Fatty acid metabolism in marine fish: Low activity of fatty acyl Δ5 desaturation in gilthead sea bream ( Sparus aurata ) cells

Marine fish are known to have an absolute dietary requirement for C20 and C22 highly unsaturated fatty acids. Previous studies using cultured cell lines indicated that underlying this requirement in marine fish was either a deficiency in fatty acyl Δ5 desaturase or C18-20 elongase activity. Recently, Ghioni et al. (Biochim. Biophys. Acta, 1437, 170-181, 1999) presented evidence that in turbot cells there was low activity of C18-20 elongase whereas Δ5 desaturase had high activity. In the present study, the fatty acid desaturase/elongase pathway was investigated in a cell line (SAF-1) from another carnivorous marine fish, sea bream. The metabolic conversions of a range of radiolabelled polyunsaturated fatty acids that comprised the direct substrates for Δ6 desaturase ([1-14C]18:2n-6 and [1-14C]18:3n-3), C18-20 elongase ([U-14C]18:4n-3), Δ5 desaturase ([1-14C]20:3n-6 and [U-14C]20:4n-3) and C20-22 elongase ([1-14C]20:4n-6 and [1-14C]20:5n-3) were utilized. The results showed that fatty acyl Δ6 desaturase in SAF-1 cells was highly active and there was substantial C18-20 elongase and C20-22 elongase activities. A deficiency in the desaturation/elongation pathway was clearly identified at the level of the fatty acyl Δ5 desaturase which was very low, particularly with 20:4n-3 as substrate. In comparison, the apparent activities of Δ6 desaturase, C18-20 elongase and C20-22 elongase were approximately 94-fold, 27-fold and 16-fold greater than that for Δ5 desaturase towards their respective n-3 polyunsaturated fatty acid substrates. The evidence obtained in the SAF-1 cell line is consistent with the dietary requirement for C20 and C22 highly unsaturated fatty acids in the marine fish, the sea bream, being primarily due to a deficiency in fatty acid Δ5 desaturase activity

Effects of dietary vegetable oil on atlantic salmon hepatocyte fatty acid desaturation and liver fatty acid compositions

Fatty acyl desaturase activities, involved in the conversion of the C18 EFA, 18:2n-6 and 18:3n-3, to the highly unsaturated fatty acids (HUFA) 20:4n-6, 20:5n-3 and 22:6n-3, are known to be under nutritional regulation. Specifically, the activity of the desaturation/elongation pathway is depressed when animals, including fish, are fed fish oils rich in n-3HUFA compared to animals fed vegetable oils rich in C18 EFA. The primary aims of the present study were a) to establish the relative importance of product inhibition (n-3HUFA) versus increased substrate concentration (C18 EFA) and, b) to determine whether 18:2n-6 and 18:3n-3 differ in their effects, on the hepatic fatty acyl desaturation/elongation pathway in Atlantic salmon (Salmo salar). Smolts were fed ten experimental diets containing blends of two vegetable oils, linseed (LO) and rapeseed oil (RO), and fish oil (FO) in a triangular mixture design for 50 weeks. Fish were sampled after 32 and 50 weeks, lipid and fatty acid composition of liver determined, fatty acyl desaturation/elongation activity estimated in hepatocytes using [1-14C]18:3n-3 as substrate, and the data subjected to regression analyses. Dietary 18:2n-6 was positively correlated, and n-3HUFA negatively correlated, with lipid content of liver. Dietary 20:5n-3 and 22:6n-3 were positively correlated with liver fatty acids with a slope greater than unity suggesting relative retention and deposition of these HUFA. In contrast, dietary 18:2n-6 and 18:3n-3 were positively correlated with liver fatty acids with a slope of less than unity suggesting metabolism via β-oxidation and/or desaturation/elongation. Consistent with this, fatty acyl desaturation/elongation in hepatocytes was significantly increased by feeding diets containing vegetable oils. Dietary 20:5n-3 and 22:6n-3 levels were negatively correlated with hepatocyte fatty acyl desaturation. At 32 weeks, 18:2n-6 but not 18:3n-3, was positively correlated with hepatocyte fatty acyl desaturation activity whereas the reverse was true at 50 weeks. The data indicate that both feedback inhibition through increased n-3HUFA and decreased C18 fatty acyl substrate concentration are probably important in determining hepatocyte fatty acyl desaturation activities, and that 18:2n-6 and 18:3n-3 may differ in their effects on this pathway

Lipid and fatty acid composition of parasitic caligid copepods belonging to the genus Lepeophtheirus

Sea lice are copepod ectoparasites that constitute a major barrier to the sustainability and economic viability of marine finfish aquaculture operations worldwide. In particular, the salmon louse, Lepeophtheirus salmonis, poses a considerable problem for salmoniculture in the northern hemisphere. The free-swimming nauplii and infective copepodids of L. salmonis are lecithotrophic, subsisting principally on maternally-derived lipid reserves. However, the lipids and fatty acids of sea lice have been sparsely studied and therefore the present project aimed to investigate the lipid and fatty acid composition of sea lice of the genus Lepeophtheirus obtained from a variety of fish hosts. Total lipid was extracted from eggs and adult female Lepeophtheirus salmonis obtained from both wild and farmed Atlantic salmon (Salmo salar L.) sampled at two time points, in the mid 1990’s and in 2009. In addition, L. salmonis from wild sea trout (Salmo trutta L.) and L. hippoglossi from wild Atlantic halibut (Hippoglossus hippoglossus L.) were sampled and analyzed. The lipids of both females and egg strings of Lepeophtheirus were characterized by triacylglycerol (TAG) as the major neutral (storage) lipid with phosphatidylcholine and phosphatidylethanolamine as the major polar (membrane) lipids. The major fatty acids were 22:6n-3 (DHA), 18:1n-9 and 16:0, with lesser amounts of 20:5n-3, 22:5n-3 and 18:0. L. salmonis sourced from farmed salmon were characterized by higher levels of 18:2n-6 and 18:3n-3 than lice from wild salmon. Egg strings had higher levels of TAG and lower DHA compared to females, whereas L. hippoglossi had lower levels of TAG and higher DHA than L. salmonis. The results demonstrate that the fatty acid compositions of lice obtained from wild and farmed salmon differ and that changes to the lipid and fatty acid composition of feeds for farmed salmon influence the louse compositions

In vivo metabolism of unsaturated fatty acids in Sepia officinalis hatchlings

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Tissue distribution of lipid and fatty acid metabolism and transcription factors genes in adult Atlantic bluefin tuna (Thunnus thynnus L.)

To determine expression of the major lipid pathways in tissues of adult bluefin tuna 8 individuals were used for collecting samples for tissue expression of key lipid metabolism genes. Triplicate sets of samples of brain, gills, heart, kidney, spleen, liver, intestine, white muscle, red muscle, adipose tissue ovary and testis were collected. Expression of genes was determined by qPCR. Tissue expression profiles showed that PUFA biosynthetic pathway genes were expressed in all tissues examined, highest expression in brain, liver and testis. Elongase elovl5 showed higher expression than desaturase fads2d6 in all other tissues, with low expression of in red muscle and ovaries. Transcription factors, pparα and pparγ showed parallel expression, with adipose tissue with the highest relative copy number, followed by intestine>testis>liver. The expression of lxr was low in liver, with highest expression in testis, brain and kidney. Similarly, rxr was poorly expressed in liver with higher expression in muscle, spleen and brain. The rank order of expression of srebp1 was brain, testis, ovary, intestines, kidney, gill, liver, white muscle, spleen, heart and red muscle. For srebp2 the highest expression was shown in brain, testis and adipose with lowest expression in heart and white muscle. Expression of fabp2 was highest in intestine, brain and heart with lower levels in liver, red muscle, adipose and kidney. fabp4 showed highest expression in ovaries with liver showing the lowest. fabp7 showed highest expression levels in brain and testis with lowest values in liver. cptI expression was highest in brain and lowest in liver. Similarly, the relative copy number for fas was highest in brain followed by gonads, gill and liver, with white muscle showing lowest expression. The expression of aco was highest in adipose and intestine, followed by liver, kidney and brain. The expression of hmgcl was highest in ovary followed by adipose, brain and testis, with lowest expression in liver. Expression of lpl was highest in testis and lowest in ovary, with liver and white muscle, adipose, heart, gills and red muscle, kidney, intestine showing intermediate levels of expression. This research was supported by projects from the Junta de Andalucía, Proyecto de Excelencia de Promoción General del Conocimiento Ref. RNM 0733, and Programa Estatal de Investigación del Ministerio de Economía y Competitividad Ref. AGL2014-52003-C2-1-R. AQUACULTURE, ENDOCRINOLOGY AND TOXICOLOGY 218. VI IBERIAN CONGRESS OF ICHTHYOLOGY MURCIA, SPAIN 21st · 24th June P-04

Molecular aspects of lipid metabolism, digestibility and antioxidant status of Atlantic bluefin tuna (T. thynnus L.) larvae during first feeding

Atlantic bluefin tuna (Thunnus thynnus L.; ABT) larvae were fed on enriched rotifers Brachionus rotundiformis and copepod nauplii Acartia tonsa from first feeding to 15 days post hatching. Rotifers were enriched with five different commercial products: OG, MG, AG and RA plus selenium and vitamin E. Copepods (COP) were cultured with the algae Rhodomonas baltica. Metabolic processes were studied by determining the expression of 30 genes related to lipid metabolism (transcription factors, fatty acid metabolism and lipid homeostasis), antioxidant enzymes, myogenesis and digestive enzymes. Growth and development parameters and high expression of myogenesis genes myhc2 and tropo indicated that COP were better than enriched rotifers as live prey for first feeding ABT. COP and AG-fed larvae showed the lowest values for the transcription factors pparã and srebp2. The expression of fas showed differences among treatments, with highest relative expression in COP-fed larvae and those fed with RA rotifers. In relation to fatty acid catabolism, larvae fed RA had the highest aco expression levels, with the lowest observed in those fed COP. The expression profiles of lipid homeostasis genes showed that larvae fed COP had higher fabp2 and 4 expressions. Larvae fed AG showed the lowest lpl expression levels, with highest values observed in larvae fed OG. Regarding antioxidant enzyme gene expression, sod showed highest values in larvae fed COP and RA, with larvae fed MG rotifers showing lowest expression levels. A similar pattern was observed for the expression of cat and gpx1 and 4. The expression of genes for digestive enzymes showed that tryp expression levels were highest in COP-fed larvae but, in contrast, COP-fed larvae showed the lowest anpep and alp levels. ABT larvae fed AG displayed the lowest expression level of pla2. bal1 and bal2 presented similar expression patterns, with highest values in COP-fed ABT and lowest expression in larvae fed AG rotifers. Copepods were a superior live prey for first feeding ABT larvae compared to enriched rotifers, as indicated by the higher growth and flexion index achieved by COP-fed larvae, possibly reflecting the higher protein content of the copepods.This work was supported by the Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía, Proyecto de Excelencia de Promoción General del Conocimiento [Ref. RNM 733, 2012], and Programa Estatal de Investigación del Ministerio de Economía y Competitividad [Ref. AGL2014-52003-C2-1-R, 2014].Versión del edito

Egg quality determinants in cod (Gadus morhua L.): egg performance and lipids in eggs from farmed and wild broodstock

Lipids and essential fatty acids, particularly the highly unsaturated fatty acids, 20:5n-3 (eicosapentaenoic acid; EPA), 22:6n-3 (docosahexaenoic acid; DHA) and 20:4n-6 (arachidonic acid, AA) have been shown to be crucial determinants of marine fish reproduction directly affecting fecundity, egg quality, hatching success, larval malformation and pigmentation. In Atlantic cod (Gadus morhua L.) culture, eggs from farmed broodstock can have much lower fertilisation and hatching rates than eggs from wild broodstock. The present study aimed to test the hypothesis that potential quality and performance differences between eggs from different cod broodstock would be reflected in differences in lipid and fatty acid composition. Thus eggs were obtained from three broodstock, farmed, wild/fed and wild/unfed, and lipid content, lipid class composition, fatty acid composition and pigment content were determined and related to performance parameters including fertilisation rate, symmetry of cell division and survival to hatching. Eggs from farmed broodstock showed significantly lower fertilisation rates, cell symmetry and survival to hatching rates than eggs from wild broodstock. There were no differences in total lipid content or the proportions of the major lipid classes between eggs from the different broodstock. However, eggs from farmed broodstock were characterised by having significantly lower levels of some quantitatively minor phospholipid classes, particularly phosphatidylinositol. There were no differences between eggs from farmed and wild broodstock in the proportions of saturated, monounsaturated and total polyunsaturated fatty acids. The DHA content was also similar. However, eggs from farmed broodstock had significantly lower levels of AA, and consequently significantly higher EPA/AA ratios than eggs from wild broodstock. Total pigment and astaxanthin levels were significantly higher in eggs from wild broodstock. Therefore, the levels of AA and phosphatidylinositol, the predominant AA-containing lipid class, and egg pigment content were positively related to egg quality or performance parameters such as fertilisation and hatching success rates, and cell symmetry

Development of feeds for juvenile Atlantic bluefin tuna (Thunnus thynnus, L): effect of lipid level and source

In conclusion, the present study suggests that ABT juveniles can be grown on inert extruded dry feeds that result in good fish growth and accumulation of the health-promoting fatty acid DHA. Furthermore, a blend of VO and KO could be used as the dietary lipid source up to a dietary lipid level of 15 % without affecting fish performance. The expression of lipid metabolism genes in ABT liver showed a different response to dietary lipid level/fatty acid profile, consistent with previous data indicating limited n-3 LC-PUFA biosynthetic capability in ABT. However, gene expression showed some differences between the two trials, which highlight how the genetic background of different batches of ABT juveniles could affect the regulation of metabolic gene expression and thus be a factor in weaning success. The expression of antioxidant enzymes was also altered by diet, related to dietary contents of antioxidant nutrients. Thus, further studies are required in order to fully elucidate the lipid and fatty acid requirements of this iconic species in relation to dietary sources and production cost

Performance, feed utilization and hepatic molecular metabolic response of weaned juvenile Atlantic bluefin tuna (Thunnus thynnus, L): effect of 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

COPEPODS OR ROTIFERS? EVALUATING THE USE OF DIFFERENT FEEDING PROTOCOLS FOR LARVAE OF ATLANTIC BLUEFIN TUNA (Thunnus thynnus. L)

There are still many issues that require to be solved in larval rearing of Atlantic bluefin tuna (Thunnus thynnus; ABT) to prevent “mass-mortality” during this developmental stage. Initial data related to the feeding sequence of ABT larvae suggested that mortality observed during the first stages of life could be due partly to nutritional deficiencies. Previous studies demonstrated that copepods appeared to be a superior live prey compared to rotifers during the first two weeks of life. Our overarching aim was to evaluate different feeding strategies during first feeding of ABT larvae from a performance, compositional and molecular perspective. In order to do so, two groups of ABT larvae were fed with either copepod (Acartia tonsa; C) nauplii or rotifers (Brachionus rotundiformis; R) enriched with Algamac 3050® from mouth opening to 13 days after hatching (dah). After this, the group C-larvae was fed either Artemia enriched with Algamac 3050® (CA), Acartia nauplii and copepodites (CC) or sea bream (Sparus aurata) yolk-sac larvae (CY), while the R group passed on to being fed on Artermia enriched with Algamac 3050® (RA) up to 18 dah. After 13 dah, larvae fed C grew more than those fed R although there were no differences in survival. ABT larvae fed R accumulated highest eicosapentaenoate (EPA) but lowest docosahexaenoate (DHA) and total n-3 long-chain polyunsaturated fatty acids (LC-PUFA) than C-fed larvae, reflecting dietary contents. Indeed, there was no activation in the expression of the enzymes involved in LC-PUFA biosynthesis. However, the different live prey elicited regulation of transcription factor, digestive enzyme, lipid metabolism and oxidative stress genes. At 18 dah larvae fed CY and CA were the largest size with larvae fed RA displaying the lowest growth with no differences in survival among the dietary treatments. The highest DHA contents were found in ABT larvae fed CC and CY, whereas the lowest contents were found in RA-fed larvae. Indeed, RA-fed larvae showed the highest level of the intermediate product n-3 docosapentaenoate, which could be reflecting up-regulation in the biosynthetic pathway although this was not supported by gene expression data