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The role of subcutaneous adipose tissue adipocytes and the effects of fatty acids on carrageenan-induced skin inflammation in gilthead seabream (Sparus aurata) were studied. Fish were injected intramuscularly with phosphate-buffered saline (control) or λ-carrageenin (1%), and skin samples collected at the injection site at 3 and 6 h post-injection (p.i.) were processed for histological study. In addition, the presence and levels of lipid classes, fatty acid methyl esters (FAME) and eicosanoids were evaluated in the skin samples obtained from the injected areas. Histological results indicated an increase in adipocyte area in fish sampled at 3 h p.i. with λ-carrageenin compared to fish in the control group. Furthermore, the frequency of adipocytes between 4500 and 5000 μm

Systematic granulomatosis is a chronic disease that affects the majority of farmed meagre (A. regius). Nutritional imbalances and overcrowding can increase the risk to suffer oxidative stress, and consequently impact the incidence of granulomatosis. In order to evaluate this, juvenile meagre were fed five isolipidic (16.7 %) and isoproteic (49.6 %) fish meal and fish oil-based feeds prepared by adding different levels of vitamin C, minerals (Mn, Zn, Se) with constant vitamin E and K (300 and 35 mg kg-1, respectively): Diet KEC (100 mg kg-1C), Diet KEC+Mn/Zn/Se (100 mg kg-1C, 40 mg kg-1Mn, 200 mg kg-1Zn, 1.5 mg kg-1Se), Diet KECC (600 mg kg-1C), Diet KECCC (1,200 mg kg-1C), Diet KECCCC (3,200 mg kg-1C). All diets were tested at 3.20 kg m-3, but diets KECC and KECCCC was also tested at 6.20 kg m-3. Growth performance was only affected by stocking density, being lower at high density. Percentage of fish with granulomas was significantly lower in fish fed with the highest dietary vitamin C contents (KECCC and KECCCC) at low density. TBARS content was correlated with the percentage of granulomas in liver (R2=0.9439, y=0.003x-0.1242) denoting the involvement of an imbalance oxidative status in the appearance of granulomas. The present results show that high levels of vitamin C (1,200-3,200 mg kg-1C) and low stock density (3.20 kg m-3) favours the growth of juvenile meagre, reducing the lipid peroxidation indicators and decreasing the incidence of granulomas, which confirms that this pathology is mostly triggered by deficiency of antioxidant nutrients, particularly vitamin C.

Aquaculture, the farming of fish and seafood, is recognized as a highly efficient system for producing protein for human consumption. In contrast, many terrestrial animal protein production systems are inefficient, impacting land use and exacerbating climate change. Humankind needs to adopt a more plant-centric diet, the only exception being fish consumed as both a source of protein and essential dietary nutrients such as omega-3 fatty acids. Here we consider the implications of such a transition, and the challenges that aquaculture must overcome to increase productivity within planetary boundaries. We consider how agriculture, specifically crops, can provide solutions for aquaculture, especially the sectors that are dependent on marine ingredients. For example, agriculture can provide experience with managing monocultures and new technologies such as genetically modified crops tailored specifically for use in aquaculture. We propose that a closer connection between agriculture and aquaculture will create a resilient food system capable of meeting increasing dietary and nutritional demands without exhausting planetary resources. Aquaculture must develop within planetary boundaries. Experience from agriculture, such as in managing monocultures and using genetically modified crops, can inform sustainable solutions for aquaculture.

An 8-week feeding trial was conducted to evaluate the effects of dietary n-3 LC-PUFA levels on growth performance, tissue fatty acid profiles and relative expression of genes involved in the lipid metabolism of mud crab (Scylla paramamosain). Ten isonitrogenous diets were formulated to contain five n-3 LC-PUFA levels at 7 and 12 % dietary lipid levels. The highest weight gain and specific growth rate were observed in crabs fed the diets with 19·8 and 13·2 mg/g n-3 LC-PUFA at 7 and 12 % lipid, respectively. Moisture and lipid contents in hepatopancreas and muscle were significantly influenced by dietary n-3 LC-PUFA at the two lipid levels. The DHA, EPA, n-3 LC-PUFA contents and n-3:n-6 PUFA ratio in hepatopancreas and muscle significantly increased as dietary n-3 LC-PUFA levels increased at both lipid levels. The expression levels of -6 fatty acyl desaturase and acyl-CoA oxidase in hepatopancreas increased significantly, and expression levels of fatty acid synthase, carnitine palmitoyltransferase I and hormone-sensitive TAG lipase were down-regulated, with increased dietary n-3 LC-PUFA regardless of lipid level. Based on weight gain, n-3 LC-PUFA requirements of S. paramamosain were estimated to be 20·1 and 12·7 mg/g of diet at 7 and 12 % dietary lipid, respectively. Overall, dietary lipid level influenced lipid metabolism, and purified, high-lipid diets rich in palmitic acid reduced the n-3 LC-PUFA requirement of juvenile mud crab.

Ensuring lumpfish health and welfare in salmon farms is vital to reduce the high mortality rates reported and to guarantee a high delousing efficiency. Recent observations of farmed lumpfish livers have shown colours ranging from pale (colours 1 and 2), through bright orange (colours 3 and 4), to dark reddish-brown (colours 5 and 6), some of which may be related to welfare condition. To characterize the status of lumpfish deployed in four Faroese salmon farms, several welfare indicators were assessed: a weight-length relationship, scoring of external physical damage, and after dissection, stomach content and liver colour scoring. Liver samples were weighed, stored and analysed for lipid content, lipid classes, total pigments, fatty acid profile and histopathology to explain the differences between the mentioned liver colours. Bright orange livers, liver colours 3 and 4, were related to increased levels of carotenoid pigments rather than levels of lipids and appear to reflect good fish welfare. However, dark reddish-brown colours, liver colours 5 and 6, were associated with very low levels of triacyl glycerides in the liver, indicating use of lipid reserves and poor welfare condition. Histopathology confirmed that the dark reddish-brown livers, liver colours 5 and 6, formed a distinct group. Thus, liver colour was shown to be a good welfare indicator and should be monitored in farms.

The use of low fishmeal/fish oil in marine fish diets affects dietary essential fatty acids (EFAs) composition and concentration and, subsequently, may produce a marginal deficiency of those fatty acids with a direct impact on the fish intestinal physiology. Supplementation of essential fatty acids is necessary to cover the requirements of the different EFAs, including the ones belonging to the n-6 series, such as arachidonic acid (ARA). ARA, besides its structural role in the configuration of the lipid classes of the intestine, plays an important role in the functionality of the gut-associated immune tissue (GALT). The present study aimed to test five levels of dietary ARA (ARA0.5 (0.5%), ARA1 (1%), ARA2 (2%), ARA4 (4%), and ARA6 (6%)) for European sea bass (Dicentrarchus labrax) juveniles in order to determine (a) its effect in selected distal intestine (DI) lipid classes composition and (b) how these changes affected gut bacterial translocation rates and selected GALT-related gene expression pre and post challenge. No differences were found between distal intestines of fish fed with the graded ARA levels in total neutral lipids and total polar lipids. However, DI of fish fed with the ARA6 diet presented a higher (P

Untargeted lipidomic analysis was conducted to explore how different dietary docosahexaenoic acid (DHA)/eicosapentaenoic acid (EPA) ratio and, specifically, how an optimal ratio (2.3) compared to a suboptimum ratio (0.6) impacted lipid molecular species and the positional distribution of fatty acids in hepatopancreas of mud crab. The results indicated that major category of lipid affected by dietary DHA/EPA ratio was glycerophospholipids (GPs). The optimum dietary DHA/EPA ratio increased the contents of DHA bound to the sn-2 and sn-3 positions of phosphatidylcholine (PC) and triacylglycerol, EPA bound to the sn-2 position of phosphatidylcholine and 18:2n-6 bound to the sn-2 position of phosphatidylethanolamine (PE). Increased dietary DHA/EPA ratio also led to competition between arachidonic acid (ARA) and 18:2n-6 bound to esterified sites. Appropriate dietary DHA/EPA ratio can not only improve the growth performance and nutritional quality of mud crab, but also provide higher quality products for human consumers.

Purpose Farmed fish are increasingly raised on feeds containing vegetable oils, which affects their composition and possibly health properties. We investigated the effects of consuming farmed salmon, raised on different feeding regimes, on nutrient status and health outcomes in healthy subjects. Methods Salmon were grown on feeds containing mainly fish oil (FO) or rapeseed oil (RO), resulting in an eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) content of fillets of 2.1 or 0.9 g/100 g, respectively. In a randomized parallel controlled trial, 51 healthy subjects were allocated to consume 2 portions/week of FO salmon (n = 17), RO salmon (n = 17) or no additional salmon (Control, n = 17) as part of their habitual diet, for 18 weeks. We collected blood at 0, 9 and 18 weeks to measure omega-3 index (O3I) in red blood cells, plasma markers of cardiovascular risk, serum 25(OH)-vitamin D3 (25(OH)D3) and plasma trace elements. Results After 18 weeks, O3I was similarly increased in subjects consuming 2 portions/week of FO or RO salmon compared to control (both p 3 was significantly higher, whereas plasma triacylglycerols were significantly lower in subjects consuming RO salmon compared to control (both p p Conclusion Consuming two portions/week of salmon raised on rapeseed oil rather than fish oil increased the O3I and vitamin D status, and decreased plasma triacylglycerols. These outcomes endorse opportunities for developing more sustainable feeds within aquaculture food systems. Clinical trial registry This trial was registered at clinicaltrials.gov as NCT01916434.

The regulation of lipogenesis and lipolysis mechanisms related to consumption of lipid has not been studied in swimming crab. The aims of present study were to evaluate the effects of dietary lipid levels on growth, enzymes activities, and expression of genes of lipid metabolism in hepatopancreas of juvenile swimming crab. Three isonitrogenous diets were formulated to contain crude lipid levels at 5.8 %, 9.9 % and 15.1 %, respectively. Crabs fed the diet containing 15.1 % lipid had significantly lower weight gain, specific growth rate and survival, and higher feed conversion ratio than those fed the 5.8 % and 9.9 % lipid diets. Crabs fed 5.8 % lipid had lower malondialdehyde concentrations in the hemolymph and hepatopancreas than those fed the other diets. Highest glutathione peroxidase in hemolymph and superoxide dismutase in hepatopancreas were observed in crabs fed 5.8 % lipid. The lowest fatty acid synthase and glucose 6-phosphate dehydrogenase activities in hepatopancreas were observed in crabs fed 15.1 % lipid, whereas crabs fed 5.8 % lipid had lower carnitine palmitoyltransferase-1 activity than those fed the other diets. Crabs fed 15.1 % lipid showed lower hepatopancreas expression of genes involved in LC-PUFA biosynthesis, lipoprotein clearance, fatty acid uptake, fatty acid oxidation, lipid anabolism and lipid catabolism than those fed the other diets, whereas expression of some genes of lipoprotein assembly and fatty acid oxidation were up-regulated compared with crabs fed 5.8 % lipid. Overall, high dietary lipid level can inhibit growth, reduce feed utilization and reduce antioxidant enzyme activities. Moreover, dietary lipid influenced enzyme activities and gene expression involved in lipid metabolism of juvenile swimming crab., The present study was supported by National Key R & D Program of China (2018YFD0900400), China Agriculture Research System (CARS-48), National Natural Science Foundation of China (41476125), Nature Science Foundation of Zhejiang Province (LY17C190002), Key Research Program of Zhejiang Province of China (2018C02037), Zhejiang Aquaculture Nutrition & Feed Technology Service Team (ZJANFTST2017-2) and Major Agriculture Program of Ningbo (2017C110007). This research was also sponsored by the K. C. Wong Magna Fund in Ningbo University.

The long-chain (≥C20) polyunsaturated fatty acid biosynthesis capacity of fish varies among species, with trophic level hypothesised as a major factor. The biosynthesis capacity is largely dependent upon the presence of functionally diversified fatty acyl desaturase 2 (Fads2) enzymes, since many teleosts have lost the gene encoding a Δ5 desaturase (Fads1). The present study aimed to characterise Fads2 from four teleosts occupying different trophic levels, namely Sarpa salpa, Chelon labrosus, Pegusa lascaris and Atherina presbyter, which were selected based on available data on functions of Fads2 from closely related species. Therefore, we had insight into the variability of Fads2 within the same phylogenetic group. Our results showed that Fads2 from S. salpa and C. labrosus were both Δ6 desaturases with further Δ8 activity while P. lascaris and A. presbyter Fads2 showed Δ4 activity. Fads2 activities of herbivorous S. salpa are consistent with those reported for carnivorous Sparidae species. The results suggested that trophic level might not directly drive diversification of teleost Fads2 as initially hypothesised, and other factors such as the species' phylogeny appeared to be more influential. In agreement, Fads2 activities from P. lascaris and A. presbyter were similar to their corresponding phylogenetic counterparts Solea senegalensis and Chirostoma estor., Te present study was funded by Ministerio de Economía y Competitividad (AGL2015-70994-R). Dr Covadonga Rodríguez is a member of the Instituto de Biotecnología de Canarias (ITB).

Systemic granulomatosis has already been reported in meagre larvae with an adequate feeding protocol and enrichment media preventing its appearance in the first weeks of life. Afterwards, the control of this disease could be prevented through nutritional components of the inert food, being the antioxidants the key to success. For this reason, in the present study, meagre larvae were reared from 30 days post hatching (dph) with five isonitrogenous and isolipidic experimental microdiets with different levels of vitamin E and C: C– (40 mg kg−1 E, 100 mg kg−1C), C+ (400 mg kg−1 E, 1000 mg kg−1C), Krill (400 mg kg−1 E, 1000 mg kg−1C and substitution of fish oil by krill oil), EC (200 mg kg−1 E, 500 mg kg−1C) and EECC (800 mg kg−1 E, 2000 mg kg−1C). Prior to this, larvae were co-fed with rotifers and Artemia following a protocol which prevented the appearance of granulomas, as previously demonstrated. The substitution of fish oil by krill oil significantly increased levels of eicosapentaenoic acid (EPA, 16.6%) and docosahexaenoic acid (DHA, 17.6%) in meagre, consequently increasing the peroxidation index, which in turn translated into a higher incidence of granulomas. Although even low levels of vitamin E and C (40 mg kg−1 E, 100 mg kg−1C; C-) allowed the adequate growth of larvae, these levels were not enough to prevent the appearance of granulomas, requiring superior levels of both antioxidant vitamins (800 mg kg−1 E and 2000 mg kg−1C) to mitigate systemic granulomatosis. This mitigation was simultaneous with the reduction of thiobarbituric acid reactive substances TBARs content in larvae, which were highly correlated with the appearance of granulomas (R2 = 0.892, y = 0.0446× + 0.0756). A strong negative correlation was observed between the dietary levels of vitamin E (y = −0.0098× + 11.174, R2 = 0.8766, p value = .019, r = −0.93) and vitamin C (y = −0.0022× + 6.4777, R2 = 0.9278, p value = .003, r = −0.96) and the percentage of larvae with granulomas. The results showed that the occurrence of systemic granulomatosis seems to be associated to the larvae peroxidation status, so that high dietary levels of vitamin E and C (800 and 2000 mg kg−1, respectively; Diet EECC), reduced lipid peroxidation and completely prevented the appearance of granulomas in meagre larvae at 44 dph.

Systemic granulomatosis is a growing disease with a high morbidity, which affects to the majority of farmed meagre (Argyrosomus regius). The impossibility of isolating any infectious agents has hypothesized a nutritional origin of the disease. In order to try to elucidate the nutritional origin of granulomas, juvenile meagre were fed for 15 weeks with six diets containing different levels of vitamin E and C and with or without addition of vitamin K: Diet 0 (basal premix, no K, 150 mg kg‐−1 E, 20 mg kg‐−1 C), K (added 23 mg kg‐−1 K), EC (300 mg kg‐−1 E, 70 mg kg‐−1 C), KEC (23 mg kg‐−1 K, 300 mg kg‐−1 E, 70 mg kg‐−1 C), EECC (450 mg kg‐−1 E, 230 mg kg‐−1 C) and KEECC (23 mg kg‐−1 K, 450 mg kg‐−1 E, 230 mg kg‐−1 C). The diet EC significantly increased meagre growth in terms of final weight and length. Fish fed the highest levels of vitamin E and C presented lower percentage of granulomas in liver and heart than fish fed diet 0. The scored severity of granulomatosis in liver and kidney (main affected organs) tended to be lower with dietary increase of vitamin E, C and addition of vitamin K (from 1.83 diet 0 to 1.3 diet KEECC and from 0.91 diet 0 to 0.39 diet KEECC). In liver, the diet KEECC significantly increased catalase expression compared with diet 0. In kidney tnfα expression was significantly up-regulated in fish fed diet EECC and KEECC. In heart, low vitamin E and C levels (300 and 70 mg kg‐−1, respectively) significantly increased superoxide dismutase and glutathione peroxidase expression and high addition increased the expression of tnfα and cox-2 (0 or 23 mg kg‐−1 K, 450 mg kg‐−1 E and 230 mg kg‐−1 C, diet EECC and KEECC). The results show that combination of high dietary content of vitamin K and antioxidant vitamins E and C (23, 450 and 230 mg kg‐−1, respectively) influenced in the incidence of the granulomatosis, which suggests that this pathology could be mediated by nutritional factors.

Copper is an essential micronutrient for living organisms, but its biological function has not been sufficiently studied, especially in crustaceans. Therefore, the present study aimed to evaluate the role of copper in the maintenance of energy homeostasis by mediating lipid and energy metabolism in Litopenaeus vannamei. L. vannamei (initial weight 0.90 ± 0.00 g) were fed diets containing 12.4 (Cu12.4), 22.0 (Cu22.0), 31.0 (Cu31.0), 41.7 (Cu41.7) and 49.8 (Cu49.8) mg kg−1 Cu for 8 weeks. Lipid and energy metabolism-related biochemical and molecular parameters were determined, and ultrastructural observation performed in hepatopancreas. Growth performance, tissue Cu deposition, antioxidant parameters, and expression of genes involved in Cu homeostasis and autophagy were also evaluated. Dietary copper supplementation improved growth, Cu concentration in tissue, and antioxidant capacity compared to the Cu-deficient diet. Copper promoted lipolysis and β-oxidation, confirmed by elevated expression levels of cpt1, ampkγ, acbp, acadl, acadm, acadvl, acox1, acox3 and decreased expression of srebp, acc1, 6pgd, and markedly increased activities of LPS and CPT1 and decreased ACC activity. Expression levels of genes related to autophagy (atg13, atg3, atg12, atg9, beclin1) were up-regulated, and autolysosomes and autophagosome were observed in the hepatopancreas of shrimp fed diets supplemented with copper. Furthermore, energy metabolism related parameters (ATPase, cytochrome c oxidase, citrate synthase, adenosine 5′-triphosphate) and genes involved in tricarboxylic-acid cycle (cs, aco, idh, ogdh, sdhc, sdhd, fh, mdh) were up-regulated in shrimp fed copper supplemented diets. The present study showed that copper-mediated lipolysis and autophagy can coordinate energy generation, and provided new insight into copper nutrition and the potential beneficial effects of dietary Cu on energy metabolism in crustaceans.

© 2021 by the authors., Fish vary in their ability to biosynthesise long-chain polyunsaturated fatty acids (LC-PUFA) depending upon the complement and function of key enzymes commonly known as fatty acyl desaturases and elongases. It has been reported in Solea senegalensis the existence of a Δ4 desaturase, enabling the biosynthesis of docosahexaenoic acid (DHA) from eicosapentaenoic acid (EPA), which can be modulated by the diet. The present study aims to evaluate the combined effects of the partial replacement of fish oil (FO) with vegetable oils and reduced environmental salinity in the fatty acid composition of relevant body compartments (muscle, hepatocytes and enterocytes), the enzymatic activity over α-linolenic acid (ALA) to form n-3 LC-PUFA through the incubation of isolated hepatocytes and enterocytes with [1-14C] 18:3 n-3, and the regulation of the S. senegalensis fads2 and elovl5 in the liver and intestine. The presence of radiolabelled products, including 18:4n-3, 20:4n-3 and EPA, provided compelling evidence that a complete pathway enabling the biosynthesis of EPA from ALA, establishing S. senegalensis, has at least one Fads2 with ∆6 activity. Dietary composition prevailed over salinity in regulating the expression of fads2, while salinity did so over dietary composition for elovl5. FO replacement enhanced the proportion of DHA in S. senegalensis muscle and the combination with 20 ppt salinity increased the amount of n-3 LC-PUFA in hepatocytes., This study was funded through the project PROPUFAW3 of the Ministry of Science, Innovation and Universities, Spanish Government (AGL2015-70994-R); Agencia Canaria de Investigación, Innovación y Sociedad de la Información de la Consejería de Economía, Conocimiento y Empleo and Fondo Social Europeo (FSE) Programa Operativo Integrado de Canarias 2014-2020, Eje 3 Tema Prioritario 74 (85%) (PhD contract M.M.); CajaSiete (PhD contract A.G.).

All living organisms require copper for growth and development, but the gene expression profiles and molecular mechanisms underpinning dietary copper are poorly investigated. Therefore, the present study aimed to determine the potential metabolic changes in response to dietary copper based on analysis of hepatopancreas transcriptome in Litopenaeus vannamei. Three practical diets were formulated to supplement 0 (control diet; Csingle bondCu) and 40 mg kg−1 inorganic Cu (CuSO4·5H2O; Isingle bondCu) and copper amino acid chelate (Osingle bondCu), with analyzed Cu being 12.4, 49.8 and 50.0 mg kg−1, respectively. Shrimp fed Isingle bondCu and Osingle bondCu diets had higher percent weight gain and Cu concentration in tissues. Some essential amino acids (lysine, methionine, isoleucine, leucine, valine) and non-essential amino acids (tyrosine, glycine, aspartic acid, proline and serine) in hepatopancreas significantly increased in shrimp fed the copper supplemented diets. Transcriptome analysis indicated a total of 742 and 912 genes were differentially expressed (q < 0.001; log2fold change ≥2) in shrimp fed the Isingle bondCu and Osingle bondCu diets, respectively, in comparison to shrimp fed the control diet. Five and eight significantly changed pathways were annotated in the Csingle bondCu vs. Isingle bondCu and Csingle bondCu vs. Osingle bondCu comparisons, with metabolism the leading category for both. Similarly, the proportion of differentially expressed genes revealed that most were enriched in the category of metabolism. Further analysis revealed that dietary copper mainly affected amino acid and glycerophospholipid metabolism. Moreover, two significantly changed pathways (phagosome and IL-17 signaling pathway) related to the immune system were identified in shrimp fed the Osingle bondCu diet. The present study analyzing the hepatopancreas transcriptome identified potential roles of dietary copper on amino acid and glycerophospholipid metabolism and provided new insight that will be valuable in future studies to further elucidate the nutritional molecular basis of copper.

An 8-week feeding trial was conducted to evaluate the effects of dietary n-3 LC-PUFA levels on growth performance, tissue fatty acid profiles and relative expression of genes involved in the lipid metabolism of mud crab (Scylla paramamosain). Ten isonitrogenous diets were formulated to contain five n-3 LC-PUFA levels at 7 and 12 % dietary lipid levels. The highest weight gain and specific growth rate were observed in crabs fed the diets with 19·8 and 13·2 mg/g n-3 LC-PUFA at 7 and 12 % lipid, respectively. Moisture and lipid contents in hepatopancreas and muscle were significantly influenced by dietary n-3 LC-PUFA at the two lipid levels. The DHA, EPA, n-3 LC-PUFA contents and n-3:n-6 PUFA ratio in hepatopancreas and muscle significantly increased as dietary n-3 LC-PUFA levels increased at both lipid levels. The expression levels of -6 fatty acyl desaturase and acyl-CoA oxidase in hepatopancreas increased significantly, and expression levels of fatty acid synthase, carnitine palmitoyltransferase I and hormone-sensitive TAG lipase were down-regulated, with increased dietary n-3 LC-PUFA regardless of lipid level. Based on weight gain, n-3 LC-PUFA requirements of S. paramamosain were estimated to be 20·1 and 12·7 mg/g of diet at 7 and 12 % dietary lipid, respectively. Overall, dietary lipid level influenced lipid metabolism, and purified, high-lipid diets rich in palmitic acid reduced the n-3 LC-PUFA requirement of juvenile mud crab.

The digestive system presents daily rhythms at both physiological and histological levels. Although cell morphology rhythms in mammals have been reported, they have scarcely been investigated in fish. The aim of the present research was to investigate the existence of daily rhythms in the morphology of cells in the liver and intestine of a teleost fish, the European sea bass (Dicentrarchus labrax), and how feeding time influences them. Regarding liver, we also focused on differences between the two metabolic zones: perivenous and periportal. For this purpose, fish were divided into two groups: fish fed once a day in the mid-light phase (ML) or the mid-dark phase (MD). After 1 month under each feeding regime, liver and intestine samples were collected every 4 h during a 24-h cycle, and different parameters were studied by light microscopy and image analysis. Daily rhythms occurred in most of the parameters evaluated in the liver. The effect of feeding time depended on the metabolic zone: the rhythms in the periportal zone were synchronized mainly by the light/dark cycle regardless of feeding time, whereas in the perivenous zone, rhythms were influenced more by feeding time. In the intestine, a daily rhythm in villi height was found with acrophases coinciding with feeding time in each group. These findings show for the first time the existence of cellular morphological rhythms in fish liver and intestine, and highlight the interactions between light and feeding cycles in the different metabolic zones of the liver.

Routine gill swabbing is a non-destructive sampling method used for the downstream qPCR detection and quantitation of the pathogen Neoparamoeba perurans, a causative agent of amoebic gill disease (AGD). Three commercially available swabs were compared aiming their application for timelier AGD diagnosis (Calgiswab® (calcium alginate fibre-tipped), Isohelix® DNA buccal and cotton wool-tipped). Calcium alginate is soluble in most sodium salts, which potentially allows the total recovery of biological material, hence a better extraction of target organisms' DNA. Thus, this study consisted of (a) an in vitro assessment involving spiking of the swabs with known amounts of amoebae and additional assessment of retrieval efficiency of amoebae from agar plates; (b) in vivo testing by swabbing of gill arches (second, third and fourth) of AGD-infected fish. Both in vitro and in vivo experiments identified an enhanced amoeba retrieval with Calgiswab® and Isohelix® swabs in comparison with cotton swabs. Additionally, the third and fourth gill arches presented significantly higher amoebic loads compared to the second gill arch. Results suggest that limiting routine gill swabbing to one or two arches, instead of all, could likely lead to reduced stress-related effects incurred by handling and sampling and a timelier diagnosis of AGD.

The present study evaluated the effects of dietary Zn level on growth performance, serum and hepatopancreas metabolites, expression of genes involved in lipid and energy metabolism, and the signalling pathway of dietary Zn-induced lipolysis. Five isonitrogenous and isolipidic diets were formulated to contain different Zn levels: 46·4 (basal diet), 77·2, 87·0, 117·1 and 136·8 mg/kg, respectively. The results indicated that shrimp fed the diet containing Zn at 117·1 mg/kg had higher weight gain and specific growth rate, and the lowest feed intake and feed conversion rate, than shrimp fed the other diets. The deposition rate of Zn in whole body significantly decreased with increasing dietary Zn level. Dietary Zn prevented the accumulation of free radicals and improved antioxidant activities by increasing Cu/Zn superoxide dismutase and reducing malondialdehyde in hepatopancreas. Dietary Zn supplementation enhanced lipase activity and adiponectin, which could promote TAG breakdown and fatty acid oxidation and lead to reduced lipid in hepatopancreas. The mRNA expressions ofob-rb,adipor,camkkβ,ampk,cd36,mcdandcpt1involved in Zn-induced lipid catabolism were up-regulated, and the expressions ofsrebp,acc,fasandscd1were down-regulated. The mRNA levels of SLC39 family genes (zip3,zip9,zip11andzip14) in hepatopancreas were up-regulated with increasing dietary Zn level. The results demonstrated that dietary Zn level could significantly affect growth performance, tissue deposition of Zn, lipid metabolites and expression of genes involved in lipogenesis and lipolysis inLitopenaeus vannamei.

This study investigated whether dietary supplementation of phytogenic compounds limonene and thymol had synergistic or additive effects on growth and selected nutritional physiology pathways in Nile tilapia. A 63-day feeding experiment was conducted using fish of 1.5 ± 0.0 g (± standard error) fed on a commercial diet coated with either 0 ppm limonene and thymol (control), 400 ppm limonene (L), 500 ppm thymol, (T) or a combination of 400 ppm limonene and 500 ppm thymol (LT). Final fish weight (FW) was significantly improved to similar extents by diet LT (16.7 ± 0.3 g) and L (16.6 ± 0.4 g). Dietary thymol alone and the control did not enhance FW (15.0 ± 0.4 g and 13.7 ± 0.4 g respectively). Dietary thymol had shown a strong tendency to improve somatic growth (P = .052). The analysed candidate genes involved in the pathways of nutrient digestion, absorption and transport (muc), lipid metabolism (lpl), antioxidant enzymes (cat) and somatotropic axis growth (igfI) were also up-regulated to similar extents in Nile tilapia by diet L and LT (P < .05), above the regulation observed with the diet supplemented exclusively with thymol. This suggests lack of synergistic or additive effects on growth and nutritional physiology pathways when limonene and thymol are supplied in the diet., This work was funded by the Schlumberger Foundation PhD Training Program, and the Government of Uganda through the Agricultural Technology and Agribusiness Services (ATAAS) Project of the National Agricultural Research Organization (NARO).

Carps, barbels and other cyprinids are the major contributors to freshwater aquaculture at global scale. Nevertheless, freshwater fish aquaculture needs to diversify their production in order to offer consumers new species. Tench (Tinca tinca) is a freshwater species with great interest for the diversification of continental aquaculture. However, up to date, no commercial formulated diet exists for this species in order to optimize their nutritional requirements and the quality of its final product. Using multiple methodological approaches, the aim of this study was to evaluate the long chain polyunsaturated fatty acid (LC-PUFA) metabolism of T. tinca. Firstly, the molecular cloning and functional characterisation by heterologous expression in yeast of a desaturase (Fads2) and two elongases (Elovl2 and Elovl5) involved in LC-PUFA biosynthesis, and the analysis of gene expression among tissues were performed. Secondly, in order to confirm the LC-PUFA biosynthesis capacity of isolated hepatocytes and enterocytes, cells were incubated with [1-14C] labelled linoleic acid (18:2n-6, LA), linolenic acid (18:3n-3, ALA) and eicosapentaenoic acid (20:5n-3, EPA). In yeast, Fads2 showed a Δ6/Δ5 bifunctional activity. Elovl2 was more active over C20 and C22 substrates, whereas Elovl5 was over C18 and C20. Liver displayed the highest expression for the three target genes (fads2, elovl2 and elovl5). Incubated cells also showed Fads2 bifunctional activity as well as elongation products in concordance with yeast heterologous expression results. Importantly, our results demonstrated that tench is able to biosynthesise docosahexaenoic acid (DHA) from 18:3n-3 in both hepatocytes and enterocytes, a capacity that seems to explain in part the surprisingly high levels of DHA found in the fish flesh compared to its dietary supply. Tench is a promising freshwater species with a potential capacity to endogenously increase its flesh DHA contents, reducing the impact that the usage of fish oils from forage fisheries may have on the aquaculture industry., This study was funded by Ministerio de Economía y Competitividad (AGL2015-70994-R).

Atlantic bluefin tuna (ABT; Thunnus thynnus) is a highly regarded and consumed species, but farming is still in its infancy. Currently, nothing is known about the presence of circadian rhythmicity at central or peripheral tissues, or if there are daily rhythms in expression of genes involved in lipid metabolism. In order to elucidate clock gene regulation of genes of lipid metabolism in ABT, six clock genes (bmal1, clock, cry1, cry2, per1 and per2) were sequenced and 24 h expression of these genes determined in brain and liver of fish acclimated to a light:dark (L:D) photoperiod. Additionally, the daily expression of lipid metabolism and digestive enzyme genes in ABT was also determined in liver. All six clock genes displayed rhythmicity in the brain and liver, other than cry2, which did not show an acrophase in liver. In liver, all the transcription factors analysed other than srebp1 and srebp2 displayed rhythmicity, with lxr and pparα displaying diurnal expression, whereas pparγ was highly expressed at the end of the scotophase. Some of the target genes of lxr such as elovl5 and lpl also oscillated rhythmically, with acrophases during the photophase. In contrast, only three of the eight digestive enzyme genes studied displayed rhythmicity, at different times of the day, suggesting that either ABT display different feeding periods or the digestion of some nutrients (e.g. lipids) is prioritized over others. The present study showed that clock and lipid metabolism genes displayed strong daily rhythmicity in ABT brain and liver, which could be an area of considerable interest for the establishment of efficient feeding protocols in this new aquaculture species., SI

The use of plant-based feeds warrants the supplementation with selenium (Se) to cover the requirement for Atlantic salmon. Depending on its chemical form, Se is a trace element with a narrow range between requirement and toxicity for most vertebrates. Information on safe upper limit for Atlantic salmon feed supplementation is lacking. Atlantic salmon (147 g) were fed a low natural background organic Se diet (0.45 mg Se kg−1, wet weight (ww)) fortified with 5 graded levels of inorganic sodium selenite (0.45, 5.4, 11.0, 29.4, or 60.0 mg kg−1 ww) or organic selenomethionine (SeMet) (0.45, 6.2, 16.2, 21, or 39 mg kg−1 ww), in triplicate for 3 months. Excess Se supplementation was assessed by targeted biomarkers of Se toxicity pathways (e.g. markers of oxidative stress and lipid metabolism), as well as general adverse effect parameters (plasma biochemistry, hematology, liver histopathology, and growth). Safe limits were set by model-fitting the effect data in a dose-response (lower bound) bench mark dose (BMDL) evaluation. Fish fed the two highest selenite levels showed mortality while fish fed SeMet had no mortality. Fish fed 5.4–11 mg selenite kg−1 feed showed significantly (ANOVA, Tukey's t-test, p

Phytogenic compounds such as limonene and thymol have been shown to have growth-promoting properties in farmed animals but studies in fish are scarce. Two experiments (Experiments I and II) were carried out to investigate the individual effects of dietary limonene and thymol on the growth and nutritional physiology of juvenile Nile tilapia (Oreochromis niloticus). In Experiment I, the fish were fed on a commercial diet coated with limonene at 0 (Control), 200, 400, and 600 mg kg−1 (ppm), while in Experiment II thymol was supplemented in the diet at 0 (Control), 250 and 500 ppm. Our results showed a significant increase in fish weight and weight gain with diets supplemented with 400 and 600 ppm limonene compared to the control. Moreover, the expression of insulin growth factor I (igf-I), mucin-like protein (muc), oligo-peptide transporter I (pept1), lipoprotein lipase (lpl), alkaline phosphatase (alp) and catalase (cat) was up-regulated by dietary limonene. Our results confirm that dietary limonene can enhance the growth of Nile tilapia juveniles through the activation of key genes involved in somatotropic axis-mediated growth, nutrient digestion and antioxidant enzyme defence. Dietary thymol did not seem to influence growth or regulate the same pathways activated by limonene in Nile tilapia juveniles at inclusion levels up to 500 ppm. Overall, the present results suggest that potential growth-promoting effects are dependent upon the phytogenetic itself and its inclusion level.

Five isoproteic (54.8%) and isolipidic (24.1%) microdiets, which varied in their docosahexaenoic acid (DHA) content (0.25%, 0.75%, 1.64%, 1.99% and 3.17%; dw), were manufactured to determine its effects on longfin yellowtail Seriola rivoliana larvae in terms of fish biological performance, whole body fatty acid profile and incidence of skeletal anomalies from 30 dah (11.31 ± 1.79 Total Length, TL) to 50 dah (19.80 ± 0.58 mm TL). The inclusion of dietary DHA up to 3.17% (dw) improved larval resistance to air exposure, although DHA did not significantly affect fish final growth or final survival. Indeed, high levels of dietary DHA (1.99% and 3.17%, dw) tended to increase the incidence of skeletal anomalies in S. rivoliana larvae, albeit no significant differences were observed. Furthermore, the occurrence of severe anomalies such as kyphosis and lordosis, was mainly associated to the larvae fed the highest levels of dietary DHA. In terms of survival, increasing dietary DHA levels did not significantly affect longfin yellowtail survival rate, despite a tendency for enhanced survival. The results of the present study proved that the inclusion of dietary DHA in inert diets up to a 3.17% (dw) and a DHA/EPA ratio above 3.1 increased the final survival and stress resistance in S. rivoliana larvae.

While chromium (Cr) has been recognized as an essential nutrient for all animals, and dietary supplementation can be beneficial, it can also be toxic. The present study aimed to investigate the contrasting effects of dietary chromium in Pacific white shrimp Litopenaeus vannamei. Five experimental diets were formulated to contain Cr at levels of 0.82 (Cr0.82, unsupplemented diet), 1.01 (Cr1.01), 1.22 (Cu1.22), 1.43 (Cr1.43) and 1.63 (Cr1.63) mg/kg and were fed to shrimp for 8 weeks. Highest weight gain was recorded in shrimp fed the diet containing 1.22 mg/kg Cr. Shrimp fed the diet containing the highest level of Cr (1.63 mg/kg) showed the lowest weight gain and clear signs of oxidative stress and apoptosis as evidenced by higher levels of H2O2, malondialdehyde and 8-hydroxydeoxyguanosine, and expression of caspase 2, 3, 5, and lower contents of total and oxidized glutathione, and expression of Cu/Zn sod, cat, gpx, mt, bcl2. Chromium supplementation promoted glycolysis and inhibited gluconeogenesis as shown by increased activities of hexokinase, phosphofructokinase and pyruvate kinase, and reduced activity of phosphoenolpyruvate carboxykinase in shrimp fed the diet containing 1.43 mg/kg Cr. Shrimp fed the diet with 1.63 mg/kg Cr had lowest contents of crustacean hyperglycemic hormone and insulin like peptide in hemolymph. Expression of genes involved in insulin signaling pathway and glycose metabolism including insr, irs1, pik3ca, pdpk1, akt, acc1, gys, glut1, pk, hk were up-regulated, and foxO1, gsk-3β, g6pc, pepck were down-regulated in shrimp fed the diets supplemented with Cr. This study demonstrated that optimum dietary supplementation of Cr had beneficial effects on glucose homeostasis and growth, whereas excess caused oxidative damage and impaired growth. The results contribute to our understanding of the biological functions of chromium in shrimp.

Several causative factors have been proposed for the occurrence of skeletal anomalies in fish larvae, among which we quote nutritional factors, such as LC-PUFAs. This study aimed to investigate the effect of different dietary DHA and ARA level and ratio on pikeperch (Sander lucioperca) larval development and performance, digestive capacity, fatty acids composition, skeleton anomalies and molecular markers of oxidative stress status (sod, gpx, and cat), stress response (StAR, gr, pparα, hsl and pepck), fatty acid synthesis (fadsd6, elovl5), eicosanoids synthesis (pla2, cox2, lox5, pge2, and lta4h), and bone development (twist, mef2c, sox9, and alp). Pikeperch larvae were fed six microdiets containing two different dietary levels of DHA (0.5% and 3.5%) combined with three levels of ARA (1.2%, 0.6%, and 0.3%). Dietary fatty acid changes did not affect growth performance but significantly influenced enzymatic activities. A significant increase in skeletal anomalies with DHA intake increment was recorded. StAR, cox2, pla2 and hsl expression were significantly depressed in 2.5% DHA larvae. An opposite effect of dietary DHA elevation was recorded in gpx expression. Both DHA and ARA had a significant effect on pparα, gr, and pge2 expressions. Although no significant interactions were found, pge2, gr, and pparα displayed a differential pattern of expression between the different treatments. A strong association was found for the larval tissue amount of ARA and DHA with eicosanoid metabolism, stress response and skeleton anomaly related genes. These results denoted the effects of dietary LC-PUFAs on immune/stress gene regulation and their potential implication in skeleton development.

While tissue fatty acid compositions reflect that of the dietary lipid source, little information is available on how dietary oils modify lipid class and molecular species profiles in hepatopancreas of crustacean. Herein, an 8-week nutritional trial and untargeted lipidomic analysis were used to investigate the impacts of dietary n-3 PUFA lipid sources including fish oil, krill oil and linseed oil on the lipidomic characteristics of hepatopancreas of swimming crab (Portunus trituberculatus). Dietary krill oil significantly increased distribution of 20:5n-3 and 22:6n-3 at sn-2 in phosphatidylcholine and phosphatidylethanolamine compared to fish oil. Fish oil intake promoted the deposition of 20:5n-3 and 22:6n-3 at sn-1,2,3 in triglyceride compared to linseed oil, which significantly increased the specific accumulation of 18:3n-3 at sn-1,3 in triglyceride and sn-2 in phosphatidylcholine and phosphatidylethanolamine. The study revealed metabolic responses to different dietary n-3 PUFA in swimming crab, which provided novel insight into the lipid nutrition of crustacean.

The aim of this study was to evaluate the effects of increasing dietary arachidonic acid (ARA) levels (from 1 up to 6 % of total fatty acids) on European sea bass (Dicentrarchus labrax) juveniles growth performance, tissue fatty acid profile, liver morphology as well as long chain polyunsaturated fatty acids (LC-PUFA) biosynthesis, triglyceride and cholesterol synthesis and lipid transport. A diet with total fish oil (FO) replacement and defatted fish meal (FM) containing a 0.1 g ARA g-1 diet was added to the experimental design as a negative control diet. Dietary ARA inclusion levels below 0.2 g ARA g-1 diet significantly worsened growth even only 30 days after the start of the feeding trial, whereas dietary ARA had no effect on fish survival. Liver, muscle and whole body fatty acid profile mainly reflected dietary contents and ARA content increased accordingly with ARA dietary levels. Tissue eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) levels were positively correlated among them. Hepatic lipid vacuolization increased with reduced dietary ARA levels. Expressions of fatty acyl desaturase 2 and 3-hydroxy-3-methylglutaryl-coenzyme genes were up-regulated in fish fed the negative control diet compared to the rest of the dietary treatments denoting the influence of ARA on lipid metabolism. Results obtained highlight the need to include adequate n-6 levels and not only n-3 LC-PUFA levels in European sea bass diets. Keywords: Dicentrarchus labrax, arachidonic acid, growth performance, tissue fatty acid profile, lipid metabolism

Seven experimental isonitrogenous (50%) and isolipidic (16%) diets with different levels of α-tocopherol acetate (16, 100, 190, 285, 430, 880 and 1300 mg kg − 1 ) were tested during 72 days to evaluate growth performance, tissue composition, fillet oxidation and liver histology in meagre juveniles, Argyrosomus regius . Growth performance, feed conversion ratio (FCR) and tissue composition were similar among treatments ( P > 0.05). In the liver, no major differences were recorded in lipid and fatty acid composition but higher lipid vacuolization were observed in diets E100, E190 and E880. Muscle fatty acid profiles showed an increment of the highly unsaturated fatty acid (HUFA) and a decrease of the saturated fatty acid with the increase of dietary vitamin E, which was accompanied with a reduction of the muscle TBARS responses. Therefore, is suggested that diets for this species should be supplemented with 451 mg kg − 1 of DL-α-tocopherol acetate (496 UI of vitamin E), as determine by broken-line regression analysis of muscle TBARS, to provide good overall growth performance and improved fish quality and storage stability. Moreover, results suggest that vitamin E deficiency or excess may deteriorate fish health. Statement of relevance The optimization of the dietary vitamin E level will contribute to formulate a suitable diet for meagre, a candidate for European aquaculture diversification, that until now is being fed with diets specific for other species. This study will narrow the knowledge gap that exists regarding meagre nutritional needs.

The regulation of lipogenesis and lipolysis mechanisms related to consumption of lipid has not been studied in swimming crab. The aims of the present study were to evaluate the effects of dietary lipid levels on growth, enzymes activities and expression of genes of lipid metabolism in hepatopancreas of juvenile swimming crab. Three isonitrogenous diets were formulated to contain crude lipid levels at 5·8, 9·9 and 15·1 %. Crabs fed the diet containing 15·1 % lipid had significantly lower growth performance and feed utilisation than those fed the 5·8 and 9·9 % lipid diets. Crabs fed 5·8 % lipid had lower malondialdehyde concentrations in the haemolymph and hepatopancreas than those fed the other diets. Highest glutathione peroxidase in haemolymph and superoxide dismutase in hepatopancreas were observed in crabs fed 5·8 % lipid. The lowest fatty acid synthase and glucose 6-phosphate dehydrogenase activities in hepatopancreas were observed in crabs fed 15·1 % lipid, whereas crabs fed 5·8 % lipid had lower carnitine palmitoyltransferase-1 activity than those fed the other diets. Crabs fed 15·1 % lipid showed lower hepatopancreas expression of genes involved in long-chain-PUFA biosynthesis, lipoprotein clearance, fatty acid uptake, fatty acid oxidation, lipid anabolism and lipid catabolism than those fed the other diets, whereas expression of some genes of lipoprotein assembly and fatty acid oxidation was up-regulated compared with crabs fed 5·8 % lipid. Overall, high dietary lipid level can inhibit growth, reduce antioxidant enzyme activities and influence lipid metabolic pathways to regulate lipid deposition in crab.

Lipid sources as alternatives to fish oil could alter the nutritional value and flavor quality of crab meat affecting consumer preferences. Herein, an 8-week nutritional trial was designed to investigate the effects of dietary lipid sources including fish oil (FO), krill oil (KO), palm oil, rapeseed oil, soybean oil and linseed oil on profiles of amino acids, fatty acids and volatiles in muscle of swimming crab (Portunus trituberculatus). Volatiles of crab muscle were characterized by headspace solid-phase microextraction and gas chromatography-tandem mass spectrometry. Results revealed that crabs fed FO and KO had significantly higher levels of protein, indispensable amino acids, eicosapentaenoic acid and docosahexaenoic acid in muscle. Principal component analysis and hierarchical cluster analysis demonstrated that muscle volatiles of crabs fed different dietary oils exhibited significant variations. Dietary FO and KO significantly increased the relative levels of 3-methylbutanal, heptanal, benzaldehyde and nonanal in muscle, which may produce more pleasant flavors.

Elongation of very long-chain fatty acid 4 (Elovl4) proteins are involved in the biosynthesis of very long-chain (> C24) fatty acids and in many teleost fish species they are key enzymes in the pathway for the production of docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Therefore, Elovl4 may be particularly important in Atlantic bluefin tuna (ABT; Thunnus thynnus) characterised by having high DHA to EPA ratios. The present study cloned and characterised both the function and expression of an elovl4 cDNA from ABT. The Elovl4 had an open reading frame of 915 base pairs encoding a putative protein of 304 amino acids. Alignment and phylogenetic analyses indicated that the Elovl4 isoform identified in the present study was an Elovl4b. Functional characterisation demonstrated that the Elovl4b enzyme had elongase activity towards all the polyunsaturated fatty acid (PUFA) substrates assayed. The ABT Elovl4b contributed to DHA biosynthesis by elongation of EPA and DPA to 24:5n-3, the latter being desaturated to 24:6n-3 by the action of fads2 (Δ6 desaturase). Additionally, the ABT Elovl4b has a role in the biosynthesis of very long-chain PUFA up to C34, compounds of key structural roles in neural tissues such as eye and brain, which had high levels of elovl4b transcripts. Surprisingly, while the relative expression of fads2, required for the production of DHA from EPA, was increased in liver of ABT fed a diet with reduced levels of EPA and DHA, expression of elovl4b was reduced. Results indicated that ABT has enzymes necessary for endogenous production of DHA from EPA and demonstrate that Elovl4b can effectively compensate for absence of Elovl2., SI

Atlantic salmon (Salmo salar) possess enzymes required for the endogenous biosynthesis ofn-3 long-chain PUFA (LC-PUFA), EPA and DHA, fromα-linolenic acid (ALA). Linoleic acid (LA) competes with ALA for LC-PUFA biosynthesis enzymes leading to the production ofn-6 LC-PUFA, including arachidonic acid (ARA). We aimed to quantify the endogenous production of EPA and DHA from ALA in salmon fed from first feeding on diets that contain no EPA and DHA and to determine the influence of dietary LA and ALA:LA ratio on LC-PUFA production. Salmon were fed from first feeding for 22 weeks with three diets formulated with linseed and sunflower oils to provide ALA:LA ratios of approximately 3:1, 1:1 and 1:3. Endogenous production ofn-3 LC-PUFA was 5·9, 4·4 and 2·8 mg per g fish and that ofn-6 LC-PUFA was 0·2, 0·5 and 1·4 mg per g fish in salmon fed diets with ALA:LA ratios of 3:1, 1:1 and 1:3, respectively. The ratio ofn-3:n-6 LC-PUFA production decreased from 27·4 to 2·0, and DHA:EPA ratio increased and EPA:ARA and DHA:ARA ratios decreased, as dietary ALA:LA ratio decreased. In conclusion, with a dietary ALA:LA ratio of 1, salmon fry/parr produced about 28 μgn-3 LC-PUFA per g fish per d, with a DHA:EPA ratio of 3·4. Production ofn-3 LC-PUFA exceeded that ofn-6 LC-PUFA by almost 9-fold. Reducing the dietary ALA:LA ratio reducedn-3 LC-PUFA production and EPA:ARA and DHA:ARA ratios but increasedn-6 LC-PUFA production and DHA:EPA ratio.