R. James Henderson

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.

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.

Effects of dietary soybean and cod-liver oil levels on growth and body composition of gilthead bream ( Sparus aurata)

Abstract Gilthead bream (Sparus aurata) of 1 g mean weight were fed six purified diets supplemented with 12% lipid composed of different proportions of soybean oil (SBO) and cod-liver cil (CLO). The experiment lasted for 5 months. Fish performance and body composition, as well as the fatty acid composition of liver phospholipids, were studied. Fish performance improved with increasing dietary CLO content, approaching an optimum at about 6% CLO. Higher values for liver fat content and hepatosomatic index were observed for diets containing less than 6% CLO suggesting a deficiency of essential fatty acids in these diets. Liver fatty infiltration with low CLO diets was high although a certain degree of fatty infiltration was apparent in livers of all the dietary treatments. Body composition of fish was also affected by dietary lipid composition. Higher protein and lower fat contents were observed for the fish fed low levels of CLO. The eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content of liver phospholipids was high, approaching a constant value after inclusion of 6% CLO. The levels of 20:4n − 6 were also found to approach a plateau at this level. Saturated andn − 9 fatty acid content remained almost stable, while the level of 18:2n − 6 in liver phospholipids reflected the amount of SBO in the diet. It is concluded that, under the conditions employed, the minimum requirement of gilthead bream for EPA and DHA is around 0.9% of the diet.

Lipid conversions during enrichment of Artemia

Abstract Artemia nauplii were enriched for 24 h with radiolabelled fatty acid ethyl esters and then starved for a subsequent period of 24 h. Analyses of the distribution of radioactivity in lipids from samples taken at the end of the enrichment period and after the subsequent starvation showed that the ethyl esters were readily converted into other lipid classes, mainly triacylglycerols, during assimilation by the nauplii. The proportions of radioactivity recovered in free fatty acids and phospholipids increased during the starvation period indicating the mobilisation of fatty acids from triacylglycerols for use in catabolism and in the formation of biomembrane lipids. The distribution pattern of radioactivity from [U– 14 C ]22:6n−3 in the fatty acids of the nauplii demonstrates that Artemia are capable of retroconverting 22:6n−3 to 20:5n−3.

Patterns of variation in the lipid class and fatty acid composition of Nannochloropsis oculata (Eustigmatophyceae) during batch culture

Changes in the lipid and fatty acyl compositions of the marine microalga Nannochloropsis oculata Droop were examined during a batch culture growth cycle. During the early phase of batch culture the cellular proportion of triacylglycerols (TAG) increased. This was in addition to the increases in TAG observed in many microalgal species in the stationary-phase. Concomitant increases in the relative proportions of both saturated and monounsaturated fatty acids and decreases in the proportion of polyunsaturated fatty acids in total lipid were also associated with this phase. The separated individual lipid classes were found to have characteristic fatty acyl compositions. The relative proportion of lipid per cell, the relative proportions of the individual lipid classes and the fatty acyl compositions of the individual classes were all subject to variability during the growth cycle. The changing total lipid fatty acyl composition of N. oculata was found to be determined by the proportion of the total lipid present as TAG. The data suggest that the changes observed in the fatty acyl composition of N. oculata are a result of the partitioning of photosynthetically fixed carbon between polar and neutral lipid class biosynthesis and fatty acyl desaturation and elongation pathways. The effect of such a partitioning of carbon is discussed in relation to the effects of environmental variables and growth phase upon the balance of lipid class and polyunsaturated fatty acids (PUFA) synthesis in marine microalgae.

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.

Lipid composition and biosynthesis in the marine dinoflagellate Crypthecodinium cohnii

Abstract Triacyglycerols were the predominant lipid of the non-photosynthetic marine dinoflagellate Crypthecodinium cohnii grown heterotrophically for six d

The desaturation and elongation of 14C-labelled polyunsaturated fatty acids by pike (Esox lucius L.) in vivo

To examine the ability of pike (Esox lucius L.) to modify exogenous PUFA by desaturation and elongation, 14C-labelled 18:2(n-6), 18:3(n-3), 20:4(n-6) and 20:5(n-3) were injected intraperitoneally and the distribution of radioactivity in tissue lipid classes and liver PUFA measured. In all tissues examined, radioactivity from all 14C-PUFA was recovered in many classes of acyl lipids and the level of recovery generally reflected the relative abundance of the lipid classes. Triacylglycerols, CGP and EGP usually contained high levels of all incorporated 14C-PUFA. PI contained higher levels of radioactivity from 14C-20:4(n-6) than from other injected substrates. In liver lipid, the Δ6 desaturation products of 14C-18:2(n-6) and 14C-18:3(n-3) contained no measurable radioactivity although the elongation products of the Δ6 desaturation products were labelled, as were the direct elongation products of these injected substrates. No radioactivity from 14C-18:2(n-6) or 14C-18:3(n-3) was detected in C20 or C22 products of Δ5 and Δ4 desaturation. Almost all radioactivity from injected 14C-20:4(n-6) was recovered in this PUFA. Of the total radioactivity from 14C-20:5(n-3) incorporated into liver lipid, 7% was present as 24:5 and 16.4% was recovered in hexaenoic fatty acids. In liver, 24:5(n-3) and 24:6(n-3) each accounted for 1% of the mass of total fatty acids and were located almost exclusively in triacylglycerols. The presence of radioactivity in these C24 PUFA suggests that in pike the synthesis of 22:6(n-3) from 20:5(n-3) may proceed without Δ4 desaturase via the pathway which involves chain shortening of 24:6(n-3). It is concluded that under the circumstances employed in this study pike, do not exhibit Δ5 desaturase activity and are unable to synthesize 20:4(n-6) and 20:5(n-3) from 18:2(n-6) and 18:3(n-3), respectively. This suggests that pike may require 20:4(n-6) and 20:5(n-3) preformed in the diet.

The lipid composition of sealoch sediments underlying salmon cages

Abstract The lipid composition of sediments underlying salmon cages in a Scottish sealoch was determined along with that of the diet supplied to the fish to examine the influence of lipids originating from fish farm wastes. Sediments were taken at regular intervals along a transect line perpendicular to the line of cages and extending 50 m on either side. Lipids were extracted from regions of the sediment cores corresponding to different depth layers of sediment and analyzed for lipid class and fatty acid composition. The lipid content of the surface layer of sediment (0–5 mm) directly under the cages (2 mg/g sediment) was substantially higher than that of the deeper layers of sediment. The amount of lipid in the surface sediment decreased markedly with the distance from the cages, with the decrease being more rapid on one side of the fish farm than the other. At the 50-m sampling sites, the lipid content of the surface sediment layer (0.4 mg/g sediment) was closer to that of the underlying layers. Triacylglycerols, the main lipid class present in the diet fed to the salmon, were present in sediments in highest concentration (0.44 mg/g sediment) in surface sediments directly beneath the cages. Free fatty acids, sterols, polar lipids and a combined hydrocarbons/wax esters/cholesterol esters fraction were all present at similar levels to those of triacylglycerols in sediments beneath the cages. The distribution pattern of the amounts of individual lipid classes in sediments followed that of total lipid and showed a decrease with the distance from the cages. The lipid component of the fish feed contained a higher level of polyunsaturated fatty acids, particularly 20:5( n − 3) and 22:6( n − 3), than lipid extracted from the sediments, while branched chain and odd-chain length fatty acids were more abundant in the latter. The principal fatty acid of the fish diet, 22:1( n − 11) (14.7% total fatty acids) comprised around 9% of the fatty acids of surface sediment layer directly under the cages, but less than 5% of those at 50 m. The proportions of the other characteristic fatty acids of the diet, 20:1( n − 9), 20:5( n − 3) and 22:6( n − 3), showed a similar decrease with distance. The results show that the lipid composition of sediments underlying marine fish cages is influenced by that of waste material from the cages.

The enrichment of n−3 polyunsaturated fatty acids using aminopropyl solid phase extraction columns

A rapid, simple and reliable method is described for the preparation of concentrates of methyl or ethyl esters of n−3 polyunsaturated fatty acids by solid phase extraction using aminopropyl bonded silica columns. After applying mixtures of fatty acid esters in hexane, saturated and monounsaturated fatty acid esters are preferentially eluted with hexane whereas polyunsaturated fatty acids (PUFA) can subsequently be eluted with dichloromethane. Concentrates containing 80–90% n−3 PUFA can thus be obtained using fish oil fatty acids esters as a starting material.