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

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

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

Study of the n-3 highly unsaturated fatty acids requirement and antioxidant status of Dentex dentex larvae at the Artemia feeding stage

This study was designed to investigate the requirements of Dentex dentex larvae for (n-3) highly unsaturated fatty acids (HUFA) at the Artemia feeding stage. Artemia were enriched using mixtures of ICES Experimental Emulsions ICES 50/0.6/C (500 mg/g (n-3) HUFA, 0.6 DHA/EPA ratio, based on ethyl esters) and ICES 0/-/C (based on coconut oil) and to give five dietary treatments which contained different levels of (n-3) HUFA from 0.72 to 6.23 as dry weight percentage. Optimal growth, as evidenced by total length, individual dry weight, specific growth rate and thermal growth coefficient, was achieved when dietary (n-3) HUFA was 3.97 on a dry weight basis. Larvae fed Artemia enriched with apparently super-optimal levels of (n-3) HUFA (5.67-6.23 %) showed significantly lower vitamin E contents and higher malondialdehyde (MDA) levels combined with their eyes having maximum (n-3) HUFA values and DHA/EPA ratios. Poorer performance of larvae was associated with increased dietary and larval MDA and decreased larval vitamin E, indicating increasing oxidation of (n-3) HUFA in Artemia and larval utilization of vitamin E with increasing levels of dietary (n-3) HUFA, particularly at supraoptimal levels of enrichment. The activities of antioxidant enzymes in the larvae was generally not greatly affected by the dietary treatments in this study. A balance is required between growth-promoting essential fatty acid (EFA) qualities of (n-3) HUFA and their potentially growth-inhibiting (prooxidant) qualities which must be counter-balanced with adequate dietary antioxidants

Docosahexaenoic acid biosynthesis via fatty acyl elongase and Δ4-desaturase and its modulation by dietary lipid level and fatty acid composition in a marine vertebrate

The present study presents the first "in vivo" evidence of enzymatic activity and nutritional regulation of a Δ4-desaturase-dependent DHA synthesis pathway in the teleost Solea senegalensis. Juvenile fish were fed diets containing 2 lipid levels (8 and 18%, LL and HL) with either 100 % fish oil (FO) or 75 % of the FO replaced by vegetable oils (VO). Fatty acyl elongation (Elovl5) and desaturation (Δ4Fad) activities were measured in isolated enterocytes and hepatocytes incubated with radiolabelled a-linolenic acid (ALA; 18:3n-3) and eicosapentaenoic acid (EPA; 20:5n-3). Tissue distributions of elovl5 and Δ4fad transcripts were also determined, and the transcriptional regulation of these genes in liver and intestine was assessed at fasting and postprandially. DHA biosynthesis from EPA occurred in both cell types, although Elovl5 and Δ4Fad activities tended to be higher in hepatocytes. In contrast, no Δ6Fad activity was detected on 14C-ALA, which was only elongated to 20:3n-3. Enzymatic activities and gene transcription were modulated by dietary lipid level (LL > HL) and fatty acid (FA) composition (VO > FO), more significantly in liver than in intestine, which was reflected in tissue FA compositions. Dietary VO induced a significant up-regulation of Δ4fad transcripts in liver 6 h after feeding, whereas in fasting conditions the effect of lipid level possibly prevailed over or interacted with FA composition in regulating the expression of elovl5 and Δ4fad, which were down-regulated in liver of fish fed the HL diets. Results indicated functionality and biological relevance of the Δ4 LC-PUFA biosynthesis pathway in S. senegalensis

Molecular and functional characterization of a SCD 1b from European sea bass (Dicentrarchus labrax L.).

Fatty acid desaturation is a highly complex and regulated process involving different molecular and genetic actors. Ultimally, the fatty acid desaturase enzymes are responsible for the introduction of double bonds at different positions of specific substrates, resulting in a wide variety of mono- and poly-unsaturated fatty acids. This substrate-specificity makes it possible to meet all the functional needs of the different tissues against a wide variety of internal and external conditions, giving rise to a varied profile of expression and functionality of the different desaturases in the body. Being our main interest to study and characterize at the molecular level the fatty acid desaturation process in fishes, we have focused our effort on characterizing SCD 1b from European sea bass (Dicentrarchus labrax, L.). In this work, we have characterized a tearoyl-CoA Desaturase cDNA that codes a protein of 334 amino acids, which shares the greatest homology to marine fish SCD 1b. Northern blot analysis showed two transcripts of 3.5kb and 1.4kb. Two putative cis-acting conserved motifs are localized in the cDNA 5'-end: a polypyrimidine CT dinucleotide repeat tract and two non-palindromic putative NRL-response elements (NREs). The deduced protein presents two Delta9 FADs like domain, three His-rich motifs, a total of nine His residues acting as di‑iron coordination ligands. The SCD 1b 3D protein modelling shows a structure made up primarily of alpha-helices, four of which could be transmembrane helices. The catalytic region is oriented to the cytosolic side of the Endoplasmic Reticulum membrane, where the 9-histidine residues are arranged coordinated to two non-heme Fe2+ ions. A new His-containing motif NX3H-like includes an Asn residue that participates in the coordination of Fe2+1 through a water molecule. The protein has a large pocket with a large opening to the outside. It includes a tunnel in which the substrate-binding site is located. The external shape is reminiscent of a boathook. It shows group specificity, although a greater preference for 18C substrates. The length of the tunnel, delimited by seven amino acids that forms a pocket at the end of the tunnel, the possibility that the substrates adopt different conformations inside the tunnel as well as and the movement of acyl chain inside the tunnel, could explain the high preference for 18C fatty acids and the group specificity of the enzyme. The cDNA encodes a functional SCD enzyme, whose subcellular localization is the Endoplasmic Reticulum, which complements the ole1Delta gene-disrupted gene in DTY-11A Saccharomyces cerevisiae strain and produces an increment of palmitoleic and oleic acids. The scd 1b gene is expressed in all tested tissues, showing the liver and adipose tissue a higher level of expression against the brain, heart, gonad and intestine. Scd 1b expression was always bigger than those of the Delta6 fad gene, being especially significant in adipose tissue and liver. From our data, we conclude that, in contrast to the functional significance of SCD 1b in adipose tissue, liver and heart, Delta6 FAD seems to play a more determining role in the biosynthesis of unsaturated fatty acids in the intestine, brain and gonad in fish

Expression of genes related to lipid metabolism in Atlantic bluefin tuna (Thunnus thynnus L.) larvae fed rotifers and copepods

The study demonstrated that copepods were a better live prey for first feeding ABT larvae based on growth and survival. Differences in the expression patterns of lipid metabolism genes were observed between the two trials. Some of the responses in lipid gene expression could be due to dietary lipid and fatty acid composition, but there was no obvious direct correlation between gene expression patterns and growth or survival. Differences in performance and metabolism among larval groups between the trials could also partly reflect differences in broodstock nutrition in the two trials. Hence, further studies are required to investigate lipid requirements, lipid accumulation and metabolism during development of ABT larvae. Special importance should be given to the expression analysis of genes related to lipid metabolism and its regulation, combined with biochemical studies of tuna lipid metabolism in order to develop optimal feeds to facilitate the commercial culture of this iconic species