More Than an Antioxidant: Role of Dietary Astaxanthin on Lipid and Glucose Metabolism in the Liver of Rainbow Trout (Oncorhynchus mykiss).

This study investigated the influence of dietary astaxanthin (AX) on glucose and lipid metabolism in rainbow trout liver. Two iso-nitrogenous and iso-lipidic diets were tested for 12 weeks in rainbow trout with an initial mean weight of 309 g. The S-ASTA diet was supplemented with 100 mg of synthetic AX per kg of feed, whereas the control diet (CTRL) had no AX. Fish fed the S-ASTA diet displayed lower neutral and higher polar lipids in the liver, associated with smaller hepatocytes and lower cytoplasm vacuolization. Dietary AX upregulated adipose triglyceride lipase (atgl), hormone-sensitive lipase (hsl2) and 1,2-diacylglycerol choline phosphotransferase (chpt), and downregulated diacylglycerol acyltransferase (dgat2), suggesting the AX's role in triacylglycerol (TAG) turnover and phospholipid (PL) synthesis. Dietary AX may also affect beta-oxidation with the upregulation of carnitine palmitoyltransferase 1 (cpt1α2). Although hepatic cholesterol levels were not affected, dietary AX increased gene expression of sterol regulatory element-binding protein 2 (srebp2). Dietary AX upregulated the expression of 6-phosphogluconate dehydrogenase (6pgdh) and downregulated pyruvate kinase (pkl). Overall, results suggest that dietary AX modulates the oxidative phase of the pentose phosphate pathway and the last step of glycolysis, affecting TAG turnover, ß-oxidation, PL and cholesterol synthesis in rainbow trout liver.

Long-term effect of parental selenium supplementation on the one-carbon metabolism in rainbow trout (Oncorhynchus mykiss) fry exposed to hypoxic stress.

This study evaluated how different forms of selenium (Se) supplementation into rainbow trout broodstock diets modified the one-carbon metabolism of the progeny after the beginning of exogenous feeding and followed by hypoxia challenge. The progeny of three groups of rainbow trout broodstock fed either a control diet (Se level: 0·3 µg/g) or a diet supplemented with inorganic sodium selenite (Se level: 0·6 µg/g) or organic hydroxy-selenomethionine (Se level: 0·6 µg/g) was cross-fed with diets of similar Se composition for 11 weeks. Offspring were sampled either before or after being subjected to an acute hypoxic stress (1·7 mg/l dissolved oxygen) for 30 min. In normoxic fry, parental Se supplementation allowed higher glutathione levels compared with fry originating from parents fed the control diet. Parental hydroxy-selenomethionine treatment also increased cysteine and cysteinyl-glycine concentrations in fry. Dietary Se supplementation decreased glutamate-cysteine ligase (cgl) mRNA levels. Hydroxy-selenomethionine feeding also lowered the levels of some essential free amino acids in muscle tissue. Supplementation of organic Se to parents and fry reduced betaine-homocysteine S-methyltransferase (bhmt) expression in fry. The hypoxic stress decreased whole-body homocysteine, cysteine, cysteinyl-glycine and glutathione levels. Together with the higher mRNA levels of cystathionine beta-synthase (cbs), a transsulphuration enzyme, this suggests that under hypoxia, glutathione synthesis through transsulphuration might have been impaired by depletion of a glutathione precursor. In stressed fry, S-adenosylmethionine levels were significantly decreased, but S-adenosylhomocysteine remained stable. Decreased bhmt and adenosylmethionine decarboxylase 1a (amd1a) mRNA levels in stressed fry suggest a nutritional programming by parental Se also on methionine metabolism of rainbow trout.

Organic Selenium (OH-MetSe) Effect on Whole Body Fatty Acids and Mx Gene Expression against Viral Infection in Gilthead Seabream (Sparus aurata) Juveniles.

The supplementation of fish diets with OH-SeMet reduces oxidative stress and modulates immune response against bacterial infection. However, despite the importance of essential polyunsaturated fatty acids in fish nutrition and their high risk of oxidation, the potential protective effect of OH-SeMet on these essential fatty acids has not been studied in detail. Moreover, while viral infection is very relevant in seabream production, no studies have focused the Se effects against viral infection. The aim of the present study was to assess the impact of dietary supplementation with OH-SeMet on gilthead seabream fatty acid profiles, growth performance and response against viral infection. Gilthead seabream juveniles (21.73 ± 0.27 g) were fed for 91 days with three experimental diets, a control diet without supplementation of Se (0.29 mg Se kg diet-1) and two diets supplemented with OH-SeMet (0.52 and 0.79 mg Se kg diet-1). A crowding stress test was performed at week 7 and an anti-viral response challenge were conducted at the end of the feeding trial. Selenium, proximate and fatty acid composition of diets and body tissues were analyzed. Although fish growth was not affected, elevation in dietary Se proportionally raised Se content in body tissues, increased lipid content in the whole body and promoted retention and synthesis of n-3 polyunsaturated fatty acids. Specifically, a net production of DHA was observed in those fish fed diets with a higher Se content. Additionally, both monounsaturated and saturated fatty acids were significantly reduced by the increase in dietary Se. Despite the elevation of dietary Se to 0.79 mg kg-1 not affecting basal cortisol levels, 2 h post-stress plasma cortisol levels were markedly increased. Finally, at 24 h post-stimulation, dietary OH-SeMet supplementation significantly increased the expression of the antiviral response myxovirus protein gene, showing, for the first time in gilthead seabream, the importance of dietary Se levels on antiviral defense.

Tissue localization of selenium of parental or dietary origin in rainbow trout (Oncorhynchus mykiss) fry using LA-ICP MS bioimaging.

In relation to the decrease of selenium (Se) content in aquafeeds, the impact of level and form of parental and dietary Se supplementation was investigated in rainbow trout fry using laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS) bioimaging. The offspring of rainbow trout broodstock, fed either a control diet without any Se supplementation (0.3 mg Se/kg diet) or a diet supplemented with Se (0.6 mg Se/kg diet) either as sodium selenite or hydroxy-selenomethionine, were sampled at swim-up fry stage or after 11 weeks of cross-feeding. Total body Se levels were influenced by parental Se nutrition in swim-up fry and by direct Se feeding in 11-week fry with higher levels in the Se-supplemented groups compared with the control and the highest levels in the hydroxy-selenomethionine treatment. The Se retention was lower for dietary sodium selenite. Selenomethionine levels increased when Se was provided as hydroxy-selenomethionine. LA-ICP MS maps revealed yolk in swim-up fry and intestine, liver, and kidney in 11-week fed fry as tissues with high Se abundance. In swim-up fry, muscle Se was the highest abundant when parents were fed hydroxy-selenomethionine. In 11-week fed fry, muscle Se abundance was higher in the head part of fry fed both Se-supplemented diets, but only in the tail part of fry fed hydroxy-selenomethionine. Liver Se abundance was higher in fry fed sodium selenite compared with the control diet supporting the hypothesis that tissue Se distribution can be influenced by parental and dietary Se forms and levels.

Reproductive performance of gilthead seabream (Sparus aurata) broodstock showing different expression of fatty acyl desaturase 2 and fed two dietary fatty acid profiles.

Previous studies have shown that it is possible to nutritionally program gilthead seabream offspring through fish oil (FO) replacement by vegetable oils (VO) in the broodstock diet, to improve their ability to grow fast when fed low fish meal (FM) and FO diets during grow-out phase. However, in those studies broodstock performance was reduced by the VO contained diet. Therefore, the present study aimed to determine if it is possible to replace FO by a mixture of FO and rapeseed oil (RO) with a specific fatty acid profile in broodstock diets, without altering gilthead seabream broodstock reproductive performance. Besides, the study also aimed to evaluate the reproductive performance of broodstock with different expression of fatty acid desaturase 2 gene (fads2) a key enzyme in synthesis of long chain polyunsaturated fatty acids. For that purpose, broodfish having either a high (HD) or low (LD) expression of fads2 were fed for three months during the spawning season with two diets containing different fatty acid profiles and their effects on reproductive hormones, fecundity, sperm and egg quality, egg biochemical composition and fads2 expression were studied. The results showed that blood fads2 expression in females, which tended to be higher than in males, was positively related to plasma 17ß-estradiol levels. Moreover, broodstock with high blood fads2 expression showed a better reproductive performance, in terms of fecundity and sperm and egg quality, which was correlated with female fads2 expression. Our data also showed that it is feasible to reduce ARA, EPA and DHA down to 0.43, 6.6 and 8.4% total fatty acids, respectively, in broodstock diets designed to induce nutritional programming effects in the offspring without adverse effects on spawning quality. Further studies are being conducted to test the offspring with low FM and FO diets along life span.

Parental Selenium Nutrition Affects the One-Carbon Metabolism and the Hepatic DNA Methylation Pattern of Rainbow Trout (Oncorhynchus mykiss) in the Progeny.

Selenium is an essential micronutrient and its metabolism is closely linked to the methionine cycle and transsulfuration pathway. The present study evaluated the effect of two different selenium supplements in the diet of rainbow trout (Onchorhynchus mykiss) broodstock on the one-carbon metabolism and the hepatic DNA methylation pattern in the progeny. Offspring of three parental groups of rainbow trout, fed either a control diet (NC, basal Se level: 0.3 mg/kg) or a diet supplemented with sodium selenite (SS, 0.8 mg Se/kg) or hydroxy-selenomethionine (SO, 0.7 mg Se/kg), were collected at swim-up fry stage. Our findings suggest that parental selenium nutrition impacted the methionine cycle with lower free methionine and S-adenosylmethionine (SAM) and higher methionine synthase (mtr) mRNA levels in both selenium-supplemented treatments. DNA methylation profiling by reduced representation bisulfite sequencing (RRBS) identified differentially methylated cytosines (DMCs) in offspring livers. These DMCs were related to 6535 differentially methylated genes in SS:NC, 6890 in SO:NC and 7428 in SO:SS, respectively. Genes with the highest methylation difference relate, among others, to the neuronal or signal transmitting and immune system which represent potential targets for future studies.

Oxidative stress and antioxidant response in rainbow trout fry exposed to acute hypoxia is affected by selenium nutrition of parents and during first exogenous feeding.

Selenium (Se) deficiency is a problem widely encountered in humans and terrestrial livestock production with increasing attention also in aquaculture. Se supports the antioxidant system, which becomes especially important during stressful conditions. In the present study, the effect of Se-supplementation in broodstock and fry diets on the performance and antioxidant metabolism of rainbow trout fry under acute hypoxia was investigated. Rainbow trout broodstock were fed plant-ingredient based diets either without any Se-supplementation (Se level: 0.3 mg/kg) or supplemented with Se supplied as sodium selenite or as hydroxy-selenomethionine (Se level: 0.6 mg/kg respectively) for 6 months prior to spawning. The progenies were subdivided into three triplicate feeding groups and fed diets with similar Se levels compared to the parental diets, resulting in a 3x3 factorial design. After 11 weeks of feeding, the fry were either sampled or subjected to a hypoxic stress challenge. One hundred fish were transferred to tanks containing water with a low oxygen level (1.7 ± 0.2 ppm) and monitored closely for 30 min. When a fish started to faint it was recorded and transferred back to normoxic water. Direct fry feeding of the hydroxy-selenomethionine supplemented diet improved the resistance towards the hypoxic stress. On the contrary, fry originating from parents fed Se-supplemented diets showed a lower stress resistance compared to fry originating from parents fed the control diet. Fry subjected to hypoxia showed elevated oxidative stress with reduced glutathione (GSH) levels and increased isoprostanes (IsoP) and phytoprostanes (PhytoP) levels produced by lipid peroxidation of polyunsaturated fatty acids (PUFA), arachidonic and α-linolenic acids respectively. Increased mRNA expression of transcription factors (nrf2, nfκb, keap1X2) and decreased mRNA expression of antioxidant enzymes (trxr, sod, gstπ) indicated a transcriptional regulation of the antioxidant response. In stressed fry, the mRNA expression of several antioxidant genes including gr, msr and gstπ was found to be higher when fed the control diet compared to the sodium selenite treatment, with a contrary effect for parental and direct Se nutrition on gpx. The long-term parental effect becomes of greater importance in stressed fry, where more than half of the genes were significantly higher expressed in the control compared to the selenite supplemented group.

Variable impacts of L-arginine or L-NAME during early life on molecular and cellular markers of muscle growth mechanisms in rainbow trout.

Two experiments were conducted to test if manipulations of the Arginine-Nitric oxide pathway during the early life of rainbow trout would act on its early myogenic process. In experiment 1, trout embryos were immersed at 72° days post-fertilization (°dpf) or 104°dpf in water alone (control treatment, C) or containing 2 mM/L L-Arg (treatment A) or 1 mM/L of L-NAME, a NOS inhibitor (treatment N). We observed the beginning of expression of myf5 and fmhc genes at 72°dpf and 96°dpf, respectively. "A" treatment doubled the free Arg content of eggs but did not affect either the pattern of expression of myf5 and fmhc, nor white muscle cross-sectional area and number of white muscle fibres at hatching, nor embryo survival and fry growth. "N" treatment also did not affect these markers. In experiment 2, trout fry were fed from first feeding onwards and during 20 days either a control diet (C) or the same diet supplemented with L-NAME (0.1 g/100 g diet, N-diet). In C-fed fry, distribution of a single meal after overnight fasting induced changes in pcna, myod1, myog, fmhc, inos, nnos and ctsd gene expressions. N-feeding decreased fry growth but did not change their growth trajectory or survival. Twenty days of N-feeding led, compared to C-feeding, to changes in kinetics of transcription of pcna, myod1, myog, fmhc, inos, nnos, ctsd genes and to decreased white muscle cross-sectional area, total number of white muscle fibres, and number of large muscle fibres. L-NAME feeding thus decreased fry muscle growth by altering both hyperplasia and hypertrophy.

Meeting Global Feed Protein Demand: Challenge, Opportunity, and Strategy.

Feed protein supplements are one of the most expensive and limiting feed ingredients. This review offers a comprehensive analysis of how the expected expansion of animal production, driven by the rising world population and living standards for more animal-sourced foods, is creating a global shortage of feed protein supply. Because ruminants, chickens, and pigs contribute to 96% of the global supply of animal protein and aquaculture is growing fast, means of meeting the feed protein requirements of these species are elaborated. Geographic variation and interdependence among China, Europe, and North America in the demand and supply of feed protein are compared. The potential and current state of exploration into alternative feed proteins, including microalgae, insects, single-cell proteins, and coproducts, are highlighted. Strategic innovations are proposed to upgrade feed protein processing and assessment, improve protein digestion by exogenous enzymes, and genetically select feed-efficient livestock breeds. An overall successful and sustainable solution in meeting global feed protein demands will lead to a substantial net gain of human-edible animal protein with a minimal environmental footprint.

Comprehensive biometric, biochemical and histopathological assessment of nutrient deficiencies in gilthead sea bream fed semi-purified diets.

Seven isoproteic and isolipidic semi-purified diets were formulated to assess specific nutrient deficiencies in sulphur amino acids (SAA), n-3 long-chain PUFA (n-3 LC-PUFA), phospholipids (PL), P, minerals (Min) and vitamins (Vit). The control diet (CTRL) contained these essential nutrients in adequate amounts. Each diet was allocated to triplicate groups of juvenile gilthead sea bream fed to satiety over an 11-week feeding trial period. Weight gain of n-3 LC-PUFA, P-Vit and PL-Min-SAA groups was 50, 60-75 and 80-85 % of the CTRL group, respectively. Fat retention was decreased by all nutrient deficiencies except by the Min diet. Strong effects on N retention were found in n-3 LC-PUFA and P fish. Combined anaemia and increased blood respiratory burst were observed in n-3 LC-PUFA fish. Hypoproteinaemia was found in SAA, n-3 LC-PUFA, PL and Vit fish. Derangements of lipid metabolism were also a common disorder, but the lipodystrophic phenotype of P fish was different from that of other groups. Changes in plasma levels of electrolytes (Ca, phosphate), metabolites (creatinine, choline) and enzyme activities (alkaline phosphatase) were related to specific nutrient deficiencies in PL, P, Min or Vit fish, whereas changes in circulating levels of growth hormone and insulin-like growth factor I primarily reflected the intensity of the nutritional stressor. Histopathological scoring of the liver and intestine segments showed specific nutrient-mediated changes in lipid cell vacuolisation, inflammation of intestinal submucosa, as well as the distribution and number of intestinal goblet and rodlet cells. These results contribute to define the normal range of variation for selected biometric, biochemical, haematological and histochemical markers.

Influence of the forms and levels of dietary selenium on antioxidant status and oxidative stress-related parameters in rainbow trout (Oncorhynchus mykiss) fry.

Se is an essential micronutrient required for normal growth, development and antioxidant defence. The objective of the present study was to assess the impact of dietary Se sources and levels on the antioxidant status of rainbow trout (Oncorhynchus mykiss) fry. First-feeding fry (initial body weight: 91 mg) were fed either a plant- or fishmeal-based diet containing 0·5 or 1·2 mg Se/kg diet supplemented or not with 0·3 mg Se/kg diet supplied as Se-enriched yeast or sodium selenite for 12 weeks at 17°C. Growth and survival of rainbow trout fry were not significantly affected by dietary Se sources and levels. Whole-body Se was raised by both Se sources and to a greater extent by Se-yeast. The reduced:oxidised glutathione ratio was raised by Se-yeast, whereas other lipid peroxidation markers were not affected by dietary Se. Whole-body Se-dependent glutathione peroxidase (GPX) activity was enhanced in fish fed Se-yeast compared to fish fed sodium selenite or non-supplemented diets. Activity and gene expression of this enzyme as well as gene expression of selenoprotein P (SelP) were reduced in fish fed the non-supplemented plant-based diet. Catalase, glutamate-cysteine ligase and nuclear factor-erythroid 2-related factor 2 (Nrf2) gene expressions were reduced by Se-yeast. These results suggest the necessity to supplement plant-based diets with Se for rainbow trout fry, and highlight the superiority of organic form of Se to fulfil the dietary Se requirement and sustain the antioxidant status of fish. GPX and SelP expression proved to be good markers of Se status in fish.

Mucins as diagnostic and prognostic biomarkers in a fish-parasite model: transcriptional and functional analysis.

Mucins are O-glycosylated glycoproteins present on the apex of all wet-surfaced epithelia with a well-defined expression pattern, which is disrupted in response to a wide range of injuries or challenges. The aim of this study was to identify mucin gene sequences of gilthead sea bream (GSB), to determine its pattern of distribution in fish tissues and to analyse their transcriptional regulation by dietary and pathogenic factors. Exhaustive search of fish mucins was done in GSB after de novo assembly of next-generation sequencing data hosted in the IATS transcriptome database (www.nutrigroup-iats.org/seabreamdb). Six sequences, three categorized as putative membrane-bound mucins and three putative secreted-gel forming mucins, were identified. The transcriptional tissue screening revealed that Muc18 was the predominant mucin in skin, gills and stomach of GSB. In contrast, Muc19 was mostly found in the oesophagus and Muc13 was along the entire intestinal tract, although the posterior intestine exhibited a differential pattern with a high expression of an isoform that does not share a clear orthologous in mammals. This mucin was annotated as intestinal mucin (I-Muc). Its RNA expression was highly regulated by the nutritional background, whereas the other mucins, including Muc2 and Muc2-like, were expressed more constitutively and did not respond to high replacement of fish oil (FO) by vegetable oils (VO) in plant protein-based diets. After challenge with the intestinal parasite Enteromyxum leei, the expression of a number of mucins was decreased mainly in the posterior intestine of infected fish. But, interestingly, the highest down-regulation was observed for the I-Muc. Overall, the magnitude of the changes reflected the intensity and progression of the infection, making mucins and I-Muc, in particular, reliable markers of prognostic and diagnostic value of fish intestinal health.

Dietary vegetable oils do not alter the intestine transcriptome of gilthead sea bream (Sparus aurata), but modulate the transcriptomic response to infection with Enteromyxum leei.

BACKGROUND: Studies conducted with gilthead sea bream (Sparus aurata L.) have determined the maximum dietary replacement of fish meal and oil without compromising growth or product quality. The present study aimed to analyze the effect of the nutritional background on fish health and fish fed plant protein-based diets with fish oil (FO diet) or a blend of vegetable oils (66VO diet) were exposed for 102 days to the intestinal myxosporean parasite Enteromyxum leei, and the intestine transcriptome was analyzed with a customized oligo-microarray of 7,500 annotated genes. RESULTS: Infection prevalence was high and similar in the two diet groups, but the outcome of the disease was more pronounced in fish fed the 66VO diet. No differences were found in the transcriptome of both diet control groups, whereas the number of differentially expressed genes in infected groups was considerable. K-means clustering of these differentially expressed genes identified four expression patterns that reflected the progression of the disease with the magnitude of the fold-change being higher in infected 66VO fish. A positive correlation was found between the time of infection and the magnitude of the transcriptional change within the 66VO group, being higher in early infected animals. Within this diet group, a strong up-regulation of many components of the immune specific response was evidenced, whereas other genes related to complement response and xenobiotic metabolism were down-regulated. CONCLUSIONS: The high replacement of fish oil by vegetable oils in practical fish feeds did not modify the intestine transcriptome of gilthead sea bream, but important changes were apparent when fish were exposed to the myxosporean E. leei. The detected changes were mostly a consequence rather than a cause of the different disease progression in the two diet groups. Hence, the developed microarray constitutes an excellent diagnostic tool to address changes associated with the action of intestinal pathogens, but lacks a prognostic value to predict in advance the different susceptibility of growing fish to the current pathogen.

Modulation of the IgM gene expression and IgM immunoreactive cell distribution by the nutritional background in gilthead sea bream (Sparus aurata) challenged with Enteromyxum leei (Myxozoa).

The aim of the present work was to determine if a plant protein-based diet containing vegetable oils (VO) as the major lipid source could alter the distribution of IgM immunoreactive cells (IRCs) and the IgM expression pattern in the intestine and haematopoietic tissues of gilthead sea bream (GSB) (Sparus aurata) challenged with the myxosporean Enteromyxum leei. In a first trial (T1), GSB fed for 9 months either a fish oil (FO) diet or a blend of VO at 66% of replacement (66VO diet) was challenged by exposure to parasite-contaminated water effluent. All fish were periodically and non-lethally sampled to know their infection status. After 102 days of exposure, samples of intestine and head kidney were obtained for IgM expression and immunohistochemical detection (IHC). Additional samples of spleen were taken for IHC. Fish were categorized as control (C, not exposed), and early (E), or late (L) infected. The 66VO diet had no effect on the number of IgM-IRCs in any of the tissues or on IgM expression in C fish, whereas the infection with E. leei had a strong effect on the intestine. A combined time-diet effect was also observed, since the highest expression and IRCs values were registered in the posterior intestine (Pi) of E-66VO fish. A positive correlation was found between IgM expression and the presence of IgM-IRCs in the Pi. The effect of the time of infection was studied more in detail in a second trial (T2) in which samples of Pi were taken at 0, 24, 51, 91 and 133 days after exposure to the parasite. A significant increase of the IgM expression was detected only in parasitized fish, and very late after exposure. These results show that the duration of the exposure to the parasite is the most determinant factor for the observed intestinal IgM increased phenotype which gets magnified by the feeding of a high VO-based diet.

Macronutrient-induced differences in food intake relate with hepatic oxidative metabolism and hypothalamic regulatory neuropeptides in rainbow trout (Oncorhynchus mykiss).

This study examines how dietary macronutrient-induced changes in voluntary food intake (FI) relate to changes in markers of hepatic oxidative metabolism and in the expression of FI regulatory neuropeptides in a teleost model, the rainbow trout. Rainbow trout were fed for 6weeks with one of four iso-energetic diets (2×2 factorial design), containing either a high (HP, ~500 g·kg(-1) DM) or a low (LP, ~250 g·kg(-1) DM) protein level (PL) with, at each PL, fat (diets HP-F and LP-F) being substituted by an iso-energetic amount of gelatinized corn starch (diets HP-St and LP-St) as non-protein energy source (ES). Irrespective of the dietary PL, FI (g·kg(-0.8)·d(-1)) and digestible energy intake (DEI, kJ·kg(-0.8)·d(-1)) were significantly (P<0.05) reduced by the iso-energetic replacement of fat by starch as non-protein ES. Interestingly, trout fed these St-diets had higher gene expression of markers of hepatic oxidative phosphorylation (OxPhos), i.e., ubiquinol-cytochrome c reductase subunit 2 (UCR2) and cytochrome oxidase subunit 4 (COX4) and of aerobic oxidative capacity (CS, citrate synthase), which paralleled glucokinase (GK) transcription. This positive relation suggests that glucose phosphorylation and markers of mitochondrial OxPhos are linked at the hepatic level and possibly triggered the observed reduction in FI. Moreover, trout displaying the reduced FI had higher cocaine amphetamine regulator transcript (CART) mRNA in hypothalamus, whereas neuropeptide Y (NPY) mRNA did not follow the macronutrient-induced changes in FI. Further studies are needed to unravel the mechanisms by which diet-induced changes in hepatic metabolism inform central feeding centers involved in the regulation of FI in fish.

Link between lipid metabolism and voluntary food intake in rainbow trout fed coconut oil rich in medium-chain TAG.

We examined the long-term effect of feeding coconut oil (CO; rich in lauric acid, C12) on voluntary food intake and nutrient utilisation in rainbow trout (Oncorhynchus mykiss), with particular attention to the metabolic use (storage or oxidation) of ingested medium-chain TAG. Trout were fed for 15 weeks one of the four isoproteic diets containing fish oil (FO) or CO as fat source (FS), incorporated at 5% (low fat, LF) or 15% (high fat, HF). Fat level or FS did not modify food intake (g/kg(0·8) per d), despite higher intestinal cholecystokinin-T mRNA in trout fed the HF-FO diet. The HF diets relative to the LF ones induced higher growth and adiposity, whereas the replacements of FO by CO resulted in similar growth and adiposity. This, together with the substantial retention of C12 (57% of intake), suggests the relatively low oxidation of ingested C12. The down-regulation of carnitine palmitoyl-transferase-1 (CPT-1) confirms the minor dependency of medium-chain fatty acids (MCFA) on CPT-1 to enter the mitochondria. However, MCFA did not up-regulate mitochondrial oxidation evaluated using hepatic hydroxyacyl-CoA dehydrogenase as a marker, in line with their high retention in body lipids. At a low lipid level, MCFA increased mRNA levels of fatty acid synthase, elongase and stearoyl-CoA desaturase in liver, showing the hepatic activation of fatty acid synthesis pathways by MCFA, reflected by increased 16 : 0, 18 : 0, 16 : 1, 18 : 1 body levels. The high capacity of trout to incorporate and transform C12, rather than to readily oxidise C12, contrasts with data in mammals and may explain the absence of a satiating effect of CO in rainbow trout.

Molecular characterization and expression analysis of six peroxiredoxin paralogous genes in gilthead sea bream (Sparus aurata): insights from fish exposed to dietary, pathogen and confinement stressors.

The aim of this work was to underline the physiological role of the antioxidant peroxiredoxin (PRDX) family in gilthead sea bream (Sparus aurata L.), a perciform fish extensively cultured in the Mediterranean area. First, extensive BLAST searches were done on the gilthead sea bream cDNA database of the AQUAMAX European Project (www.sigenae.org/iats), and six contigs were unequivocally identified as PRDX1-6 after sequence completion by RT-PCR. The phylogenetic analysis evidenced three major clades corresponding to PRDX1-4 (true 2-Cyst PRDX subclass), PRDX5 (atypical 2-Cys PRDX subclass) and PRDX6 (1-Cys PRDX subclass) that reflected the present hierarchy of vertebrates. However, the PRDX2 branch of modern fish including gilthead sea bream was related to the monophyletic PRDX1 node rather than to PRDX2 cluster of mammals and primitive fish, which probably denotes the acquisition of novel functions through vertebrate evolution. Transcriptional studies by means of quantitative real-time PCR evidenced a ubiquitous PRDX gene expression that was tissue specific for each PRDX isoform. In a second set of transcriptional studies, liver and head kidney were chosen as target tissues in fish challenged with i) the intestinal parasite Enteromyxum leei, ii) a plant oil (VO) diet with deficiencies in essential fatty acids and iii) prolonged exposure to high-rearing densities. These studies showed that PRDX genes were highly and mostly constitutively expressed in the liver and were not affected by dietary intervention or high density. In contrast, head kidney was highly sensitive to the different experimental challenges: significantly lower values were found for PRDX5 in the three trials, for PRDX6 in parasitized and high density fish and for PRDX1 in parasitized and VO fish. PRDX2, 3 and 5 were decreased only in VO, high density and parasitized animals, respectively. These findings would highlight the role of PRDXs as integrative and highly predictive biomarkers of health and welfare in fish and gilthead sea bream in particular.

The nutritional background of the host alters the disease course in a fish-myxosporean system.

The aim of the present work was to determine if a practical plant protein-based diet containing vegetable oils (VO) as the major lipid source could alter the disease course when challenged with the myxosporean Enteromyxum leei, a wide-spread parasite in the Mediterranean basin causing heavy economic losses. Gilthead sea bream (Sparus aurata) fed for 9 months either a fish oil (FO) diet or a blend of VOs at 66% of replacement (66VO diet) were challenged by exposure to parasite-contaminated water effluent. All fish were periodically and non-lethally sampled to obtain biometrical data and to know their infection status. After 102 days of exposure, fish were euthanized and haematological, biometrical, histological, immunological, glutathione and anti-oxidant data were obtained from tissue, blood and serum samples. Anorexia appeared in both exposed groups, but feed intake reduction was higher in 66VO fish. The signs of disease (lower growth, condition factor, specific growth rate, haematocrit) as well as the disease course were worse in fish from 66VO group, with a higher prevalence and intensity of infection, a higher percentage of fish harbouring the parasite in the entire intestinal tract, and a faster establishment of the parasite. Parasite intensity of infection was negatively correlated with growth parameters and haematocrit in both groups, and with complement, lysozyme and hepatic total glutathione in 66VO fish.

Hepatic protein kinase B (Akt)-target of rapamycin (TOR)-signalling pathways and intermediary metabolism in rainbow trout (Oncorhynchus mykiss) are not significantly affected by feeding plant-based diets.

The aim of the present study was to analyse the effects of partial or total replacement of fish meal (FM) and fish oil (FO) by a mixture of plant protein (PP) and a mixture of vegetable oils (VO) on the hepatic insulin-nutrient-signalling pathway and intermediary metabolism-related gene expression in rainbow trout (Oncorhynchus mykiss). Triplicate groups of fish were fed four practical diets containing graded levels of replacement of FM and FO by PP and VO for 12 weeks: diet 0/0 (100 % FM, 100 % FO); diet 50/50 (50 % FM and 50 % PP, 50 % FO and 50 % VO); diet 50/100 (50 % FM and 50 % PP, 100 % VO); diet 100/100 (100 % PP, 100 % VO). Samplings were performed on trout starved for 5 d then refed with their allocated diet. In contrast to partial substitution (diet 50/50), total substitution of FM and FO (diet 100/100) led to significantly lower growth compared with diet 0/0. The insulin-nutrient-signalling pathway (protein kinase B (Akt), target of rapamycin (TOR), S6 protein kinase 1 (S6K1) and S6) was characterised in trout liver and found to be activated by refeeding. However, changes in diet compositions did not differentially affect the Akt-TOR-signalling pathway. Moreover, expression of genes encoding fructose-1,6-biphosphatase, mitochondrial phosphoenolpyruvate carboxykinase, glucokinase, pyruvate kinase and carnitine palmitoyl transferase 1 were not affected by refeeding or by dietary changes. Refeeding down- and up-regulated the expression of gluconeogenic glucose-6-phosphatase isoform 1 and lipogenic fatty acid synthase genes, respectively. Expression of both genes was also increased with partial replacement of FM and total replacement of FO (diet 50/100). These findings indicate that plant-based diets barely affect glucose and lipid metabolism in trout.