Gut physiology of rainbow trout (Oncorhynchus mykiss) is influenced more by short-term fasting followed by refeeding than by feeding fishmeal-free diets.

Supplementing a fishmeal-free diet with yeast extract improves rainbow trout (Oncorhynchus mykiss) growth performance and modulates the hepatic and intestinal transcriptomic response. These effects are often observed in the long term but are not well documented after short periods of fasting. Fasting for a few days is a common practice in fish farming, especially before handling the fish, such as for short sorting, tank transfers, and vaccinations. In the present study, rainbow trout were subjected to a 4-day fast and then refed, for 8 days, a conventional diet containing fishmeal (control diet) or alternative diets composed of terrestrial animal by-products supplemented or not with a yeast extract. During the refeeding period alone, most of the parameters considered did not differ significantly in response to the different feeds. Only the expression of claudin-15 was upregulated in fish fed the yeast-supplemented diet compared to the control diet. Conversely, fasting followed by refeeding significantly influenced most of the parameters analyzed. In the proximal intestine, the surface area of villi significantly increased, and the density of goblet cell tended to decrease during refeeding. Although no distinct plasma immune response or major signs of gut inflammation were observed, some genes involved in the structure, complement pathway, antiviral functions, coagulation, and endoplasmic reticulum stress response of the liver and intestine were significantly regulated by refeeding after fasting. These results indicate that short-term fasting, as commonly practiced in fish farming, significantly alters the physiology of the liver and intestine regardless of the composition of the diet.

Early starvation in European seabass (Dicentrarchus labrax) larvae has no drastic effect on hepatic intermediary metabolism in juveniles.

The present study aims to investigate nutritional programming through early starvation in the European seabass (Dicentrarchus labrax). European seabass larvae were fasted at three different developmental periods for three durations from 60 to 65 dph (F1), 81 to 87 dph (F2), and 123 to 133 dph (F3). Immediate effects were investigated by studying gene expression of npy (neuropeptide Y) and avt (Arginine vasotocin) in the head, while potential long-term effects (i.e., programming) were evaluated on intermediary metabolism later in life (in juveniles). Our findings indicate a direct effect regarding gene expression in the head only for F1, with higher avt mRNA level in fasted larved compared to controls. The early starvation periods had no long-term effect on growth performance (body weight and body length). Regarding intermediary metabolism, we analyzed related key plasma metabolites which reflect the intermediary metabolism: no differences for glucose, triglycerides, and free fatty acids in the plasma were observed in juveniles irrespective of the three early starvation stimuli. As programming is mainly linked to molecular mechanisms, we then studied hepatic mRNA levels for 23 key actors of glucose, lipid, amino acid, and energy metabolism. For many of the metabolic genes, there was no impact of early starvation in juveniles, except for three genes involved in glucose metabolism (glut2-glucose transporter and pk-pyruvate kinase) and lipid metabolism (acly-ATP citrate lyase) which were higher in F2 compared to control. Together, these results highlight that starvation between 81 to 87 dph may have more long-term impact, suggesting the existence of a developmental window for programming by starvation. In conclusion, European seabass appeared to be resilient to early starvation during larvae stages without drastic impacts on intermediary metabolism later in life.

Exploring the effects of dietary inulin in rainbow trout fed a high-starch, 100% plant-based diet.

BACKGROUND: High dietary carbohydrates can spare protein in rainbow trout (Oncorhynchus mykiss) but may affect growth and health. Inulin, a prebiotic, could have nutritional and metabolic effects, along with anti-inflammatory properties in teleosts, improving growth and welfare. We tested this hypothesis in rainbow trout by feeding them a 100% plant-based diet, which is a viable alternative to fishmeal and fish oil in aquaculture feeds. In a two-factor design, we examined the impact of inulin (2%) as well as the variation in the carbohydrates (CHO)/plant protein ratio on rainbow trout. We assessed the influence of these factors on zootechnical parameters, plasma metabolites, gut microbiota, production of short-chain fatty acids and lactic acid, as well as the expression of free-fatty acid receptor genes in the mid-intestine, intermediary liver metabolism, and immune markers in a 12-week feeding trial. RESULTS: The use of 2% inulin did not significantly change the fish intestinal microbiota, but interestingly, the high CHO/protein ratio group showed a change in intestinal microbiota and in particular the beta diversity, with 21 bacterial genera affected, including Ralstonia, Bacillus, and 11 lactic-acid producing bacteria. There were higher levels of butyric, and valeric acid in groups fed with high CHO/protein diet but not with inulin. The high CHO/protein group showed a decrease in the expression of pro-inflammatory cytokines (il1b, il8, and tnfa) in liver and a lower expression of the genes coding for tight-junction proteins in mid-intestine (tjp1a and tjp3). However, the 2% inulin did not modify the expression of plasma immune markers. Finally, inulin induced a negative effect on rainbow trout growth performance irrespective of the dietary carbohydrates. CONCLUSIONS: With a 100% plant-based diet, inclusion of high levels of carbohydrates could be a promising way for fish nutrition in aquaculture through a protein sparing effect whereas the supplementation of 2% inulin does not appear to improve the use of CHO when combined with a 100% plant-based diet.

Long-term regulation of fat sensing in rainbow trout (Oncorhynchus mykiss) fed a vegetable diet from the first feeding: focus on free fatty acid receptors and their signalling.

Taste plays a fundamental role in an animal's ability to detect nutrients and transmits key dietary information to the brain, which is crucial for its growth and survival. Providing alternative terrestrial ingredients early in feeding influences the growth of rainbow trout (RT, Oncorhynchus mykiss). Thus, the present study aimed to assess the influence, via long-term feeding (from the first feeding to 8 months), of alternative plant ingredients (V diet for vegetable diet v. C diet for a control diet) in RT on the mechanism of fat sensing at the gustatory level. After the feeding trial, we studied the pathways of the fat-sensing mechanism in tongue tissue and the integrated response in the brain. To this end, we analysed the expression pattern of free fatty acid receptors (ffar) 1 and 2, markers of calcium-signalling pathways (phospholipase Cß, Orai, Stim or Serca), the serotonin level (a key neurotransmitter in taste buds) and the expression pattern of appetite-regulating neuropeptides in the hypothalamus (central area of appetite regulation). The results revealed that the V diet modified the expression pattern of ffar1 and paralogs of ffar2 genes in tongue tissue, along with differential regulation of calcium-signalling pathways and a defect in serotonin level and brain turnover, without influencing neuropeptide expression. This study is the first to support that changes in feeding behaviour of RT fed a V diet could be due to the difference in nutrient sensing and a decrease in hedonic sensation. We revealed that RT have similar fat-detection mechanisms as mammals.

New alternative ingredients and genetic selection are the next game changers in rainbow trout nutrition: a metabolomics appraisal.

The formulation of sustainable fish feeds based on plant ingredients supplemented by alternative ingredients to plant (insect, micro-algae, yeast) and genetic selection of fish for plant-based diets were tested on rainbow trout in two separate experiments. Plant-based diets and corresponding diets supplemented with an ingredient mix: insect, micro-algae and yeast in Experiment A, and insect and yeast in Experiment B were compared to commercial-like diets. In experiment A, the mix-supplemented diet was successful in compensating the altered growth performance of fish fed their respective plant-based diet compared to those fed the commercial diet, by restoring feed conversion. In experiment B, the selected line demonstrated improved growth performances of fish fed mix-supplemented and plant-based diets compared to the non-selected line. Metabolomics demonstrated a plasma compositional stability in fish fed mix-supplemented and basal plant-based diets comprising an amino acid accumulation and a glucose depletion, compared to those fed commercial diets. The selected line fed mix-supplemented and commercial diets showed changes in inositol, ethanol and methanol compared to the non-selected line, suggesting an involvement of microbiota. Changes in plasma glycine-betaine content in fish fed the mix-supplemented diet suggest the ability of the selected line to adapt to alternative ingredients.

Tissue origin of circulating microRNAs and their response to nutritional and environmental stress in rainbow trout (Oncorhynchus mykiss).

Stresses associated with changes in diet or environmental disturbances are common situations that fish encounter during their lifetime. The stability and ease of measuring microRNAs (miRNAs) present in biological fluids make these molecules particularly interesting biomarkers for non-lethal assessment of stress in animals. Rainbow trout were exposed for four weeks to abiotic stress (moderate hypoxia) and/or nutritional stress (a high-carbohydrate/low-protein diet). Blood plasma and epidermal mucus were sampled at the end of the experiment, and miRNAs were assessed using small RNA sequencing. We identified four miRNAs (miR-122-5p, miR-184-3p, miR-192-5p and miR-194a-5p) and three miRNAs (miR-210-3p, miR-153a-3p and miR-218c-5p) that accumulated in response to stress in blood plasma and epidermal mucus, respectively. In particular, the abundance of miR-210-3p, a hypoxamiR in mammals, increased strongly in the epidermal mucus of rainbow trout subjected to moderate hypoxia, and can thus be considered a relevant biomarker of hypoxic stress in trout. We explored the contribution of 22 tissues/organs to the abundance of circulating miRNAs (c-miRNAs) in blood plasma and epidermal mucus influenced by the treatments. Some miRNAs were tissue-specific, while others were distributed among several tissues. Some c-miRNAs (e.g., miR-210-3p, miR184-3p) showed similar variations in both tissues and fluids, while others showed an inverse trend (e.g., miR-122-5p) or no apparent relationship (e.g. miR-192-5p, miR-194a-5p. Overall, these results demonstrate that c-miRNAs can be used as non-lethal biomarkers to study stress in fish. In particular, the upregulation of miR-210-3p in epidermal mucus induced by hypoxia demonstrates the potential of using epidermal mucus as a matrix for identifying non-invasive biomarkers of stress. This study provides information about the tissue sources of c-miRNAs and highlights the potential difficulty in relating variations in miRNA abundance in biological fluids to that in tissues.

Physical Enrichment Triggers Brain Plasticity and Influences Blood Plasma Circulating miRNA in Rainbow Trout (Oncorhynchus mykiss).

Physical enrichment is known to improve living conditions of fish held in farming systems and has been shown to promote behavioral plasticity in captive fish. However, the brain's regulatory-mechanism systems underlying its behavioral effects remain poorly studied. The present study investigated the impact of a three-month exposure to an enriched environment (EE vs. barren environment, BE) on the modulation of brain function in rainbow trout (Oncorhynchus mykiss) juveniles. Using high-throughput RT-qPCR, we assessed mRNA genes related to brain function in several areas of the trout brain. These included markers of cerebral activity and plasticity, neurogenesis, synaptogenesis, or selected neurotransmitters pathways (dopamine, glutamate, GABA, and serotonin). Overall, the fish from EE displayed a series of differentially expressed genes (neurotrophic, neurogenesis, and synaptogenesis markers) essentially localized in the telencephalon, which could underpin the beneficial effects of complexifying the environment on fish brain plasticity. In addition, EE significantly affected blood plasma c-miRNA signatures, as revealed by the upregulation of four c-miRNAs (miR-200b/c-3p, miR-203a-3p, miR-205-1a-5p, miR-218a-5p) in fish blood plasma after 185 days of EE exposure. Overall, we concluded that complexifying the environment through the addition of physical structures that stimulate and encourage fish to explore promotes the trout's brain function in farming conditions.

Effect of micro-algae Schizochytrium sp. supplementation in plant diet on reproduction of female rainbow trout (Oncorhynchus mykiss): maternal programming impact of progeny.

BACKGROUND: The broodstock diet, and in particular the lipid and fatty acid composition of the diet, is known to play a key role in reproductive efficiency and survival of the progeny in fish. A major problem when replacing both fish meal and fish oil by plant sources is the lack of n-3 long chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). To address this problem, we studied the effect of the plant-based diet supplemented with Schizochytrium sp. microalgae, source of DHA, compared to a conventional commercial diet rich in fish meal and fish oil on reproductive performance and egg quality and the consequences on progeny, in female rainbow trout broodstock. RESULTS: The results demonstrated that DHA-rich microalgae supplementation in a plant-based diet allowed for the maintenance of reproductive performance and egg quality comparable to a conventional commercial feed rich in fish meal and fish oil and led to an increased significant fry survival after resorption. Moreover, when females were fed a plant-based diet supplemented with micro-algae, the 4-month-old progenies showed a significant higher growth when they were challenged with a similar diet as broodstock during 1 month. We provide evidence for metabolic programming in which the maternal dietary induced significant protracted effects on lipid metabolism of progeny. CONCLUSIONS: The present study demonstrates that supplementation of a plant-based diet with DHA-rich microalgae can be an effective alternative to fish meal and fish oil in rainbow trout broodstock aquafeed.

Circulating miRNA repertoire as a biomarker of metabolic and reproductive states in rainbow trout.

BACKGROUND: Circulating miRNAs (c-miRNAs) are found in most, if not all, biological fluids and are becoming well-established non-invasive biomarkers of many human pathologies. However, their features in non-pathological contexts and whether their expression profiles reflect normal life history events have received little attention, especially in non-mammalian species. The aim of the present study was to investigate the potential of c-miRNAs to serve as biomarkers of reproductive and metabolic states in fish. RESULTS: The blood plasma was sampled throughout the reproductive cycle of female rainbow trout subjected to two different feeding regimes that triggered contrasting metabolic states. In addition, ovarian fluid was sampled at ovulation, and all samples were subjected to small RNA-seq analysis, leading to the establishment of a comprehensive miRNA repertoire (i.e., miRNAome) and enabling subsequent comparative analyses to a panel of RNA-seq libraries from a wide variety of tissues and organs. We showed that biological fluid miRNAomes are complex and encompass a high proportion of the overall rainbow trout miRNAome. While sharing a high proportion of common miRNAs, the blood plasma and ovarian fluid miRNAomes exhibited strong fluid-specific signatures. We further revealed that the blood plasma miRNAome significantly changed depending on metabolic and reproductive states. We subsequently identified three evolutionarily conserved muscle-specific miRNAs or myomiRs (miR-1-1/2-3p, miR-133a-1/2-3p, and miR-206-3p) that accumulated in the blood plasma in response to high feeding rates, making these myomiRs strong candidate biomarkers of active myogenesis. We also identified miR-202-5p as a candidate biomarker for reproductive success that could be used to predict ovulation and/or egg quality. CONCLUSIONS: Together, these promising results reveal the high potential of c-miRNAs, including evolutionarily conserved myomiRs, as physiologically relevant biomarker candidates and pave the way for the use of c-miRNAs for non-invasive phenotyping in various fish species.

Why Do Some Rainbow Trout Genotypes Grow Better With a Complete Plant-Based Diet? Transcriptomic and Physiological Analyses on Three Isogenic Lines.

Within the context of a growing aquaculture production coupled with a plateau of the production in the main components of aquafeeds (fish oil and fishmeal), recent studies have typically focused on replacing these feedstuffs with terrestrial plant ingredients for cultured carnivorous aquatic species, such as rainbow trout (Oncorhynchus mykiss). Substitution rates without adverse effects have, however, reached their limit. One potential way forward would be to take advantage of the genetic variability that exists in the salmonid population. However, to date, little is known about the underlying molecular mechanisms responsible for this genetic variability. The aim of the present research was to understand why some genotypes are better able to utilize plant-based diets devoid of marine resources. In this regard, three isogenic lines of rainbow trout (R23h, AB1h, and A22h), with similar growth when fed marine resources-based diets and which differ greatly in their responses to a plant-based diet, were fed with either a complete plant-based diet (V diet) or a marine resources-based diet (M diet) since first-feeding. Fish traits and the hepatic transcriptome of these three genotypes were compared after 5 months of feeding. First, differences in the ability to grow with the V diet observed amongst genotypes was not due to higher feed intake, but instead due to differences in feed efficiency. The comparison of transcriptome profiles revealed 575 (R23h vs. AB1h), 1,770 (R23h vs. A22h), and 2,973 (AB1h vs. A22h) probes differentially expressed amongst the three genotypes when fed the V diet. Interestingly, R23h and AB1h fish, which were the least affected by the V diet, exhibited the highest growth. These results demonstrate that these fish were able to maintain a high level of energy production and protein synthesis. Moreover, these genotypes were also able to activate pathways linked to lipid and cholesterol metabolisms, such as the biosynthesis of long-chain polyunsaturated fatty acids. Finally, as previously, immunity seems to also play an important role in the ability of fish to use the V diet, and further studies are needed to understand the mechanisms by which immunity interacts with growth.

Sustainable plant-based diets promote rainbow trout gut microbiota richness and do not alter resistance to bacterial infection.

BACKGROUND: Farmed fish food with reduced fish-derived products are gaining growing interest due to the ecological impact of fish-derived protein utilization and the necessity to increase aquaculture sustainability. Although different terrestrial plant proteins could replace fishmeal proteins, their use is associated with adverse effects. Here, we investigated how diets composed of terrestrial vegetal sources supplemented with proteins originating from insect, yeast or terrestrial animal by-products affect rainbow trout (Onchorynchus mykiss) gut microbiota composition, growth performance and resistance to bacterial infection by the fish pathogen Flavobacterium psychrophilum responsible for frequent outbreaks in aquaculture settings. RESULTS: We showed that the tested regimes significantly increased gut bacterial richness compared to full vegetal or commercial-like diets, and that vegetal diet supplemented with insect and yeast proteins improves growth performance compared to full vegetal diet without altering rainbow trout susceptibility to F. psychrophilum infection. CONCLUSION: Our results demonstrate that the use of insect and yeast protein complements to vegetal fish feeds maintain microbiota functions, growth performance and fish health, therefore identifying promising alternative diets to improve aquaculture's sustainability.

Proton-NMR Metabolomics of Rainbow Trout Fed a Plant-Based Diet Supplemented with Graded Levels of a Protein-Rich Yeast Fraction Reveal Several Metabolic Processes Involved in Growth.

BACKGROUND: Plant raw materials are commonly used in aquafeeds, as marine resources are unsustainable. However, full plant-based diets lead to poorer fish growth performance. OBJECTIVE: We aimed to understand the metabolic effects of a yeast fraction as a protein supplement in a plant-based diet and to integrate such effects with phenotypic traits as a new approach to assess the interest of this raw material. METHODS: Juvenile (49 g) rainbow trout (Oncorhynchus mykiss) were fed graded levels of a yeast protein-rich fraction (5% YST05, 10% YST10, 15% YST15) in a plant-based diet (PB) for 84 d. Final body weight, feed conversion ratio, and hepatosomatic and viscerosomatic indexes were measured. Plasma, liver, and muscle 1H-NMR fingerprints were analyzed with principal component analyses, and their metabolite patterns were clustered according to the yeast level to identify concomitant metabolic effects. A regression modeling approach was used to predict tissue metabolite changes from plasma fingerprints. RESULTS: In tissues, the patterns of metabolite changes followed either linear trends with the gradual inclusion of a yeast fraction (2 patterns out of 6 in muscle, 1 in liver) or quadratic trends (4 patterns in muscle, 5 in liver). Muscle aspartate and glucose (395 and 138% maximum increase in relative content compared with PB, respectively) revealing modification in energy metabolism, as well as modification of liver betaine (163% maximum increase) and muscle histidine (57% maximum decrease) related functions, indicates that the yeast fraction could improve growth in several ways. The highest correlation between measured and predicted metabolite intensities in a tissue based on plasma fingerprints was observed for betaine in liver (r = 0.80). CONCLUSIONS: These findings herald a new approach to assess the plurality of metabolic effects induced by diets and establish the optimal level of raw materials. They open the way for using plasma as a noninvasive matrix in trout nutrition studies.

Impact on cerebral function in rainbow trout fed with plant based omega-3 long chain polyunsaturated fatty acids enriched with DHA and EPA.

Characterization and modulation of cerebral function by ω-3 long chain polyunsaturated fatty acids (ω-3 LC-PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) enrichment in plant based-diet were studied in rainbow trout (Oncorhynchus mikyss). We hypothesized that ω-3 LC-PUFAs are involved in the regulation of cerebral function in fish. During nine weeks, we examined the growth performance of rainbow trout for three experimental plant based-diets containing distinct levels of EPA and DHA. Using RT-qPCR, we assessed mRNA genes related to feeding behavior regulated by the central nervous system of humans, rodents and fish. These include markers of neuropeptides, indicators of cellular specification, animal stress, oxidant status, cytokines and genes regulating animal behaviour. ω-3 LC-PUFAs enrichment decreased daily food intake and induced a simultaneous mRNA expression increase in orexigenic transcript npy peptide and a decrease in anorexigen transcript pomcA peptide in the hypothalamus. Overall transcript genes related to proinflammatory cytokines, inflammation, antioxidant status, cortisol pathway, serotoninergic pathways and dopaminergic pathways were down-regulated in the juveniles fed the high ω-3 LC-PUFAs diet. However, the mRNA expression of transcripts related to cell specification were down regulated, namely tmem119 markers of microglial cell in forebrain and midbrain, gfap markers of astrocyte in the midbrain, and rbfox3 markers of neurons in the midbrain and hindbrain in juveniles fed high ω-3 experimental diet. In conclusion, this study revealed that a diet rich in ω-3 LC-PUFAs affected a relatively high proportion of the brain function in juvenile rainbow trout through mechanisms comparable to those characterized previously in mammals.

Integrative Metabolomics for Assessing the Effect of Insect (Hermetia illucens) Protein Extract on Rainbow Trout Metabolism.

Nutrition of high trophic species in aquaculture is faced with the development of sustainable plant-based diets. Insects seem particularly promising for supplementing plant-based diets. However, the complex effect of whole insect meal on fish metabolism is not well understood, and even less is known about insect meal extracts. The purpose of this work was to decipher the metabolic utilization of a plant-based diet supplemented with the gradual addition of an insect protein extract (insect hydrolysate at 0%, 5%, 10% and 15%). 1H-NMR profiling was used to assess metabolites in experimental diets and in fish plasma, liver and muscle. A significant dose-dependent increase in growth and feed efficiency with increasing insect extract amounts was observed. The incremental incorporation of the insect extract in diet had a significant and progressive impact on the profile of dietary soluble compounds and trout metabolome. The metabolites modulated by dietary insect extracts in plasma and tissues were involved in protein and energy metabolism. This was associated with the efficient metabolic use of dietary free amino acids toward protein synthesis through the concomitant supply of balanced free amino acids and energy substrates in muscle. The findings provide new insights into how the dietary food metabolome affects fish metabolism.

Cholesterol metabolism regulation mediated by SREBP-2, LXRα and miR-33a in rainbow trout (Oncorhynchus mykiss) both in vivo and in vitro.

Cholesterol metabolism is greatly affected in fish fed plant-based diet. The regulation of cholesterol metabolism is mediated by both transcriptional factors such as sterol regulatory element-binding proteins (SREBPs) and liver X receptors (LXRs), and posttranscriptional factors including miRNAs. In mammals, SREBP-2 and LXRα are involved in the transcriptional regulation of cholesterol synthesis and elimination, respectively. In mammals, miR-33a is reported to directly target genes involved in cholesterol catabolism. The present study aims to investigate the regulation of cholesterol metabolism by SREBP-2 and LXRα and miR-33a in rainbow trout using in vivo and in vitro approaches. In vivo, juvenile rainbow trout of ~72 g initial body weight were fed a total plant-based diet (V) or a marine diet (M) containing fishmeal and fish oil. In vitro, primary cell culture hepatocytes were stimulated by graded concentrations of 25-hydroxycholesterol (25-HC). The hepatic expression of cholesterol synthetic genes, srebp-2 and miR-33a as well as miR-33a level in plasma were increased in fish fed the plant-based diet, reversely, their expression in hepatocytes were inhibited with the increasing 25-HC in vitro. However, lxrα was not affected neither in vivo nor in vitro. Our results suggest that SREBP-2 and miR-33a synergistically enhance the expression of cholesterol synthetic genes but do not support the involvement of LXRα in the regulation of cholesterol elimination. As plasma level of miR-33a appears as potential indicator of cholesterol synthetic capacities, this study also highlights circulating miRNAs as promising noninvasive biomarker in aquaculture.

Kinetic study of the expression of genes related to hepatic steatosis, glucose and lipid metabolism, and cellular stress during overfeeding in mule ducks.

Induced by overfeeding, hepatic steatosis is a process exploited for the "foie gras" production in mule ducks. To better understand the mechanisms underlying its development, the physiological responses of mule ducks overfed with corn for a duration of 11 days were analyzed. A kinetic analysis of glucose and lipid metabolism and cell protection mechanisms was performed on 96 male mule ducks during overfeeding with three sampling times (after the 4th, the 12th, and the 22nd meal). Gene expression and protein analysis realized on the liver, muscle, and abdominal fat showed an activation of a cholesterol biosynthetic pathway during the complete overfeeding period mainly in livers with significant correlations between its weight and its cholesterolemia (r = 0.88; P < 0.0001) and between the liver weight and the hmgcr and soat1 expression (r = 0.4, P < 0.0001 and r = 0.67; P < 0.0001, respectively). Results also revealed an activation of insulin and amino acid cells signaling a pathway suggesting that ducks boost insulin sensitivity to raise glucose uptake and use via glycolysis and lipogenesis. Cellular stress analysis revealed an upregulation of key autophagy-related gene expression atg8 and sqstm1(P < 0.0001) during the complete overfeeding period, mainly in the liver, in contrast to an induction of cyp2e1(P < 0.0001), suggesting that autophagy could be suppressed during steatosis development. This study has highlighted different mechanisms enabling mule ducks to efficiently handle the starch overload by keeping its liver in a nonpathological state. Moreover, it has revealed potential biomarker candidates of hepatic steatosis as plasma cholesterol for the liver weight.

Higher glycolytic capacities in muscle of carnivorous rainbow trout juveniles after high dietary carbohydrate stimulus at first feeding.

BACKGROUND: Rainbow trout is a "glucose-intolerant" carnivorous species. Using the metabolic programming strategy, we used early nutritional stimuli in order to modify carbohydrate utilization in trout juveniles. METHOD: Fish were fed two diets during the first feeding, namely HP (no carbohydrate / high protein) diet and LP (high carbohydrate / low protein) diet. HP diet was used as the control diet and LP diet as an early stimulus diet. We also used another early stimulus with fish fed HP diet every other day during the first feeding (HP restriction feeding - HPR). After the first-feeding stage (4 weeks), all fish were subsequently subjected to a growth trial with a commercial diet followed by a challenge test with the LP diet (11 weeks). At the end of the first feeding stimulus and of the challenge test, we investigated growth performance, glucose metabolism-related parameters and global DNA CmCGG methylation in trout. RESULTS: LP and HPR dietary stimuli have been a success as shown by the direct modifications of growth performance and mRNA levels for glucose metabolism-related genes at the end of first feeding compared to alevins fed the HP diet. At the end of the challenge trial, no variation in growth performance and hepatic metabolism of LP-history and HPR-history in trout juveniles were observed. However, in muscle of trout juvenile subjected to LP diet at the first feeding, we found an up-regulation of mRNA levels of some glucose metabolism (glucose transport and glycolysis)-related genes and an increase of activities of important glycolysis-related enzymes (hexokinase, phosphofructokinase and pyruvate kinase). These observations are associated with a decrease in the content of glycogen compared to fish fed the HP diet. Moreover, global CmCGG DNA methylation in the muscle of fish with LP history was significantly lower than those fed the HP diet. CONCLUSION: Dietary LP stimulus at first feeding could permanently modify glucose metabolism and global CmCGG DNA methylation level in muscle of trout juveniles, showing that the first feeding stage is efficient for programming the glucose metabolism in fish.

Circulating miRNA measurements are reflective of cholesterol-based changes in rainbow trout (Oncorhynchus mykiss).

MicroRNAs (miRNAs) are a class of small non-coding RNAs which are known to posttranscriptionally regulate the expression of most genes in both animals and plants. Meanwhile, studies have shown that numbers of miRNAs are present in body fluids including the plasma. Despite the mode of action of these circulating miRNAs still remains unknown, they have been found to be promising biomarkers for disease diagnosis, prognosis and response to treatment. In order to evaluate the potential of miRNAs as non-invasive biomarkers in aquaculture, a time-course experiment was implemented to investigate the postprandial regulation of miRNAs levels in liver and plasma as well as the hepatic expression of genes involved in cholesterol metabolism. We showed that miR-1, miR-33a, miR-122, miR-128 and miR-223 were expressed in the liver of rainbow trout and present at detectable level in the plasma. We also demonstrated that hepatic expression of miR-1, miR-122 and miR-128 were regulated by feed intake and reached their highest levels 12 hours after the meal. Interestingly, we observed that circulating levels of miR-128 and miR-223 are subjected to postprandial regulations similar to that observed in their hepatic counterparts. Statistical correlations were observed between liver and plasma for miR-128 and miR-223 and between hepatic and circulating miR-122, miR-128 and miR-223 and expression of genes related to cholesterol synthesis and efflux or glucose phosphorylation. These results demonstrated that circulating miR-122, miR-128 and miR-223 are potential biomarkers of cholesterol metabolism in rainbow trout.

Regulation by Dietary Carbohydrates of Intermediary Metabolism in Liver and Muscle of Two Isogenic Lines of Rainbow Trout.

Rainbow trout (Oncorhynchus mykiss) is recognized as a typical "glucose-intolerant" fish, and the limits of dietary carbohydrate utilization have been investigated for many years. In this study, the objective was to test the molecular effects of dietary carbohydrates on intermediary metabolism in two major metabolic tissues, liver and muscle. Another objective was also to study if the response to carbohydrate intake depended on the genetic background. We fed two isogenic lines of rainbow trout (named A22h and N38h) with high carbohydrate diet (carbohydrate, 22.9%) or low carbohydrate diet (carbohydrate, 3.6%) for 12 weeks. Carbohydrates were associated with higher feed utilization owned by the well-known protein-sparing effect, with better fish growth performance. However, atypical regulation of glycolysis and gluconeogenesis in liver and absence of hk and glut4 induction in muscle, were also observed. Regarding the effects of carbohydrates on other metabolism, we observed an increased, at a molecular level, of hepatic cholesterol biosynthesis, fatty acid oxidation and mitochondrial energy metabolism. Genetic variability (revealed by the differences between the two isogenic lines) was observed for some metabolic genes especially for those involved in the EPA and DHA biosynthetic capacity. Finally, our study demonstrates that dietary carbohydrate not only affect glucose metabolism but also strongly impact the lipid and energy metabolism in liver and muscle of trout.

Composition of Intestinal Microbiota in Two Lines of Rainbow Trout (Oncorhynchus Mykiss) Divergently Selected for Muscle Fat Content.

BACKGROUND: Recently, studies suggest that gut microbiota contributes to the development of obesity in mammals. In rainbow trout, little is known about the role of intestinal microbiota in host physiology. OBJECTIVE: The aim of this study was to investigate the link between intestinal microbiota and adiposity, by high-throughput 16S RNA gene based illumina Miseq sequencing in two rainbow trout lines divergently selected for muscle lipid content. Fish from these two lines of rainbow trout are known to have a differing lipid metabolism. METHODS: Samples from the two lines (L for lean and F for fat) were collected from Midgut (M) and Hindgut (H) in juvenile fish (18 months) to compare intestinal microbiota diversity. RESULTS: Whatever the lines and intestinal localisation, Proteobacteria, Firmicutes and Actinobacteria are the dominant phyla in the bacterial community of rainbow trout (at least 97%). The results indicate that richness and diversity indexes as well as bacterial composition are comparable between all groups even though 6 specific OTUs were identified in the intestinal microbiota of fish from the fat line and 2 OTUs were specific to the microbiota of fish from the lean line. Our work contributes to a better understanding in microbial diversity in intestinal microbiota of rainbow trout. CONCLUSION: Altogether, our study indicates that no major modification of the intestinal microbiota is induced by selection for muscle lipid content and associated metabolic changes. Finally, we identified members of core microbiota in rainbow trout.