Douglas R. Tocher

Metabolism and Functions of Lipids and Fatty Acids in Teleost Fish

Lipids and their constituent fatty acids are, along with proteins, the major organic constituents of fish, and they play major roles as sources of metabolic energy for growth including reproduction and movement, including migration. Furthermore, the fatty acids of fish lipids are rich in ω3 long chain, highly unsaturated fatty acids (n-3 HUFA) that have particularly important roles in animal nutrition, including fish and human nutrition, reflecting their roles in critical physiological processes. Indeed, fish are the most important food source of these vital nutrients for man. Thus, the longstanding interest in fish lipids stems from their abundance and their uniqueness. This review attempts to summarize our present state of knowledge of various aspects of the basic biochemistry, metabolism, and functions of fatty acids, and the lipids they constitute part of, in fish, seeking where possible to relate that understanding as much to fish in their natural environment as to farmed fish. In doing so, it highlights the areas that require investigation in greater depth and also the increasing application of molecular technologies in fish lipid metabolism, which will further fascilitate advances through molecular biological and genetic techniques, including genomics and proteomics.

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.

Fatty acid compositions of the major phosphoglycerides from fish neural tissues; (n-3) and (n-6) polyunsaturated fatty acids in rainbow trout (Salmo gairdneri) and cod (Gadus morhua) brains and retinas.

The fatty acid compositions of brain phosphoglycerides from a freshwater fish, the rainbow trout (Salmo gairdneri), and a marine fish, the cod (Gadus morhua), were determined and compared with those from a terrestrial mammal, the rat. Fish brain lipids were characterized by a higher degree of unsaturation encompassing increased percentages of (n−3)PUFA (22∶6 and 20∶5) and lower percentages of (n−6)PUFA (20∶4 and 22∶4). However the distribution of fatty acids and specific PUFA between different phosphoglycerides was essentially similar in rat and fish brain tissue. PE and PS contained the highest percentages of 22∶6(n−3), PI was characterized by higher 18∶0 and 20∶4(n−6)/20∶5(n−3), and PC had higher 16∶0 and the lowest percentage of PUFA in all species. A generally similar pattern was found in the fish retinal phosphoglycerides except that PC was also rich in 22∶6(n−3). Overall trout brain phosphoglycerides were slightly more unsaturated than the cod lipids but with lower (n−3)/(n−6) ratios whereas cod retinal lipids were more unsaturated than the trout retinal lipids.

Analyses of lipids and fatty acids in ripe roes of some Northwest European marine fish

Lipid class analyses and fatty acid analyses of neutral and polar lipids were carried out on ripe roes of herring, cod, haddock, whiting, saithe, sand eel and capelin. Total lipid was 10–26% of roe dry weight. The species with the highest total lipid, sand eel and capelin, also had the highest percentage of neutral lipid in total lipid, 77% and 49% respectively. In the other species, phospholipids accounted for 62–77% of roe total lipid. Both the neutral lipids, and especially the phospholipids, of all species were very unsaturated because of high concentrations of (n−3) polyunsaturated fatty acids (PUFA), frequently amounting to 50% of the total egg lipid. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) had similar fatty acid compositions in all species, with an average ratio (n−3)/(n−6) of ca. 20∶1. Phosphatidylinositol (PI) consistently had high concentrations of 18∶0 and 20∶4 (n−6) with an average ratio of (n−3)/(n−6) of 1.8∶1. Requirements for high levels of (n−3) PUFA in the embryonic and early larval development stages of marine fish are suggested as is a special role for the 20∶4(n−6) in PI.

A vertebrate fatty acid desaturase with Δ5 and Δ6 activities

Δ5 and Δ6 fatty acid desaturases are critical enzymes in the pathways for the biosynthesis of the polyunsaturated fatty acids arachidonic, eicosapentaenoic, and docosahexaenoic acids. They are encoded by distinct genes in mammals and Caenorhabditis elegans. This paper describes a cDNA isolated from zebrafish (Danio rerio) with high similarity to mammalian Δ6 desaturase genes. The 1,590-bp sequence specifies a protein that, in common with other fatty acid desaturases, contains an N-terminal cytochrome b5 domain and three histidine boxes, believed to be involved in catalysis. When the zebrafish cDNA was expressed in Saccharomyces cerevisiae it conferred the ability to convert linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3) to their corresponding Δ6 desaturated products, 18:3n-6 and 18:4n-3. However, in addition it conferred on the yeast the ability to convert di-homo-γ-linoleic acid (20:3n-6) and eicosatetraenoic acid (20:4n-3) to arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3), respectively, indicating that the zebrafish gene encodes an enzyme having both Δ5 and Δ6 desaturase activity. The zebrafish Δ5/Δ6 desaturase may represent a component of a prototypic vertebrate polyunsaturated fatty acids biosynthesis pathway.

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.

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.

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.