions between the three factors (time, oil and vitamin E) showed that the relationships were complicated. In conclusion, the present study showed that feeding diets containing oxidised oil significantly affected the activities of liver antioxidant defence enzymes and that dietary vitamin E partially abrogated these effects. Growth and survival of the fish were relatively unaffected suggesting that the responses in gilthead sea bream offered effective protection. However, the duration of feeding the diets of high pro-oxidative stress was observed to have a hitherto unknown effect, possibly the result of an adaptive process, but which requires further investigation.

Dietary sunflower, linseed and fish oils affect phospholipid fatty acid composition, development of cardiac lesions, phospholipase activity and eicosanoid production in Atlantic salmon (Salmo salar)

Atlantic salmon (Salmo salar) post-smolts were fed practical-type diets in which the lipid was supplied either as fish oil (FO), sunflower oil (SFO) or linseed oil (LO) for 12 weeks. In general, the heart phospholipids from SFO-fed fish had increased 18:2n-6, 20:2n-6, 20:3n-6 and 20:4n-6 but decreased 20:5n-3 compared to both other dietary treatments. This was reflected in a decreased n-3/n-6 polyunsaturated fatty acid (PUFA) ratio and an increased 20:4n-6/20:5n-3 or eicosanoid precursor ratio in SFO-fed fish. While heart phospholipids of fish fed LO had increased levels of 18:2n-6, 20:2n-6 and 20:3n-6 compared to fish fed FO, 20:4n-6 levels were reduced, although only significantly in phosphatidylcholine (PC). Dietary-induced changes in phospholipid fatty acid compositions of blood leucocytes were similar to those in heart, although fish fed LO had increased 20:5n-3 compared to fish fed FO. Thromboxane B2 (TXB2) produced by stimulated blood cells was reduced in fish fed LO compared to those fed SFO. Prostaglandin E2 (PGE2) production was reduced in LO-fed fish compared to both other dietary treatments. Fish fed LO had reduced PC in heart membranes compared to the other two dietary treatments, resulting in a ratio of PC:PE (phosphatidylethanolamine) less than unity. Fish fed SFO developed a marked cardiac histopathology which, while present in FO-fed fish albeit in a less severe form, was virtually absent in fish fed LO. Fish fed SFO had increased heart phospholipase A activity compared to those given either FO or LO.

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.

Lipid and fatty acid composition of normal and malpigmented Atlantic halibut (Hippoglossus hippoglossus) fed enriched Artemia: a comparison with fry fed wild copepods

Atlantic halibut larvae originating from a single spawn of one female and one male were fed either Super Selco™ enriched, enrichment-grade Artemia nauplii or extensively grown zooplankton. The feeding experiment was conducted in 1.5-m3 outdoor tanks to evaluate if diet affected pigmentation. Pigmentation rates were found to be higher in the zooplankton-fed fish: 99.2% compared to 66.4% in the Artemia-fed halibut. Lipid and fatty acid analyses were performed on five malpigmented Artemia-fed fry, five normally pigmented Artemia-fed fry and 10 copepod-fed fry after 43 days feeding. Lipid class analysis was carried out on the eyes and carcasses of the halibut fry by HPTLC and densitometry. Fatty acid analyses of the same tissues were performed by gas liquid chromatography. Copepod-fed fry showed significantly higher levels of docosahexaenoic acid (22:6n−3; DHA) than their Artemia-fed counterparts (54.9% and 15.8% DHA in eye phosphatidylethanolamine (PE), respectively). Ratios of DHA: eicosapentaenoic acid (20:5n−3; EPA) were also higher in the copepod-fed fish: 11.1, 4.1 and 2.3 in, respectively, PE, phosphatidylcholine (PC) and triglyceride (TAG) in the eye lipid fractions of copepod-fed fry compared to 0.7, 0.3 and 0.1 in the same lipid of eyes of Artemia-fed fry. Within the Artemia-fed population, DHA and EPA levels were slightly, but significantly, raised in the eye PC fractions of normally pigmented fish compared to malpigmented ones. However, there was no significant difference in DHA:EPA ratios between normal and malpigmented fish fed Artemia. EPA: arachidonic acid (ARA) ratios were higher in zooplankton-fed fish but there was no significant difference in these ratios between normal and malpigmented Artemia-fed halibut.

Hepatocyte fatty acid desaturation and polyunsaturated fatty acid composition of liver in salmonids: effects of dietary vegetable oil

The desaturation and elongation of [1-(14)C]18:3n-3 was investigated in hepatocytes from different populations and three different species of salmonids indigenous to Scotland, brown trout, Atlantic salmon and Arctic charr. Two groups of fish were sampled, before and after they were fed two experimental diets, a control diet containing fish oil and a diet containing vegetable oil (a 1:1 blend of linseed and rapeseed oils) for 12 weeks. At each sampling time, fatty acyl desaturation and elongation activity was determined in isolated hepatocytes, and samples of liver were also collected for lipid compositional analysis. At the initiation of the dietary trial, the liver polar-lipid fatty acid compositions of salmon and brown trout were very similar to each other, and the two charr populations were similar to each other, having lower total n-3 polyunsaturated fatty acids (PUFA) and 22:6n-3, but higher 20:5n-3 than the other salmonids. Initially, hepatocyte desaturation activity varied, with the highest activity in brown trout, followed by salmon and then charr. Production of 20:5n-3 was particularly high in brown trout. Desaturation of [1-(14)C]18:3n-3 was significantly greater in all fish fed the diet containing vegetable oil compared to fish fed the diet containing fish oil. The increase in activity was less in brown trout compared to the other groups of fish. Feeding the vegetable oil diet increased the levels of 18:2n-6, 20:3n-6, total n-6 PUFA, 18:3n-3, 18:4n-3, 20:3n-3 and 20:4n-3, and decreased 22:6n-3 and the n-3/n-6 ratio in salmon and brown trout. By contrast, in charr fed the vegetable oil diet, there was no increase in 18:3n-3, 18:4n-3, 20:3n-3 or 20:4n-3 in liver polar lipids and the level of 22:6n-3 was not decreased. In addition, there was only a modest increase in the levels of 18:2n-6 and total n-6 PUFA, and so the n-3/n-6 ratio was only slightly decreased. The percentage of 20:4n-6, which was not increased in salmon and brown trout fed vegetable oil, was increased in charr fed the vegetable oil diet. Overall, the results indicated that there were significant differences in liver PUFA metabolism between Arctic charr and the other salmonids, which could have important consequences, both physiologically and in their ability to be successfully cultured on diets containing vegetable oils.

Dietary lipid enhancement of broodstock reproductive performance and egg and larval quality in Atlantic halibut (Hippoglossus hippoglossus)

Abstract Two series of experiments were conducted to assess whether the trash fish-based diet traditionally used by the industry for broodstock halibut could be replaced by a formulated pelleted feed and to investigate the effects of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (AA) on halibut fecundity, blastomere morphology and fertilisation and hatching rates. In the first experiment, two formulated broodstock feeds (one containing Krill meal and one supplemented with tuna orbital oil, which provided higher dietary levels of DHA and AA), gave similar performances (Relative fecundity 9557±3462 and 10,551±2166, fertilisation rate 40.0±6.4% and 40.0±9.1%, respectively) to that of a fish fed a trash fish-based diet (Relative fecundity 11,782±1873, fertilisation rate 41.0±4.0%). In a second experiment, the spawning performance and egg quality of two groups of broodstock maintained on two formulated diets, each containing different levels of AA (1.8% and 0.4%) was compared over two successive spawning seasons. The diet with the higher (1.8%) level of AA resulted in significantly higher ( p