Adverse effects of excessive dietary arachidonic acid on survival, PUFA-derived enzymatic and non-enzymatic oxylipins, stress response in rainbow trout fry.

Arachidonic acid (C20: 4n-6, AA) plays a fundamental role in fish physiology, influencing growth, survival and stress resistance. However, imbalances in dietary AA can have detrimental effects on fish health and performance. Optimal AA requirements for rainbow trout have not been established. This study aimed to elucidate the effects of varying dietary AA levels on survival, growth, long-chain polyunsaturated fatty acid (LC-PUFA) biosynthetic capacity, oxylipin profiles, lipid peroxidation, and stress resistance of rainbow trout fry. Over a period of eight weeks, 4000 female rainbow trout fry at the resorptive stage (0.12 g) from their first feeding were fed diets with varying levels of AA (0.6%, 1.1% or 2.5% of total fatty acids) while survival and growth metrics were closely monitored. The dietary trial was followed by an acute confinement stress test. Notably, while the fatty acid profiles of the fish reflected dietary intake, those fed an AA-0.6% diet showed increased expression of elongase5, highlighting their inherent ability to produce LC-PUFAs from C18 PUFAs and suggesting potential AA or docosapentaenoic acidn-6 (DPAn-6) biosynthesis. However, even with this biosynthetic capacity, the trout fed reduced dietary AA had higher mortality rates. The diet had no effect on final weight (3.38 g on average for the three diets). Conversely, increased dietary AA enhanced eicosanoid production from AA, suggesting potential inflammatory and oxidative consequences. This was further evidenced by an increase in non-enzymatic lipid oxidation metabolites, particularly in the AA-2.5% diet group, which had higher levels of phytoprostanes and isoprostanes, markers of cellular oxidative damage. Importantly, the AA-1.1% diet proved to be particularly beneficial for stress resilience. This was evidenced by higher post-stress turnover rates of serotonin and dopamine, neurotransmitters central to the fish's stress response. In conclusion, a dietary AA intake of 1.1% of total fatty acids appears to promote overall resilience in rainbow trout fry.

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.

Remodeling of lipid landscape in high fat fed very-long chain acyl-CoA dehydrogenase null mice favors pro-arrhythmic polyunsaturated fatty acids and their downstream metabolites.

Very-long chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial step of mitochondrial long chain (LC) fatty acid ß-oxidation (FAO). Inherited VLCAD deficiency (VLCADD) predisposes to neonatal arrhythmias whose pathophysiology is still not understood. We hypothesized that VLCADD results in global disruption of cardiac complex lipid homeostasis, which may set conditions predisposing to arrhythmia. To test this, we assessed the cardiac lipidome and related molecular markers in seven-month-old VLCAD-/- mice, which mimic to some extent the human cardiac phenotype. Mice were sacrificed in the fed or fasted state after receiving for two weeks a chow or a high-fat diet (HFD), the latter condition being known to worsen symptoms in human VLCADD. Compared to their littermate counterparts, HFD/fasted VLCAD-/- mouse hearts displayed the following lipid alterations: (1) Lower LC, but higher VLC-acylcarnitines accumulation, (2) higher levels of arachidonic acid (AA) and lower docosahexaenoic acid (DHA) contents in glycerophospholipids (GPLs), as well as (3) corresponding changes in pro-arrhythmogenic AA-derived isoprostanes and thromboxane B2 (higher), and anti-arrythmogenic DHA-derived neuroprostanes (lower). These changes were associated with remodeling in the expression of gene or protein markers of (1) GPLs remodeling: higher calcium-dependent phospholipase A2 and lysophosphatidylcholine-acyltransferase 2, (2) calcium handling perturbations, and (3) endoplasmic reticulum stress. Altogether, these results highlight global lipid dyshomeostasis beyond FAO in VLCAD-/- mouse hearts, which may set conditions predisposing the hearts to calcium mishandling and endoplasmic reticulum stress and thereby may contribute to the pathogenesis of arrhythmias in VLCADD in mice as well as in humans.

Sex dimorphism of glucosensing parameters and appetite-regulating peptides in the hypothalamus of rainbow trout broodstocks.

Rainbow trout (Oncorhynchus mykiss) is traditionally considered as a poor user of digestible carbohydrates harbouring persistent postprandial hyperglycaemia and decreased growth performances when fed a diet containing more than 20% of digestible carbohydrates. While this glucose-intolerant phenotype is well-described in juveniles, evidence points to a particular regulation of glucose metabolism in rainbow trout broodstrocks. By detecting changes in glucose levels and triggering a specific metabolic response, the hypothalamus plays a key role in the regulation of peripheral glucose metabolism. Therefore, our objective was to assess, for the first time in fish, the short-term consequences in hypothalamus, the glucose sensing and feed intake regulation of feeding mature female and male, and neomale rainbow trout with a diet containing either no or a 33% carbohydrate. The hypothalamic glucosensing capacity was assessed through mRNA levels of glucosensing related-genes and feed intake regulation through appetite-regulating peptides. Our data indicate that a brief period of carbohydrate intake (5 meals at 8 °C) did not induce specific changes in glucosensing capacity and appetite-regulating peptides in the hypothalamus of rainbow trout broodstock. Our results did however demonstrate, for the first time in fish, the existence of sex dimorphism of glucosensing-related genes and appetite-regulating peptides.

Characterization of free fatty acid receptor family in rainbow trout (Oncorhynchus mykiss): towards a better understanding of their involvement in fatty acid signalisation.

Since 20 years of research, free fatty acids receptors (FFARs) have received considerable attention in mammals. To date, four FFARs (FFAR1, FFAR2, FFAR3 and FFAR4) are especially studied owing to their physiological importance in various biological processes. This ubiquitist group of G protein-coupled receptors (GPCRs) are majors reports in the key physiological functions such as the regulation of energy balance, metabolism or fatty acid sensing. However, up till date, even some studies were interested in their potential involvement in fatty acid metabolism, no genome investigation of these FFARs have been carried out in teleost fish. Through genome mining and phylogenetic analysis, we identified and characterised 7 coding sequences for ffar2 in rainbow trout whereas no ffar3 nor ffar4 gene have been found. This larger repertoire of ffar2 genes in rainbow trout results from successive additional whole-genome duplications which occurred in early teleosts and salmonids, respectively. A syntenic analysis was used to assign a new nomenclature to the salmonid ffar2 and showed a clear conservation of genomic organisation, further supporting the identity of these genes as ffar2. RT-qPCR was then used to examine, firstly during ontogenesis and secondly on feeding response the expression pattern of ffar1 and ffar2 genes in proximal gut and brain of all trout ffar genes. Overall, this study presents a comprehensive overview of the ffar family in rainbow trout.

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.

Detection and Modulation of Olfactory Sensing Receptors in Carnivorous Rainbow Trout (Oncorhynchus mykiss) Fed from First Feeding with Plant-Based Diet.

Sense of smell is mediated by diverse families of olfactory sensing receptors, conveying important dietary information, fundamental for growth and survival. The aim of this study was to elucidate the role of the sensory olfactory pathways in the regulation of feeding behavior of carnivorous rainbow trout (RT, Oncorhynchus mykiss), from first feeding until 8 months. Compared to a commercial diet, RT fed with a total plant-based diet showed drastically altered growth performance associated with feed intake from an early stage. Exhaustive examination of an RT genome database identified three vomeronasal type 1 receptor-like (ORA), 10 vomeronasal type 2 receptor-like (OLFC) and 14 main olfactory receptor (MOR) genes, all highly expressed in sensory organs, indicating their potential functionality. Gene expression after feeding demonstrated the importance in olfactory sensing perception of some OLFC (olfcg6) and MOR (mor103, -107, -112, -113, -133) receptor family genes in RT. The gene ora1a showed evidence of involvement in olfactory sensing perception for fish fed with a commercial-like diet, while ora5b, mor118, mor124 and olfch1 showed evidence of involvement in fish fed with a plant-based diet. Results indicated an impact of a plant-based diet on the regulation of olfactory sensing pathways as well as influence on monoaminergic neurotransmission in brain areas related to olfactory-driven behaviors. The overall findings suggest that feeding behavior is mediated through olfactory sensing detection and olfactory-driven behavior pathways in RT.

Effects of Low Stocking Densities on Zootechnical Parameters and Physiological Responses of Rainbow Trout (Oncorhynchus mykiss) Juveniles.

The present study investigated the effect of low stocking density on growth, survival, feed parameters and physiological responses (blood metabolites, welfare indicators, immune biomarkers, and transcriptomic responses of stress and immune-related genes) on juvenile rainbow trout (Oncorhynchus mykiss) reared under a recirculating aquaculture system during 12 weeks. Fish (average weight 29.64 g) were reared in triplicate under four initial densities: nine fish per tank (D9, 3.76 ± 0.06 kg/m3), 18 fish per tank (D18, 7.66 ± 0.18 kg/m3), 27 fish per tank (D27, 9.67 ± 0.01 kg/m3) and 36 fish per tank (D36, 12.94 ± 0.14 kg/m3). Results showed that lower stocking density D9 significantly altered survival with several fish dying during the experiment and an alteration of growth and feed efficiency for the remaining fish. In parallel, the study revealed that low stocking density induced a chronic stress altering the physiological responses of trout by dysregulation of the inflammatory, immune system, and indolamine/catecholamine brain levels. In conclusion, regarding all the variables observed, low stocking density (D9) alters survival, growth and feed efficiency of rainbow trout with alteration of their physiological responses. Selecting appropriate fish density relating to rearing conditions proved to be an essential concern to improve welfare in an aquaculture context.

Polylactide Nanocapsules Attenuate Adverse Cardiac Cellular Effects of Lyso-7, a Pan-PPAR Agonist/Anti-Inflammatory New Thiazolidinedione.

Lyso-7 is a novel synthetic thiazolidinedione, which is a receptor (pan) agonist of PPAR α,ß/δ,γ with anti-inflammatory activity. We investigated the cardiotoxicity of free Lyso-7 in vitro (4.5-450 nM), and Lyso-7 loaded in polylactic acid nanocapsules (NC) in vivo (Lyso-7-NC, 1.6 mg/kg). In previous work, we characterized Lyso-7-NC. We administered intravenously Lyso-7, Lyso-7-NC, control, and blank-NC once a day for seven days in mice. We assessed cell contraction and intracellular Ca2+ transients on single mice cardiomyocytes enzymatically isolated. Lyso-7 reduced cell contraction and accelerated relaxation while lowering diastolic Ca2+ and reducing Ca2+ transient amplitude. Lyso-7 also promoted abnormal ectopic diastolic Ca2+ events, which isoproterenol dramatically enhanced. Incorporation of Lyso-7 in NC attenuated drug effects on cell contraction and prevented its impact on relaxation, diastolic Ca2+, Ca2+ transient amplitude, Ca2+ transient decay kinetics, and promotion of diastolic Ca2+ events. Acute effects of Lyso-7 on cardiomyocytes in vitro at high concentrations (450 nM) were globally similar to those observed after repeated administration in vivo. In conclusion, we show evidence for off-target effects of Lyso-7, seen during acute exposure of cardiomyocytes to high concentrations and after repeated treatment in mice. Nano-encapsulation of Lyso-7 in polymeric NC attenuated the unwanted effects, particularly ectopic Ca2+ events known to support life-threatening arrhythmias favored by stress or exercise.

Long chain omega-3 fatty acids and their oxidized metabolites are associated with reduced prostate tumor growth.

INTRODUCTION: Cancer has been associated with increased oxidative stress and deregulation of bioactive oxylipins derived from long-chain polyunsaturated fatty acids (LC-PUFA) like arachidonic acid (AA). There is a debate whether ω-3 LC-PUFA could promote or prevent prostate tumor growth through immune modulation and reduction of oxidative stress. Our aim was to study the association between enzymatically or non-enzymatically produced oxidized-LC-PUFA metabolites and tumor growth in an immune-competent eugonadal and castrated C57BL/6 male mice injected with TRAMP-C2 prostate tumor cells, fed with ω-3 or ω-6 LC-PUFA-rich diets. MATERIALS AND METHODS: Tumor fatty acids were profiled by gas chromatography and 26 metabolites derived from either AA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were assessed by liquid chromatography-mass spectrometry. RESULTS: The enriched ω-3 diet did not reduce oxidative stress overall in tumors but favored the formation of ω-3 rather than ω-6 derived isoprostanoids. We discovered that EPA and its oxidized-derivatives like F3-isoprostanes and prostaglandin (PG)F3α, were inversely correlated with tumor volume (spearman correlations and T-test, p<0.05). In contrast, F2-isoprostanes, adrenic acid, docosapentaenoic acid (DPAω-6) and PGE2 were positively correlated with tumor volume. Interestingly, F4-neuroprostanes, PGD2, PGF2α, and thromboxane were specifically increased in TRAMP-C2 tumors of castrated mice compared to those of eugonadal mice. DISCUSSION: Decreasing tumor growth under ω-3 diet could be attributed in part to increased levels of EPA and its oxidized-derivatives, a reduced level of pro-angiogenic PGE2 and increased levels of F4-neuroprostanes and resolvins content in tumors, suspected of having anti-proliferative and anti-inflammatory effects.

Characterization and modulation of brain lipids content of rainbow trout fed with 100% plant based diet rich in omega-3 long chain polyunsaturated fatty acids DHA and EPA.

Brain functions are known to be mainly modulated by adequate dietary intake. Inadequate intake as can be an excess or significant deficiency affect cognitive processes, behavior, neuroendocrine functions and synaptic plasticity with protective or harmful effects on neuronal physiology. Lipids, in particular, ω-6 and ω-3 long chain polyunsaturated fatty acids (LC-PUFAs) play structural roles and govern the different functions of the brain. Hence, the goal of this study was to characterize the whole brain fatty acid composition (precursors, enzymatic and non-enzymatic oxidation metabolites) of fish model of rainbow trout fed with three experimental plant-based diet containing distinct levels of eicosapentaenoic acid (EPA, 20:5 ω-3) and docosahexaenoic acid (DHA, 22:6 ω-3) (0% for low, 15.7% for medium and 33.4% for high, total fatty acid content) during nine weeks. Trout fed with the diet devoid of DHA and EPA showed reduced brain content of total ω-3 LC-PUFAs, with diminution of EPA and DHA. Selected enzymatic (cyclooxygenases and lipoxygenases) oxidation metabolites of arachidonic acid (AA, 20:4 ω-6) decrease in medium and high ω-3 LC-PUFAs diets. On the contrary, total selected enzymatic oxidation metabolites of DHA and EPA increased in high ω-3 LC-PUFAs diet. Total selected non-enzymatic oxidation metabolites of DHA (not detected for EPA) increased in medium and high ω-3 LC-PUFAs diets. In conclusion, this work revealed for the first time in fish model the presence of some selected enzymatic and non-enzymatic oxidation metabolites in brain and the modulation of brain lipid content by dietary DHA and EPA levels.

Fish oil supplementation alleviates metabolic and anxiodepressive effects of diet-induced obesity and associated changes in brain lipid composition in mice.

OBJECTIVE: Obesity significantly elevates the odds of developing mood disorders. Chronic consumption of a saturated high-fat diet (HFD) elicits anxiodepressive behavior in a manner linked to metabolic dysfunction and neuroinflammation in mice. Dietary omega-3 polyunsaturated fatty acids (n-3 PUFA) can improve both metabolic and mood impairments by relieving inflammation. Despite these findings, the effects of n-3 PUFA supplementation on energy homeostasis, anxiodepressive behavior, brain lipid composition, and gliosis in the diet-induced obese state are unclear. METHODS: Male C57Bl/6J mice were fed a saturated high-fat diet (HFD) or chow for 20 weeks. During the last 5 weeks mice received daily gavage ("supplementation") of fish oil (FO) enriched with equal amounts of docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) or control corn oil. Food intake and body weight were measured throughout while additional metabolic parameters and anxiety- and despair-like behavior (elevated-plus maze, light-dark box, and forced swim tasks) were evaluated during the final week of supplementation. Forebrain lipid composition and markers of microglia activation and astrogliosis were assessed by gas chromatography-mass spectrometry and real-time PCR, respectively. RESULTS: Five weeks of FO supplementation corrected glucose intolerance and attenuated hyperphagia in HFD-induced obese mice without affecting adipose mass. FO supplementation also defended against the anxiogenic and depressive-like effects of HFD. Brain lipids, particularly anti-inflammatory PUFA, were diminished by HFD, whereas FO restored levels beyond control values. Gene expression markers of brain reactive gliosis were supressed by FO. CONCLUSIONS: Supplementing a saturated HFD with FO rich in EPA and DHA corrects glucose intolerance, inhibits food intake, suppresses anxiodepressive behaviors, enhances anti-inflammatory brain lipids, and dampens indices of brain gliosis in obese mice. Together, these findings support increasing dietary n-3 PUFA for the treatment of metabolic and mood disturbances associated with excess fat intake and obesity.

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.

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.

Rainbow trout prefer diets rich in omega-3 long chain polyunsaturated fatty acids DHA and EPA.

The control of feed intake in fish in aquaculture requires the development of new techniques to improve diet composition, feed conversion efficiency and growth. The aim must be sustainability and an effective use of resources. The effect of replacing traditional aqua-feed ingredients (fishmeal and fish oil) by a 100% plant-based diet is known to drastically decrease fish performance (survival and growth). The present study examined the feed preference of rainbow trout Oncorhynchus mykiss for three diets containing distinct levels of omega-3 long chain polyunsaturated fatty acids (ω-3 LCPUFA): eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (0% for low, 5% for medium and 20% for high, total fatty acid content). Feed preference values for each group (low v. medium ω-3 diets, medium v. high ω-3 diets and low v. high ω-3 diets) were observed using two self-feeders positioned at opposite sides of the tank. The hypothesis was that the decrease of fish growth and survival rate of fish fed with 100% plant-based diet could be explained by the absence of ω-3 LCPUFA relating to decrease of food intake. This could explain the tasting role of ω-3 LCPUFA in the feeding behavior of rainbow trout (which reflects the motivation to consume feed). The results showed that rainbow trout could discriminate between the diets containing different level of ω-3 LCPUFA even if unable to differentiate between level of 5% (no preference observed in low v. medium ω-3 diets). Overall they had a preference for diet high in ω-3 LCPUFA: 59.5% preference for high ω-3 diet in high v. low ω-3 diets, and 75.6% preference for high ω-3 diet in medium v. high ω-3 diets respectively. This preference was repeated after 21 days and for a further 21 days when the feeds were exchanged between the two self- feeders in each tank: 63.3% preference for high ω-3 diet in high v. low ω-3 diets, and 69,5% preference for high ω-3 diet in medium v. high ω-3 diets respectively. The tests also indicated a difference in the extent of food waste of each of the three diets revealed by uneaten pellets after feed demands. During two periods of test, high ω-3 diet was the most appreciated, the least wasted and the most eaten (all choice groups) whereas the most uneaten feed remained the least appreciated diet in three choices diets (low ω-3 diet in low v. medium ω-3 diets, medium in medium v. high ω-3 diets and low in low v. high ω-3). In conclusion, this study highlighted the influence of ω-3 LCPUFA in the feeding behavior of juvenile rainbow trout, levels of ω-3 LCPUFA drove dietary choices in the fish.

Early calcium handling imbalance in pressure overload-induced heart failure with nearly normal left ventricular ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a common clinical syndrome associated with high morbidity and mortality. Therapeutic options are limited due to a lack of knowledge of the pathology and its evolution. We investigated the cellular phenotype and Ca2+ handling in hearts recapitulating HFpEF criteria. HFpEF was induced in a portion of male Wistar rats four weeks after abdominal aortic banding. These animals had nearly normal ejection fraction and presented elevated blood pressure, lung congestion, concentric hypertrophy, increased LV mass, wall stiffness, impaired active relaxation and passive filling of the left ventricle, enlarged left atrium, and cardiomyocyte hypertrophy. Left ventricular cell contraction was stronger and the Ca2+ transient larger. Ca2+ cycling was modified with a RyR2 mediated Ca2+ leak from the sarcoplasmic reticulum and impaired Ca2+ extrusion through the Sodium/Calcium exchanger (NCX), which promoted an increase in diastolic Ca2+. The Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2a) and NCX protein levels were unchanged. The phospholamban (PLN) to SERCA2a ratio was augmented in favor of an inhibitory effect on the SERCA2a activity. Conversely, PLN phosphorylation at the calmodulin-dependent kinase II (CaMKII)-specific site (PLN-Thr17), which promotes SERCA2A activity, was increased as well, suggesting an adaptive compensation of Ca2+ cycling. Altogether our findings show that cardiac remodeling in hearts with a HFpEF status differs from that known for heart failure with reduced ejection fraction. These data also underscore the interdependence between systolic and diastolic "adaptations" of Ca2+ cycling with complex compensative interactions between Ca2+ handling partner and regulatory proteins.

Biological activities of non-enzymatic oxygenated metabolites of polyunsaturated fatty acids (NEO-PUFAs) derived from EPA and DHA: New anti-arrhythmic compounds?

ω3 Polyunsaturated fatty acids (ω3 PUFAs) have several biological properties including anti-arrhythmic effects. However, there are some evidences that it is not solely ω3 PUFAs per se that are biologically active but the non-enzymatic oxygenated metabolites of polyunsaturated fatty acids (NEO-PUFAs) like isoprostanes and neuroprostanes. Recent question arises how these molecules take part in physiological homeostasis, show biological bioactivities and anti-inflammatory properties. Furthermore, they are involved in the circulations of childbirth, by inducing the closure of the ductus arteriosus. In addition, oxidative stress which can be beneficial for the heart in given environmental conditions such as the presence of ω3 PUFAs on the site of the stress and the signaling pathways involved are also explained in this review.

Primary microglia isolation from mixed cell cultures of neonatal mouse brain tissue.

BACKGROUND: Microglia are the main resident immunological cells of the central nervous system (CNS) that are functionally equivalent to macrophages. However, due to the cellular heterogeneity of the brain, it is technically challenging to obtain highly specific, healthy microglia with the desired phenotype in sufficient yield for in vivo experiments. NEW METHOD: This study presents a new and easy method for the isolation of microglia cells from mouse pups (P1-P3). This method consists of a 20-day protocol, divided in three sections: mixed cell culture, culture maintaining (astrocytes growing), and isolation after astrocytes confluence. RESULTS: This procedure produces microglia with no astrocyte, neuron and oligodendrocyte precursors cells contamination that are functionally active to answer inflammatory responses based on the measurement of cell and inflammatory markers. This technique requires approximately three hours for the isolation of neonatal mixed cell culture, 20 to 22 days for microglia growing and two days before starting experiments from pure and healthy microglia. COMPARISON WITH EXISTING METHODS AND CONCLUSIONS: This study presents an isolation protocol that is adapted from existing methods and is economic, rapid, not tedious, with little manipulation time and work. This method also allows to isolate large amount of high specific microglia cells with no specific phenotype and with great reproducibility and efficiently. This study provides a detailed description of the methods that is routinely used in our laboratory for the isolation and the culture of microglia, with emphasis on the steps that are deemed most critical for obtaining large amount of pure and healthy cultures.