Development of a fish cell culture model to investigate the impact of fish oil replacement on lipid peroxidation

Fish oils are rich in omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), predominantly 20:5n-3 and 22:6n-3, whereas vegetable oils contain abundant C18-PUFA, predominantly 18:3n-3 or 18:2n-6. We hypothesized that replacement of fish oils with vegetable oils would increase the oxidative stability of fish lipids. Here we have used the FHM cell line to test this hypothesis. The FHM cells were readily able to synthesize 20:5n-3 and 24:6n-3 from 18:3n-3 but 22:6n-3 synthesis was negligible. Also, they were readily able to synthesize 20:3n-6 from 18:2n-6 but 20:4n-6 synthesis was negligible. Mitochondrial β-oxidation was greatest for 18:3n-3 and 20:5n-3 and the rates for 16:0, 18:2n-6, 22:6n-3 and 18:1n-9 were significantly lower. Fatty acid incorporation was predominantly into phospholipids (79-97%) with very little incorporation into neutral lipids. Increasing the fatty acid concentration in the growth medium substantially increased the concentrations of 18:3n-3 and 18:2n-6 in the cell phospholipids but this was not the case for 20:5n-3 or 22:6n-3. When they were subjected to oxidative stress, the FHM cells supplemented with either 20:5n-3 or 22:6n-3 (as compared with 18:3n-3 or saturated fatty acids) exhibited significantly higher levels of thiobarbituric reactive substances (TBARS) indicating higher levels of lipid peroxidation. The results are discussed in relation to the effects of fatty acid unsaturation on the oxidative stability of cellular lipids and the implications for sustainable aquaculture

Effects of dietary eicosapentaenoic acid on growth, survival, pigmentation and fatty acid composition in Senegal sole (Solea senegalensis) larvae during the Artemia feeding period

We examined the effect of dietary eicosapentaenoic acid (20:5n-3, EPA) on growth, survival, pigmentation and fatty acid composition of Senegal sole larvae using a dose-response design. From 3 to 40 days post hatch (dph), larvae were fed live food that had been enriched using one of four experimental emulsions containing graduated concentrations of EPA and constant docosahexaenoic acid (22:6n-3, DHA) and arachidonic acid (20:4n-6, ARA). Proportions of EPA in the enriched Artemia nauplii were described as “nil” (EPA-N, 0.5% total fatty acids, TFA), “low” (EPA-L, 10.7% TFA), “medium” (EPA-M, 20.3% TFA) or “high” (EPA-H, 29.5% TFA). Significant differences among dietary treatments in larval length were observed at 25, 30 and 40 dph, and in dry weight at 30 and 40 dph, although no significant correlation could be found between dietary EPA content and growth. The stage of eye migration at 17 and 25 dph was significantly affected by dietary levels of EPA. Significantly lower survival was observed in fish fed EPA-H enriched nauplii. A significantly lower percentage of fish fed EPA-N (82.7%) and EPA-L (82.9%) diets were normally pigmented compared to the fish fed EPA-M (98.1%) and EPA-H (99.4%) enriched nauplii. Tissue fatty acid concentrations reflected the corresponding dietary composition. Arachidonic and docosahexaenoic acid levels in all the tissues examined were inversely related to dietary EPA. There was an increase in the proportion of docosapentaenoic acid (22:5n-3, DPA) in the tissues relative to the diet, which is indicative of chain elongation of EPA. This work concluded that Senegal sole larvae have a very low EPA requirement during the live feeding period

The efficiency of indicator groups for the conservation of amphibians in the Brazilian Atlantic Forest

The adequate selection of indicator groups of biodiversity is an important aspect of the systematic conservation planning. However, these assessments differ in the spatial scale, in the methods used and in the groups considered to accomplish this task, which generally produces contradictory results. The quantification of the spatial congruence between species richness and complementarity among different taxonomic groups is a fundamental step to identify potential indicator groups. Using a constructive approach, the main purposes of this study were to evaluate the performance and efficiency of eight potential indicator groups representing amphibian diversity in the Brazilian Atlantic Forest. Data on the geographic range of amphibian species that occur in the Brazilian Atlantic Forest was overlapped to the full geographic extent of the biome, which was divided into a regular equal-area grid. Optimization routines based on the concept of complementarily were applied to verify the performance of each indicator group selected in relation to the representativeness of the amphibians in the Brazilian Atlantic Forest as a whole, which were solved by the algorithm"simulated annealing", through the use of the software MARXAN. Some indicator groups were substantially more effective than others in regards to the representation of the taxonomic groups assessed, which was confirmed by the high significance of data (F = 312.76; p < 0.01). Leiuperidae was considered as the best indicator group among the families analyzed, as it showed a good performance, representing 71% of amphibian species in the Brazilian Atlantic Forest (i.e. 290 species), which may be associated with the diffuse geographic distribution of its species. This study promotes understanding of how the diversity standards of amphibians can be informative for systematic conservation planning on a regional scale

A description of the origins, design and performance of the TRAITS-SGP Atlantic salmon Salmo salar L. cDNA microarray

The origins, design, fabrication and performance of an Atlantic salmon microarray are described. The microarray comprises 16 950 Atlantic salmon-derived cDNA features, printed in duplicate and mostly sourced from pre-existing expressed sequence tag (EST) collections [SALGENE and salmon genome project (SGP)] but also supplemented with cDNAs from suppression subtractive hybridization libraries and candidate genes involved in immune response, protein catabolism, lipid metabolism and the parr–smolt transformation. A preliminary analysis of a dietary lipid experiment identified a number of genes known to be involved in lipid metabolism. Significant fold change differences (as low as 1.2x) were apparent from the microarray analysis and were confirmed by quantitative real-time polymerase chain reaction analysis. The study also highlighted the potential for obtaining artefactual expression patterns as a result of cross-hybridization of similar transcripts. Examination of the robustness and sensitivity of the experimental design employed demonstrated the greater importance of biological replication over technical (dye flip) replication for identification of a limited number of key genes in the studied system. The TRAITS (TRanscriptome Analysis of Important Traits of Salmon)–salmon genome project microarray has been proven, in a number of studies, to be a powerful tool for the study of key traits of Atlantic salmon biology. It is now available for use by researchers in the wider scientific community

Dietary micronutrient composition affects fillet texture and muscle cell size in Atlantic salmon (Salmo salar)

During the past 20 years, plant ingredients have taken over as the main constituents in feed for Atlantic salmon. This has changed the nutrient composition of the feeds, the bioavailability of nutrients and perhaps nutrient metabolism. Plant‐based diets also contain more anti‐nutrients. The EU‐funded project ARRAINA re‐evaluated recommendations for micronutrient supplementation to Atlantic salmon feeds, and the present study compared a diet supplemented with micronutrients according to NRC (2011) (control diet, 100% NP (nutrient package)) with a diet supplemented according to the new recommendations (New NP). Tissue concentrations of pyridoxine, pantothenic acid, niacin, vitamin C, Zn and Se were significantly higher; and Cu was lower in Atlantic salmon fed the diet with the New NP compared to the control fish. The New NP also gave a near significant effect on growth, decreased muscle firmness and increased muscle cell size, and it affected metabolism of nitrogen‐containing metabolites in the muscle. While we cannot be certain which micronutrient(s) gave these effects, the B vitamins are probable candidates, since they are mediators of intermediary metabolism and have been shown to influence some of the affected metabolites

Functional Desaturase Fads1 (Δ5) and Fads2 (Δ6) Orthologues Evolved before the Origin of Jawed Vertebrates

Long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are essential components of biomembranes, particularly in neural tissues. Endogenous synthesis of ARA, EPA and DHA occurs from precursor dietary essential fatty acids such as linoleic and α-linolenic acid through elongation and Δ5 and Δ6 desaturations. With respect to desaturation activities some noteworthy differences have been noted in vertebrate classes. In mammals, the Δ5 activity is allocated to the Fads1 gene, while Fads2 is a Δ6 desaturase. In contrast, teleosts show distinct combinations of desaturase activities (e.g. bifunctional or separate Δ5 and Δ6 desaturases) apparently allocated to Fads2-type genes. To determine the timing of Fads1-Δ5 and Fads2-Δ6 evolution in vertebrates we used a combination of comparative and functional genomics with the analysis of key phylogenetic species. Our data show that Fads1 and Fads2 genes with Δ5 and Δ6 activities respectively, evolved before gnathostome radiation, since the catshark Scyliorhinus canicula has functional orthologues of both gene families. Consequently, the loss of Fads1 in teleosts is a secondary episode, while the existence of Δ5 activities in the same group most likely occurred through independent mutations into Fads2 type genes. Unexpectedly, we also establish that events of Fads1 gene expansion have taken place in birds and reptiles. Finally, a fourth Fads gene (Fads4) was found with an exclusive occurrence in mammalian genomes. Our findings enlighten the history of a crucially important gene family in vertebrate fatty acid metabolism and physiology and provide an explanation of how observed lineage-specific gene duplications, losses and diversifications might be linked to habitat-specific food web structures in different environments and over geological timescales

Highly Unsaturated Fatty Acid Synthesis in Atlantic Salmon: Characterization of ELOVL5- and ELOVL2-like Elongases

Fish species vary in their capacity to biosynthesize the n-3 long-chain polyunsaturated fatty acids (LC-PUFA) eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of LC-PUFA involves enzyme-mediated fatty acyl desaturation and elongation. Previously, a cDNA for an elongase, now termed elovl5a, had been cloned from Atlantic salmon. Here we report on the cloning of two new elongase cDNAs: a second elovl5b elongase, corresponding to a 294 aa protein, and an elovl2-like elongase, coding for a 287 aa protein, characterized for the first time in a non-mammalian vertebrate. Heterologous expression in yeast showed that the salmon Elovl5b elongated C18 and C20 PUFA, with low activity towards C22, while Elovl2 elongated C20 and C22 PUFA with lower activity towards C18 PUFA. All three transcripts showed predominant expression in the intestine and liver, followed by the brain. Elongase expression showed differential nutritional regulation. Levels of elovl5b and particularly of elovl2, but not of elovl5a, transcripts were significantly increased in liver of salmon fed vegetable oils (VO) compared to fish fed fish oil (FO). Intestinal expression showed a similar pattern. Phylogenetic comparisons indicate that, in contrast to salmon and zebrafish, Acanthopterygian fish species lack elovl2 which is consistent with their neglible ability to biosynthesise LC-PUFA and to adapt to VO dietary inclusion, compared to predominantly freshwater salmonids. Thus the presence of elovl2 in salmon explains the ability of this species to biosynthesise LC-HUFA and may provide a biotechnological tool to produce enhanced levels of LC-PUFA, particularly DHA, in transgenic organisms

Growth, flesh adiposity and fatty acid composition of Atlantic salmon (Salmo salar) families with contrasting flesh adiposity: effects of replacement of dietary fish oil with vegetable oils

The present study compared the effects of diets formulated with reduced fishmeal (FM) content and either 100% fish oil (FO) or 100% of a vegetable oil (VO) blend in post-smolts of three family groups of Atlantic salmon. Two groups were selected as being either &ldquo;Lean&rdquo; or &ldquo;Fat&rdquo; based on estimated breeding values (EBV) for flesh adiposity of their parents derived from a breeding programme, while the third group (CAL) was a mix of non-pedigreed commercial families unrelated to the two groups above. The VO blend comprised rapeseed, palm and a new product, Camelina oil in a ratio of 5/3/2, and diets were fed to duplicate pens of each salmon group. After an ongrowing period of 55 weeks, to reach a mean weight of 3kg, the fish from all treatments were switched to a decontaminated FO for a further 24 weeks to follow restoration of long-chain n-3 polyunsaturated fatty acids (LC-PUFA) in the fish previously fed VO. Final weights were significantly affected by family group and there was also an interaction between diet and group with Fat and Lean FO fish being larger than the same fish fed VO. Specific growth rate (SGR) was highest in CAL fish (1.01), feed conversion ratio (FCR) was highest in the Lean fish but there were no significant effects on thermal growth coefficient (TGC). Condition Factor (CF) was lowest in CAL fish while the hepato-somatic index (HSI) was highest in Lean fish and viscero-somatic index (VSI) highest in Fat fish. Flesh and viscera lipid content was affected by both family group and diet with a significant interaction between the two. Flesh lipid in fish fed FO was in the order Fat &gt; CAL &gt; Lean although this order was Fat = Lean &gt; CAL when fed VO. Flesh fatty acid compositions were affected mainly by diet although some minor fatty acids were also influenced by group. Fish fed VO had n-3 LC-PUFA reduced by ~65% compared to fish fed FO but this could be restored by a 16 week FO finishing diet phase. The differences observed in lipid and fatty acid deposition suggested that genetics affected lipid deposition and metabolism and that breeding programmes could select for fish that retained more n-3 LC-PUFA in their flesh, particularly when fed diets low in these fatty acids

Modelling the predictable effects of dietary lipid sources on the fillet fatty acid composition of one-year-old gilthead sea bream (Sparus aurata L.)

The present study aimed to ascertain the different fatty acid (FA) descriptors linking dietary and muscle FA composition in one-year-old gilthead sea bream. For that purpose, our own published data along with additional data from the present study were compiled and analysed. High linear correlations (r2 = 0.90, P &lt; 0.001) between dietary and muscle fatty acid composition were reported for monoenes, C18 polyunsaturated FA (PUFA) and long-chain PUFA. Prediction deviations due to changes in muscle fatness were analyzed in an independent trial with two different feeding levels (full ration size, 30% restriction ration). Regardless of feeding regimen, predicted values for muscle FA at low concentrations deviated (P &lt; 0.001) from observed values, but good predictions with less than 6% deviations were found for abundant fatty acids (16:1n-7, 18:1n-9, 18:2n-6, 18:3n-3, 20:4n-6, 20:5n-3, 22:6n-3). All this highlights the predictable effects of dietary oils in the muscle FA composition of gilthead sea bream, although further research is needed to cover all the range of commercial fish size and for the up-scaling of laboratory results to different fish farming conditions

Performance, feed utilization, and hepatic metabolic response of weaned juvenile Atlantic bluefin tuna (Thunnus thynnus L.): effects of dietary lipid level and source

The development of formulated diets and feeds is essential to increase production of farmed tuna species. There is limited knowledge of this topic, mainly on Pacific Bluefin tuna (Thunnus orientalis) in Japan, whereas no major attempts have been made with Atlantic Bluefin tuna (Thunnus thynnus; ABT). In the present study, two trials were performed using inert formulated diets as on-growing feeds for weaned ABT juvenile in order to establish adequate dietary levels of both lipid and omega-3 long-chain polyunsaturated fatty acids (LC-PUFA). In a first trial, ABT (initial weight = 2.9±0.9g) were fed for 10 days with either a commercial (Magokoro®, MGK) or two experimental feeds with two different lipid levels (15 or 20%) using krill oil (KO) as the single lipid source in order to estimate the suitable lipid content. Fish fed MGK displayed the highest growth, followed by 15KO, with no differences in fish survival. Thus, a lipid content of 15% was considered better than 20% for ABT juveniles. In the second trial, fish (initial weight = 3.3 ± 0.6g) were fed either MGK, 15KO or a feed containing 15% lipid with a combination (1:1, v/v) KO and rapeseed oil (RO) (15KORO). Fish fed 15KO and 15KORO showed the highest growth in terms of weight and fork length (including weight gain and SGR). Increasing dietary lipid level or adding RO to the feeds did not increase liver lipid content. The liver fatty acid profile largely reflected dietary intake confirming very limited LC-PUFA biosynthetic activity for this teleost species. In this respect, liver of fish fed 15KO and 20KO displayed the highest contents of docosahexaenoic acid (DHA). The hepatic expression of genes of lipid and fatty acid metabolism, transcription factors, and antioxidant enzymes was investigated with many of the genes showing regulation by both dietary lipid and LC-PUFA contents. The present study showed promising results that suggested ABT juveniles can be on grown on inert dry feeds that supported good fish growth and the accumulation of the health-promoting fatty acid DHA. Further studies are required in order to fully elucidate lipid and fatty acid requirements of this iconic species regarding dietary sources and production costs.En prensa1,52