Lipid classes and their content of n-3 highly unsaturated fatty acids (HUFA) in <i>Artemia franciscana</i> after hatching, HUFA-enrichment and subsequent starvation

The distribution of n-3 highly unsaturated fatty acids (HUFA) over the major neutral and polar lipid classes was determined for two predominant types of live food used in the larviculture of marine fish and shrimp, i.e. freshly hatched and HUFA-enriched Artemia, and compared with data reported in the literature for wild copepods, representing the natural diet of these larvae. Lipid class composition and their content of n-3 HUFA, particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), were assessed in freshly hatched, HUFA-enriched and subsequently starved Artemia franciscana. The n-3 HUFA enrichment was based on feeding Artemia a lipid emulsion in which either fatty acid ethyl esters (EE, diluted with olive oil) or triacylglycerol (TAG) provided a level of 30% n-3 HUFA. Enrichment of Artemia with either type of the lipid emulsions resulted in an increase of total lipid content from 20.0 to 28.2-28.7% of dry matter mainly due to the accumulation of neutral lipid, primarily TAG (from 82 to 158 mg g(-1) dry wt in freshly hatched and 24-h enriched Artemia). Enriched brine shrimp utilized up to 27-30% of their TAG content during 72 h of starvation at 12 degrees C. The absolute tissue concentrations of polar lipids remained constant at 71 to 79 mg g(-1) dry wt throughout the enrichment and subsequent starvation. The level of n-3 HUFA increased drastically during enrichment from 6.3% of total fatty acids (8.2 mg g(-1) dry wt) in freshly hatched nauplii to between 20.4 and 21.8% (40.4 to 43.2 mg g(-1) dry wt) in 24-h enriched Artemia and was not significantly affected by the source of n-3 HUFA. During starvation, 18:0, 20:4n-6 and 20:5n-3 were retained, whereas 18:4n-3, 22:5n-3 and 22:6n-3 were specifically catabolized. The major polar lipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), of freshly hatched Artemia showed very low levels of DHA ( 64% of the EPA and > 91% of the total DHA present. This is in sharp contrast with the high levels of n-3 HUFA, in particular DHA, in the polar lipid fraction reported for wild copepods. The contrasting distribution of DHA in the neutral and polar lipid fractions of enriched brine shrimp compared to the natural diet may influence the efficacy of this essential fatty acid for marine fish larvae in aquaculture systems

An approach to study the nutritional requirements of the bluefin tuna (Thunnus thynnus thynnus L.)

The research required in relation to the nutrition of bluefin tuna (BFT) is great. In previous trials in the domestication of large scombrids and in current capture-fattening schemes, nutritional studies have been few. Therefore, virtually nothing is presently known about the nutritional requirements for these species. Nutritional factors are important in any domestication programme in a variety of areas. Correct nutrition is a vital factor in the production of a successful broodstock with high fecundity and fertility producing large numbers of high quality eggs. Reproductive control itself is affected by nutritional factors such as lipid/energy content which can influence sexual maturation in other fish species. Successful larval rearing of marime fish is highly dependent upon suitable diets, whether live prey species or artificial, and their precise composition particularly in relation to fatty acids is an area that still demands much research for all marine species. Clues to the nutritional requirements of any animal can be obtained by looking at the natural food for that animal, in this instance, the natural prey species of the BFT and also by determining the composition of wild caught animals, both prey and predator. The latter certainly being a source of information in terms of lipids and fatty acid requirements. The few data available from previous trials can also add to the overall view. In this section we will briefly review the literature on what is known about the nutrition of the large scombrids and also, importantly, place the problems of their feeding and nutrition in a global perspective

Tuna nutrition and feeds: current status and future perspectives

Aquaculture is providing an ever-increasing proportion of fish in the human food basket prompting a search for new species to expand the range available to consumers. Large tunids/scombrids have long-since been a very valuable resource providing not only high quality protein, but also a rich source of the highly beneficial omega-3 (or n-3) long-chain polyunsaturated fatty acids including eicosapentaenoic and, especially docosahexaenoic acids in the human diet. Consequently, there is considerable interest worldwide in developing the culture of large tunids, including Atlantic northern bluefin tuna (Thunnus thynnus), Pacific bluefin tuna (Thunnus orientalis), southern bluefin tuna (Thunnus maccoyii) and yellowfin tuna (Thunnus albacares). Nutrition is vital to this development, playing key roles in reproductive success, including the establishment of successful broodstock producing high quality eggs and larvae, and ultimately the cost-effective production of nutritious seafood. This review summarises the rather fragmentary data that compromise the current state-of-the-art in relation to tuna nutrition and the development of artificial, formulated feeds for these species. In highlighting the various considerable challenges that feed development will pose, we discuss the future perspectives for tuna culture in terms of both fish and human nutrition and welfare, against the background of diminishing global marine resources

Effect of partial substitution of dietary fish oil by vegetable oils on desaturation and beta-oxidation of [1-14C]18:3n-3 (LNA) and [1-14C]20:5n-3 (EPA) in hepatocytes and enterocytes of European sea bass (Dicentrarchus labrax L.)

The increasing worldwide aquaculture output and concomitant decrease in the stocks of feed-grade fish used for fish oil production has made fish oil replacement in feeds a priority for the aquaculture industry. The regulation of fatty acid metabolism in fish is important in order to determine strategies for the best use of plant oils in diets for commercially important cultured fish species. We have studied the desaturation/elongation and &beta;-oxidation of 14C-linolenic (LNA) and 14C-eicosapentaenoic (EPA) acids in hepatocytes and pyloric caecal enterocytes in European sea bass fed diets with partial substitution (60%) of fish oil (FO) with vegetable oils (rapeseed, linseed and palm oil) blended in different proportions, for 64 weeks. The rate of desaturation of 14C‑LNA was very low in hepatocytes from all treatments and no significant differences were observed among treatments. The rate of desaturation of 14C‑LNA in enterocytes was higher than that in hepatocytes but still low (less than 5% of total radioactivity recovered). The desaturation of 14C‑EPA in enterocytes was also higher than in hepatocytes, but again was low and no significant differences were found among treatments. The rates of &szlig;‑oxidation of 14C-LNA and 14C-EPA were much higher than the rates of desaturation in both hepatocytes and enterocytes, however, no significant differences were observed in either hepatocytes or enterocytes among treatments. The rates of &beta;-oxidation of 14C-LNA were considerably higher than those of 14C-EPA in both hepatocytes and enterocytes. In conclusion, European sea bass (a carnivorous marine fish), showed very low desaturation and elongation of LNA to EPA and DHA, and EPA to DHA, higher &beta;&minus;oxidation of LNA than EPA, and all desaturation and oxidation activities were significantly higher in enterocytes than in hepatocytes. A second major conclusion is that no clear quantitative nutritional effects on the desaturation/elongation and &beta;&minus;oxidation activities in either hepatocytes or enterocytes of sea bass were observed upon the inclusion of vegetable oils in the diet

Effects of partial substitution of dietary fish oil with blends of vegetable oils, on blood leukocyte fatty acid compositions, immune function and histology in European sea bass, (Dicentrarchus labrax L.)

Within a decade or so insufficient fish oil (FO) will be available to meet the requirements for aquaculture growth. Consequently, alternative sources are being investigated to reduce reliance on wild fish as a source of FO. Vegetable oils (VO) are a feasible alternative to FO. However, it is important to establish that alternative dietary lipids are not only supplied in the correct quantities and balance for optimal growth, but can maintain immune function and prevent infection, since it is known that the nutritional state of the fish can influence their immune function and disease resistance. A way of maintaining immune function, while replacing dietary FO, is by using a blend of VOs rather than a single oil. In this study, juvenile European sea bass, Dicentrarchus labrax, were fed diets with a 60 % substitution of FO with a blend of rapeseed (RO), linseed (LO) and palm oils (PO). Two oil blends were used to achieve a fatty acid composition similar to FO, in terms of energy content and provide a similar balance of saturates, monounsaturates and polyunsaturated fatty acids. Fish were fed the diets for 64 weeks, after which time growth and fatty acid compositions of liver and blood leukocytes were monitored. The impact of the dietary blends on selected innate immune responses and histopathology were also assessed, together with levels of plasma prostaglandin E2. The results suggest that potential exists for replacing FO with a VO blend farmed sea bass feeds without compromising growth, non-specific immune function or histology

Effects of dietary polyunsaturated fatty acid/vitamin E (PUFA/tocopherol) ratio on antioxidant defence mechanisms of juvenile gilthead sea bream (Sparus aurata L., Osteichthyes, Sparidae)

Lipid peroxidation, specifically polyunsaturated fatty acid (PUFA) oxidation is highly deleterious, resulting in damage to cellular biomembranes, and may be a principal cause of several diseases in fish including jaundice and nutritional muscular dystrophy. Tissue lipid PUFA content and composition are critical factors in lipid peroxidation, as is the level of endogenous antioxidant molecules such as vitamin E. The primary objective of the present study was the characterization of antioxidant systems in a cultured juvenile marine fish, gilthead sea bream (Sparus aurata) with the underlying aim to understand how to avoid oxidation problems that may cause pathologies and disease and so to enhance growth and quality of early ongrowing stages. Juvenile sea bream were fed diets having either high or low levels of fish oil and supplemented or basal levels of vitamin E with PUFA/vitamin E ratios ranging from 117 ± 12 in the diet with low PUFA supplemented with vitamin E to 745 ± 48 in the diet with high PUFA with no additional vitamin E. None of the diets had serious deliterious effects on growth or survival of the fish, but the different dietary regimes were effective in significantly altering the PUFA/vitamin E ratios in the fish livers with values ranging from 5.7 ± 0.4 in fish fed the diet with low PUFA supplemented with vitamin E to 91.1 ± 13.2 in fish fed the diet with high PUFA with no additional vitamin E. This had effects on the peroxidation status of the fish as indicated by the significantly altered levels of in vivo lipid peroxidation products measured in liver, with fish fed the diet rich in PUFA and low in vitamin E showing significantly higher values of thiobarbituric acid reactive substances (TBARS) and isoprostanes. The isoprostane levels generally followed the same pattern as the TBARS levels supporting its value as an indicator of in vivo oxidative stress in fish, as it is in mammals. However, few significant effects on antioxidant enzyme activities were observed suggesting that more severe conditions may be required to affect these activities such as increasing the PUFA/vitamin E ratio or by increasing peroxidative stress through the feeding of oxidized oils

Effects of substitution of dietary fish oil with a blend of vegetable oils on liver and peripheral blood leucocyte fatty acid composition, plasma prostaglandin E2 and immune parameters in three strains of Atlantic salmon (Salmo salar)

Duplicate groups of three genetic strains of Atlantic salmon smolts were cultured on diets containing either fish oil (FO) or a blend of vegetable oils (VO). Fatty acid compositions of liver and peripheral blood leukocytes were compared. The effect of different strains and diets on innate immune parameters and plasma prostaglandin E2 were also measured. Two strains were selected as being either “fat” or “lean” in terms of muscle adiposity. The third strain was a commercial stock (MH). Total replacement of dietary FO with VO resulted in reduced docosahexaenoic (DHA; 22:6n-3) and eicosapentanoic acids (EPA; 20:5n-3) in liver, while oleic (18:1n-9), linoleic (18:2n-6) and α-linolenic (18:3n-3) acids were all increased in VO-fed fish. Fatty acid compositions of blood leucocytes showed similar changes. Evaluation of innate immune function showed that in the fat strain circulating leucocytes were significantly lower in VO fish. The lean strain also had significantly higher serum lysozyme activity than MH fish. Reduced haematocrit was seen in VO lean fish compared to FO lean fish. This study provides evidence of strain-induced differences in liver and leukocyte fatty acid compositions and innate immunity in Atlantic salmon fed either FO or VO-based diets

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

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 (a-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-Stransferase (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

Molecular and Functional Characterization and Expression Analysis of a Delta6 Fatty Acyl Desaturase cDNA of European Sea Bass (Dicentrarchus labrax L.)

The extent to which fish species can produce highly unsaturated fatty acids (HUFA) from C18 fatty acids varies with their complement of fatty acyl desaturase (FAD) enzymes. Marine fish are unable to produce HUFA at a significant rate due to apparent deficiencies in one or more enzymatic steps in the desaturation/ elongation pathway. It is not known if this is due to a lack of the genes or to tight regulation of the enzymatic activity in some of the transformation steps. In the present study, we report molecular cloning, cDNA, protein and functional analysis of a Δ6 FAD of European sea bass (Dicentrarchus labrax L.), and describe its tissue expression and nutritional regulation. An FAD cDNA contig sequence from brain tissue of sea bass was obtained by gene walking, and full-length cDNA was obtained by amplification using 5′end forward and 3′ end reversed primers. The full length of the sea bass FAD cDNA was 2089 bp, which included a 5′-UTR (untranslated region) of 267 bp, a 3′-UTR of 484 bp and an open reading frame (ORF) of 1338 bp, which specified a protein of 445 amino acids. The mRNA size, estimated by northern blot analysis was 2.1 kb, consistent with the cDNA. Transient expression of Δ6-FAD-EGFP in HeLa cells showed the protein compartmentalized to the endoplasmic reticulum. Functional expression in yeast showed the sea bass cDNA encoded a unifunctional Δ6 FAD enzyme. The sea bass FAD was more active towards 18:3n-3 with 14.5% being converted to 18:4n-3 compared to 5.6% of 18:2n-6 converted to 18:3n-6. Expression of the Δ6 FAD gene in the sea bass tissues showed a rank order of heart, brain, ovary, kidney, adipose tissue and liver as determined by RT-qPCR. Nutritional regulation of gene expression was studied. Diets containing partial substitution of fish oil with rapeseed or linseed oils induced up-regulation of the Δ6 FAD gene; whereas, a diet containing olive oil did not influence the expression. Similarly, when fish oil was partially replaced by blends of vegetable oils, one increased expression and one did not